Research Items (114)
- Jun 2018
This study examines the action of hypoxia on integrity, fluidity and protein composition of red blood cell (RBC) membrane. Twenty-min exposure to oxygen-free environment decreases rat RBC integrity documented by 3-fold elevation of hemoglobin release without any action on the membrane fluidity estimated by electron magnetic resonance spectroscopy of spin-labeled stearic acid analogues. The proteomics technology in combination with relative label free quantification analysis revealed a dozen of membrane-bound proteins, including elevated content of hemoglobin, reproducibly affected by hypoxia. Mapping the identified proteins in the KEGG pathway database we found that the proteins of multi subunit Cullin-Rbx E3 ubiquitin ligase complex are presented in normoxic RBC ghosts but not in the hypoxic samples. Our results suggest that Cullin-Rbx E3 complex, associated with RBC membrane in normoxia, provides detection and deletion of membrane proteins damaged by reactive oxygen species. In hypoxic conditions, deoxy-Hb binds to band 3 protein, resulting in dissociation of Cullin-Rbx E3 complex from RBC membrane and impaired clearance of damaged cytoskeleton proteins. These rearrangements of membrane proteins might be involved in attenuated membrane integrity revealed in hypoxic RBC. Significance: This study demonstrate that sustained deoxygenation of rat erythrocytes alters the composition of membrane-bound proteins including elevation of the content of hemoglobin without any changes in the viscosity of erythrocyte membrane lipid bilayer. These results suggest that the changes the composition of membrane proteins result in attenuated membrane integrity and contribute to augment release of hemoglobin seen in hypoxic conditions.
- May 2018
A method to simultaneously assess the changes in intracellular calcium concentration and cell volume in single cells was developed using the Ca²⁺-sensitive fluorescent probe Fura-2 and a three-dimensional image-surface reconstruction technique, respectively. Studies with this method showed that Fura-2 loading had no significant effect on the kinetics of A549 human epithelial cell swelling in a hypotonic solution, as well as the volume restoration kinetics. Significant changes in intracellular Ca²⁺ concentration were not observed in the examined volume modulation range. The results suggest that Ca²⁺-mediated signaling pathways are not involved in the autoregulation of the cell volume in A549 cells exposed to hypotonic conditions.
- Apr 2018
Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat erythrocytes. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5- and 2.5-fold, respectively. In rat erythrocytes a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL 67156. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia-induced ATP release from rat erythrocytes. The obtained results indicate a key role of hemolysis in the ATP release from red blood cells.
- Jan 2018
Red blood cells are involved not only in transportation of oxygen and carbon dioxide but also in autoregulation of vascular tone by ATP release in hypoxic conditions. Molecular mechanisms of the ATP release from red blood cells in response to a decrease in partial oxygen pressure still remain to be elucidated. In this work we have studied effects of hypoxia on red blood cell hemolysis in humans and rats and compared the effects of inhibitors of ecto-ATPase and pannexin on the release of ATP and hemoglobin from rat red blood cells. The 20-min hypoxia at 37°C increased hemolysis of red blood cells in humans and rats 1.5 and 2.5 times, respectively. In rat red blood cells a significant increase in hypoxia-induced extracellular ATP level was found only in the presence of ecto-ATPase inhibitor ARL67166. In these conditions we observed a positive correlation (R² = 0.5003) between the increase in free hemoglobin concentration and the ATP release. Neither carbenoxolon, nor probenecid, the inhibitors of low-selectivity pannexin channels, altered the hypoxia- induced ATP release from rat red blood cells. The obtained results indicate the key role of hemolysis in the ATP release from red blood cells.
- Jan 2018
The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD). The downstream signaling pathways include reorganization of 2D cytoskeleton and altered composition of polyphosphoinositides located on the inner surface of the plasma membrane. In addition to its action on physico-chemical properties of cytoplasmic hydrogel, cell volume changes in anisosmotic conditions affect the ionic strength of the cytoplasm and the [Na⁺]i/[K⁺]i ratio. Elevated intracellular ionic strength evoked by long term exposure of cells to hypertonic environment resulted in the activation of TonEBP and augmented expression of genes controlling intracellular organic osmolyte levels. The role of Na⁺i/K⁺i -sensitive, Ca²⁺i -mediated and Ca²⁺i-independent mechanisms of excitation-transcription coupling in cell volume-adjustment remains unknown.
Abnormalities of mucus viscosity play a critical role in the pathogenesis of several respiratory diseases, including cystic fibrosis. Currently, there are no approaches to assess the rheological properties of mucin granule matrices in live cells. This is the first example of the use of a molecular rotor, a BODIPY dye, to quantitatively visualize the viscosity of intragranular mucin matrices in a large population of individual granules in differentiated primary bronchial epithelial cells using fluorescence lifetime imaging microscopy.
Intravascular hemolysis occurs in hereditary, acquired, and iatrogenic hemolytic conditions but it could be also a normal physiological process contributing to intercellular signaling. New evidence suggests that intravascular hemolysis and the associated release of adenosine triphosphate (ATP) may be an important mechanism for in vivo local purinergic signaling and blood flow regulation during exercise and hypoxia. However, the mechanisms that modulate hypoxia-induced RBC membrane fragility remain unclear. Here, we provide an overview of the role of RBC ATP release in the regulation of vascular tone and prevailing assumptions on the putative release mechanisms. We show importance of intravascular hemolysis as a source of ATP for local purinergic regulation of blood flow and discuss processes that regulate membrane propensity to rupture under stress and hypoxia.
- Feb 2017
Cell swelling is a potent ATP secretion stimulus. While the sources of ATP are known, it's mechanisms of secretion in epithelial cells remain unclear. Two secretion pathways have been proposed: exocytosis of nucleotide-containing vesicles or conductive release through pore-forming channels, like pannexins or connexins. In this project, we have tested whether ATP is released or not through a conductive pathway in A549 lung epithelial cells. Real-time ATP secretion from glass-adherent A549 cells was imaged with a custom-designed lens system, combining a large field of view and a high light-gathering power, mounted directly on a Photometrics® EvolveTM 512 EMCCD camera. ATP was detected with a light-emitting luciferin-luciferase reaction. Secretion was stimulated by acute cell swelling or exposure to ionomycin (10 µM). Plasma membrane permeability to large molecules was assessed in parallel by propidium iodide (PI) uptake with epifluorescence Zeiss Axio Observer microscope. Cells were grown in 10% (control cells) or 0.1% FBS (fragile cells). Our novel imaging system revealed a strong homogeneous ATP secretion lasting several minutes from control A549 cells stimulated with 50% hypotonic shock. Yet, no PI-stained cells were detected under these conditions. In contrast, cells rendered fragile, and thus prone to cell lysis, showed an appreciable amount of PI-stained cells post-hypotonic shock and a punctuate pattern of ATP secretion co-localizing with PI-positive cells. Sudden intracellular calcium elevation with ionomycin provoked a rapid, albeit small, ATP secretion. Based on the fact that hypotonically-induced ATP release in normal conditions was not accompanied with detectable permeation of PI, we conclude that ATP in A549 cells is released by a non-conductive pathway.
- Jan 2017
An upregulation of Egr-1 expression has been reported in models of atherosclerosis and intimal hyperplasia and, various vasoactive peptides and growth promoting stimuli have been shown to induce the expression of Egr-1 in vascular smooth muscle cells (VSMC). Angiotensin-II (Ang-II) is a key vasoactive peptide that has been implicated in the pathogenesis of vascular diseases. Ang-II elevates intracellular Ca(2+) through activation of the store-operated calcium entry (SOCE) involving an inositol-3-phosphate receptor (IP3R)-coupled depletion of endoplasmic reticular Ca(2+) and a subsequent activation of the stromal interaction molecule 1 (STIM-1) /Orai-1 complex. However, the involvement of IP3R/STIM-1/Orai-1-Ca(2+-) dependent signaling in Egr-1 expression in VSMC remains unexplored. Therefore, in the present studies, we have examined the role of Ca(2+) signaling in Ang-II-induced Egr-1 expression in VSMC and investigated the contribution of STIM-1 or Orai-1 in mediating this response. 2-aminoethoxydiphenyl borate (2-APB), a dual non-competitive antagonist of IP3R and inhibitor of SOCE, decreased Ang-II-induced Ca(2+) release and attenuated Ang-II-induced enhanced expression of Egr-1 protein and mRNA levels. Egr-1 upregulation was also suppressed following blockade of calmodulin and CaMKII. Furthermore, RNA interference-mediated depletion of STIM-1 or Orai-1 attenuated Ang-II-induced Egr-1 expression as well as Ang-II-induced phosphorylation of ERK1/2 and CREB. In addition, siRNA-induced silencing of CREB resulted in a reduction in the expression of Egr-1 stimulated by Ang-II. In summary, our data demonstrate that Ang-II-induced Egr-1 expression is mediated by STIM-1/Orai-1/Ca(2+-) dependent signaling pathways in A-10 VSMC. This article is protected by copyright. All rights reserved.
- Oct 2016
Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca(2+) signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50 % hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca(2+) concentration ([Ca(2+)]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20 min and followed, during the next 20 min, by RVD of 60 ± 7 % of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca(2+)]i. Loading with intracellular Ca(2+) chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82 % of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca(2+)-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.
- Sep 2016
Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucocilliary clearance, surfactant secretion and local blood flow. Cellular ATP release is mechanosensitive, however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real-time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex-vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into airspaces brief inflation of such edematous lung (1-s, ~20 cmH2O) induced transient (<30s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ~10-6 M, relevant for autocrine/paracrine signaling but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus-dependent, prolonged (100-s) inflation evoked long-lasting ATP release which terminated upon alveoli deflation/de-recruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patch-like areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP-release imaging in ex-vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung airspaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.
- Jul 2016
Background/aims: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. Methods: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. Results: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. Conclusion: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.
- Sep 2015
In this issue of Blood, 2 independent reports by Rapetti-Mauss et al and Glogowska et al describe the first example of a human disease: a novel type of hemolytic hereditary xerocytosis (HX) that is caused by a genetic defect in the Gardos channel, a Ca2+-sensitive K+ channel critical for red cell volume regulation, survival, and function.
Extensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K(+) channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before. Wound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair. Taken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.
Recently we found that cytoplasm of permeabilized mammalian cells behaves as a hydrogel displaying intrinsic osmosensitivity. This study examined the role of microfilaments and microtubules in the regulation of hydrogel osmosensitivity, volume-sensitive ion transporters, and their contribution to volume modulation of intact cells. We found that intact and digitonin-permeabilized A549 cells displayed similar rate of shrinkage triggered by hyperosmotic medium. It was significantly slowed-down in both cell preparations after disruption of actin microfilaments by cytochalasin B, suggesting that rapid water release by intact cytoplasmic hydrogel contributes to hyperosmotic shrinkage. In hyposmotic swelling experiments, disruption of microtubules by vinblastine attenuated the maximal amplitude of swelling in intact cells and completely abolished it in permeabilized cells. The swelling of intact cells also triggered ~10-fold elevation of furosemide-resistant (86)Rb(+) (K(+)) permeability and the regulatory volume decrease (RVD), both of which were abolished by Ba(2+). Interestingly, RVD and K(+) permeability remained unaffected in cytocholasin/vinblastine treated cells demonstrating that cytoskeleton disruption has no direct impact on Ba(2+)-sensitive K(+)-channels involved in RVD. Our results show, for the first time, that the cytoskeleton network contributes directly to passive cell volume adjustments in anisosmotic media via the modulation of the water retained by the cytoplasmic hydrogel. Copyright © 2015. Published by Elsevier B.V.
