[Show abstract][Hide abstract] ABSTRACT: The Ca(2+) sensor synaptotagmin-1 is thought to trigger membrane fusion by binding to acidic membrane lipids and SNARE proteins. Previous work has shown that binding is mediated by electrostatic interactions that are sensitive to the ionic environment. However, the influence of divalent or polyvalent ions, at physiological concentrations, on synaptotagmin's binding to membranes or SNAREs has not been explored. Here we show that binding of rat synaptotagmin-1 to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP2) is regulated by charge shielding caused by the presence of divalent cations. Surprisingly, polyvalent ions such as ATP and Mg(2+) completely abrogate synaptotagmin-1 binding to SNAREs regardless of the presence of Ca(2+). Altogether, our data indicate that at physiological ion concentrations Ca(2+)-dependent synaptotagmin-1 binding is confined to PIP2-containing membrane patches in the plasma membrane, suggesting that membrane interaction of synaptotagmin-1 rather than SNARE binding triggers exocytosis of vesicles.
[Show abstract][Hide abstract] ABSTRACT: Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP2 binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP2, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP2-PLC interaction. This charge shielding of PIP2 was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP2 analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP2 via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP2 was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP2 hydrolysis by PLC as triggered by M1 muscarinic receptors with a potency order of Mg2+ < spermine4+ < neomycin6+. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg2+ and polyamines modulate the activity of PLCs by controlling the amount of free PIP2 available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.
Preview · Article · Jan 2015 · Biophysical Journal
[Show abstract][Hide abstract] ABSTRACT: Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single-cell and subcellular levels, and can be extended to other cell types with minor modifications.
[Show abstract][Hide abstract] ABSTRACT: Melatonin secretion from the pineal gland is triggered by norepinephrine released from sympathetic terminals at night. In contrast, cholinergic and parasympathetic inputs, by activating nicotinic cholinergic receptors (nAChR), have been suggested to counterbalance the noradrenergic input. Here we investigated whether adrenergic signaling regulates nAChR channels in rat pinealocytes. Acetylcholine or the selective nicotinic receptor agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), activated large nAChR currents in whole-cell patch clamp experiments. Norepinephrine (NE) reduced the nAChR currents, an effect partially mimicked by a β-adrenergic receptor agonist, isoproterenol, and blocked by a β-adrenergic receptor antagonist, propranolol. Increasing intracellular cAMP levels using membrane-permeable 8-Br-cAMP or cBiMPS also reduced nAChR activity, mimicking the effects of NE and isoproterenol. Further, removal of ATP from the intracellular pipette solution blocked the reduction of nAChR currents, suggesting involvement of protein kinases. Indeed protein kinase A inhibitors, H-89 and Rp-cAMPS, blocked the modulation of nAChR by adrenergic stimulation. After the down-modulation by NE, nAChR channels mediated a smaller Ca(2+) influx and less membrane depolarization from the resting potential. Together these results suggest that NE released from sympathetic terminals at night attenuates nicotinic cholinergic signaling.
Preview · Article · Feb 2014 · AJP Cell Physiology
[Show abstract][Hide abstract] ABSTRACT: Alcohol abuse is a major cause of pancreatitis. However alcohol toxicity has not been fully elucidated in the pancreas and little is known about the effect of alcohol on pancreatic ducts. We report the molecular mechanisms of ethanol-induced damage of pancreatic duct epithelial cells (PDEC). Ethanol treatment for 1, 4, and 24 h resulted in cell death in a dose-dependent manner. The ethanol-induced cell damage was mainly apoptosis due to generation of reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (MMP), and activation of caspase-3 enzyme. The antioxidant N-acetylcysteine (NAC) attenuated these cellular responses and reduced cell death significantly, suggesting a critical role for ROS. Acetaldehyde, a metabolic product of alcohol dehydrogenase, induced significant cell death, depolarization of MMP, and caspase-3 activation as ethanol and this damage was also averted by NAC. Reverse transcription-polymerase chain reaction revealed the expression of several subtypes of alcohol dehydrogenase and acetaldehyde dehydrogenase. Nuclear magnetic resonance spectroscopy data confirmed the accumulation of acetaldehyde in ethanol-treated cells, suggesting that acetaldehyde formation can contribute to alcohol toxicity in PDEC. Finally, ethanol increased the leakage of PDEC monolayer which was again attenuated by NAC. In conclusion, ethanol induces apoptosis of PDEC and thereby may contribute to the development of alcohol-induced pancreatitis.
