Mobilization of intracellular Ca(2+) by endothelin-1 in rat intrapulmonary arterial smooth muscle cells

Endothelin-1 (ET-1) increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs); however, the mechanisms for Ca(2+) mobilization are not clear. We determined the contributions of extracellular influx and intracellular release to the ET-1-induced Ca(2+) response using Indo 1 fluorescence and electrophysiological techniques. Application of ET-1 (10(-10) to 10(-8) M) to transiently (24-48 h) cultured rat PASMCs caused concentration-dependent increases in [Ca(2+)](i). At 10(-8) M, ET-1 caused a large, transient increase in [Ca(2+)](i) (>1 microM) followed by a sustained elevation in [Ca(2+)](i) (<200 nM). The ET-1-induced increase in [Ca(2+)](i) was attenuated (<80%) by extracellular Ca(2+) removal; by verapamil, a voltage-gated Ca(2+)-channel antagonist; and by ryanodine, an inhibitor of Ca(2+) release from caffeine-sensitive stores. Depleting intracellular stores with thapsigargin abolished the peak in [Ca(2+)](i), but the sustained phase was unaffected. Simultaneously measuring membrane potential and [Ca(2+)](i) indicated that depolarization preceded the rise in [Ca(2+)](i). These results suggest that ET-1 initiates depolarization in PASMCs, leading to Ca(2+) influx through voltage-gated Ca(2+) channels and Ca(2+) release from ryanodine- and inositol 1,4,5-trisphosphate-sensitive stores.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Rapid effects of calciotropic hormones on female rat enterocytes: combined actions of 1,25(OH)2-vitamin D3, PTH and 17beta-estradiol on intracellular Ca2+ regulation.

1,25(OH)(2)-Vitamin D(3) [1,25(OH)(2)D(3)], PTH and 17beta-estradiol increase intracellular Ca(2+) levels ([Ca(2+)](i)) in rat enterocytes by stimulating inner Ca(2+) store mobilization and voltage-dependent Ca(2+) channels through non-genomic activation of second-messenger cascades. The participation of store-operated Ca(2+) (SOC) channels in 17beta-estradiol regulation of enterocyte [Ca(2+)](i) has also been suggested. The aim of this work was to investigate whether PTH and/or 17beta-estradiol exert additive or synergistic effects acting in concert with the classic intestinal calciotropic hormone 1,25(OH)(2)D(3). Fura-2-loaded rat duodenal cells were stimulated using rPTH (10 nM), 17beta-estradiol (0.1 nM) or 1,25(OH)(2)D(3) (0.1 nM). The resulting Ca(2+) signal was characterized by an almost immediate rise in [Ca(2+)](i) (within 30 s) rapidly reaching peak levels, followed by a plateau phase that remained sustained as long as the cells were exposed to the stimulus. The addition of PTH at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, the addition of the secosteroid after the PTH-induced effect, did not induce additional increases in [Ca(2+)](i). Simultaneous treatment with both hormones resulted in an elevation of [Ca(2+)](i) equivalent to the maximal level caused by either agonist alone, suggesting common components for [Ca(2+)]i stimulation by PTH and 1,25(OH)(2)D(3). Treatment with 17beta-estradiol at the sustained phase induced by 1,25(OH)(2)D(3) or, conversely, treatment with the secosteroid after the 17beta-estradiol effect, induced additional increments in [Ca(2+)](i) (58 % and 63 %, respectively). Simultaneous treatment of enterocytes with both steroids potentiated their individual effects to the same extent as when added sequentially, also indicative of additive actions mediated by different sources of calcium signaling cascades. Moreover, 17beta-estradiol failed to further increase the 1,25(OH)(2)D(3)-induced initial Ca(2+) elevation in Ca(2+)-free medium, thus suggesting that extracellular influx mechanisms rather than intracellular Ca(2+) mobilization account for estrogen potentiation of 1,25(OH)(2)D(3) modulation of [Ca(2+)](i) in duodenal cells.     

Progesterone metabolites rapidly stimulate calcium influx in human platelets by a src-dependent pathway

The effects of several steroids and their metabolites were examined for their ability to rapidly alter intracellular free calcium ([Ca(2+)](i)) in the anucleate human platelet. Earlier studies suggested that steroids had direct and rapid non-genomic effects to alter platelet physiology. The rationale for performing this study was to investigate the signal transduction events being activated by steroids. Super-physiologic concentrations (1.0-10.0microM) of beta-estradiol and several estradiol metabolites and analogs potentiated (approximately twofold) the action of thrombin to elevate [Ca(2+)](i) in platelets, whereas 10.0microM progesterone inhibited the action of thrombin by 10-15%. Progesterone and beta-estradiol by themselves did not affect [Ca(2+)](i). Progesterone metabolites can achieve high blood concentrations. Some progesterone metabolites, particularly those in the beta-conformation, were potent stimulators of Ca(2+) influx and intracellular Ca(2+) mobilization in platelets. They activated phospholipase C because their ability to increase [Ca(2+)](i) was inhibited by the phospholipase C inhibitor U-73122. The ability of pregnanediol and collagen to increase [Ca(2+)](i) was inhibited by the src tyrosine kinase inhibitor PP1, whereas the actions of thrombin and thapsigargin to increase [Ca(2+)](i) were not affected by PP1. The effects of progesterone metabolites to increase [Ca(2+)](i) were observed with concentrations as low as 0.1microM. Pregnanolone synergized with thrombin to increase [Ca(2+)](i). It is hypothesized that human platelets possess receptors for progesterone metabolites. These receptors when stimulated will activate platelets by causing a rapid increase in [Ca(2+)](i). Pregnanolone, isopregnanediol and pregnanediol were the most effective stimulators of this newly identified src-dependent signal transduction system in platelets. Progesterone metabolites may regulate platelet aggregation and hence thrombosis in vivo.     

17beta-estradiol rapidly mobilizes intracellular calcium from ryanodine-receptor-gated stores via a PKC-PKA-Erk-dependent pathway in the human eccrine sweat gland cell line NCL-SG3

We describe a novel rapid non-genomic effect of 17beta-estradiol (E2) on intracellular Ca(2+) ([Ca(2+)](i)) signalling in the eccrine sweat gland epithelial cell line NCL-SG3. E2 had no observable effect on basal [Ca(2+)](i), however exposure of cells to E2 in the presence of the microsomal Ca(2+) ATPase pump inhibitor, thapsigargin, produced a secondary, sustained increase in [Ca(2+)](i) compared to thapsigargin treatment alone, where cells responded with a transient single spike-like increase in [Ca(2+)](i). The E2-induced increase in [Ca(2+)](i) was not dependent on the presence of extracellular calcium and was completely abolished by ryanodine (100muM). The estrogen receptor antagonist ICI 182,780 (1muM) prevented the E2-induced effects suggesting a role for the estrogen receptor in the release of [Ca(2+)](i) from ryanodine-receptor-gated stores. The E2-induced effect on [Ca(2+)](i) could also be prevented by the protein kinase C delta (PKCdelta)-specific inhibitor rottlerin (10muM), the protein kinase A (PKA) inhibitor Rp-adenosine 3',5'-cyclic monophosphorothioate (200muM) and the MEK inhibitor PD98059 (10muM). We established E2 rapidly activates the novel PKC isoform PKCvarepsilon, PKA and Erk 1/2 MAPK in a PKCdelta and estrogen-receptor-dependent manner. The E2-induced effect was specific to 17beta-estradiol, as other steroids had no effect on [Ca(2+)](i). We have demonstrated a novel mechanism by which E2 rapidly modulates [Ca(2+)](i) release from ryanodine-receptor-gated intracellular Ca(2+) stores. The signal transduction pathway involves the estrogen receptor coupled to a PKC-PKA-Erk 1/2 signalling pathway.     

Mechanisms of membrane estrogen receptor-alpha-mediated rapid stimulation of Ca2+ levels and prolactin release in a pituitary cell line