A drop in oxygen partial pressure results in elevation of blood vessel diameter. It has been demonstrated that isolated vessels exhibit this unique feature only when they are perfused in the presence of erythrocytes. More recently, it was shown that haemoglobin plays a key role in oxygen sensing. Its deoxygenated form interacts with band 3 protein, triggering the cascade of non-identified intracellular signals involved in nitric oxide production and release of ATP interacting with P2Y purinergic receptors in endothelial cells. In this review, we summarize the data on mechanisms of ATP release from erythrocytes, as well as on its physiological and pathophysiological implications.
To investigate the role of adenosine tri-phosphate (ATP) purinergic signaling in mast cells (MCs) modulated by heat to further understand the molecular mechanisms of moxibustion. Skin temperatures induced by monkshood cake moxibustion were evaluated by measuring the Neiguan acupoint (PC 6) from 31 participants with a digital thermocouple thermometer. Temperatures of 43 °C and 52 °C were applied to cultured human leukemia mast cell line HMC-1 in vitro. Calcium fluorescence was applied to detect intracellular Ca2+ ([Ca2+]). Extracellular ATP contents were measured by luciferin-luciferase assay. Maximum skin temperatures mostly ranged from 40-45 °C , but some reached up to 50 °C. Both 43 °C and 52 °C induced MC degranulation, which was accompanied by an increase in [Ca2+] and ATP release. Complexing extracellular Ca2+ with 5 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the noxious, heat-induced elevation of [Ca2+]i and prevented the enhanced ATP secretion by those. cells at 52 °C, but not 43 °C. Monkshood cake moxibustion can generate heat sufficient to trigger cellular events of MCs, including degranulation, [Ca2+]i elevation, and ATP release, suggesting that purinergic signals originating from MCs are possibly the initiating response of acupoints to moxibustion.
Low-level-laser therapy (LLLT) is an effective complementary treatment, especially for anti-inflammation and wound healing in which dermis or mucus mast cells (MCs) are involved. In periphery, MCs crosstalk with neurons via purinergic signals and participate in various physiological and pathophysiological processes. Whether extracellular ATP, an important purine in purinergic signaling, of MCs and neurons could be modulated by irradiation remains unknown. In this study, effects of red-laser irradiation on extracellular ATP content of MCs and dorsal root ganglia (DRG) neurons were investigated and underlying mechanisms were explored in vitro. Our results show that irradiation led to elevation of extracellular ATP level in the human mast cell line HMC-1 in a dose-dependent manner, which was accompanied by elevation of intracellular ATP content, an indicator for ATP synthesis, together with [Ca(2+)]i elevation, a trigger signal for exocytotic ATP release. In contrast to MCs, irradiation attenuated the extracellular ATP content of neurons, which could be abolished by ARL 67156, a nonspecific ecto-ATPases inhibitor. Our results suggest that irradiation potentiates extracellular ATP of MCs by promoting ATP synthesis and release and attenuates extracellular ATP of neurons by upregulating ecto-ATPase activity. The opposite responses of these two cell types indicate complex mechanisms underlying LLLT.
Osmotic perturbations, occurring frequently under physiological and pathological conditions, alter cell size/volume and function. To protect cellular homeostasis, cell osmo- and volume-sensing mechanisms activate volume compensatory processes. The plasma membrane plays a prominent role in cell volume regulation by mediating the selective transport of extra- and intracellular osmolytes. The function of the membrane-enclosed cytoplasm in osmosensing and cell volume homeostasis is much less appreciated. We present current concepts and discuss evidence of cell volume sensors with emphasis on the hydrogel nature of the mammalian cytoplasm and its intrinsic osmosensitivity.
The hypothesis that regulated ATP release from red blood cells (RBCs) contributes to nitric oxide-dependent control of local blood flow has sparked much interest in underlying release mechanisms. Several stimuli, including shear stress and hypoxia, have been found to induce significant RBC ATP release attributed to activation of ATP-conducting channels. In the present study, we first evaluated different experimental approaches investigating stimulated RBC ATP release and quantifying hemolysis. We then measured ATP and free hemoglobin in each and every RBC supernatant sample to directly assess the contribution of hemolysis to ATP release. Hypotonic shock, shear stress, hypoxia, but not cAMP agonists, significantly enhanced ATP release. It tightly correlated, however, with free hemoglobin in RBC supernatants, indicating that lysis was responsible for most, if not all, ATP release. Luminescence ATP imaging combined with simultaneous infrared cell imaging showed that ATP was released exclusively from lysing cells with no contribution from intact cells. In summary, with all stimuli tested, we found no evidence of regulated ATP release from intact RBCs other than by cell lysis. Such a release mechanism might be physiologically relevant in vivo, e.g., during exercise and hypoxia where intravascular hemolysis, predominantly of senescent cells, is augmented.
Upstream intermediates of intracellular signaling involved in cell volume regulation remain poorly explored. Recently, we demonstrated that osmolarity-induced volume changes in permeabilized cells were several-fold higher than those observed with intact cells, indicating the osmosensing properties of cytoplasmic gel. To further examine the role of cytoplasmic biogel in cell volume regulation, we compared the action of short-term heat treatment on volume changes in intact and permeabilized A549 cells. Pretreatment of A549 cells at 48 °C suppressed swelling triggered by dissipation of Donnan's equilibrium as well as by hyposmotic medium. Significantly, heat treatment completely abolished the action of hyposomotic medium on volume changes in permeabilized cells, showing that temperature elevation suppresses osmosensing properties via its effect on biogel rather than on plasma membrane water permeability. Identical heat treatment blocked the regulatory volume decrease (RVD) as well as the increment of Ba(2+)-sensitive K(+)-channel activity seen in control cells exposed to hyposmotic swelling. Unlike swelling, hyperosmotic shrinkage was decreased by twofold in cells subjected to 10-min preincubation at 50 °C. Our results disclose that osmosensing by cytoplasmic gel is a key event in the RVD triggered by hypotonic swelling. The role of biogel and plasma membrane in intracellular signaling triggered by hyperosmotic shrinkage should be further investigated.
- Feb 2014
Typically the signal-to-background ratio is the limiting aspect of fluorescence-based detecting and imaging. The background signal can be composed of a variety of sources-excitation scattering, contaminants, and autofluorescence from cellular constituents. Most of these sources have a short-lived lifetime (ps to ns range). In order to increase the signal-to-background ratio, fluorophores with high brightness or in large concentrations are typically used along with time-gated detection. This unfortunately sacrifices the probe's signal unless it has a very long lifetime. Herein we are presenting a simple method to enhance the detection of widely available and well-known mid-range lifetime (~20 ns) fluorophores' signal against short-lived backgrounds. This requires a repetition rate of ~300 MHz to pump a 20 ns probe sufficiently. Typical laser sources today are not equipped with repetition rates above 80 MHz. However, this multipulse method allows these rates to be attainable for nearly any pulsed laser source. Multiple pulses of excitation are separated by a variable temporal length, which is short compared to the lifetime of the long-lived fluorophore, to increase the excited state population of a long-lived fluorophore, while the short-lived background decays almost completely between pulses. This is accomplished by simply redirecting the pulsed excitation beam through glass and then a delay length any number of times and lengths as desired to control the number of pulses and separation times.
- Sep 2013
Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 μM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4-30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98 ±0.07 x10(-7) cm(2)/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.
Alveolar epithelial cells are involved in Na(+) absorption via the epithelial Na(+) channel (ENaC), an important process for maintaining an appropriate volume of liquid lining the respiratory epithelium and for lung oedema clearance. Here, we investigated how a 20% hypotonic shock modulates the ionic current in these cells. Polarized alveolar epithelial cells isolated from rat lungs were cultured on permeant filters and their electrophysiological properties recorded. A 20% bilateral hypotonic shock induced an immediate, but transient 52% rise in total transepithelial current and a 67% increase in the amiloride-sensitive current mediated by ENaC. Amiloride pre-treatment decreased the current rise after hypotonic shock, showing that ENaC current is involved in this response. Since Cl(-) transport is modulated by hypotonic shock, its contribution to the basal and hypotonic-induced transepithelial current was also assessed. Apical NPPB, a broad Cl(-) channel inhibitor and basolateral DIOA a potassium chloride co-transporter (KCC) inhibitor reduced the total and ENaC currents, showing that transcellular Cl(-) transport plays a major role in that process. During hypotonic shock, a basolateral Cl(-) influx, partly inhibited by NPPB is essential for the hypotonic-induced current rise. Hypotonic shock promoted apical ATP secretion and increased intracellular Ca(2+). While apyrase, an ATP scavenger, did not inhibit the hypotonic shock current response, W7 a calmodulin antagonist completely prevented the hypotonic current rise. These results indicate that a basolateral Cl(-) influx as well as Ca(2+)/calmodulin, but not ATP, are involved in the acute transepithelial current rise elicited by hypotonic shock.
- Aug 2013
Extracellular ATP and other purines are ubiquitous mediators of local intercellular signaling within the body. While the last two decades have witnessed enormous progress in uncovering and characterizing purinergic receptors and extracellular enzymes controlling purinergic signals, our understanding of the initiating step in this cascade, i.e., ATP release, is still obscure. Imaging of extracellular ATP by luciferin-luciferase bioluminescence offers the advantage of studying ATP release and distribution dynamics in real time. However, low-light signal generated by bioluminescence reactions remains the major obstacle to imaging such rapid processes, imposing substantial constraints on its spatial and temporal resolution. We have developed an improved microscopy system for real-time ATP imaging, which detects ATP-dependent luciferin-luciferase luminescence at ∼10 frames/s, sufficient to follow rapid ATP release with sensitivity of ∼10 nM and dynamic range up to 100 μM. In addition, simultaneous differential interference contrast cell images are acquired with infra-red optics. Our imaging method: (1) identifies ATP-releasing cells or sites, (2) determines absolute ATP concentration and its spreading manner at release sites, and (3) permits analysis of ATP release kinetics from single cells. We provide instrumental details of our approach and give several examples of ATP-release imaging at cellular and tissue levels, to illustrate its potential utility.
In Chinese medicine acupuncture points are treated by physical stimuli to counteract various diseases. These stimuli include mechanical stress as applied during the needle manipulation or tuina, high temperatures as applied during moxibustion, and red laser light applied during laser acupuncture. This study aimed to investigate cellular responses to stimuli that might occur in the tissue of acupuncture points. Since they have a characteristically high density of mast cells that degranulate in response to acupuncture, we asked whether these processes lead to ATP release. We tested in in vitro experiments on mast cells of the human mast-cell line HMC-1 the effects of the physical stimuli; mechanical stress was applied by superfusion of the cells with hypotonic solution, heat was applied by incubation of the cells at 52°C, and red laser light of 657 nm was used for irradiation. We demonstrate that all the stimuli induce ATP release from model human mast HMC-1 cells, and this release is associated with an intracellular free Ca(2+) rise. We hypothesize that ATP released from mast cells supplements the already known release of ATP from keratinocytes and, by acting on P2X receptors, it may serve as initial mediator of acupuncture-induced analgesia.