[Show abstract][Hide abstract] ABSTRACT: Store-operated Ca(2+) channels (SOCs) are activated by depletion of intracellular Ca(2+) stores following agonist-mediated Ca(2+) release. Previously we demonstrated that Ca(2+) influx through SOCs elicits exocytosis efficiently in pancreatic duct epithelial cells (PDEC). Here we describe the biophysical, pharmacological, and molecular properties of the duct epithelial SOCs using Ca(2+) imaging, whole-cell patch-clamp, and molecular biology. In PDEC, agonists of purinergic, muscarinic, and adrenergic receptors coupled to phospholipase C activated SOC-mediated Ca(2+) influx as Ca(2+) was released from intracellular stores. Direct measurement of [Ca(2+)] in the ER showed that SOCs greatly slowed depletion of the ER. Using IP3 or thapsigargin in the patch pipette elicited inwardly rectifying SOC currents. The currents increased ∼8-fold after removal of extracellular divalent cations, suggesting competitive permeation between mono- and divalent cations. The current was completely blocked by high doses of La(3+) and 2-aminoethoxydiphenyl borate (2-APB) but only partially depressed by SKF-96365. In polarized PDEC, SOCs were localized specifically to the basolateral membrane. RT-PCR screening revealed the expression of both STIM and Orai proteins for the formation of SOCs in PDEC. By expression of fluorescent STIM1 and Orai1 proteins in PDEC, we confirmed that colocalization of the two proteins increases after store depletion. In conclusion, basolateral Ca(2+) entry through SOCs fills internal Ca(2+) stores depleted by external stimuli and will facilitate cellular processes dependent on cytoplasmic Ca(2+) such as salt and mucin secretion from the exocrine pancreatic ducts.
[Show abstract][Hide abstract] ABSTRACT: Here we present a convenient method for easy hand selection of enzymatically isolated small tissues such as islets of Langerhans. Islets are continuously collected in a micropipette tip connected to a peristaltic pump. After entering the conical micropipette tip, the islets are quickly dragged up by solution flow, but this movement subsequently decreases as the flow rate decreases. Thus, the islets are trapped at a specific height where downward gravitation balances upward buoyancy and the drag provided by solution flow. Our device allows more efficient isolation of islets compared to conventional manual collection methods.
[Show abstract][Hide abstract] ABSTRACT: In most eukaryotic cells, microtubules and filamentous actin (F-actin) provide tracks on which intracellular organelles move using molecular motors. Here we report that cytoplasmic movement of both mitochondria and lysosomes is slowed by F-actin meshwork formation in pancreatic duct epithelial cells (PDEC). Mitochondria and lysosomes were labeled with fluorescent Mitotracker Red CMXRos and Lysotracker Red DND-99, respectively, and their movements were monitored using epi-fluorescence and confocal microscopy. Mitochondria and lysosomes moving actively at rest stopped rapidly within several seconds after an intracellular Ca(2+) rise induced by activation of P2Y(2) purinergic receptors. The 'freezing' of the organelles was inhibited by blocking the Ca(2+) rise or by pretreatment with latrunculin B, an inhibitor of F-actin formation. Indeed, this freezing effect on the organelles was accompanied by the formation of F-actin in the whole cytoplasm as stained with Alexa 488-phalloidin in fixed PDEC. For real-time monitoring of F-actin formation in live cells, we expressed sGFP-fimbrin actin binding domain2 (fABD2) in PDEC. Rapid recruitment of the fluorescent probe near the nucleus and lysosomes suggested dense F-actin formation around intracellular structures. The development of F-actin paralleled that of organelle freezing. We conclude that rapid Ca(2+)-dependent F-actin formation physically restrains intracellular organelles and reduces their mobility non-selectively in PDEC.
[Show abstract][Hide abstract] ABSTRACT: Glucose supply fluctuates between meal and fasting periods and its consumption by the body varies greatly depending on bodily metabolism. Pancreatic islets of Langerhans secrete various endocrine hormones including insulin and glucagon to keep blood glucose level relatively constant. Additionally, islet hormones regulate activity of neighboring cells as local autocrine or paracrine modulators. Moreover, islet cells release neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) to gain more precise regulation of hormones release kinetics. Excitatory glutamate is co-released with glucagon from α-cells and activates glutamate receptors in the neighboring cells. GABA released from β-cells was shown to inhibit α-cells but to activate β-cells by acting GABA(A) receptors. This review summarizes the recent progress in understanding the paracrine/autocrine interactions in islets.