The role of membrane estrogen receptor-alpha (mERalpha) in rapid nongenomic responses to 17beta-estradiol (E(2)) was tested in sublines of GH3/B6 rat prolactinoma cells selected for high (GH3/B6/F10) and low (GH3/B6/D9) mERalpha expression. E(2) elicited rapid, concentration-dependent intracellular Ca(2+) concentration ([Ca(2+)](i)) increases in the F10 subline. Lack of inhibition by thapsigargin depletion of intracellular Ca(2+) pools, together with abrogation of the response in Ca(2+)-free medium, suggested an extracellular source of Ca(2+) for this response. The participation of voltage-dependent channels in the E(2)-induced [Ca(2+)](i) increase was confirmed by the specific L-type Ca(2+) channel inhibitor nifedipine. For comparison, the D9 mERalpha-depleted subline was insensitive to steroid action via this signaling mechanism. [Ca(2+)](i) elevation was correlated with prolactin (PRL) release in the F10 cell line in as little as 3 min. E(2) caused a much higher PRL release than KCl treatment (which caused maximal Ca(2+) elevation), suggesting that secretion was also controlled by additional mechanisms. Participation of mERalpha in these effects was confirmed by the ability of E(2)-peroxidase (a cell-impermeable analog of E(2)) to cause these responses, blockage of the responses with the ER antagonist ICI 182 780, and the inability of the E(2) stereoisomer 17alpha-E(2) to elicit a response. Thus rapid exocytosis of PRL is regulated in these cells by mERalpha signaling to specific Ca(2+) channels utilizing extracellular Ca(2+) sources and additional signaling mechanisms.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

Intracellular Ca(2+) signaling in endothelial cells by the angiogenesis inhibitors endostatin and angiostatin

Intracellular signaling mechanisms by the angiogenesis inhibitors endostatin and angiostatin remain poorly understood. We have found that endostatin (2 microg/ml) and angiostatin (5 microg/ml) elicited transient, approximately threefold increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Acute exposure to angiostatin or endostatin nearly abolished subsequent endothelial [Ca(2+)](i) responses to carbachol or to thapsigargin; conversely, thapsigargin attenuated the Ca(2+) signal elicited by endostatin. The phospholipase C inhibitor U-73122 and the inositol trisphosphate (IP(3)) receptor inhibitor xestospongin C both inhibited endostatin-induced elevation in [Ca(2+)](i), and endostatin rapidly elevated endothelial cell IP(3) levels. Pertussis toxin and SB-220025 modestly inhibited the endostatin-induced Ca(2+) signal. Removal of extracellular Ca(2+) inhibited the endostatin-induced rise in [Ca(2+)](i), as did a subset of Ca(2+)-entry inhibitors. Peak Ca(2+) responses to endostatin and angiostatin in endothelial cells exceeded those in epithelial cells and were minimal in NIH/3T3 cells. Overnight pretreatment of endothelial cells with endostatin reduced the subsequent acute elevation in [Ca(2+)](i) in response to vascular endothelial growth factor or to fibroblast growth factor by approximately 70%. Intracellular Ca(2+) signaling may initiate or mediate some of the cellular actions of endostatin and angiostatin.     

Effects of chronic exercise on calcium signaling in rat vascular endothelium

Chronic exercise enhances endothelium-dependent vasodilating responses. To investigate whether this is due to a change in endothelial Ca(2+) signaling, we examined intracellular Ca(2+) concentration ([Ca(2+)](i)) level in rat aortic endothelium in response to acetylcholine (ACh) or ATP. Four-week-old male Wistar rats were divided into control and exercise groups. The exercised animals ran on a treadmill at a moderate intensity for 60 min/day, 5 day/wk, for 10 wk. Rat aortas were then excised and loaded with fura 2. After the aortas were mounted on a flow chamber, these specimens were observed under an epifluorescence microscope equipped with ratio-imaging capability. Our results showed that 1) chronic exercise increased both ACh- and ATP-induced [Ca(2+)](i) responses; 2) ACh induced heterogeneous [Ca(2+)](i) elevation in individual endothelial cells; and 3) the exercise effect on ACh-evoked endothelial [Ca(2+)](i) elevation was inhibited by the Ca(2+) influx blocker SKF-96365, by a Ca(2+)-free buffer, or by high concentrations of extracellular K(+). We conclude that chronic exercise increases ACh-induced [Ca(2+)](i) elevation in rat aortic endothelium in situ, possibly by facilitating Ca(2+) influx.     