Cell death is accompanied by the dissipation of electrochemical gradients of monovalent ions across the plasma membrane that, in turn, affects cell volume via modulation of intracellular osmolyte content. In numerous cell types, apoptotic and necrotic stimuli caused cell shrinkage and swelling, respectively. Thermodynamics predicts a cell type-specific rather than an ubiquitous impact of monovalent ion transporters on volume perturbations in dying cells, suggesting their diverse roles in the cell death machinery. Indeed, recent data show that apoptotic collapse may occur in the absence of cell volume changes and even follow cell swelling rather than shrinkage. Moreover, side-by-side with cell volume adjustment, monovalent ion transporters contribute to cell death machinery engagement independently of volume regulation via cell type-specific signalling pathways. Thus, inhibition of Na(+),K(+)-ATPase by cardiotonic steroids (CTS) rescues rat vascular smooth muscle cells from apoptosis via a novel Na(+)i,K(+)i-mediated, Ca(2+)i-independent mechanism of excitation-transcription coupling. In contrast, CTS kill renal epithelial cells independently of Na(+),K(+)-ATPase inhibition and increased [Na(+)]i/[K(+)]i ratio. The molecular origin of [Na(+)]i/[K(+)]i sensors involved in the inhibition of apoptosis as well as upstream intermediates of Na(+)i/K(+)i-independent death signalling triggered by CTS remain unknown.
- Mar 2013
Pathophysiological conditions challenge cell volume homeostasis and perturb cell volume regulatory mechanisms leading to alterations of cell metabolism, active transepithelial transport, cell migration and death. We report that inhibition of the 5-lipoxygenase (5-LO) with AA861 or ETH 615-139, the cysteinyl leukotriene 1 receptor (CysLT1) with the anti-asthmatic drug Zafirlukast, or the volume-sensitive organic anion channel (VSOAC) with DIDS, blocks the release of organic osmolytes (taurine, meAIB) and the concomitant cell volume restoration following hypoosmotic swelling of human type II-like lung epithelial cells (A549). Reactive oxygen species (ROS) are produced in A549 cells upon hypotonic cell swelling by a diphenylene iodonium sensitive NADPH oxidase. The swelling-induced taurine release is suppressed by ROS scavenging (butylated hydroxytoluene, N-acetyl cysteine) and potentiated by H2O2. Ca(2+) mobilization with ionomycin or ATP stimulates the swelling-induced taurine release whereas calmodulin inhibition (W7) inhibits the release. Chelation of the extracellular Ca(2+) (EGTA) had no effect on swelling-induced taurine release but prevented ATP-induced stimulation. H2O2, ATP and ionomycin were unable to stimulate the taurine release in the presence of AA861 or Zafirlukast, placing 5-LO and CysLT1 as essential elements in the swelling-induced activation of VSOAC with ROS and Ca(2+) as potent modulators. Inhibition of tyrosine kinases (genistein, cucurbitacin) reduces volume-sensitive taurine release, adding tyrosine kinases (Janus kinase) as regulators of VSOAC activity. Caspase 3 activity during hypoxia is unaffected by inhibition of 5-LO/CysLT1 but reduced when swelling-induced taurine loss via VSOAC is prevented by DIDS excess extracellular taurine, indicating a beneficial role of taurine under hypoxia.
- Feb 2013
Previously, we reported that in mammalian erythrocytes irreversible annealing of spectrin heterodimers at 49-50 °C abolished cell volume-dependent regulation of ion carriers, thus suggesting an implication of a two-dimensional (2D) membrane carcass in volume sensing and/or signal transduction. To further examine this hypothesis, we employed atomic force microscopy. This method revealed folded membrane relief of fixed human erythrocytes with an average wave height of 3-5 nm covered by globular structures with a diameter of 40-50 nm and an average height of 1-2 nm. Erythrocyte swelling caused by reduction of medium osmolality decreased the height of membrane surface waves by 40 % and increased K(+),Cl(-) cotransport by approximately sixfold. Both volume-sensitive changes of membrane relief and activity of K(+),Cl(-) cotransporter were abolished by a 10-min preincubation at 50 °C. Our results strongly suggest that volume-dependent alterations of the human erythrocyte membrane relief are caused by reorganization of the 2D spectrin-actin network contributing to regulation of the activity of volume-sensitive ion transporters.
- Dec 2012
Mechano-transduction at the cellular and tissue levels often involves ATP release and activation of the purinergic signaling cascade. In the lungs, stretch is an important physical stimulus but its impact on ATP release, the underlying release mechanisms and transduction pathways are poorly understood. Here, we investigated the effect of unidirectional stretch on ATP release from human alveolar A549 cells by real-time luciferin-luciferase bioluminescence imaging coupled with simultaneous infrared imaging, to monitor the extent of cell stretch and to identify ATP-releasing cells. In sub-confluent (<90%) cell cultures, single 1-s stretch (10%-40%) induced transient ATP release from a small fraction (≤1.5%) of cells that grew in number dose-dependently with increasing extent of stretch. ATP concentration in the proximity (≤150μm) of releasing cells often exceeded 10 μM, sufficient for autocrine/paracrine purinoreceptor stimulation of neighboring cells. ATP release responses were insensitive to the putative ATP channel blockers carbenoxolone and 5-nitro-2-(3-phenylpropyl-amino) benzoic acid, but were inhibited by N-ethylmaleimide and bafilomycin. In confluent cell cultures, the maximal fraction of responding cells dropped to <0.2%, but was enhanced several-fold in the wound/scratch area after it was re-populated by new cells during the healing process. Fluo8 fluorescence experiments revealed 2 types of stretch-induced intracellular Ca(2+) responses, rapid sustained Ca(2+) elevations in a limited number of cells, and delayed secondary responses in neighboring cells, seen as Ca(2+) waves whose propagation was consistent with extracellular diffusion of released ATP. Our experiments revealed that single >10% stretch was sufficient to initiate intercellular purinergic signaling in alveolar cells, which may contribute to the regulation of surfactant secretion and wound healing.
- Feb 2012
In the present work, we compared the outcome of hyperosmotic and isosmotic shrinkage on ion transport and protein phosphorylation in C11-MDCK cells resembling intercalated cells from collecting ducts and in vascular smooth muscle cells (VSMC) from the rat aorta. Hyperosmotic shrinkage was triggered by cell exposure to hypertonic medium, whereas isosmotic shrinkage was evoked by cell transfer from an hypoosmotic to an isosmotic environment. Despite a similar cell volume decrease of 40%-50%, the consequences of hyperosmotic and isosmotic shrinkage on cellular functions were sharply different. In C11-MDCK and VSMC, hyperosmotic shrinkage completely inhibited Na(+),K(+)-ATPase and Na(+),P(i) cotransport. In contrast, in both types of cells isosmotic shrinkage slightly increased rather than suppressed Na(+),K(+)-ATPase and did not change Na(+),P(i) cotransport. In C11-MDCK cells, phosphorylation of JNK1/2 and Erk1/2 mitogen-activated protein kinases was augmented in hyperosmotically shrunken cells by ∼7- and 2-fold, respectively, but was not affected in cells subjected to isosmotic shrinkage. These results demonstrate that the data obtained in cells subjected to hyperosmotic shrinkage cannot be considered as sufficient proof implicating cell volume perturbations in the regulation of cellular functions under isosmotic conditions.
- Jan 2012
Mechanotransduction at the cellular/tissue level often involves release of signaling molecules. Among them, purines appear to be the most primitive and widespread chemical messengers in the animal and plant kingdoms. Their release is highly mechano-sensitive, but the release pathways and regulatory mechanisms are not well understood. Here, we investigated the effect of unidirectional stretch on ATP release from human lung A549 alveolar cells grown on a flexible substrate. We used real-time luciferin-luciferase bioluminescence imaging combined with IR imaging to simultaneously monitor cellular ATP release and extend of cell stretch. Single 1-s stretch of 15-30% induced transient ATP release that ceased in 2-3 min and was restricted to a limited number of cells. The number of responding cells increased dose-dependently with the extent of stretch but did not involve cell damage. Calibration of the ATP response showed that local ATP concentration in the close proximity (≤150 μm) to stretch-activated cells may exceed 1 μM or even 10 μM. These concentrations are sufficient for autocrine/paracrine stimulation of cell surface purinergic receptors on the neighboring cells. ATP responses were insensitive to putative ATP channel blockers carbenoxolone or NPPB (100 μM), inhibitors of pannexin or anion channels respectively, but were abolished by N-ethylmaleimide. Fluo8 fluorescence measurement of stretch-induced intracellular Ca2+ responses revealed that limited number of cells displayed rapid responses, which peaked in <1-s and ceased in 1-3 min. This is similar to stretch-induced ATP responses and suggests functional connection between the two signals. Experiments show that cell stretch induces ATP release via cell-regulated process, likely exocytosis. Mechano-sensitive ATP release, via autocrine/paracrine effects, initiates purinergic signaling cascade in other cells and may function as a general intercellular mechanotransduction paradigm in the lung and other tissues.
Contrasting cell volume behaviours (swelling vs. shrinkage) are considered as criteria to distinguish necrosis from apoptosis. In this study, we employed a time-lapse, dual-image surface reconstruction technique to assess the volume of single vascular smooth muscle cells transfected with E1A-adenoviral protein (E1A-VSMC) and undergoing rapid apoptosis in the absence of growth factors or in the presence of staurosporine. After 30- to 60-min lag-phase, serum-deprived E1A-VSMC volume was increased by ~40%, which preceded maximal increments of caspase-3 activity and chromatin cleavage. Swollen cells underwent rapid apoptotic collapse, documented by plasma membrane budding, and terminated in 10-15 min by the formation of numerous apoptotic bodies. Suppression of apoptosis by inhibition of Na(+),K(+)-ATPase and activation of cAMP signalling with ouabain and forskolin, respectively, completely abolished the swelling of serum-deprived E1A-VSMC. In contrast to serum deprivation, apoptotic collapse of staurosporine-treated E1A-VSMC preceded attenuation of their volume by ~30%. Neither transient hyposmotic swelling nor isosmtotic shrinkage triggered apoptosis. Our results show that cell shrinkage can not be considered as ubiquitous hallmark of apoptosis. The involvement of stimulus-specific cell volume perturbations in initiation and progression of apoptosis in vascular smooth muscle cells should be examined further.
Nucleotide release constitutes the first step of the purinergic signaling cascade, but its underlying mechanisms remain incompletely understood. In alveolar A549 cells much of the experimental data is consistent with Ca(2+)-regulated vesicular exocytosis, but definitive evidence for such a release mechanism is missing, and alternative pathways have been proposed. In this study, we examined ATP secretion from A549 cells by total internal reflection fluorescence microscopy to directly visualize ATP-loaded vesicles and their fusion with the plasma membrane. A549 cells were labeled with quinacrine or Bodipy-ATP, fluorescent markers of intracellular ATP storage sites, and time-lapse imaging of vesicles present in the evanescent field was undertaken. Under basal conditions, individual vesicles showed occasional quasi-instantaneous loss of fluorescence, as expected from spontaneous vesicle fusion with the plasma membrane and dispersal of its fluorescent cargo. Hypo-osmotic stress stimulation (osmolality reduction from 316 to 160 mOsm) resulted in a transient, several-fold increment of exocytotic event frequency. Lowering the temperature from 37°C to 20°C dramatically diminished the fraction of vesicles that underwent exocytosis during the 2-min stimulation, from ~40% to ≤1%, respectively. Parallel ATP efflux experiments with luciferase bioluminescence assay revealed that pharmacological interference with vesicular transport (brefeldin, monensin), or disruption of the cytoskeleton (nocodazole, cytochalasin), significantly suppressed ATP release (by up to ~80%), whereas it was completely blocked by N-ethylmaleimide. Collectively, our data demonstrate that regulated exocytosis of ATP-loaded vesicles likely constitutes a major pathway of hypotonic stress-induced ATP secretion from A549 cells.