[Show abstract][Hide abstract] ABSTRACT: This article describes a perfusion system for biophysical single cell experiments at the physiological temperature. Our system regulates temperature of test solutions using a small heat exchanger that includes several capillaries. Water circulating inside the heat exchanger warms or cools test solutions flowing inside the capillaries. Temperature-controlled solutions are delivered directly to a single cell(s) through a multibarreled manifold that switches solutions bathing a cell in less than 1s. This solution exchange is optimal for patch clamp, single-cell microamperometry, and microfluorometry experiments. Using this system, we demonstrate that exocytosis from pancreatic β cells and activation of TRPV1 channels are temperature sensitive. We also discuss how to measure local temperature near a single cell under investigation.
[Show abstract][Hide abstract] ABSTRACT: Exocytosis is evoked by intracellular signals, including Ca(2+) and protein kinases. We determined how such signals interact to promote exocytosis in exocrine pancreatic duct epithelial cells (PDECs). Exocytosis, detected using carbon-fiber microamperometry, was stimulated by [Ca(2+)](i) increases induced either through Ca(2+) influx using ionomycin or by activation of P2Y2 or protease-activated receptor 2 receptors. In each case, the exocytosis was strongly potentiated when cyclic AMP (cAMP) was elevated either by activating adenylyl cyclase with forskolin or by activating the endogenous vasoactive intestinal peptide receptor. This potentiation was completely inhibited by H-89 and partially blocked by Rp-8-Br-cAMPS, inhibitors of protein kinase A. Optical monitoring of fluorescently labeled secretory granules showed slow migration toward the plasma membrane during Ca(2+) elevations. Neither this Ca(2+)-dependent granule movement nor the number of granules found near the plasma membrane were detectably changed by raising cAMP, suggesting that cAMP potentiates Ca(2+)-dependent exocytosis at a later stage. A kinetic model was made of the exocytosis stimulated by UTP, trypsin, and Ca(2+) ionophores with and without cAMP increase. In the model, without a cAMP rise, receptor activation stimulates exocytosis both by Ca(2+) elevation and by the action of another messenger(s). With cAMP elevation the docking/priming step for secretory granules was accelerated, augmenting the releasable granule pool size, and the Ca(2+) sensitivity of the final fusion step was increased, augmenting the rate of exocytosis. Presumably both cAMP actions require cAMP-dependent phosphorylation of target proteins. cAMP-dependent potentiation of Ca(2+)-induced exocytosis has physiological implications for mucin secretion and, possibly, for membrane protein insertion in the pancreatic duct. In addition, mechanisms underlying this potentiation of slow exocytosis may also exist in other cell systems.
Full-text · Article · May 2010 · The Journal of General Physiology
[Show abstract][Hide abstract] ABSTRACT: Pancreatic islet cells use neurotransmitters such as l-glutamate to regulate hormone secretion. We determined which cell types in mouse pancreatic islets express ionotropic glutamate receptor channels (iGluRs) and describe the detailed biophysical properties and physiological roles of these receptors. Currents through iGluRs and the resulting membrane depolarization were measured with patch-clamp methods. Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+)-evoked exocytosis were detected by Ca(2+) imaging and carbon-fiber microamperometry. Whereas iGluR2 glutamate receptor immunoreactivity was detected using specific antibodies in immunocytochemically identified mouse alpha- and beta-cells, functional iGluRs were detected only in the alpha-cells. Fast application of l-glutamate to cells elicited rapidly activating and desensitizing inward currents at -60 mV. By functional criteria, the currents were identified as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. They were activated and desensitized by AMPA, and were activated only weakly by kainate. The desensitization by AMPA was inhibited by cyclothiazide, and the currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Islet iGluRs showed nonselective cation permeability with a low Ca(2+) permeability (P(Ca)/P(Na) = 0.16). Activation of the AMPA receptors induced a sequence of cellular actions in alpha-cells: 1) depolarization of the membrane by 27 +/- 3 mV, 2) rise in intracellular Ca(2+) mainly mediated by voltage-gated Ca(2+) channels activated during the membrane depolarization, and 3) increase of exocytosis by the Ca(2+) rise. In conclusion, iGluRs expressed in mouse alpha-cells resemble the low Ca(2+)-permeable AMPA receptor in brain and can stimulate exocytosis.