Nongenomic inhibition of catecholamine secretion by 17beta-estradiol in PC12 cells

We investigated the effects of 17beta-estradiol, an estrogen, on [(3)H]norepinephrine ([(3)H]NE) secretion in PC12 cells. Pretreatment with 17beta-estradiol reduced 70 mM K(+)-induced [(3)H]NE secretion in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) of 2 +/- 1 microM. The 70 mM K(+)-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) rise was also reduced when the cells were treated with 17beta-estradiol (IC(50) = 15 +/- 2 microM). Studies with voltage-sensitive calcium channel (VSCC) antagonists such as nifedipine and omega-conotoxin GVIA revealed that both L- and N-type VSCCs were affected by 17beta-estradiol treatment. The 17beta-estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17beta-estradiol. 17beta-Estradiol also inhibited the ATP-induced [(3)H]NE secretion and [Ca(2+)](i) rise. In PC12 cells, the ATP-induced [Ca(2+)](i) rise is known to occur through P2X(2) receptors, the P2Y(2)-mediated phospholipase C (PLC) pathway, and VSCCs. 17beta-Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP-induced [Ca(2+)](i) rise. Our results suggest that 17beta-estradiol inhibits catecholamine secretion by inhibiting L- and N-type Ca(2+) channels and P2X(2) receptors in a nongenomic manner.     

Stimulation of human spermatozoa with progesterone gradients to simulate approach to the oocyte. Induction of [Ca(2+)](i) oscillations and cyclical transitions in flagellar beating

Progesterone is present at micromolar concentrations in the cumulus matrix, which surrounds mammalian oocytes. Exposure of human spermatozoa to a concentration gradient of progesterone (0-3 microM) to simulate approach to the oocyte induced a slowly developing increase in [Ca(2+)](i) upon which, in many cells, slow oscillations were superimposed. [Ca(2+)](i) oscillations often started at very low progesterone (<10 nm), and their frequency did not change during the subsequent rise in concentration. Oscillations also occurred, but in a much smaller proportion of cells, in response to stepped application of progesterone (3 microM). When progesterone was removed, [Ca(2+)](i) oscillations often persisted or quickly resumed. Superfusion with low-Ca(2+) bathing medium (no added Ca(2+)) did not prevent [Ca(2+)](i) oscillations, but they could be abolished by addition of EGTA or La(3+). Inhibitors of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases or inositol trisphosphate signaling had no effect on [Ca(2+)](i) oscillations, but pharmacological manipulation of ryanodine receptors affected both their frequency and amplitude. Staining of live spermatozoa with BODIPY FL-X ryanodine showed localization of ryanodine binding primarily to the caudal part of the head and mid-piece. [Ca(2+)](i) oscillations did not induce acrosome reaction, but in cells generating oscillations, the flagellar beat mode alternated in synchrony with the oscillation cycle. Flagellar bending and lateral movement of the sperm head during [Ca(2+)](i) peaks were markedly increased compared with during [Ca(2+)](i) troughs. This alternating pattern of activity is likely to facilitate zona penetration. These observations show that progesterone initiates unusual and complex store-mediated [Ca(2+)](i) signaling in human spermatozoa and identify a previously unrecognized effect of progesterone in regulating sperm "behavior" during fertilization.     

Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells

The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores.     

Increased cytosolic Ca(2+) concentration in endothelial cells by calmodulin antagonists

Many functions of endothelial cells are Ca(2+)/calmodulin dependent, whereas the role of calmodulin in the regulation of cytosolic Ca(2+) ([Ca(2+)](i)) remains largely unexplained. In the present study, effects of various calmodulin antagonists on [Ca(2+)](i) were investigated in cultured aortic endothelial cells loaded with the Ca(2+)-sensitive dye fura-2/AM, and were compared with those of calmodulin-dependent protein kinase II (CaM kinase II) inhibitors. The calmodulin antagonists W-7, calmidazolium and fendiline provoked dose-dependent increases in [Ca(2+)](i). However, the CaM kinase II inhibitors KN-93 and lavendustin C had no effect on [Ca(2+)](i). In the absence of extracellular Ca(2+), pretreatment of cells with bradykinin (BK) and thapsigargin completely prevented W-7-stimulated increase in [Ca(2+)](i). Alternatively, pretreatment with W-7 also completely blocked BK- and thapsigargin-stimulated increases in [Ca(2+)](i). The time course of the Ca(2+)-response in W-7 treated cells was identical to that in thapsigargin-treated cells, but not that in BK-stimulated cells, suggesting that calmodulin antagonists could share a common signaling pathway with thapsigargin to increase [Ca(2+)](i) in endothelial cells. These findings indicate that calmodulin is involved in the regulation of [Ca(2+)](i), and may play an important role in the uptake of Ca(2+) to intracellular stores.     

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.     

Endothelin-1 increases intracellular Ca(2+) in rabbit pulmonary artery smooth muscle cells through phospholipase C

In freshly isolated rabbit pulmonary artery smooth muscle cells, endothelin (ET)-1 induced a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) followed by a return to the initial [Ca(2+)](i). This response was not abolished by the voltage-dependent Ca(2+) channel blocker nicardipine or removal of Ca(2+) from the bath solution but was inhibited by ryanodine and thapsigargin. This finding suggested that the increase in [Ca(2+)](i) induced by ET-1 was attributable to release of Ca(2+) from ryanodine- and inositol 1,4,5-trisphosphate-sensitive intracellular Ca(2+) stores. The transient increase in [Ca(2+)](i) induced by ET-1 was also inhibited by pretreatment with antagonists of ET type A and B (ET(A) and ET(B)) receptors (BQ-123 and BQ-788, respectively). Furthermore, the ET(B) receptor agonist IRL-1620 induced an increase in [Ca(2+)](i) that was followed by a sustained increase in [Ca(2+)](i); the sustained increase in [Ca(2+)](i) was blocked by nicardipine. Using the nystatin-perforated patch-clamp technique, we found that IRL-1620 caused an increase in Ca(2+) current that was inhibited by addition of ET-1. ET-1 did not inhibit Ca(2+) current when cells were pretreated with BQ-123. These results suggested that when both receptor types are activated, the opposing responses lead to abolition of the sustained [Ca(2+)](i) increases induced by ET(B) receptor activation. Western blot analysis confirmed expression of ET(A) and ET(B) receptors. Finally, U-73122 inhibited the ET-1-induced [Ca(2+)](i) increase, indicating that phospholipase C was involved in modulation of the ET-1-induced [Ca(2+)](i) increase in rabbit pulmonary artery smooth muscle cells.     

Synergistic effects of tumor necrosis factor-alpha and thrombin in increasing endothelial permeability

Because activation of the coagulation cascade and the generation of thrombin coexist with sepsis and the release of tumor necrosis factor (TNF)-alpha, we determined the effects of TNF-alpha on the mechanism of thrombin-induced increase in endothelial permeability. We assessed Ca(2+) signaling in human umbilical vein endothelial cells. In human umbilical vein endothelial cells exposed to TNF-alpha for 2 h, thrombin produced a rise in the intracellular Ca(2+) concentration ([Ca(2+)](i)) lasting up to 10 min. In contrast, thrombin alone produced a rise in [Ca(2+)](i) lasting for 3 min, whereas TNF-alpha alone had no effect on [Ca(2+)](i.) Thrombin-induced inositol 1,4,5-trisphosphate generation was not different between control and TNF-alpha-exposed cells. In the absence of extracellular Ca(2+), thrombin produced similar increases in [Ca(2+)](i) in both control and TNF-alpha-exposed cells. In TNF-alpha-exposed cells, the thrombin-induced Ca(2+) influx after intracellular Ca(2+) store depletion was significantly greater and prolonged compared with control cells. Increased Ca(2+) entry was associated with an approximately fourfold increase in Src activity and was sensitive to the Src kinase inhibitor PP1. After TNF-alpha exposure, thrombin caused increased tyrosine phosphorylation of junctional proteins and actin stress fiber formation as well as augmented endothelial permeability. These results suggest that TNF-alpha stimulation of endothelial cells results in amplification of the thrombin-induced Ca(2+) influx by an Src-dependent mechanism, thereby promoting loss of endothelial barrier function.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Involvement of intracellular and extracellular Ca2+ in tetramethylpyrazine-induced colonic anion secretion

In the present study we investigated the role of Ca(2+) in tetramethylpyrazine (TMP)-induced anion secretion in the human colonic epithelial cell line, Caco-2, using the short-circuit current (I(SC)) technique in conjunction with intracellular Ca(2+) measurements. The results showed that TMP-induced I(SC) response was significantly reduced by 58.8% and 38.3% after inhibiting Ca(2+) ATPase of endoplasmic reticulum (ER) with thapsigargin and mobilizing ER stored Ca(2+) release with ATP, respectively. Conversely, thapsigargin- and ATP-evoked I(SC) responses were also significantly reduced by pretreatment with TMP by 43.2% and 38.5%, respectively. Conversely, removal of extracellular Ca(2+), apical but not basolateral, or the presence of the Ca(2+) chelator (EGTA) significantly increased TMP-induced I(SC) by 47.1% and 37.8%, respectively. Similar to TMP, thapsigargin-induced current increase was also enhanced by chelating extracellular Ca(2+) or in Ca(2+) free solution; however, removal of extracellular Ca(2+) did not significantly affect 3-isobutyl-1-methylxanthine (IBMX)- and forskolin-induced transepithelial current. Measurement of the intracellular concentration of free Ca(2+) ([Ca(2+)](i)) with fura-2/AM showed that TMP could induce an increase in [Ca(2+)](i) but pretreatment with TMP significantly reduced thapsigargin-evoked, but not ATP-induced, [Ca(2+)](i) increase. These results suggest that the effect of TMP on colonic anion secretion is partly mediated by TMP-increased [Ca(2+)](i) by acting on a target similar to thapsigargin. The observed inhibitory effect of extracellular Ca(2+) on Ca(2+)-dependent anion secretion represents a novel mechanism by which Ca(2+)-dependent regulation of epithelial electrolyte transport may be fine-tuned by extracellular Ca(2+) in the apical domain.     

The essential role of H2O2 in the regulation of intracellular Ca2+ by epidermal growth factor in rat-2 fibroblasts

We have investigated a new mechanism by which epidermal growth factor (EGF) increases intracellular Ca(2+) ([Ca(2+)](i)) in Rat-2 fibroblasts. EGF induced a transient increase of [Ca(2+)](i), and sustained Ca(2+) increase disappeared in the absence of extracellular Ca(2+). However, EGF had no effect on the formation of inositol phosphates. Expression of N17Rac or scrape-loading of C3 transferase blocked the elevation of [Ca(2+)](i) by EGF, but not by lysophosphatidic acid (LPA). EGF increased intracellular H(2)O(2), with a maximal increase at 5 min, which was blocked by catalase, scrape-loading of C3 transferase, or expression of N17Rac. H(2)O(2) scavengers, catalase and N-acetyl-L-cysteine, also blocked the Ca(2+) response to EGF, but not to LPA. In the presence of EGTA, preincubation with EGF completely inhibited subsequent Ca(2+) response to extracellular H(2)O(2) and vice versa. Incubation with EGF or phosphatidic acid abolished subsequent elevation of [Ca(2+)](i) by phosphatidic acid or EGF, respectively. Furthermore, preincubation with LPA inhibited the subsequent Ca(2+) response to EGF, but not vice versa. These results suggested that intracellular H(2)O(2) regulated by Rac and RhoA, but not inositol phosphates, was responsible for the EGF-stimulated elevation of [Ca(2+)](i). It was also suggested that EGF cross talked with LPA in the regulation of [Ca(2+)](i) by producing intracellular H(2)O(2).     

Dopamine-induced graded intracellular Ca2+ elevation via the Na+Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts

Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca(2+) influx from the extracellular space. Additionally, dopamine induces a massive [Na(+)](i) elevation via the Na(+)K(+)2Cl(-) cotransporter (NKCC). We have reasoned that Ca(2+)-entry is mediated by the Na(+)Ca(2+) exchanger (NCE) operating in the Ca(2+)-entry mode. To test this hypothesis, [Ca(2+)](i) and [Na(+)](i) were measured by using the fluorescent dyes Fura-2, Fluo-3, and SBFI. Inhibition of Na(+)-entry from the extracellular space by removal of extracellular Na(+) or inhibition of the NKCC by 10 microM bumetanide did not influence resting [Ca(2+)](i) but completely abolished the dopamine-induced [Ca(2+)](i) elevation. Simultaneous recordings of [Ca(2+)](i) and [Na(+)](i) revealed that the dopamine-induced [Na(+)](i) elevation preceded the [Ca(2+)](i) elevation. During dopamine stimulation, the generation of an outward Na(+) concentration gradient by removal of extracellular Na(+) boosted the [Ca(2+)](i) elevation. Furthermore, prolonging the dopamine-induced [Na(+)](i) rise by blocking the Na(+)/K(+)-ATPase reduced the recovery from [Ca(2+)](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na(+)](i), which reverses the NCE activity into the reverse mode causing a graded [Ca(2+)](i) elevation in the duct cells.