Store-operated Calcium Entry in Vascular Endothelial Cells Is Inhibited by cGMP via a Protein Kinase G-dependent Mechanism
2000; Elsevier BV; Volume: 275; Issue: 10 Linguagem: Inglês
10.1074/jbc.275.10.6758
ISSN1083-351X
AutoresHiu Yee Kwan, Yü Huang, Xiaoqiang Yao,
Tópico(s)Phosphodiesterase function and regulation
ResumoStore-operated Ca2+ entry in vascular endothelial cells not only serves to refill the intracellular Ca2+ stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca2+ entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca2+-ATPase, were used to induce store-operated Ca2+ entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca2+ entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 μm) or H-8 (10 μm), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca2+ entry. Addition ofS-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca2+ influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca2+-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca2+ entry in vascular endothelial cells. Store-operated Ca2+ entry in vascular endothelial cells not only serves to refill the intracellular Ca2+ stores, but also acts to stimulate the synthesis of nitric oxide, a key vasodilatory factor. In this study, we examined the role of cGMP in regulating the store-operated Ca2+ entry in aortic endothelial cells. Cyclopiazonic acid (CPA) and thapsigargin, two selective inhibitors of endoplasmic reticulum Ca2+-ATPase, were used to induce store-operated Ca2+ entry. 8-Bromo-cGMP, an activator of protein kinase G, inhibited the CPA- or thapsigargin-induced Ca2+ entry in a concentration-dependent manner. An inhibitor of protein kinase G, KT5823 (1 μm) or H-8 (10 μm), abolished the inhibitory action of 8-bromo-cGMP and resumed Ca2+ entry. Addition ofS-nitroso-N-acetylpenicillamine (a nitric oxide donor) or dipyridamole (a cGMP phosphodiesterase inhibitor) during CPA treatment elevated cellular cGMP levels, stimulated protein kinase G activity, and at the same time reduced Ca2+ influx due to CPA. Patch clamp study confirmed the existence of a CPA-activated Ca2+-permeable channel sensitive to cGMP inhibition. These results suggest that cGMP via a protein kinase G-dependent mechanism may play a key role in the regulation of the store-operated Ca2+ entry in vascular endothelial cells. cytoplasmic free calcium concentration protein kinase G bromo cyclopiazonic acid phosphate-buffered saline normal physiological saline solution calcium-free physiological saline solution S-nitroso-N-acetylpenicillamine Ca2+ influx in nonexcitable cells regulates such diverse processes as gene regulation, contraction, exocytosis, and apoptosis. In these cells, the predominant Ca2+ entry pathway is the store-operated one (1.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1287) Google Scholar), in which Ca2+ entry is governed by Ca2+ content of intracellular Ca2+ stores. Release of Ca2+ from intracellular stores causes store depletion, which then activates Ca2+entry from extracellular space. This Ca2+ entry, which has been termed the store-operated Ca2+ entry, may be the basis by which these cells maintain elevated [Ca2+]i 1and replenish their intracellular Ca2+ stores in response to agonist stimulation (1.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1287) Google Scholar). The importance of this signaling pathway has been recognized in numerous investigations and has received a great deal of attention (1.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1287) Google Scholar). However, up to now, the mechanism by which this Ca2+ entry pathway is regulated has still been poorly understood.At least in some cell types, activation of store-operated Ca2+ entry appears to involve a soluble messenger that is generated at the endoplasmic reticulum and that is capable of modulating plasma membrane Ca2+ permeability. A putative mediator termed the Ca2+ influx factor has been identified in Jurkat T lymphocytes (2.Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (785) Google Scholar). Store-operated Ca2+ entry may also be regulated by several other factors, including small molecular weight G proteins (3.Bird G. St J. Putney Jr., J.W. J. Biol. Chem. 1993; 268: 21486-21488Abstract Full Text PDF PubMed Google Scholar, 4.Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar), tyrosine kinase (5.Vostal J.G. Jackson W.L. Shulman N.R. J. Biol. Chem. 1991; 266: 16911-16916Abstract Full Text PDF PubMed Google Scholar), and tyrosine phosphatase (6.Jenner S. Farndale R.W. Sage S.O. Biochem. J. 1994; 303: 337-339Crossref PubMed Scopus (46) Google Scholar).There is a controversy as to whether cGMP plays a key regulatory role in store-operated Ca2+ entry. Several research groups proposed that cGMP could activate the store-operated Ca2+entry in pancreatic acinar cells (7.Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 8.Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar) and colonic epithelial cells (9.Bischof G. Brenman J. Bredt D.S. Machen T.E. Cell Calcium. 1995; 17: 250-262Crossref PubMed Scopus (48) Google Scholar). However, subsequent studies by Putney and co-workers (10.Bian X. Bird G. St J. Putney Jr., J.W. Cell Calcium. 1996; 19: 351-354Crossref PubMed Scopus (15) Google Scholar, 11.Gilon P. Obie J.F. Bian X. Bird G.S. Dagorn J.C. Putney Jr., J.W. Biochem. J. 1995; 311: 645-656Google Scholar) failed to observe any effect of cGMP on thapsigargin-evoked Ca2+ influx in both pancreatic acinar cells and Jurkat T cells. Similarly, in Xenopus oocytes and rat basophilic leukemia cells, cGMP had no effect on store-operated Ca2+influx (4.Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar, 12.Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (154) Google Scholar, 13.Parekh A.B. Terlau H. Stuehmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (319) Google Scholar).Vascular endothelial cells in vivo form an interface between flowing blood and vascular tissue, responding to numerous humoral and physical stimuli to secrete relaxing and contracting factors, which modulate the contractility of vascular smooth muscle cells. In many cases, the initial response of endothelial cells to these diverse signals involves Ca2+ release from intracellular stores (14.Himmel H.M. Whorton A.R. Strauss H.C. Hypertension (Dallas). 1993; 21: 112-127Crossref PubMed Scopus (234) Google Scholar). Like in other nonexcitable cells, depletion of intracellular stores activates Ca2+ entry in vascular endothelial cells (15.Pasyk E. Inazu M. Daniel E.E. Am. J. Physiol. 1995; 268: H138-H146Crossref PubMed Google Scholar, 16.Schilling W.P. Cabello O.A. Rajan L. Biochem. J. 1992; 284: 521-530Crossref PubMed Scopus (164) Google Scholar, 17.Sharma N.R. Davis M.J. Am. J. Physiol. 1996; 270: H267-H274PubMed Google Scholar, 18.Vaca L. Kunze D.L. Am. J. Physiol. 1994; 267: C920-C925Crossref PubMed Google Scholar). An increase in intracellular Ca2+ may then elevate cellular cGMP levels in endothelial cells (19.Luckhoff A. Pohl U. Mulsch A. Busse R. Br. J. Pharmacol. 1988; 95: 189-196Crossref PubMed Scopus (254) Google Scholar, 20.Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 108-143Google Scholar, 21.White D.G. Martin W. Br. J. Pharmacol. 1989; 97: 683-690Crossref PubMed Scopus (36) Google Scholar). However, it is not known whether this elevation in cellular cGMP may in any way influence the store-activated Ca2+ entry in vascular endothelial cells.This study was performed to assess the role of cGMP and protein kinase G (PKG) in the regulation of the store-operated Ca2+ entry in vascular endothelial cells. We found that an elevated cGMP level attenuated the store-operated Ca2+ entry and that this inhibitory effect might be mediated by a PKG-dependent mechanism. These results suggest that cGMP and PKG may play a key role in the regulation of the store-operated Ca2+ entry in vascular endothelial cells.RESULTSCPA is a mycotoxin from Aspergillus andPenicillium. It selectively inhibits endoplasmic reticulum Ca2+-ATPase and prevents Ca2+ re-uptake, thereby stimulating Ca2+ entry by a mechanism independent of receptor stimulation and inositol 1,4,5-trisphosphate formation. This chemical has become a major tool for depleting intracellular Ca2+ stores and activating store-operated Ca2+entry (1.