[Show abstract][Hide abstract] ABSTRACT: Stochastic resonance (SR) is a novel cooperative phenomenon occurring in
nonlinear systems due to coupling of an ambient noise and an external
signal. Biological systems may use SR mechanism to detect the signal
efficiently from an external environment. A number of studies have addressed
the SR in artificial ion channels considering external voltages as noises.
More important than these external noises is the internal, thermal noise
which changes the channel conformations essential for biological functions.
In this work, we consider that the channel gating rates follow a
non-Arrhenius temperature dependence derived from experimental data of a
real biological channel. Using the Monte-Carlo simulations, we find that in
this channel SR occurs near a physiological temperature in a very
distinctive manner compared with that for the Arrhenius gating model.
Full-text · Article · May 2009 · Physics of Condensed Matter
[Show abstract][Hide abstract] ABSTRACT: Elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) triggers exocytosis of secretory granules in pancreatic duct epithelia. In this study, we find that the signal also controls granule movement. Motions of fluorescently labeled granules stopped abruptly after a [Ca(2+)](i) increase, kinetically coincident with formation of filamentous actin (F-actin) in the whole cytoplasm. At high resolution, the new F-actin meshwork was so dense that cellular structures of granule size appeared physically trapped in it. Depolymerization of F-actin with latrunculin B blocked both the F-actin formation and the arrest of granules. Interestingly, when monitored with total internal reflection fluorescence microscopy, the immobilized granules still moved slowly and concertedly toward the plasma membrane. This group translocation was abolished by blockers of myosin. Exocytosis measured by microamperometry suggested that formation of a dense F-actin meshwork inhibited exocytosis at small Ca(2+) rises <1 microm. Larger [Ca(2+)](i) rises increased exocytosis because of the co-ordinate translocation of granules and fusion to the membrane. We propose that the Ca(2+)-dependent freezing of granules filters out weak inputs but allows exocytosis under stronger inputs by controlling granule movements.
[Show abstract][Hide abstract] ABSTRACT: Glucose homeostasis is controlled by the islets of Langerhans which are equipped with α-cells increasing the blood glucose level, β-cells decreasing it, and δ-cells the precise role of which still needs identifying. Although intercellular communications between these endocrine cells have recently been observed, their roles in glucose homeostasis have not been clearly understood. In this study, we construct a mathematical model for an islet consisting of two-state α-, β-, and δ-cells, and analyze effects of known chemical interactions between them with emphasis on the combined effects of those interactions. In particular, such features as paracrine signals of neighboring cells and cell-to-cell variations in response to external glucose concentrations as well as glucose dynamics, depending on insulin and glucagon hormone, are considered explicitly. Our model predicts three possible benefits of the cell-to-cell interactions: First, the asymmetric interaction between α- and β-cells contributes to the dynamic stability while the perturbed glucose level recovers to the normal level. Second, the inhibitory interactions of δ-cells for glucagon and insulin secretion prevent the wasteful co-secretion of them at the normal glucose level. Finally, the glucose dose–responses of insulin secretion is modified to become more pronounced at high glucose levels due to the inhibition by δ-cells. It is thus concluded that the intercellular communications in islets of Langerhans should contribute to the effective control of glucose homeostasis.
Full-text · Article · Mar 2009 · Journal of Theoretical Biology
[Show abstract][Hide abstract] ABSTRACT: Glutamate transporters are expressed throughout the CNS where their major role is to clear released glutamate from presynaptic terminals. Here, we report a novel function of the transporter in rat pinealocytes. This electrogenic transporter conducted inward current in response to L-glutamate and L- or D-aspartate and depolarized the membrane in patch-clamp experiments. Ca2+ imaging demonstrated that the transporter-mediated depolarization induced a significant Ca2+ influx through voltage-gated Ca2+ channels. The Ca2+ rise finally evoked glutamate exocytosis as detected by carbon-fiber amperometry and by HPLC. In pineal slices with densely packed pinealocytes, glutamate released from the cells effectively activated glutamate transporters in neighboring cells. The Ca2+ signal generated by KCl depolarization or acetylcholine propagated through several cell layers by virtue of the regenerative "glutamate-induced glutamate release." Therefore, we suggest that glutamate transporters mediate synchronized elevation of L-glutamate and thereby efficiently downregulate melatonin secretion via previously identified inhibitory metabotropic glutamate receptors in the pineal gland.
Full-text · Article · Nov 2008 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience