-
[show abstract]
[hide abstract]
ABSTRACT: Store-operated Ca(2+) entry (SOCE), and its major determinants are known to be important for cell migration, but mechanism of their involvement in this complex process is unknown. This study presents a detailed characterization of distinct roles of Orai1, STIM1 and PLA2g6 in focal adhesion (FA) formation and migration. Using HEK293 cells, we discovered that while molecular knock-down of Orai1, STIM1 or PLA2g6 resulted in a similar reduction in migration velocity, there were profound differences in their effects on number, localization, and lifetime of FAs. Knock-down of STIM1 caused an increase in lifetime and number of FAs, their re-distribution towards lamellae region, and increase in cell tail length. In contrast, the number of FAs in Orai1- or PLA2g6-deficient cells was significantly reduced, and FAs accumulated closer to the leading edge. Assembly rate and Vinculin phosphorylation of FAs was similarly reduced in Orai1, PLA2g6 or STIM1-deficient cells. While Orai1 and PLA2g6 accumulated and co-localized at the leading edge, STIM1 distribution was more complex. We found STIM1 protrusions in lamellipodia which co-localized with FAs, while major accumulation could be seen in central and retracting parts of the cell. Interestingly, knock-down of Orai1 and PLA2g6 produced similar and non-additive effect on migration, while knock-down of STIM1 simultaneously with either Orai1 or PLA2g6 produced additional inhibition. Together these data suggest that while Orai1, PLA2g6 and STIM1 play major role in formation of new FAs at the leading edge, STIM1 may also be involved in Orai1 and PLA2g6-independent disassembly of FAs in the back of cells.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Orai1 and STIM1 have been identified as the main determinants of the store-operated Ca(2+) entry (SOCE). Their specific roles in SOCE and their molecular interactions have been studied extensively following heterologous overexpression or molecular knockdown and extrapolated to the endogenous processes in naïve cells. Using molecular and imaging techniques, we found that variation of expression levels of Orai1 or STIM1 can significantly alter expression and role of some endogenous regulators of SOCE. Although functional inhibition of Ca(2+)-independent phospholipase A(2) β (iPLA(2)β or PLA2g6A), or depletion of plasma membrane cholesterol caused a dramatic loss of endogenous SOCE in HEK293 cells, these effects were attenuated significantly when either Orai1 or STIM1 were overexpressed. Molecular knockdown of iPLA(2)β impaired SOCE in both control cells and cells overexpressing STIM1. We also discovered important cross-talk between expression of Orai1 and a specific plasma membrane variant of iPLA(2)β but not STIM1. These data confirm the role of iPLA(2)β as an essential mediator of endogenous SOCE and demonstrate that its physiological role can be obscured by Orai1 and STIM1 overexpression.
Journal of Biological Chemistry 05/2012; 287(27):22865-72. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Store-operated Ca(2+) entry (SOCE) is important for multiple functions of vascular smooth muscle cells (SMC), which, depending of their phenotype, can resemble excitable and nonexcitable cells. Similar to nonexcitable cells, Orai1 was found to mediate Ca(2+)-selective (CRAC-like) current and SOCE in dedifferentiated cultured SMC and smooth muscle-derived cell lines. However, the role of Orai1 in cation-selective store-operated channels (cat-SOC), which are responsible for SOCE in primary SMC, remains unclear. Here we focus on primary SMC, and assess the role of Orai1 and Ca(2+)-independent phospholipase A(2) (iPLA(2)β, or PLA2G6) in activation of cat-SOC current (I(cat-SOC)), SOCE, and SMC proliferation. Using molecular, electrophysiological, imaging, and functional approaches, we demonstrate that molecular knockdown of either Orai1 or iPLA(2)β leads to similar inhibition of the whole cell cat-SOC current and SOCE in primary aortic SMC and results in significant reduction in DNA synthesis and impairment of SMC proliferation. This is the first demonstration that Orai1 and iPLA(2)β are equally important for cat-SOC, SOCE, and proliferation of primary aortic SMC.
AJP Cell Physiology 11/2011; 302(5):C748-56. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane. To test some of the predictions of a conformational coupling model, we assessed how tight the relationships are between STIM1 and Orai1 expression, puncta formation, and SOCE activation. Here we present evidence that STIM1 accumulates in puncta equally well in the presence or absence of Orai1 expression, that STIM1 accumulation is not sufficient for Orai1 accumulation in the same areas, and that normal Ca(2+) release-activated Ca(2+) current (I(CRAC)) can be activated in STIM1-deficient cells. These data challenge the idea of direct conformational coupling between STIM1 and Orai1 as a viable mechanism of puncta formation and SOCE activation and uncover greater complexity in their relationship, which may require additional intermediate elements.
AJP Cell Physiology 10/2008; 295(5):C1133-40. · 3.54 Impact Factor
-
Victoria M Bolotina
[show abstract]
[hide abstract]
ABSTRACT: The mechanism of store-operated Ca(2+) entry (SOCE) remains one of the intriguing mysteries in the field of Ca(2+) signalling. Recent discoveries have resulted in the molecular identification of STIM1 as a Ca(2+) sensor in endoplasmic reticulum, Orai1 (CRACM1) as a plasma membrane channel that is activated by the store-operated pathway, and iPLA(2)beta as an essential component of signal transduction from the stores to the plasma membrane channels. Numerous studies have confirmed that molecular knock-down of any one of these three molecules impair SOCE in a wide variety of cell types, but their mutual relations are far from being understood. This report will focus on the functional roles of Orai1, STIM1 and iPLA(2)beta, and will address some specific questions about Orai1 and TRPC1, and their relation to SOC channels in excitable and non-excitable cells. Also, it will analyse the novel role of STIM1 as a trigger for CIF production, and the complex relationship between STIM1 and Orai1 expression, puncta formation and SOCE activation. It will highlight some of the most recent findings that may challenge simple conformational coupling models of SOCE, and will offer some new perspectives on the complex relationships between Orai1, STIM1 and iPLA(2)beta in the SOCE pathway.
The Journal of Physiology 08/2008; 586(13):3035-42. · 4.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.
AJP Cell Physiology 07/2008; 294(6):C1499-508. · 3.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: STIM1 has been recently identified as a Ca(2+) sensor in endoplasmic reticulum (ER) and an initiator of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of SOCE activation remains controversial. Here we focus on the early ER-delimited steps of the SOCE pathway and demonstrate that STIM1 is critically involved in initiating of production of calcium influx factor (CIF), a diffusible messenger that can deliver the signal from the stores to plasma membrane and activate SOCE. We discovered that CIF production is tightly coupled with STIM1 expression and requires functional integrity of its intraluminal sterile alpha-motif (SAM) domain. We demonstrate that 1) molecular knockdown or overexpression of STIM1 results in corresponding impairment or amplification of CIF production and 2) inherent deficiency in the ER-delimited CIF production and SOCE activation in some cell types can be a result of their deficiency in STIM1 protein; expression of a wild-type STIM1 in such cells was sufficient to fully rescue their ability to produce CIF and SOCE. We found that glycosylation sites in the ER-resident SAM domain of STIM1 are essential for initiation of CIF production. We propose that after STIM1 loses Ca(2+) from EF hand, its intraluminal SAM domain may change conformation, and via glycosylation sites it can interact with and activate CIF-producing machinery. Thus, CIF production appears to be one of the earliest STIM1-dependent events in the ER lumen, and impairment of this process results in impaired SOCE response.