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1287) Google Scholar, 15.Pasyk E. Inazu M. Daniel E.E. Am. J. Physiol. 1995; 268: H138-H146Crossref PubMed Google Scholar). In this experiment, CPA was applied to induce store depletion. Ca2+ influx was then initiated by changing the extracellular medium from a Ca2+-free solution (0Ca2+-PSS) to a Ca2+-containing solution (N-PSS). Control cells without CPA pretreatment were washed in and then maintained briefly in 0Ca2+-PSS. For the control cells, there was no change in [Ca2+]i when external Ca2+ was elevated (Fig.1 A). In contrast, for those cells pretreated with 10 μm CPA, an elevation in external Ca2+ drastically increased [Ca2+]i(Fig. 1 A). These results suggest that depletion of intracellular Ca2+ stores activates Ca 2+influx. A prolonged exposure of cultured cells to a Ca2+-free medium may also result in store depletion. We examined the relationship between cell exposure time in 0Ca2+-PSS and the subsequent Ca2+ influx triggered by elevation of extracellular Ca2+. Elevation of extracellular Ca2+ caused no change in [Ca2+]i for those cells incubated in 0Ca2+-PSS for up to 30 min. An incubation of 60 min in 0Ca2+-PSS resulted in the Ca2+ influx evoked by extracellular Ca2+. However, such a prolonged incubation in 0Ca2+-PSS often led to cell detachment from cultureware.To confirm that the CPA-induced increase in [Ca2+]i is indeed caused by Ca2+influx instead of Ca2+ release from CPA-insensitive intracellular stores, we used two known blockers of Ca2+entry. Ni2+ (3 mm), a potent blocker of Ca2+ entry that competes for Ca2+-binding sites (29.Winegar B.D. Kelly R. Lansman J.B. J. Physiol. (Lond.). 1991; 97: 351-367Google Scholar), completely blocked the increase in [Ca2+]i(Fig. 1 B). SKF-96365 (50 μm), an inhibitor of receptor-mediated Ca2+ entry (30.Cabello O.A. Schilling W.P. Biochem. J. 1993; 295: 357-366Crossref PubMed Scopus (56) Google Scholar), also abolished the rise in [Ca2+]i (Fig. 1 B). We also tested the effect of membrane depolarization on the rise in [Ca2+]i. 80 mm extracellular K+ completely suppressed the [Ca2+]irise due to CPA (Fig. 1 B). Membrane depolarization is known to reduce the driving force for Ca2+ entry (31.Adams D.J. Barakeh J. Laskey R. van Breemen C.V. FASEB J. 1989; 3: 2389-2400Crossref PubMed Scopus (375) Google Scholar). Therefore, these results are consistent with the concept that the CPA-induced rise in [Ca2+]i was caused by Ca2+influx.8-Br-cGMP was used to examine the effect of cGMP on store-operated Ca2+ entry. Application of 8-Br-cGMP reduced the [Ca2+]i rise due to CPA in a concentration-dependent manner with an IC50 of 180 μm (Fig.2 B). 8-Br-cGMP at 2 mm completely blocked the Ca2+ entry (Fig. 2,A and B). Since cGMP is an intracellular second messenger that activates PKG, we next examined the possible involvement of PKG. KT5823, a potent and highly specific PKG inhibitor (32.Grider J.R. Am. J. Physiol. 1993; 264: G334-G340PubMed Google Scholar), and H-8, another PKG inhibitor, were used for this purpose. 1 μm KT5823 or 10 μm H-8 abolished the inhibitory action of 8-Br-cGMP and resumed the Ca2+ entry due to CPA (Fig. 2 A and B). These results suggest that store-operated Ca2+ entry in endothelial cells is regulated by a PKG-dependent mechanism. It appears that when PKG activity is blocked by KT5823 or H-8, 8-Br-cGMP is no longer able to stimulate PKG. The overall effect is an inhibition of PKG by KT5823 or H-8. The inhibition of PKG may then open the pathway for store-operated Ca2+ entry.Figure 2Effects of PKG modulators on CPA-induced rise in [Ca2+]i. Cells were placed in 0Ca2+-PSS with CPA for 40 min. Chemicals were introduced 5 min prior to the experiments. At the time indicated by thearrow, the media were changed to the respective media containing 1 mm CaCl2 without EGTA.A, effects of PKG modulators. ○, control; ●, 2 mm 8-Br-cGMP; ▿, 2 mm 8-Br-cGMP + 1 μm KT5823; ▾, 2 mm 8-Br-cGMP + 10 μm H-8. B, concentration-dependent inhibition by 8-Br-cGMP of the CPA-induced rise in [Ca2+]i. The peak amplitude of [Ca2+]i was plotted versus 8-Br-cGMP concentration. The peak value in the absence of cGMP was normalized to 100. Each point represents the mean ± S.E. (n = 6–12).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then tested whether CPA-induced Ca2+ influx could be augmented by prior inhibition of PKG. In the presence of 1 μm KT5823 or 10 μm H-8, CPA-induced Ca2+ influx was significantly increased (Fig.3). For those cells treated with PKG inhibitors, an initial [Ca2+]i peak was followed by a rapid decrease in [Ca2+]i (Fig. 3). We speculate that quicker refillings of intracellular Ca2+stores under this condition may subsequently suppress Ca2+influx.Figure 3Effects of KT5823 and H-8 on CPA-induced rise in [Ca2+]i. Cells were placed in 0Ca2+-PSS in the presence of CPA + KT5823 or CPA + H-8 for 40 min. At the time indicated by the arrow, the media were changed to the respective media containing 1 mmCaCl2 without EGTA. ○, control; ●, 1 μmKT5823; ▾, 10 μm H-8. Each point represents the mean ± S.E. (n = 8–12). *, p < 0.05 compared with the control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)It has been reported that PKG and protein kinase A have similarities in structure and substrate specificity (33.Beebe S.J. Corbin J.D. Boyer P.D. Krebs E.G. The Enzymes. 17. Academic Press, New York1986: 43-106Google Scholar). We thus tested the effect of 8-Br-cAMP on CPA-induced Ca2+ entry. Unlike cGMP, 2 mm 8-Br-cAMP had no effect on CPA-induced Ca2+entry (n = 15), suggesting that PKA is not involved.Store depletion has been reported to increase cellular cGMP in other nonexcitable cells (8.Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar). To further explore the possible regulatory role of cGMP in store-operated Ca2+ entry, we tested the effect of CPA on cellular cGMP levels in vascular endothelial cells. Fig.4 illustrates that CPA (10 μm) stimulated cGMP production in vascular endothelial cells. Maximal stimulation occurred at 10 min after addition of CPA. At this time point, CPA increased cGMP levels by 13-fold. SNAP (100 μm), a NO donor (34.Southam E. Garthwaite J. Neurosci. Lett. 1991; 130: 107-111Crossref PubMed Scopus (158) Google Scholar), and dipyridamole (10 μm), an inhibitor of cGMP phosphodiesterase (35.Weishaar R.E. Burrows S.D. Kobylarz D.C. Quade M.M. Evans D.B. Biochem. Pharmacol. 1986; 35: 787-800Crossref PubMed Scopus (245) Google Scholar), were also used to elevate cellular cGMP levels. Addition of SNAP or dipyridamole during CPA treatment further raised cellular cGMP (Fig.5 B) and almost abolished the Ca2+ influx due to CPA (Fig. 5 A). Endogenous PKG was also stimulated, with its activity raised to ∼95% of its maximal activity (Fig. 5 C).Figure 4Effect of CPA on cGMP content of rat aortic endothelial cells. Cells were treated with CPA. At the indicated times, samples were extracted with ethanol to determine cGMP content. The cGMP content in the control cells without CPA treatment was normalized to 1. Values represent means ± S.E. (n= 6).View Large Image Figure ViewerDownload Hi-res image Download (PPT)For all the above experiments, we also used thapsigargin, another selective inhibitor of endoplasmic reticulum Ca2+-ATPase (1.Parekh A.B. Penner R. Physiol. Rev. 1997; 77: 901-930Crossref PubMed Scopus (1287) Google Scholar, 36.Thastrup O. Cullen P.J. Drobak B.K. Hanley M.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2466-2470Crossref PubMed Scopus (2986) Google Scholar), as an independent tool to evaluate our findings obtained from CPA experiments. When thapsigargin was used to activate Ca2+ influx, results similar to those with CPA were observed (data not shown).These data strongly suggest that a PKG-sensitive Ca2+ entry pathway may play an important role in store-operated Ca2+entry. However, they give no indication as to whether this pathway plays a role in the maintenance of [Ca2+]i under basal unstimulated conditions. An attempt was therefore made to address this issue. Cultured endothelial cells were bathed in a normal physiological solution containing 1 mm Ca2+without CPA pretreatment. Application of 2 mm 8-Br-cGMP caused only a slight decrease in intracellular Ca2+(n = 10). On the other hand, treatment of the cells with 1 μm KT5823 caused a large increase in [Ca2+]i (Fig.6 A). The KT5823-induced rise in [Ca2+]i resulted from Ca2+ influx since it was rapidly blocked by 3 mm Ni2+ (Fig.6 B). These data suggest that the basal Ca2+entry through the PKG-sensitive pathway may be low. On the other hand, opening of this Ca2+ entry pathway can significantly increase [Ca2+]i.Figure 6Effect of KT5823 on resting [Ca2+]i. Cells were placed in N-PSS, which contained 1 mm CaCl2. At the time indicated by the upward arrows, 1 μm KT5823 was added. Thedownward arrow illustrates the point at which 3 mm NiCl2 was added. A, effect of KT5823; B, effect of Ni2+ on KT5823-induced rise in [Ca2+]i. Each point represents the mean ± S.E. (n = 4–12).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also tested the effect of H-8 on resting [Ca2+]i of the cells bathed in normal physiological solution without CPA pretreatment. However, unlike KT5823, H-8 (10 μm) did cause any immediate rise in [Ca2+]i. One possibility is that the inhibition of PKG by H-8 might be slow, probably due to slow diffusion of the chemical across the plasma membrane to its targeted site. Any increase in [Ca2+]i due to slow opening of this PKG-sensitive Ca2+ influx pathway may be cancelled out by other [Ca2+]i-reducing mechanisms such as plasma membrane Ca2+-ATPase and endoplasmic reticulum Ca2+-ATPase. Therefore, the net change in [Ca2+]i might be very little.A patch clamp technique was used to search for store depletion-activated channels in isolated aortic endothelial cells. A Ca2+-permeable channel was identified in the cell-attached mode with the bath solution being KCl saline and the pipette solution being CaCl2 saline. Fig. 7(A–C) shows the single channel current traces recorded in a typical experiment. Fig. 7 (E–G) shows the corresponding amplitude histograms. The current traces were recorded with the patch potential held at −100 mV. A very low level of channel activity could be observed immediately after the formation of a gigaohm seal (Fig. 7,A and E). CPA treatment drastically increased the channel activity (Fig. 7, B and F). A subsequent application of 1 mm 8-Br-cGMP in the presence of CPA completely abolished the channel activity (Fig. 7, C andG). A unitary current amplitude of −1.0 ± 0.5 pA was calculated by fitting with two gaussian functions. The single channelI-V relationship from the same patch is displayed in Fig. 7 D. This CPA-activated channel was observed in 11 out of 62 cell-attached membrane patches. Among these 11 patches, seven had long-lasting good seals, which made the subsequent cGMP treatment possible. cGMP treatment abolished CPA-induced channel activity in all seven membrane patches. For three membrane patches that lasted even longer, we applied 1 μm KT5823. KT5823 resumed the channel activity in all three patches. The slope conductance and the extrapolated reversal potential were obtained by linear regression. The average slope conductance and the reversal potential from seven different patches were 9.1 ± 1.4 picosiemens and 22 ± 5 mV, respectively. The conductance of this channel was not changed after application of CPA. Since this channel reversed at a positive voltage under our experimental conditions, it should be a Ca2+-permeable channel.Figure 7A CPA-inducible Ca2+-permeable channel sensitive to the inhibition of cGMP. The pipette/bath solutions contained CaCl2 saline/KCl saline.A–C are single channel current traces. E–G are current amplitude histograms for A–C, respectively. Each histogram was derived from current activities during a period of 60 s. A and E, control; B andF, CPA treatment; C and G, 8-Br-cGMP treatment in the presence of CPA. The patch potential was held at −100 mV. D, single channel current-voltage relationship.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn this study, we used CPA and thapsigargin, two potent inhibitors of endoplasmic reticulum Ca2+-ATPase, to deplete intracellular Ca2+ stores. CPA or thapsigargin treatment activated Ca2+ entry from extracellular space as indicated by an elevation in intracellular Ca2+ (Fig. 1 A). Several lines of evidence suggest that the rise in [Ca2+]i is caused by Ca2+ influx instead of Ca2+ release from CPA- or thapsigargin-insensitive intracellular stores. 1) The increase in [Ca2+]i is evoked by application of Ca2+ to extracellular solution. 2) It is blocked by Ni2+ and SKF-96365. 3) It is abolished by membrane depolarization induced by 80 mm external K+(Fig. 1 B).Fig. 2 illustrates that the store-operated Ca2+ entry in endothelial cells can be inhibited by 8-Br-cGMP in a concentration-dependent manner. PKG inhibitors KT5823 and H-8 reverse the inhibitory effect of 8-Br-cGMP. These data suggest that the store-operated Ca2+ entry is subjected to modulation by cGMP through a PKG-dependent mechanism. We propose that cGMP and PKG may be crucial components in controlling store-operated Ca2+ entry at least in aortic endothelial cells.In pancreatic acinar cells, depletion of intracellular Ca2+stores is known to stimulate production of NO, which subsequently activates guanylate cyclase, leading to an elevation of cellular cGMP (8.Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar). The results from Fig. 4 indicate that store depletion elevates cellular cGMP levels in cultured endothelial cells as well. Our proposed model is that the elevated cGMP may in turn inhibit Ca2+ entry via a PKG-dependent pathway, therefore providing a negative feedback mechanism through which Ca2+ influx is finely regulated depending upon intracellular contents of Ca2+, cGMP, and NO. NO is a crucial signal in the regulation of vascular tone. It is conceivable that vascular endothelial cells may need such a fine regulatory mechanism for a better control of vascular tone. This feedback mechanism also allows the cells to avoid detrimental effects of excessive [Ca2+]i and/or NO. Excessive [Ca2+]i or NO may lead to apoptosis and cell death (37.Murphy M.P. Biochim. Biophys. Acta. 1999; 1411: 401-414Crossref PubMed Scopus (360) Google Scholar, 38.Balligand J.L. Cannon P.J. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1845-1858Crossref Scopus (220) Google Scholar). Our model predicts that the inhibition of PKG will substantially augment CPA-induced Ca2+ influx. This is indeed confirmed by the experiments in Fig. 3, which show that, in the presence of KT5823 (1 μm) or H-8 (10 μm), CPA-induced Ca2+ influx is augmented.Although CPA elevates cellular cGMP levels and stimulates PKG activity in endothelial cells, PKG is not maximally activated (Fig.5 C). The elevated PKG activity is not enough to offset the Ca2+ influx triggered by CPA-induced store depletion. SNAP and dipyridamole were used to further increase the cellular cGMP levels in the presence of CPA. These treatments cause additional increases in cellular cGMP and PKG activity (Fig. 5, B and C) and almost abolish the Ca2+ influx due to CPA (Fig.5 A).It is likely that this PKG-sensitive Ca2+ entry plays only a limited role in the maintenance of [Ca2+]iunder basal unstimulated conditions. PKG activity is substantial even without CPA treatment (Fig. 5 C). Treatment of cultured endothelial cells with 2 mm 8-Br-cGMP causes no significant change in [Ca2+]i, whereas the inhibition of PKG by 1 μm KT5823 causes a rapid and significant increase in Ca2+ entry (Fig. 6. A and B). These results suggest that this Ca2+ influx pathway is mostly closed by PKG phosphorylation under basal unstimulated conditions. However, this Ca2+ influx pathway may open under the condition of store depletion, presumably through a PKG-independent pathway such as production of the Ca2+ influx factor or activation of tyrosine kinase (2.Randriamampita C. Tsien R.Y. Nature. 1993; 364: 809-814Crossref PubMed Scopus (785) Google Scholar, 3.Bird G. St J. Putney Jr., J.W. J. Biol. Chem. 1993; 268: 21486-21488Abstract Full Text PDF PubMed Google Scholar, 4.Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar, 5.Vostal J.G. Jackson W.L. Shulman N.R. J. Biol. Chem. 1991; 266: 16911-16916Abstract Full Text PDF PubMed Google Scholar, 6.Jenner S. Farndale R.W. Sage S.O. Biochem. J. 1994; 303: 337-339Crossref PubMed Scopus (46) Google Scholar). On the other hand, store depletion may stimulate the production of cGMP (Fig. 4), which then initiates the PKG-dependent feedback mechanism. The presence of two separate control mechanisms, one that positively stimulates Ca2+ entry via the Ca2+ influx factor or tyrosine kinase and the other that negatively attenuates Ca2+ entry through PKG, allows intracellular Ca2+ to be finely regulated.With the use of the patch clamp technique, we have recorded a CPA-activated Ca2+-permeable channel from isolated endothelial cells in the cell-attached mode (Fig. 7). Its properties appear to be similar to those of the channel previously reported by Zhang et al. (39.Zhang H. Inazu M. Weir B. Buchnan M. Daniel E. Eur. J. Pharmacol. 1994; 251: 119-125Crossref PubMed Scopus (49) Google Scholar). Both channels are stimulated by CPA. When the pipette is filled with high concentrations of CaCl2, both channels reverse at a positive voltage with the single channel conductance around 7–9.5 picosiemens. In this study, we demonstrated that the activity of this channel is inhibited by bath application of 1 mm 8-Br-cGMP (Fig. 7, C and G). A subsequent application of KT5823 (1 μm) can resume the channel activity. It is likely that this particular Ca2+-permeable nonselective cation channel might be the target for CPA stimulation and PKG phosphorylation.Our conclusion that cGMP inhibits the store-operated Ca2+entry in vascular endothelial cells is not consistent with previously published data in other nonexcitable cells. Available reports on other nonexcitable cells suggest that an increase in cGMP from the basal level either activates store-operated Ca2+ entry or has no effect (4.Fasolato C. Hoth M. Penner R. J. Biol. Chem. 1993; 268: 20737-20740Abstract Full Text PDF PubMed Google Scholar, 7.Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar, 8.Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar, 9.Bischof G. Brenman J. Bredt D.S. Machen T.E. Cell Calcium. 1995; 17: 250-262Crossref PubMed Scopus (48) Google Scholar, 10.Bian X. Bird G. St J. Putney Jr., J.W. Cell Calcium. 1996; 19: 351-354Crossref PubMed Scopus (15) Google Scholar, 11.Gilon P. Obie J.F. Bian X. Bird G.S. Dagorn J.C. Putney Jr., J.W. Biochem. J. 1995; 311: 645-656Google Scholar, 12.Parekh A.B. Penner R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7907-7911Crossref PubMed Scopus (154) Google Scholar, 13.Parekh A.B. Terlau H. Stuehmer W. Nature. 1993; 364: 814-818Crossref PubMed Scopus (319) Google Scholar). In pancreatic acinar cells, Xu et al. (8.Xu X. Star R.A. Tortorici G. Muallem S. J. Biol. Chem. 1994; 269: 12645-12653Abstract Full Text PDF PubMed Google Scholar) described a "biphasic response," in which they proposed that a low concentration of cGMP enhanced Ca2+ entry, whereas a high concentration inhibited Ca2+ entry. No quantitative concentration of cGMP was given in their report. However, their hypothesis was not supported by Bahnson et al. (7.Bahnson T.D. Pandol S.J. Dionne V.E. J. Biol. Chem. 1993; 268: 10808-10812Abstract Full Text PDF PubMed Google Scholar) and Bischof et al. (9.Bischof G. Brenman J. Bredt D.S. Machen T.E. Cell Calcium. 1995; 17: 250-262Crossref PubMed Scopus (48) Google Scholar), who reported that a high concentration of 8-Br-cGMP (1 mm) activated store-operat
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