This study examined the role of cell volume modulation in plasma membrane rupture and death documented in ouabain-treated renal epithelial cells. Long-term exposure to ouabain caused massive death of C11-MDCK (Madin-Darby canine kidney) epithelial cells, documented by their detachment, chromatin cleavage and complete loss of lactate dehydrogenase (LDH), but did not affect the survival of vascular smooth muscle cells (VSMCs) from the rat aorta. Unlike the distinct impact on cell survival, 2-h exposure to ouabain led to sharp elevation of the [Na+]i/[K+]i ratio in both cell types. A similar increment of Na i + content was evoked by sustained inhibition of Na+,K+-ATPase in K+-free medium. However, in contrast to ouabain, C11-MDCK cells survived perfectly during 24-h exposure to K+-free medium. At 3 h, the volume of ouabain-treated C11-MDCK cells and VSMCs, measured by the recently developed dual-image surface reconstruction technique, was increased by 16 and 12%, respectively, whereas 5–10 min before the detachment of ouabain-treated C11-MDCK cells, their volume was augmented by ~30–40%. To examine the role of modest swelling in the plasma membrane rupture of ouabain-treated cells, we compared actions of hypotonic medium on volume and LDH release. We observed that LDH release from hyposmotically swollen C11-MDCK cells was triggered when their volume was increased by approximately fivefold. Thus, our results showed that the rupture of plasma membranes in ouabain-treated C11-MDCK cells was not directly caused by cell volume modulation evoked by Na+,K+-ATPase inhibition and inversion of the [Na+]i/[K+]i ratio.
Purinergic receptors activate diverse signaling cascades and regulate the activity of cell volume-sensitive ion transporters. However, the effects of ATP and other agonists of P2 receptors on cell volume dynamics are only scarcely studied. In the present work, we used the recently developed dual-image surface reconstruction technique to explore the influence of purinergic agonists on cell volume in the C11-Madin-Darby canine kidney cell line resembling intercalated cells from kidney collecting ducts. Unexpectedly, we found that ATP and UTP triggered very robust (55-60%) cell shrinkage that lasted up to 2 h after agonist washout. Purinergic regulation of cell volume required increases in intracellular Ca(2+) and could be partially mimicked by the Ca(2+)-ionophore ionomycin or activation of protein kinase C by 4β-phorbol 12-myristate 13-acetate. Cell shrinkage was accompanied by strong reductions in intracellular K(+) and Cl(-) content measured using steady-state (86)Rb(+) and (36)Cl(-) distribution. Both shrinkage and ion efflux in ATP-treated cells were prevented by the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and by the BK(Ca) channel inhibitors charybdotoxin, iberiotoxin, and paxilline. To evaluate the significance of cell-volume changes in purinergic signaling, we measured the impact of ATP on the expression of the immediate-early gene c-Fos. Thirty-minute treatment with ATP increased c-Fos immunoreactivity by approximately fivefold, an effect that was strongly inhibited by charybdotoxin and completely prevented by NPPB. Overall, our findings suggest that ATP-induced cell-volume changes are partially responsible for the physiological actions of purinergic agonists.
Extracellular nucleotides regulate mucociliary clearance in the airways and surfactant secretion in alveoli. Their release is exquisitely mechanosensitive and may be induced by stretch as well as airflow shear stress acting on lung epithelia. We hypothesized that, in addition, tension forces at the air-liquid interface (ALI) may contribute to mechanosensitive ATP release in the lungs. Local depletion of airway surface liquid, mucins, and surfactants, which normally protect epithelial surfaces, facilitate such release and trigger compensatory mucin and fluid secretion processes. In this study, human bronchial epithelial 16HBE14o(-) and alveolar A549 cells were subjected to tension forces at the ALI by passing an air bubble over the cell monolayer in a flow-through chamber, or by air exposure while tilting the cell culture dish. Such stimulation induced significant ATP release not involving cell lysis, as verified by ethidium bromide staining. Confocal fluorescence microscopy disclosed reversible cell deformation in the monolayer part in contact with the ALI. Fura 2 fluorescence imaging revealed transient intracellular Ca(2+) elevation evoked by the ALI, which did not entail nonspecific Ca(2+) influx from the extracellular space. ATP release was reduced by ∼40 to ∼90% from cells loaded with the Ca(2+) chelator BAPTA-AM and was completely abolished by N-ethylmalemide (1 mM). These experiments demonstrate that in close proximity to the ALI, surface tension forces are transmitted directly on cells, causing their mechanical deformation and Ca(2+)-dependent exocytotic ATP release. Such a signaling mechanism may contribute to the detection of local deficiency of airway surface liquid and surfactants on the lung surface.
Fluorescence can be greatly enhanced near metal surfaces due to many-fold increased brightness and photostability of fluorophores located near metallic nanoparticles or nanostructures. Further more, fluorophores deposited on a plane of a 50 nm thick silver or gold mirrors, show directional fluorescence in a form of hollow cone. These favorable properties of fluorophore-plasmonic interaction can be utilized in high-sensitivity imaging of cellular processes. However, the cell growth strongly depends on the nature of the substrate and is often very difficult on bare metal surfaces. In our study we examined suitability of different metal surface coatings for growing lung A549 epithelial cells. Six different surfaces were tested - glass, silver mirror, silver mirror coated with SiO2, gold mirror, gold mirror coated with SiO2 and silver fractals on glass. The glass coverslips with five different metallic surface coatings and one control were placed in a tissue culture plates containing DMEM-High Glucose media. Suspension of 0.5x106 cells/plate was deposited on the slides and the cell cultures were placed in 37°C, 5%CO2 incubator. Cell growth was monitored every 24 hours. No substantial differences in cell morphology were found whether they were on the regular microscopic glass slide or slide covered with metals, but cells initially grew significantly slower on fractals compared to other “smooth” surfaces. The findings demonstrate feasibility of growing A549 cells on metal-coated glass surfaces and opens the opportunity for imaging live A549 cells using metal enhanced fluorescence.Supported by the Canadian Institutes of Health Research and the Canadian Cystic Fibrosis Foundation (R.G), and Emerging Technologies Fund Grant Texas (Z.G.).
Extracellular nucleotides, via interaction with cell surface purinergic receptors, regulate multiple physiological processes in the lungs, including airway mucociliary clearance and surfactant secretion. Release of ATP from nonexcitable cells can be provoked by mechanical perturbations and cell-swelling, but the underlying mechanisms remain incompletely understood. We have shown previously that cell-swelling induced ATP secretion from A549 cells tightly correlates with intracellular Ca2+ elevations and sought to establish whether Ca2+-dependent exocytosis is involved. In this study, 50% hypotonic shock-induced ATP release from A549 cells was examined by total internal reflection fluorescence (TIRF) microscopy in an attempt to directly visualize ATP-loaded vesicle movement, recruitment and fusion with the plasma membrane. Cells were loaded with quinacrine, a fluorescent marker of ATP intracellular storage sites, and time-lapse imaging was performed using through the objective TIRF system. The time-course of fluorescence intensity changes of individual quinacrine-stained vesicles was evaluated during 1-2-min following hypoosmotic stimulation. Approximately 20%-30% of vesicles visible by TIRF at the cell base showed a quasi-instantaneous disappearance during the first minute post-stimulation, as expected for vesicle fusion and dispersal of their content. This was accompanied by recruitment of ∼10% new vesicles into the evanescent field followed by their exocytosis. The hypotonic stimulus significantly (∼5-fold) increased rate of exocytotic events compared to rate of spontaneous events in unstimulated cells. Exocytotic release mechanism is also consistent with ATP efflux measurements using luciferin-luciferase luminescence assay. Agents known to disrupt exocytotic process (brefeldin, monensin), or cytoskeleton (nocodazole, cytocholasin) reduced ATP release significantly (by up to 80%), while the release was completely blocked by N-ethylmaleimide (1 mM), and low (10oC) temperature. Thus, hypotonic shock-induced ATP secretion from A549 cells occurs mainly via Ca2+-dependent exocytosis. Supported by CIHR and CCFF (RG), Emerging Technologies Fund Grant Texas (Z.G.), NIH-HL090786 (JB).
Pseudomonas aeruginosa is a gram-negative bacterium that causes chronic infection in cystic fibrosis patients. We reported recently that P. aeruginosa modulates epithelial Na(+) channel (ENaC) expression in experimental chronic pneumonia models. For this reason, we tested whether LPS from P. aeruginosa alters ENaC expression and activity in alveolar epithelial cells. We found that LPS induces a approximately 60% decrease of ENaC apical current without significant changes in intracellular ENaC or surface protein expression. Because a growing body of evidence reports a key role for extracellular nucleotides in regulation of ion channels, we evaluated the possibility that modulation of ENaC activity by LPS involves extracellular ATP signaling. We found that alveolar epithelial cells release ATP upon LPS stimulation and that pretreatment with suramin, a P2Y(2) purinergic receptor antagonist, inhibited the effect of LPS on ENaC. Furthermore, ET-18-OCH3, a PLC inhibitor, and Go-6976, a PKC inhibitor, were able to partially prevent ENaC inhibition by LPS, suggesting that the actions of LPS on ENaC current were mediated, in part, by the PKC and PLC pathways. Together, these findings demonstrate an important role of extracellular ATP signaling in the response of epithelial cells to LPS.
We report that 38% of primary large B-cell lymphoma (DLBCL) tested expressed active Src family kinases, which are targeted by dasatinib. The expression of active Src family of kinases (SFK) in primary DLBCL tumors correlated with unfavorable prognostic markers such as Ki67 and Mum1. Using four DLBCL cell lines we found that: (1) sensitivity to dasatinib (but not imatinib) varied 400-fold; (2) dasatinib resistance was associated with distinct signaling profiles downstream of BCR activation. In particular, although Src family kinase phosphorylation was inhibited by 100-150 nM dasatinib in all cell lines, this failed to inhibit BCR-mediated Blnk phosphorylation, calcium signaling and proliferation in a dasatinib resistant cell line.
- Jun 2009
Cytoplasm is thought to have many hydrogel-like characteristics, including the ability to absorb large amounts of water and change volume in response to alterations in external environment, as well as having limited leakage of ions and proteins. Some gel-like behaviors have not been rigorously confirmed in mammalian cells, and others should be examined under conditions where gel volume can be accurately monitored. Thus, possible contributions of cytoplasm hydrogel properties to cellular processes such as volume sensing and regulation remain unclear. We used three-dimensional imaging to measure volume of single substrate-attached cells after permeabilization of their plasma membrane. Permeabilized cells swelled or shrinked reversibly in response to variations of external osmolality. Volume changes were 3.7-fold greater than observed with intact cells, consistent with cytoplasm's high water-absorbing capacity. Volume was maximal at neutral pH and shrunk at acidic or alkaline pH, consistent with pH-dependent changes of protein charge density and repulsive forces within cellular matrix. Volume shrunk with increased Mg(2+) concentration, as expected for increased charge screening and ionic crosslinking effects. Findings demonstrate that mammalian cytoplasm resembles hydrogel and functions as a highly sensitive osmosensor and extracellular pH sensor. Its high water-absorbing capacity may allow rapid modulation of local fluidity, macromolecular crowding, and activity of intracellular environment.