Journal of Biological Chemistry 06/2008; 283(21):14524-31. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Store-operated channels (SOC) and store-operated Ca2+ entry are known to play a major role in agonist-induced constriction of smooth muscle cells (SMC) in conduit vessels. In microvessels the role of SOC remains uncertain, in as much as voltage-gated L-type Ca2+ (Ca2+L) channels are thought to be fully responsible for agonist-induced Ca2+ influx and vasoconstriction. We present evidence that SOC and their activation via a Ca2+-independent phospholipase A2 (iPLA2)-mediated pathway play a crucial role in agonist-induced constriction of cerebral, mesenteric, and carotid arteries. Intracellular Ca2+ in SMC and intraluminal diameter were measured simultaneously in intact pressurized vessels in vitro. We demonstrated that 1) Ca2+ and contractile responses to phenylephrine (PE) in cerebral and carotid arteries were equally abolished by nimodipine (a Ca2+L) inhibitor) and 2-aminoethyl diphenylborinate (an inhibitor of SOC), suggesting that SOC and Ca2+L channels may be involved in agonist-induced constriction of cerebral arteries, and 2) functional inhibition of iPLA2beta totally inhibited PE-induced Ca2+ influx and constriction in cerebral, mesenteric, and carotid arteries, whereas K+-induced Ca2+ influx and vasoconstriction mediated by Ca2+L channels were not affected. Thus iPLA2-dependent activation of SOC is crucial for agonist-induced Ca2+ influx and vasoconstriction in cerebral, mesenteric, and carotid arteries. We propose that, on PE-induced depletion of Ca2+ stores, nonselective SOC are activated via an iPLA2-dependent pathway and may produce a depolarization of SMC, which could trigger a secondary activation of Ca2+L channels and lead to Ca2+ entry and vasoconstriction.
AJP Heart and Circulatory Physiology 04/2008; 294(3):H1183-7. · 3.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2beta and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species) mimics the effects of endogenous CIF and activates iPLA2beta when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2beta prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2beta, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2beta activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2beta, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry.
Journal of Biological Chemistry 12/2006; 281(46):34926-35. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Here we tested the role of calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2) in activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry in rat basophilic leukemia (RBL-2H3) cells. We demonstrate that 1) endogenous CIF production may be triggered by Ca2+ release (net loss) as well as by simple buffering of free Ca2+ within the stores, 2) a specific 82-kDa variant of iPLA2β and its corresponding activity are present in membrane fraction of RBL cells, 3) exogenous CIF (extracted from other species)
mimics the effects of endogenous CIF and activates iPLA2β when applied to cell homogenates but not intact cells, 4) activation of ICRAC can be triggered in resting RBL cells by dialysis with exogenous CIF, 5) molecular or functional inhibition of iPLA2β prevents activation of ICRAC, which could be rescued by cell dialysis with a human recombinant iPLA2β, 6) dependence of ICRAC on intracellular pH strictly follows pH dependence of iPLA2β activity, and 7) (S)-BEL, a chiral enantiomer of suicidal substrate specific for iPLA2β, could be effectively used for pharmacological inhibition of ICRAC and store-operated Ca2+ entry. These findings validate and significantly advance our understanding of the CIF-iPLA2-dependent mechanism of activation of ICRAC and store-operated Ca2+ entry.
Journal of Biological Chemistry 11/2006; 281(46):34926-34935. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The molecular mechanism of the store-operated Ca2+-entry (SOCE) pathway remains one of the most intriguing and long lasting mysteries of Ca2+ signaling. The elusive calcium influx factor (CIF) that is produced upon depletion of Ca2+ stores has attracted growing attention, triggered by new discoveries that filled the gap in the chain of reactions leading to activation of store-operated channels and Ca2+ entry. Ca2+-independent phospholipase A2 emerged as a target of CIF, and a major determinant of the SOCE mechanism. Here, we present our viewpoint on CIF and conformational-coupling models of SOCE from a historical perspective, trying to resolve some of the problem areas, and summarizing our present knowledge on how depletion of intracellular Ca2+ stores signals to plasma membrane channels to open and provide Ca2+ influx that is required for many important physiological functions.
Trends in Biochemical Sciences 08/2005; 30(7):378-87. · 10.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have recently found that diethylstilbestrol (DES), a synthetic estrogen agonist, inhibits thrombin-induced Ca(2+) influx in human platelets, but it remains unclear to what extend this effect might be related to the store-operated Ca(2+) influx pathway. To study the effect of DES on store-operated channels and capacitative Ca(2+) influx, we used rat basophilic leukemia (RBL) cells, vascular smooth muscle cells (SMC), and human platelets, and recorded whole-cell Ca(2+) release-activated Ca(2+) (CRAC) currents and thapsigargin (TG)-induced capacitative Ca(2+) influx. In this study, we demonstrate that extracellular DES produces a dose-dependent and reversible inhibition of CRAC currents in RBL cells (IC(50), approximately 0.5 microM), whereas intracellular DES (25 microM) has no effect. Extracellular DES (up to 30 microM) inhibited only CRAC but did not affect a whole-cell monovalent cation current mediated by TRPM7 channels. DES effectively inhibited TG-induced capacitative Ca(2+) influx in a dose-dependent manner with an IC(50) values of approximately 0.1 microM in RBL cells, <0.1 microM in SMC, and approximately 1 microM in human platelets. It is noteworthy that trans-stilbene, a close structural analog of DES that lacks hydroxyl and ethyl groups, had no effect on CRAC current and on store-operated Ca(2+) influx. Thus, we found DES to be a very effective inhibitor of store-operated channels and Ca(2+) influx in a variety of cell types.
Molecular Pharmacology 10/2004; 66(3):702-7. · 4.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cardiac tissue expresses several TRP proteins as well as a Mg2+ -inhibited, non-selective cation current (IMIC) that bears many characteristics of TRP channel currents. We used the whole-cell voltage clamp technique in pig and rat ventricular myocytes to characterize the permeation, blockage properties and regulation of the cardiac IMIC channels in order to compare them with TRP channels, in particular with Mg2+ -sensitive TRPM6 and TRPM7. We show that removing extracellular divalent cations unmasks large inward and outward monovalent currents, which can be inhibited by intracellular Mg2+. Inward currents are suppressed upon replacing extracellular Na+ by NMDG+. Divalent cations block monovalent IMIC and, at 10-20 mm, carry measurable currents. Their efficacy sequence in decreasing outward IMIC (Ni2+ = Mg2+ > Ca2+ > Ba2+) and in inducing inward IMIC (Ni2+ > Mg2+ = Ca2+ approximately Ba2+), and their permeabilities calculated from reversal potentials are similar to those of TRPM6 and TRPM7 channels. The trivalent cations Gd3+ and Dy3+ also block IMIC in a voltage-dependent manner (delta = 0.4-0.5). In addition they inhibit the inward current carried by divalent cations. IMIC is regulated by pH. Decreasing or increasing extracellular pH decreased and increased IMIC, respectively (pH0.5 = 6.9, nH = 0.98). Qualitatively similar results were obtained on IMIC in rat basophilic leukaemia cells. These effects in cardiac myocytes were absent in the presence of high intracellular buffering by 40 mm Hepes. Our results suggest that IMIC in cardiac cells is due to TRPM channels, most probably to TRPM6 or TRPM7 channels or to their heteromultimeres.
The Journal of Physiology 10/2004; 559(Pt 3):761-76. · 4.72 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cardiac tissue expresses several TRP proteins as well as a Mg2+-inhibited, non-selective cation current (IMIC) that bears many characteristics of TRP channel currents. We used the whole-cell voltage clamp technique in pig and rat ventricular myocytes to characterize the permeation, blockage properties and regulation of the cardiac IMIC channels in order to compare them with TRP channels, in particular with Mg2+-sensitive TRPM6 and TRPM7. We show that removing extracellular divalent cations unmasks large inward and outward monovalent currents, which can be inhibited by intracellular Mg2+. Inward currents are suppressed upon replacing extracellular Na+ by NMDG+. Divalent cations block monovalent IMIC and, at 10–20 mm, carry measurable currents. Their efficacy sequence in decreasing outward IMIC (Ni2+= Mg2+ > Ca2+ > Ba2+) and in inducing inward IMIC (Ni2+≫ Mg2+= Ca2+≈ Ba2+), and their permeabilities calculated from reversal potentials are similar to those of TRPM6 and TRPM7 channels. The trivalent cations Gd3+ and Dy3+ also block IMIC in a voltage-dependent manner (δ= 0.4–0.5). In addition they inhibit the inward current carried by divalent cations. IMIC is regulated by pH. Decreasing or increasing extracellular pH decreased and increased IMIC, respectively (pH0.5= 6.9, nH= 0.98). Qualitatively similar results were obtained on IMIC in rat basophilic leukaemia cells. These effects in cardiac myocytes were absent in the presence of high intracellular buffering by 40 mm Hepes. Our results suggest that IMIC in cardiac cells is due to TRPM channels, most probably to TRPM6 or TRPM7 channels or to their heteromultimeres.
The Journal of Physiology 09/2004; 559(3):761 - 776. · 4.72 Impact Factor
-
Victoria M Bolotina
[show abstract]
[hide abstract]
ABSTRACT: This perspective addresses two questions: How many store-operated channels (SOCs) are there, and how many mechanisms can account for SOC activation by depleted stores? Accumulating evidence suggests that the SOC family is not limited to the calcium-selective SOC that is responsible for ICRAC (Ca2+-SOC), but includes poorly selective cation SOCs (cat-SOCs) that may satisfy physiological needs in diverse excitable and nonexcitable cells. A growing number of studies in different cell types support the idea that all the members of SOC family (Ca2+-SOC and cat-SOC) may be activated by depletion of the stores through the same mechanism, which is mediated by calcium influx factor (CIF) and calcium-independent phospholipase A2 (iPLA2). A conformational coupling model is also discussed. To account for the most recent findings, we propose that two distinct classes of calcium-conducting channels may exist in plasma membrane, which respond to different signals: SOCs, which are activated by depletion of calcium stores through the CIF-iPLA2 mechanism [no inositol triphosphate (IP3) needed]; and IP3 receptor-operated channels (IP3ROCs), which are activated by IP3 receptor through a direct coupling mechanism (no store depletion is needed). This model, with two separate mechanisms linked to different channels, may resolve many conflicting findings and interpretations and may give a new perspective on the diversity of calcium influx pathways.
Science s STKE 08/2004; 2004(243):pe34.
-
[show abstract]
[hide abstract]
ABSTRACT: Activation of store-operated channels (SOCs) and capacitative calcium influx are triggered by depletion of intracellular calcium stores. However, the exact molecular mechanism of such communication remains unclear. Recently, we demonstrated that native SOC channels can be activated by calcium influx factor (CIF) that is produced upon depletion of calcium stores, and showed that Ca(2+)-independent phospholipase A(2) (iPLA(2)) has an important role in the store-operated calcium influx pathway. Here, we identify the key plasma-membrane-delimited events that result in activation of SOC channels. We also propose a novel molecular mechanism in which CIF displaces inhibitory calmodulin (CaM) from iPLA(2), resulting in activation of iPLA(2) and generation of lysophospholipids that in turn activate soc channels and capacitative calcium influx. Upon refilling of the stores and termination of CIF production, CaM rebinds to iPLA(2), inhibits it, and the activity of SOC channels and capacitative calcium influx is terminated.
Nature Cell Biology 03/2004; 6(2):113-20. · 19.49 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Store-operated cation (SOC) channels and capacitative Ca(2+) entry (CCE) play very important role in cellular function, but the mechanism of their activation remains one of the most intriguing and long lasting mysteries in the field of Ca(2+) signaling. Here, we present the first evidence that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is a crucial molecular determinant in activation of SOC channels and store-operated Ca(2+) entry pathway. Using molecular, imaging, and electrophysiological techniques, we show that directed molecular or pharmacological impairment of the functional activity of iPLA(2) leads to irreversible inhibition of CCE mediated by nonselective SOC channels and by Ca(2+)-release-activated Ca(2+) (CRAC) channels. Transfection of vascular smooth muscle cells (SMC) with antisense, but not sense, oligonucleotides for iPLA(2) impaired thapsigargin (TG)-induced activation of iPLA(2) and TG-induced Ca(2+) and Mn(2+) influx. Identical inhibition of TG-induced Ca(2+) and Mn(2+) influx (but not Ca(2+) release) was observed in SMC, human platelets, and Jurkat T-lymphocytes when functional activity of iPLA(2) was inhibited by its mechanism-based suicidal substrate, bromoenol lactone (BEL). Moreover, irreversible inhibition of iPLA(2) impaired TG-induced activation of single nonselective SOC channels in SMC and BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-induced activation of whole-cell CRAC current in rat basophilic leukemia cells. Thus, functional iPLA(2) is required for activation of store-operated channels and capacitative Ca(2+) influx in wide variety of cell types.
Journal of Biological Chemistry 05/2003; 278(14):11909-15. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Platelets, on activation by endothelial damage, release ADP, ATP, serotonin, epinephrine, and norepinephrine. Although ATP is known to augment the action of norepinephrine in cardiovascular and endocrine systems, the possible interaction between ATP and catecholamines in regulation of platelet reactivity has not been reported. The addition of ATP (1-5 microM) to human platelet-rich plasma did not induce platelet aggregation; however, it selectively augmented the aggregatory response to norepinephrine and epinephrine, but not to serotonin. This potentiating action of ATP was dose dependent and was not due to contamination by, or hydrolysis to, ADP. The action of ATP was blocked by 10 microM of adenosine 3'-phosphate 5'-phosphosulfate, a selective P(2)Y(1) receptor antagonist. ATP alone did not cause release of intracellular Ca(2+), but produced a significant Ca(2+) response in the presence of norepinephrine. In contrast, the P(2)X(1) receptor agonists P(1),P(6)-diadenosine-5' hexophosphate and alpha,beta-methylene-ATP had no effect on norepinephrine-induced platelet aggregation even when added at 100 microM. This synergistic interaction between ATP and norepinephrine in stimulating platelet aggregation may have significant clinical implications and suggests a prothrombotic role for ATP in stress.
AJP Heart and Circulatory Physiology 03/2003; 284(2):H619-25. · 3.71 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: 1 Previously we have described a monovalent cation (MC) current that could be unmasked by the removal of extracellular divalent cations in vascular smooth muscle cells (SMC) and cardiac myocytes, but specific and potent inhibitors of MC current have not been found, and the mechanism of its intracellular regulation remains obscure. 2 Here we show that small MC current is present in intact cells and could be dramatically up-regulated during cell dialysis. MC current in dialyzed cells strongly resembled monovalent cation current attributed to Ca(2+) release-activated Ca(2+)-selective (CRAC) channels, but its activation did not require depletion of Ca(2+) stores, and was observed when the cells were dialyzed with, or without BAPTA. 3 Intracellular free Mg(2+) inhibits MC current with K(d)=250 microM. 4 Extracellular (but not intracellular) spermine effectively blocked MC current with K(d) =3-10 microM, while store-operated cations (SOC) channels and capacitative Ca(2+) influx were not affected. 5 Spermine effectively inhibited MC current-induced SMC depolarization, and prevented Ca(2+) paradox-induced vascular contracture. 6 Both, MC and SOC currents were inhibited by 2-aminoethoxydiphenyl borate (2-APB). 7 It is concluded that MC current could be regulated by intracellular Mg(2+), and low concentrations of extracellular spermine could be used to discriminate it from SOC current, and to assess its role in cellular function.
British Journal of Pharmacology 02/2003; 138(1):234-44. · 4.41 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Store-operated channels (SOC) are known to be physiologically activated following agonist-induced IP3 production and depletion of Ca2+ stores. Here we present molecular,biophysical and mechanistic evidence that two ubiquitously expressed plasma membrane channels may be responsible for creating a complex and sometimes controversial SOC image: one being a real SOC encoded by Orai1 and activated exclusively upon depletion of Ca2+ stores (via iPLA2beta -dependent pathway), while the second one is an IP3 receptor-operated channel (IP3ROC) encoded by TRPC1 and activated via its conformational coupling with IP3 receptor. In RBL-2H3 cells endogenously expressing Orai1 and TRPC1, we unmasked and characterized whole-cell current through IP3ROC channels that was hiding behind some familiar fingerprints of ICRAC, a current through the classical Ca2+-selective SOC (CRAC) channels. We discriminated these currents by their molecular identity, selectivity and different requirements for store depletion, IP3, iPLA2beta and conformational coupling to IP3 receptor. New knowledge on the properties and coexistence of Orai1-encoded SOC and TRPC1-encoded IP3ROC, and the use of experimental approaches introduced in this manuscript should help avoid further confusion about these channels, and open new exciting possibilities for their independent studies
Channels (Austin, Tex.) 1(4):246-52. · 1.91 Impact Factor