This study examines the action of agonists and antagonists of P2 receptors on mouse mesenteric artery contractions and the possible involvement of these signaling pathways in myogenic tone (MT) evoked by elevated intraluminal pressure. Both ATP and its non-hydrolyzed analog alpha,beta-ATP triggered transient contractions that were sharply decreased in the presence of NF023, a potent antagonist of P2X(1) receptors. In contrast, UTP and UDP elicited sustained contractions which were suppressed by MRS2567, a selective antagonist of P2Y(6) receptors. Inhibition of Na(+), K(+), 2Cl(-) cotransport (NKCC) with bumetanide led to attenuation of contractions in UTP- but not ATP-treated arteries. Both UTP-induced contractions and MT were suppressed by MRS2567 and bumetanide but were insensitive to NF023. These data implicate a P2Y(6)-mediated, NKCC-dependent mechanism in MT of mesenteric arteries. The action of heightened intraluminal pressure on UTP release from mesenteric arteries and its role in the triggering of P2Y(6)-mediated signaling should be examined further.
The expression of the epithelial Na+ channel (ENaC) is tissue-specific and dependent on a variety of mediators and interacting proteins. Here we examined the role of intracellular Na+ ([Na+](i)) as a modulator of the expression of rat ENaC in Xenopus laevis oocytes. We manipulated [Na+](i) of ENaC-expressing oocytes in the range of 0-20 mM by incubating in extracellular solutions of different [Na+](o). Electrophysiological, protein biochemical and fluorescence optical methods were used to determine the effects of different [Na+]i on ENaC expression and membrane abundance. In voltage-clamp experiments we found that amiloride-sensitive ENaC current (Iami) and conductance (Gami) peak at a [Na+](i) of approximately 10 mM Na+, but were significantly reduced in 5 mM and 20 mM [Na+](i). Fluorescence intensity of EGFP-ENaC-expressing oocytes also followed a bell-shaped curve with a maximum at approximately 10 mM [Na+](i). In Western blot experiments with specific anti-ENaC antibodies the highest protein expression was found in ENaC-expressing oocytes with [Na+](i) of 10-15 mM. Since ENaC is also highly permeable for Li+, we incubated ENaC-expressing oocytes in different Li+ concentrations and found a peak of Iami and Gami with 5 mM Li+. The influence of [Na+](i) on the expression is not ENaC-specific, since expression of a Cl(-) channel (CFTR) and a Na+/glucose cotransporter (SGLT1) showed the same dependence on [Na+](i). We conclude that specific concentrations of Na+ and Li+ influence the expression and abundance of ENaC and other transport proteins in the plasma membrane in Xenopus laevis oocytes. Furthermore, we suggest the existence of a general mechanism dependent on monovalent cations that optimizes the expression of membrane proteins.
Extracellular nucleotides play an important role in lung defense, but the release mechanism and relative abundance of different nucleotide species secreted by lung epithelia are not well defined. In this study, to minimize cell surface hydrolysis, we used a low-volume, flow-through chamber and examined adenosine and uridine nucleotide concentrations in perfusate aliquots of human lung A549 cells challenged by 50% hypotonic shock. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine (Ado) were quantified in high-performance liquid chromatography (HPLC) analysis of fluorescent etheno derivatives, and uridine triphosphate (UTP) and uridine diphosphate (UDP) were measured using HPLC-coupled radioenzymatic assays. After the onset of hypotonic shock, ATP, ADP, UTP, and UDP in the perfusates increased markedly and peaked at approximately 2.5 min, followed by a gradual decay in the next 15-20 min; peak changes in Ado and AMP were relatively minor. The peak concentrations and fold increment (in parentheses) were: 34 +/- 13 nM ATP (5.6), 11 +/- 5 nM ADP (3.7), 3.3 +/- 1.2 nM AMP (1.4), 23 +/- 7 nM Ado (2.1), 21 nM UTP (>7), and 11 nM UDP (27). Nucleotide release was almost completely abolished from cells loaded with the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Under isotonic conditions, elevation of intracellular calcium with the calcium ionophore ionomycin (5 muM, 3 min) also released nucleotides with kinetics and relative abundance as above, albeit less robust. ADP:ATP (1:3) and UDP:UTP (1:2) ratios in perfusates from stimulated cells were markedly higher than the cytosolic ratios of these species, suggesting that a nucleotide diphosphate (NDP)-rich compartment, e.g., the secretory pathway, contributed to nucleotide release. Laser confocal microscopy experiments illustrated increased FM1-43 uptake into the plasma membrane upon hypotonic shock or ionomycin treatment, consistent with enhanced vesicular exocytosis under these conditions. In summary, our results strongly suggest that calcium-dependent exocytosis is responsible, at least in most part, for adenosine and uridine nucleotide release from A549 cells.
- Nov 2007
Extracellular ATP is a potent surfactant secretagogue but its origin in the alveolus, its mechanism(s) of release and its regulatory pathways remain unknown. Previously, we showed that hypotonic swelling of alveolar A549 cells induces Ca2+-dependent secretion of several adenosine and uridine nucleotides, implicating regulated exocytosis. In this study, we examined sources of Ca2+ for the elevation of intracellular Ca2+ concentration ([Ca2+](i)) evoked by acute 50% hypotonic stress and the role of autocrine purinergic signalling in Ca2+-dependent ATP release. We found that ATP release does not directly involve Ca2+ influx from extracellular spaces, but depends entirely on Ca2+ mobilization from intracellular stores. The [Ca2+](i) response consisted of slowly rising elevation, representing mobilization from thapsigargin (TG)-insensitive stores and a superimposed rapid spike due to Ca2+ release from TG-sensitive endoplasmic reticulum (ER) Ca2+ stores. The latter could be abolished by hydrolysis of extracellular triphospho- and diphosphonucleotides with apyrase; blocking P2Y(2)/P2Y(6) receptors of A549 cells with suramin; blocking UDP receptors (P2Y(6)) with pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS); emptying TG-sensitive stores downstream with TG or caffeine in Ca2+-free extracellular solution; or blocking the Ca2+-release inositol 1,4,5-triphosphate receptor channel of the ER with 2-aminoethyldiphenylborinate. These data demonstrate that the rapid [Ca2+], spike results from the autocrine stimulation Of IP3/Ca2+-coupled P2Y, predominantly P2Y(6), receptors, accounting for similar to 70% of total Ca2+-dependent ATP release evoked by hypotonic shock. Our study reveals a novel paradigm in which stress-induced ATP release from alveolar cells,is amplified by the synergistic autocrine/paracrine action of coreleased uridine and adenosine nuclecitides. We suggest that a similar mechanism of purinergic signal propagation operates in other cell types.
Despite the existence of a functional arginine vasopressin (AVP) system in the adult heart and evidence that AVP induces myogenesis, its significance in cardiomyogenesis is currently unknown. In the present study, we hypothesized a role for AVP in cardiac differentiation of D3 and lineage-specific embryonic stem (ES) cells expressing green fluorescent protein under the control of atrial natriuretic peptide (Anp) or myosin light chain-2V (Mlc-2V) promoters. Furthermore, we investigated the nitric oxide (NO) involvement in AVP-mediated pathways. AVP exposure increased the number of beating embryoid bodies, fluorescent cells, and expression of Gata-4 and other cardiac genes. V1a and V2 receptors (V1aR and V2R) differentially mediated these effects in transgenic ES cells, and exhibited a distinct developmentally regulated mRNA expression pattern. A NO synthase inhibitor, L-NAME, powerfully antagonized the AVP-induced effects on cardiogenic differentiation, implicating NO signaling in AVP-mediated pathways. Indeed, AVP elevated the mRNA and protein levels of endothelial NO synthase (eNOS) through V2R stimulation. Remarkably, increased beating activity was found in AVP-treated ES cells with down-regulated eNOS expression, indicating the significant involvement of additional pathways in cardiomyogenic effects of AVP. Finally, patch clamp recordings revealed specific AVP-induced changes of action potentials and increased L-type Ca2+ (ICa,L) current densities in differentiated ventricular phenotypes. Thus, AVP promotes cardiomyocyte differentiation of ES cells and involves Gata-4 and NO signaling. AVP-induced action potential prolongation appears likely to be linked to the increased ICa,L current in ventricular cells. In conclusion, this report provides new evidence for the essential role of the AVP system in ES cell-derived cardiomyogenesis.
Oxytocin (OT), a hormone recently identified in the heart, induces embryonic and cardiac somatic stem cells to differentiate into cardiomyocytes (CM), possibly through nitric oxide (NO). We verified this hypothesis using P19 cells and P19 Clone 6 derivatives expressing a green fluorescent protein (GFP) reporter linked to cardiac myosin light chain-2v promoter. OT treatment of these cells induced beating cell colonies that were fully inhibited by N,G-nitro-L-arginine-methyl-ester (L-NAME), an inhibitor of NO synthases (NOS), partially reduced by 1400W, an inhibitor of inducible NOS, and ODQ, an inhibitor of NO-sensitive guanylyl cyclases. The NO generator S-nitroso-N-acetylpenicillamine (SNAP) reversed the L-NAME inhibition of cell beating and GFP expression. In OT-induced cells, L-NAME significantly decreased transcripts of the cardiac markers Nkx2.5, MEF2c, alpha-myosin heavy chain, and less, GATA4, endothelial NOS, and atrial natriuretic peptide, as well as the skeletal myocyte (SM) marker myogenin. Image analysis of OT-induced P19Cl6-GFP cells revealed ventricular CM coexpressing sarcomeric alpha-actinin and GFP, with some cells exclusively expressing alpha-actinin, most likely of the SM phenotype. The OT-mediated production of CM, but not SM, was diminished by L-NAME. In P19 cells, exogenously added OT stimulated the expression of its own transcript, which was reduced in the presence of L-NAME. Surprisingly, L-NAME alone decreased the expression of anti-stage specific embryonic antigen-1 marker of the undifferentiated state and induced some beating colonies as well as GFP in P19Cl6-GFP cells. Collectively, our data suggest that the pleiotropic action of NO is involved in the initiation of CM differentiation of P19 cells and maintenance of their undifferentiated state.
- Feb 2007
- Biomedical Optics (BiOS) 2007
We report new approach to Fluorescence Correlation Spectroscopy (FCS) and Single Molecule Detection (SMD) based on Surface Plasmon-Coupled Emission (SPCE) technology. The use of SPCE offers significant reduction of fluorescence volume (detection volume) reduction decreasing background contribution. Fluorophore interaction with surface plasmons increases the rate of photon detection and makes fluorescence very sensitive to change in a position of emitting molecule. The effective thickness of the fluorescence volume in SPCE experiments depends on two factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. The excitation with the laser beam at Surface Plasmon Resonance (SPR) angle (Kretschmann configuration) through the high numerical aperture objective makes observation volume very shallow below 100 nm. The layer thickness is further reduced by the metal quenching of excited fluorophores immediately close to the interface (~10 nm). The fluorescence light is emitted through the metal film only at the SPCE angle. Any fluorescence occurring at the distances greater than the coupling distance is effectively reflected (~92%) by the metal film and not transmitted to the objective. The thickness of the detected volume can be 20-50 nm, depending on the prism dielectric constants and orientation of the excited dipoles. In addition the signal is very sensitive to the change in fluorophore position and orientation. Such strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles opens new possibilities to study conformational changes of macromolecules in real time.
To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o(-), CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (approximately 6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 +/- 0.2-fold and V 10.7 +/- 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10 degrees C) reduced SA and V increases of A549 cells to 1.7 +/- 0.3-fold and 4.4 +/- 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.
- Jan 2006
- Conference on Ultrasensitive and Single-Molecule Detection Technologies
Surface plasmon-coupled emission (SPCE) has been used to reduce the detection volume in fluorescence measurements. The effective fluorescence volume (detection volume) in SPCE experiments depends on two near-field factors: the depth of evanescent wave excitation and a distance-dependent coupling of excited fluorophores to the surface plasmons. With the excitation through the glass prism at SPR angle (Kretschmann configuration), the detection volume is a composition (product) of evanescent wave penetration depth and distance-dependent coupling. In addition, the detection volume is further reduced by a metal quenching of excited fluorophores at a close proximity (below 10 nm). The height of the detected volume size is 40-70 nm, depending on the orientation of the excited dipoles. We show that using Kretchmann configuration in a microscope with high numerical aperture objective (1.45) together with confocal detection, the detection volume can be reduced to 1-2 attoL, which is necessary to observe a single cross-bridge in the muscle. The strong dependence of the coupling to the surface plasmons on the orientation of excited dipoles can be also used to study the small conformational changes of macromolecules.
- Jun 2005
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a crucial role in regulating fluid secretion by the airways, intestines, sweat glands and other epithelial tissues. It is well established that the CFTR is a cAMP-activated, nucleotide-dependent anion channel, but additional functions are often attributed to it, including regulation of the epithelial sodium channel (ENaC). The absence of CFTR-dependent ENaC inhibition and the resulting sodium hyperabsorption were postulated to be a major electrolyte transport abnormality in cystic fibrosis (CF)-affected epithelia. Several ex vivo studies, including those that used the Xenopus oocyte expression system, have reported ENaC inhibition by activated CFTR, but contradictory results have also been obtained. Because CFTR-ENaC interactions have important implications in the pathogenesis of CF, the present investigation was undertaken by our three independent laboratories to resolve whether CFTR regulates ENaC in oocytes and to clarify potential sources of previously reported dissimilar observations. Using different experimental protocols and a wide range of channel expression levels, we found no evidence that activated CFTR regulates ENaC when oocyte membrane potential was carefully clamped. We determined that an apparent CFTR-dependent ENaC inhibition could be observed when resistance in series with the oocyte membrane was not low enough or the feedback voltage gain was not high enough. We suggest that the inhibitory effect of CFTR on ENaC reported in some earlier oocyte studies could be attributed to problems arising from high levels of channel expression and suboptimal recording conditions, that is, large series resistance and/or insufficient feedback voltage gain.
- Jan 2005
Mechanical stresses release ATP from a variety of cells by a poorly defined mechanism(s). Using custom-designed flow-through chambers, we investigated the kinetics of cell swelling-induced ATP secretion, cell volume and intracellular calcium changes in epithelial A549 and 16HBE14o- cells, and NIH/3T3 fibroblasts. Fifty per cent hypotonic shock triggered transient ATP release from cell confluent monolayers, which consistently peaked at around 1 min 45 s for A549 and NIH/3T3, and at 3 min for 16HBE14o- cells, then declined to baseline within the next 15 min. Whereas the release time course had a similar pattern for the three cell types, the peak rates differed significantly (294 +/- 67, 70 +/- 22 and 17 +/- 2.8 pmol min(-1) (10(6) cells)(-1), for A549, 16HBE14o- and NIH/3T3, respectively). The concomitant volume changes of substrate-attached cells were analysed by a 3-dimensional cell shape reconstruction method based on images acquired from two perpendicular directions. The three cell types swelled at a similar rate, reaching maximal expansion in 1 min 45 s, but differed in the duration of the volume plateau and regulatory volume decrease (RVD). These experiments revealed that ATP release does not correlate with either cell volume expansion and the expected activation of stretch-sensitive channels, or with the activation of volume-sensitive, 5-nitro-2-(3-phenylpropylamino) benzoic acid-inhibitable anion channels during RVD. By contrast, ATP release was tightly synchronized, in all three cell types, with cytosolic calcium elevations. Furthermore, loading A549 cells with the calcium chelator BAPTA significantly diminished ATP release (71% inhibition of the peak rate), while the calcium ionophore ionomycin triggered ATP release in the absence of cell swelling. Lowering the temperature to 10 degrees C almost completely abolished A549 cell swelling-induced ATP release (95% inhibition of the peak rate). These results strongly suggest that calcium-dependent exocytosis plays a major role in mechanosensitive ATP release.
- Oct 2004
Precise measurement of rapid volume changes of substrate-adherent cells is essential to understand many aspects of cell physiology, yet techniques to evaluate volume changes with sufficient precision and high temporal resolution are limited. Here, we describe a novel imaging method that surveys the rapid morphology modifications of living, substrate-adherent cells based on phase-contrast, digital video microscopy. Cells grown on a glass substrate are mounted in a custom-designed, side-viewing chamber and subjected to hypotonic swelling. Side-view images of the rapidly swelling cell, and at the end of the assay, an image of the same cell viewed from a perpendicular direction through the substrate, are acquired. Based on these images, off-line reconstruction of 3D cell morphology is performed, which precisely measures cell volume, height and surface at different points during cell volume changes. Volume evaluations are comparable to those obtained by confocal laser scanning microscopy (DeltaVolume < or = 14%), but our method has superior temporal resolution limited only by the time of single-image acquisition, typically approximately 100 ms. The advantages of using standard phase-contrast microscopy without the need for cell staining or intense illumination to monitor cell volume make this system a promising new tool to investigate the fundamentals of cell volume physiology.
The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the fetal lung, but during lung development it gradually disappears in cells of future alveolar spaces. Recent studies have implicated the CFTR in fluid transport by the adult alveolar epithelium, but its presence has not been demonstrated directly. This study re-evaluated CFTR expression and activity in the adult pulmonary epithelium by using freshly isolated rat alveolar type II (ATII) cells. CFTR mRNA was detected by semiquantitative polymerase chain reaction on the day of cell isolation but was rapidly reduced by 60% after 24 h of cell culture. This was paralleled by a similar decrease of surfactant protein A expression and alkaline phosphatase staining, markers of the ATII cell phenotype. CFTR expression increased significantly on day 4 in cells grown on filters at the air-liquid interface compared with cells submerged or grown on plastic. Significantly higher CFTR expression was detected in distal lung tissue compared with the trachea. The CFTR was also found at the protein level in Western blot experiments employing lysates of freshly isolated alveolar cells. Whole cell patch-clamp experiments revealed cAMP-stimulated, 5-nitro-2-(3-phenylpropylamino)-benzoate-sensitive Cl(-) conductance with a linear current-voltage relationship. In cell-attached membrane patches with 100 microM amiloride in pipette solution, forskolin stimulated channels of approximately 4 pS conductance. Our results indicate that 50-250 of functional CFTR Cl(-) channels occur in adult alveolar cells and could contribute to alveolar liquid homeostasis.
- Jan 2004
More than 20 years ago, it was shown that the addition of EGTA increases the affinity of the plasma membrane Ca2+ pump for Ca2+ by an order of magnitude. The left-hand shift of Ca2+-dependencies in the presence of EGTA has been also documented in studies of the sarcoplasmic reticulum Ca2+ pump, mitochondrial Ca2+-transporter as well as Ca2+-binding by calmodulin and troponin C. These data allow us to hypothesise that this effect is caused by an admixture of di- and trivalent cations possessing high affinity for EGTA and interacting with Ca2+-transporting and binding proteins. Here, we propose that polyvalent cations affect the estimation of absolute values of free intracellular Ca2+ concentration. Indeed, EGTA sharply increases the apparent affinity of the fluorescent Ca2+ indicators quin-2 and fluo-3 for Ca2+. The impact of polyvalent cations on Ca2+ measurement was further confirmed by the study showing the high sensitivity of Ca2+-induced fluo-3 fluorescence to Mn2+, Fe2+, Cu2+, and Co2+ seen in the absence of EGTA.
- Oct 2002
In this study, we examined the effect of Na(+)-K(+) pump inhibition on the expression of early response genes in vascular smooth muscle cells (VSMC) as possible intermediates of the massive RNA synthesis and protection against apoptosis seen in ouabain-treated VSMC in our previous experiments. Incubation of VSMC with ouabain resulted in rapid induction of c-Fos protein expression with an approximately sixfold elevation after 2 h of incubation. c-Jun expression was increased by approximately fourfold after 12 h, whereas expression of activating transcription factor 2, cAMP/Ca(2+) response element binding protein (CREB)-1 and c-Myc was not altered. Markedly augmented c-Fos expression was also observed under Na(+)-K(+) pump inhibition in potassium-depleted medium. Na(+)-K(+) pump inhibition triggered c-Fos expression via elevation of the [Na(+)](i)/[K(+)](i) ratio. This conclusion follows from experiments showing the lack of effect of ouabain on c-Fos expression in high-potassium-low-sodium medium and from the comparison of dose responses of Na(+)-K(+) pump activity, [Na(+)](i) and [K(+)](i) content and c-Fos expression to ouabain. A fourfold increment of c-Fos mRNA was revealed 30 min following addition of ouabain to the incubation medium. At this time point, treatment with ouabain resulted in an approximately fourfold elevation of [Na(+)](i) but did not affect [K(+)](i). Augmented c-Fos expression was also observed under VSMC depolarization in high-potassium medium. Increments in both c-Fos expression and (45)Ca uptake in depolarized VSMC were abolished under inhibition of L-type Ca(2+) channels with 0.1 microM nicardipine. Ouabain did not affect the free [Ca(2+)](i) or the content of exchangeable [Ca(2+)](i). Ouabain-induced c-Fos expression was also insensitive to the presence of nicardipine and [Ca(2+)](o), as well as chelators of [Ca(2+)](o) (EGTA) and [Ca(2+)](i) (BAPTA). The effect of ouabain and serum on c-Fos expression was additive. In contrast to serum, however, ouabain failed to activate the Elk-1, serum response factor, CREB and activator protein-1 transcription factors identified within the c-Fos promoter. These results suggest that Na(+)-K(+) pump inhibition triggers c-Fos expression via [Na(+)](i)-sensitive [Ca(2+)](i)-independent transcription factor(s) distinct from factors interacting with known response elements of this gene promoter.
- May 2002
This study examines purinergic modulation of short-circuit current (I(SC)) in monolayers of C7- and C11-MDCK cells resembling principal and intercalated cells from collecting ducts. In C7 monolayers, basolateral and apical application of ATP led to similar elevation of I(SC), consisting of a transient phase with maximal I(SC) increment of approximately 10 microA/cm2 terminating in 2-3 min, and a sustained phase with maximal I(SC) less than 2 microA/cm2 and terminating in 10 min. ATP-induced I(SC) was insensitive to the presence of Na+, Cl- and inhibitors of K+ (Ba2+, charibdotoxin (ChTX), clotrimazole (CLT), apamin) and Na + (amiloride) channels in the mucosal solution. Inhibitors of Cl- channels, DIDS and NPPB, added to apical membranes at a concentration of 100 microM, did not affect ATP-induced I(SC), whereas at 500 microM, NPPB inhibited it by 70-80%. Substitution of Cl- with NO3- in serosal medium decreased ATP-induced I(SC) by 2-3-fold and elevation of [K+]o from 6 to 60 mM changed its direction. Basolateral NPPB inhibited I(SC) by 10-fold with ED50 of approximately 30 microM, whereas ChTX (50 nM) and CLT (2 microM) diminished this parameter by 30-50%. Suppression of Na+, K+, Cl- cotransport with bumetanide did not affect the transient phase of ATP-induced I(SC) and slightly diminished its sustained phase. ATP increased ouabainand bumetanide-resistant K+ (86Rb) influx across the basolateral membrane by 7-8-fold, which was partially inhibited with ChTX and CLT. ATP-treated C11 cells exhibited negligible I(SC), and their presence did not affect I(SC) triggered by ATP in C7 cells. Thus, our results show that transcellular ion current in ATP-treated C7 cells is mainly caused by the coupled function of apical and basolateral anion transporters providing transient Cl- secretion. These transporters possess different sensitivities to anion channel blocker NPBB and are under the control of basolateral K+ channels(s) inhibited by ChTX and CLT.
- Feb 2002
ATP release induced by hypotonic swelling is an ubiquitous phenomenon in eukaryotic cells, but its underlying mechanisms are poorly defined. A mechanosensitive (MS) ATP channel has been implicated because gadolinium (Gd(3+)), an inhibitor of stretch-activated channels, suppressed ATP efflux monitored by luciferase bioluminescence. We examined the effect of Gd(3+) on luciferase bioluminescence and on ATP efflux from hypotonically swollen cells. We found that luciferase was inhibited by < or =10 microM Gd(3+), and this may have contributed to the previously reported inhibition of ATP release. In ATP efflux experiments, luciferase inhibition could be prevented by chelating Gd(3+) with EGTA before luminometric ATP determinations. Using this approach, we found that 10-100 microM Gd(3+), i.e., concentrations typically used to block MS channels, actually stimulated hypotonically induced ATP release from fibroblasts. Inhibition of ATP release required at least 500, 200, or 100 microM Gd(3+) for fibroblasts, A549 cells, and 16HBE14o(-) cells, respectively. Such biphasic and cell-specific effects of Gd(3+) are most consistent with its action on membrane lipids and membrane-dependent processes such as exocytosis.
- Feb 2001
This study examines the relative contributions of K-Cl cotransport and K(+) channels to swelling-induced K(+) fluxes in vascular smooth muscle cells (VSMC). DIOA known as a potent inhibitor of erythrocyte K-Cl cotransport exerts diverse side-effects on VSMC and can not be used to analyze the role of this carrier in swelling-induced K(+) fluxes. Other inhibitors of K-Cl cotransport (furosemide, okadaic acid and calyculin A) did not affect K(+) fluxes in VSMC triggered by swelling. Swelling-induced K(+) fluxes in VSMC were also not affected by K(+) channel blockers such as TEA, glibenclamide and apamin, but were blocked by Ba(2+) and charybdotoxin (ChTX), a potent inhibitor of Ca(2+)- and voltage-gated K(+) channels. Swelling-induced K(+) influx in VSMC was diminished in Ca(2+)-free medium and in cells loaded with Ca(2+) chelator BAPTA, but was not accompanied by detectable elevation of [Ca(2+)](i). In contrast to Ca(2+)-induced hyperpolarization of erythrocytes triggered by activation of intermediate conductance Ca(2+)-gated K(+) channels (IK(Ca)), neither clotrimazole nor calmodulin antagonists (R24571, trifluoroperazine, fluphenazine) affected swelling-induced K(+) influx in VSMC. In conclusion, K(+) fluxes triggered in swollen VSMC are mediated by Ba(2+)- and ChTX-sensitive K(+) channels. These channels are distinct from IK(Ca) expressed in erythrocytes. Their molecular origin and systems involved in the swelling-induced Ca(2+)(i)-independent signal transduction pathway need further investigation.
- Jul 1999
Defective regulatory interactions between the cystic fibrosis conductance regulator (CFTR) and the epithelial sodium channel (ENaC) have been implicated in the elevated Na+ transport rates across cystic fibrosis airway epithelium. It has recently been proposed that ENaC downregulation by CFTR depends on the ability of CFTR to conduct Cl- into the cell and is negligible when Cl- flows out of the cell. To study the mechanisms of this downregulation we have measured amiloride-inhibitable Na+ current (Iamil) in oocytes co-expressing rat ENaC and human wild-type CFTR. In oocytes voltage-clamped to -60 mV, stimulating CFTR with 1 mm IBMX reduced Iamil by up to 80%, demonstrating that ENaC is inhibited when Cl- is conducted out of the cell. Decreasing the level of CFTR stimulation in a single oocyte, decreased both the degree of Iamil downregulation and the CFTR-mediated plasma membrane Cl- conductance, suggesting a direct correlation. However, Iamil downregulation was not affected when Cl- flux across oocyte membrane was minimized by holding the oocyte membrane potential near the Cl- reversal potential (67% +/- 10% inhibition at -20 mV compared to 79% +/- 4% at -60 mV) demonstrating that Iamil downregulation was independent of the amount of current flow through CFTR. Studies with the Ca2+-sensitive photoprotein aequorin showed that Ca2+ is not involved in Iamil downregulation by CFTR, although Ca2+ injection into the cytoplasm did inhibit Iamil. These results demonstrate that downregulation of ENaC by CFTR depends on the degree of CFTR stimulation, but does not involve Ca2+ and is independent of the direction and magnitude of Cl- transport across the plasma membrane.
- Nov 1998
The patch-clamp technique was applied to study ion conductances in various configurations of the nuclear envelope of non-enzyme-treated red beet (Beta vulgaris L.) nuclei. With excised patches a non-selective cation channel was observed, that was activated by micromolar concentrations of Ca2+ on the nucleoplasmic side of the envelope. The channel activity was also voltage-dependent and the voltage threshold of channel activation changed with the nucleoplasmic Ca2+ concentration. The most prominent conductance level was 110+/-22 pS with 150 mM KCl in the bath and pipette. The channel was permeable to small cations: permeabilities relative to K+ were PK congruent with PNa=1, PCs=0.3, but PCl=0.09. Calcium ions also permeated the channel with PCa=0.43, estimated from reversal potential, or 0.14, estimated from conductance ratio. Zn2+ (1 mM) when applied to the cytoplasmic side of the envelope blocked the channel activity completely, while amiloride (2 mM) reduced the channel current by 86% from the nucleoplasmic side. The properties of the whole-nucleus current (voltage-, time- and Ca2+-dependence) paralleled those observed with excised patches. The channel may provide a Ca2+-regulated pathway for passive diffusion of cations across the nuclear envelope and thus may play an important role in Ca2+-dependent nuclear processes ranging from gene transcription to apoptosis.
- May 1997
In rat glomeruli and mesangial cells, the thromboxane A2 (TxA2) mimetic, U-46,619, but not 8-iso-prostaglandin F2alpha (8-iso-PGF2alpha), reduced glomerular inulin space and increased inositol 1,4,5-trisphosphate production, effects abolished by SQ-29,548. In competitive binding studies using 8-iso-[3H]PGF2alpha or [3H]SQ-29,548, mesangial cells displayed TxA2 binding sites but not ones for 8-iso-PGF2alpha. In contrast, rat aortic smooth muscle cells possessed specific binding sites for both TxA2 and 8-iso-PGF2alpha and displayed functional responses to both agonists, such as time- and dose-dependent activation of mitogen-activated protein kinases. In these cells, the mean dissociation constant value for the isoprostane receptor was 31.8 +/- 5.7 nM. When human TxA2 receptor cDNA was expressed in Xenopus oocytes injected with the Ca2+-specific photoprotein, aequorin, 8-iso-PGF2alpha gave much weaker responses than U-46,619. These studies provide the first radioligand binding characteristics of the F2-isoprostane receptor and demonstrate its specific and heterologous cellular localization. These studies support the distinct nature and biological significance of isoprostane receptors and provide a tool for their further molecular characterization.
- Apr 1997
Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated ATP efflux has been proposed as an autocrine mechanism for regulating chloride secretion through other types of chloride channels. Although we found in previous studies that wild-type CFTR channels bathed with high-ATP solutions do not conduct ATP at rates that can be measured with the patch-clamp technique, those experiments would not have detected very small or electroneutral ATP fluxes through CFTR or ATP efflux through other pathways that might be regulated by CFTR. To examine these possibilities, we have now used a sensitive luciferase luminometric assay to measure ATP efflux from epithelial and nonepithelial cell lines. Adenosine 3',5'-cyclic monophosphate (cAMP) stimulation did not raise external ATP concentration above the background noise in any of the cell lines tested [T84, Calu-3, 9HTEo- and sigma CFTE29o- (colonic and airway human epithelial cells, respectively), NIH/3T3 fibroblasts, and Chinese hamster ovary cells], and variations in ATP release were not correlated with CFTR expression. The rate of ATP release was unaffected by cAMP but was exquisitely sensitive to mechanical perturbations in both CFTR expressing and nonexpressing cells. Mechanically induced, CFTR-independent ATP release may be a physiologically relevant mechanism of epithelial regulation, which has not previously been fully appreciated.
- Aug 1996
Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.
- Jul 1996
The cystic fibrosis transmembrane conductance regulator (CFTR) in an ATP-dependent channel which mediates cAMP-stimulated chloride secretion by epithelia, particularly those of the pancreas, airways, and intestine. CFTR homologues have been found in all higher vertebrates examined to date and also in some lower vertebrates, although only the human, shark, and Xenopus genes have been heterologously expressed and shown to generate protein kinase A-activated Cl channels. Once phosphorylated, CFTR channels require hydrolyzable nucleotides to be active, but they can be locked in an open burst state when exposed to mixtures of ATP and its hydrolysis-resistant analogue AMP-PNP. This locking requires low-level phosphorylation at unidentified sites that are not among the ten "strong" (dibasic) PKA consensus sequences on CFTR. Mutagenesis of the dibasic PKA sites, which reduces in vitro phosphorylation by > 98%, reduces open probability (Po) by about 50% whilst having no effect on burst duration. Thus, incremental phosphorylation of these sites under normal conditions does not increase Po by slowing down ATP hydrolysis and stabilizing the open burst state, although locking does strictly require low-level phosphorylation at one or more cryptic sites. In addition to serving as a Cl channel, there is compelling evidence that CFTR inhibits the amiloride-sensitive, epithelial sodium channel (ENaC). The mechanism of coupling is not known but most likely involves physical interactions between the channels, perhaps mediated by an intermediate protein that impinges on other transport proteins. CFTR does not function as a conductive channel for ATP; however, extracellular ATP does regulate epithelial channels through activation of P2U purinergic receptors and, after being hydrolyzed extracellularly, through activation of adenosine receptors which elevate cAMP.
- Jun 1996
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-dependent chloride channel which may have additional functions. Recent reports that CFTR mediates substantial electrodiffusion of ATP from epithelial cells have led to the proposal that CFTR regulates other ion channels through an autocrine mechanism involving ATP. The aim of this study was to determine the ATP conductance of wild-type CFTR channels stably expressed in Chinese hamster ovary cells using patch clamp techniques. In the cell-attached configuration with 100 mM Mg middle dot ATP or Tris middle dot ATP solution in the pipette and 140 mM NaCl in the bath, exposing cells to forskolin caused the activation of a low-conductance channel having kinetics resembling those of CFTR. Single channel currents were negative at the resting membrane potential (Vm), consistent with net diffusion of Cl from the cell into the pipette. The transitions decreased in amplitude, but did not reverse direction, as Vm was clamped at increasingly positive potentials to enhance the driving force for inward ATP flow (>+80 mV). In excised patches, single channel currents did not reverse under essentially biionic conditions (Clin/ATPout or ATPin/Clout), although PKA-activated currents were clearly visible in the same patches at voltages where they would be carried by chloride ions. Moreover, with NaCl solution in the bath and a mixture of ATP and Cl in the pipette, the single channel I/V curve reversed at the predicted equilibrium potential for chloride. CFTR channel currents disappeared when patches were exposed to symmetrical ATP solutions and were restored by reexposure to Cl solution. Finally, in the whole-cell configuration with NaCl in the bath and 100 mM MgATP or TrisATP in the pipette, cAMP-stimulated cells had time-independent, outwardly rectifying currents consistent with CFTR selectivity for external Cl over internal ATP. Whole-cell currents reversed near Vm = -55 mV under these conditions, however the whole cell resistance measured at -100 mV was comparable to that of the gigaohm seal between the plasma membrane and glass pipette (7 Gomega). We conclude that CFTR does not mediate detectable electrodiffusion of ATP.
- Apr 1996
The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel regulated by protein kinase A and adenosine triphosphate (ATP). Loss of CFTR-mediated chloride ion conductance from the apical plasma membrane of epithelial cells is a primary physiological lesion in cystic fibrosis. CFTR has also been suggested to function an an ATP channel, although the size of the ATP anion is much larger than the estimated size of the CFTR pore. ATP was not conducted through CFTR in intact organs, polarized human lung cell lines, stably transfected mammalian cell lines, or planar lipid bilayers reconstituted with CFTR protein. These findings suggest that ATP permeation through the CFTR is unlikely to contribute to the normal function of CFTR or to the pathogenesis of cystic fibrosis.
- Jun 1995
To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.
A cDNA clone encoding the human prostaglandin (PG) E2 receptor EP2 subtype has been isolated from a human lung cDNA library. The 1.9-kilobase pair cDNA, hEP2, encodes for a 488-amino acid protein with a predicted molecular mass of 53,115 and has the seven putative transmembrane domains characteristic of G protein-coupled receptors. The specific binding of [3H]PGE2 to COS cell membranes transfected with the hEP2 cDNA was of high affinity with an equilibrium dissociation constant (Kd) of 1 nM and the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding was PGE1 = PGE2 > iloprost > PGF2 alpha > PGD2. In competition studies using more selective prostanoid-receptor agonist and antagonists, the [3H]PGE2 specific binding was competed by MB28767, an EP3 agonist, but not by the EP1-preferring antagonists AH6809 and SC19220, or by the EP2 agonist butaprost. Electrophysiological studies of Xenopus oocytes co-injected with hEP2 and cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) cDNAs detected PGE2-specific inward Cl- currents, demonstrating that the hEP2 cDNA encoded a functional receptor which produced an increase in cAMP levels. Thus, we have cloned the human EP2 receptor subtype which is functionally coupled to increase in cAMP. Northern blot analysis showed that hEP2 is expressed as a 3.8-kilobase mRNA in a number of human tissues with the highest expression levels present in the small intestine.
A cDNA clone coding for a functional human prostanoid IP receptor has been isolated from a lung cDNA library. The human IP receptor consists of 386 amino acid residues with a predicted molecular mass of 40,961, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes co-expressing the IP receptor and the cystic fibrosis transmembrane conductance regulator (cAMP-activated Cl- channel) with the stable prostacyclin analog iloprost resulted in specific inward Cl- currents, demonstrating that the cDNA encoded a functional IP prostanoid receptor coupled to elevation in cAMP. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the IP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]iloprost specific binding sites was as predicted for the IP receptor, with iloprost > carbacyclin > prostaglandin (PG) E2 > PGF 2 alpha = PGD2 = U46619. Northern blot analysis showed that IP mRNA was most abundantly expressed in kidney, with lesser amounts detected in lung and liver. In summary, we have cloned and expressed a cDNA for the human prostanoid IP receptor that is functionally coupled to a signaling pathway involving stimulation of intracellular cAMP production.
A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor, with PGF2 alpha approximately fluprostenol > PGD2 > PGE2 > U46619 > iloprost. In summary, we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway.
A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2 alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.
- Dec 1993
A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [H-3]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.
- Sep 1992
Human airway epithelial cells were obtained by nasal brushing, thus avoiding the use of proteolytic enzymes for cell isolation. Whole-cell Cl- conductances were studied in these cells by means of the patch-clamp technique. During whole-cell recordings, cell swelling activated a Cl- conductance that was blocked by indanyloxyacetic acid (48 +/- 10% inhibition at 50 microM). The swelling-induced current outwardly rectified and showed inactivation at depolarizing voltages (> or = +60 mV) and activation at hyperpolarizing voltages (< or = -30 mV). The voltage sensitivity of current activation was approximately twice that of inactivation. Another Cl- current with different kinetics was observed when nonswollen airway cells were stimulated with ionomycin (2 microM) in the presence of 1 mM Ca2+. The Ca(2+)-induced current exhibited activation during depolarizing voltage steps (> or = +40 mV) and inactivation during hyperpolarizing voltage steps (< or = -40 mV). In contrast to the swelling-induced current, the activation of Ca(2+)-induced current was less sensitive to voltage compared with its inactivation. Tail current analysis suggested that Cl- channels having a linear current-voltage relation mediate the response to Ca2+. This study indicates that brushed human nasal epithelial cells possess Cl- conductances that are regulated by cell swelling and Ca2+ and that they represent a useful in vitro model for studying ion transport in epithelia.
- Jan 1992
The cDNA encoding the voltage-gated chloride channel from the electric organ of Torpedo californica has been isolated and sequenced. The 2.7 kilobase pair cDNA encodes an 810 amino acid polypeptide which is highly homologous at both the DNA (97%) and amino acid (97%) levels to the voltage-gated chloride channel from the electric organ of T. marmorata. The majority of the 24 amino acid differences between the T. californica and T. marmorata voltage-gated chloride channels are clustered in two putative cytoplasmic domains with six differences located between residues 10-92 and 14 differences occurring between residues 576 to 708. Only one amino acid difference occurs in one of the predicted transmembrane domains. The most dramatic difference is an insertion of Asp-Val-Pro-Gly in a large cytoplasmic domain at amino acid residue 627 of the T. californica channel.
- Oct 1991
Trains of long-duration “action potentials” were induced by Ba2+ in osteoblast-like rat osteosarcoma cells (ROS 17/2.8), under current clamp and voltage clamp. Large depolarizing pulses were seen in microelectrode measurements at 37°C following the addition of 10 or 20mm Ba2+ to physiological bathing medium. Application of BAY K 8644 resulted in the onset of the pulses at earlier times and at more negative potentials. The pulses were blocked by nifedipine and Cd2+, but not by Ni2+. Large inward current pulses were seen in whole-cell patch technique voltage-clamp measurements at 37°C in the presence of from 10 to 110mm Ba2+ in the bathing medium. The current pulses were not seen at 22°C in the presence of 110mm Ba2+, but could be induced by BAY K 8644. These pulses were not blocked by TTX, but were blocked by nifedipine, Cd2+, Zn2+, Co2+, and by an increase in bathing [Ca2+]. The shape and frequency of the current pulses were the same as for voltage pulses under current clamp. A model that can explain these observations involves opening of L-type Ca2+ channels in a voltage-independent manner by cytosolic Ba2+ via a screening of Ca2+ from sites that produce either inactivation or a lower probability of opening in the activated state. There would be a closing of these channels at higher [Ba2+] as Ba2+ is forced onto these sites. A refractory period is also required to give repeated pulses of openings.
Chloride (Cl) channels have been proposed to play roles in lymphocyte functions including volume regulation and cellular cytotoxicity; however, direct studies of such channels in normal human lymphocytes are lacking. In the present study we describe a large conductance Cl channel observed in about 50% of excised, inside-out patches from normal human peripheral T lymphocytes. The channel has multiple conductance states with linear single-channel current-versus-voltage relationships in symmetrical Cl solutions. The most prevalent state is the largest, which has a conductance of about 365 pS. The channel closes in a voltage-dependent manner at both negative and positive potentials, but does not show voltage-dependent inactivation. The probability of opening is maximal between −15 mV and +15 mV and the voltage dependence is well described by two Boltzmann equations with half-maximal probabilities at −22.8 mV and +18.0 mV. The slopes of the voltage dependence suggest two gates in series with 5.7 and 9.6 equivalent charges. The channel was about 30 times more selective for Cl− than for Na+ or K+ under balanced osmolarity but less selective (approx. 11∶1) under a large osmotic gradient. The single-channel conductance increased with Cl concentration with an apparent saturation at about 581 pS and a Michaelis-Menten constant of about 120 mM. The selectivity sequence among anions, determined from changes in reversal potential was: I− > NO 3−> Br−, Cl− > F−, isethionate, HCO 3−> SO 42−> gluconate, propionate > aspartate ≫ Na+, K+ and was apparently the same for subconductance states. The sequence determined from measurable values of single-channel conductance was: I− > NO 3−> Br− > Cl− > F− > HCO 3−, isethionate. The channel was rapidly and reversibly blocked by 1 mM Zn2+ or 1 mM Ni2+ added to the cytoplasmic face. Possible roles of this maxi-Cl channel in lymphocyte function are discussed.
- Oct 1989
Whole cell patch clamp studies on osteoblast-like rat osteosarcoma cells (ROS 17/2.8) show the existence of L-type calcium channels in the cell membrane. Measurements were carried out at both 21 and 37 degrees C. With isotonic CsCl in the pipette and a bathing medium containing either 110 or 10 mM Ba2+, a strong depolarizing pulse was required to activate an inward current. The current-voltage relationship (I-V) of this inward current showed a maximum amplitude near +30 mV at 21 and 37 degrees C, with 110 mM Ba2+ in the bathing medium, and near +10 mV at 37 degrees C with 10 mM Ba2+. At both 21 and 37 degrees C the dihydropyridine, BAY K 8644 (2 microM), increased this current and shifted the I-V maximum to less positive potentials, while nifedipine (5 microM) reduced the current. Cd2+ (50 microM) and Co2+ (100 microM) blocked the current. At 21 degrees C the measured inward current showed a slow inactivation, with a time constant of some hundreds of milliseconds. At 37 degrees C, inactivation was considerably faster. The current was suppressed by holding the membrane potential more positive than -30 mV. These data are strong evidence that ROS 17/2.8 cells have a significant number of 'L-type' calcium channels.
Modulation of Ca2+-activable K+ permeability was compared with modulation of a membrane-bound oxidoreductase activity in human erythrocytes. Changes in the K+ permeability were monitored by flux measurements and single-channel recordings. The enzyme activity was detected by measuring reduction of ferricyanide. Pb2+, Atebrin and menadione had parallel effects on the channel protein and the enzyme. In contrast, propranolol stimulates K+ permeability, but is without effect on enzyme activity. The results demonstrate that the K+ channel and the enzyme are distinct membrane proteins but that the enzyme activity may influence channel gating.
Des mRNA de reticulocytes codant pour la bande 3, proteine membranaires, sont injectes dans des ovocytes de Xenopus laevis. Leur expression est mesuree, apres incubation. Une methode requerant un appareillage d'electrophysiologie est mise au point: elle permet de suivre parallelement a la synthese des transporteurs anionique, les flux de Cl - a travers la membrane plasmique de l'ovocyte