Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca²⁺ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) were observed in the absence of extracellular Ca²⁺. Capacitative Ca²⁺ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca²⁺ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca²⁺ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca²⁺ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca²⁺-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca²⁺]_cyt. In PAEC bathed in Ca²⁺-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca²⁺]_cyt but did not affect initial transient increase in [Ca²⁺]_cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca²⁺]_cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca²⁺ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca²⁺ release. intracellular calcium stores; oscillations; pulmonary hypertension     

Acute hypoxia increases intracellular [Ca2+] in pulmonary arterial smooth muscle by enhancing capacitative Ca2+ entry

Hypoxic pulmonary vasoconstriction (HPV) requires influx of extracellular Ca2+ in pulmonary arterial smooth muscle cells (PASMCs). To determine whether capacitative Ca2+ entry (CCE) through store-operated Ca2+ channels (SOCCs) contributes to this influx, we used fluorescent microscopy and the Ca2+-sensitive dye fura-2 to measure effects of 4% O2 on intracellular [Ca2+] ([Ca2+]i) and CCE in primary cultures of PASMCs from rat distal pulmonary arteries. In PASMCs perfused with Ca2+-free Krebs Ringer bicarbonate solution (KRBS) containing cyclopiazonic acid to deplete Ca2+ stores in sarcoplasmic reticulum and nifedipine to prevent Ca2+ entry through L-type voltage-operated Ca2+ channels (VOCCs), hypoxia markedly enhanced both the increase in [Ca2+]i caused by restoration of extracellular [Ca2+] and the rate at which extracellular Mn2+ quenched fura-2 fluorescence. These effects, as well as the increased [Ca2+]i caused by hypoxia in PASMCs perfused with normal salt solutions, were blocked by the SOCC antagonists SKF-96365, NiCl2, and LaCl3 at concentrations that inhibited CCE >80% but did not alter [Ca2+]i responses to 60 mM KCl. In contrast, the VOCC antagonist nifedipine inhibited [Ca2+]i responses to hypoxia by only 50% at concentrations that completely blocked responses to KCl. The increased [Ca2+]i caused by hypoxia was completely reversed by perfusion with Ca2+-free KRBS. LaCl3 increased basal [Ca2+]i during normoxia, indicating effects other than inhibition of SOCCs. Our results suggest that acute hypoxia enhances CCE through SOCCs in distal PASMCs, leading to depolarization, secondary activation of VOCCs, and increased [Ca2+]i. SOCCs and CCE may play important roles in HPV.     

Effects of hypercapnia and hypocapnia on [Ca2+]i mobilization in human pulmonary artery endothelial cells.

The hydrogen ion is an important factor in the alteration of vascular tone in pulmonary circulation. Endothelial cells modulate vascular tone by producing vasoactive substances such as prostacyclin (PGI2) through a process depending on intracellular Ca2+ concentration ([Ca2+]i). We studied the influence of CO2-related pH changes on [Ca2+]i and PGI2 production in human pulmonary artery endothelial cells (HPAECs). Hypercapnic acidosis appreciably increased [Ca2+]i from 112 +/- 24 to 157 +/- 38 nmol/l. Intracellular acidification at a normal extracellular pH increased [Ca2+]i comparable to that observed during hypercapnic acidosis. The hypercapnia-induced increase in [Ca2+]i was unchanged by the removal of Ca2+ from the extracellular medium or by the depletion of thapsigargin-sensitive intracellular Ca2+ stores. Hypercapnic acidosis may thus release Ca2+ from pH-sensitive but thapsigargin-insensitive intracellular Ca2+ stores. Hypocapnic alkalosis caused a fivefold increase in [Ca2+]i compared with hypercapnic acidosis. Intracellular alkalinization at a normal extracellular pH did not affect [Ca2+]i. The hypocapnia-evoked increase in [Ca2+]i was decreased from 242 +/- 56 to 50 +/- 32 nmol/l by the removal of extracellular Ca2+. The main mechanism affecting the hypocapnia-dependent [Ca2+]i increase was thought to be the augmented influx of extracellular Ca2+ mediated by extracellular alkalosis. Hypercapnic acidosis caused little change in PGI2 production, but hypocapnic alkalosis increased it markedly. In conclusion, both hypercapnic acidosis and hypocapnic alkalosis increase [Ca2+]i in HPAECs, but the mechanisms and pathophysiological significance of these increases may differ qualitatively.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca²⁺ stores opens store-operated Ca²⁺ channels (SOC) and causes Ca²⁺ entry. Transient receptor potential (TRP) cation channels that are permeable to Na⁺ and Ca²⁺ are believed to form functional SOC. Because sarcolemmal Na⁺/Ca²⁺ exchanger has also been implicated in regulating [Ca²⁺]_cyt, this study was designed to test the hypothesis that the Na⁺/Ca²⁺ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca²⁺]_cyt by contributing to store depletion-mediated Ca²⁺ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na⁺, which switches the Na⁺/Ca²⁺ exchanger from the forward (Ca²⁺ exit) to reverse (Ca²⁺ entry) mode, significantly increased [Ca²⁺]_cyt, whereas inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Store depletion also induced a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Removal of extracellular Na⁺ or inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 attenuated the store depletion-mediated Ca²⁺ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange contributes to store depletion-mediated increase in [Ca²⁺]_cyt, and that blockade of the Na⁺/Ca²⁺ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle     

Mitochondrial ROS-PKCepsilon signaling axis is uniquely involved in hypoxic increase in [Ca2+]i in pulmonary artery smooth muscle cells

The molecular mechanisms underlying hypoxic responses in pulmonary and systemic arteries remain obscure. Here we for the first time report that acute hypoxia significantly increased total PKC and PKCepsilon activity in pulmonary, but not mesenteric arteries, while these two tissues showed comparable PKCepsilon protein expression and activation by the PKC activator phorbol 12-myristate 13-acetate. Hypoxia induced an increase in intracellular reactive oxygen species (ROS) generation in isolated pulmonary artery smooth muscle cells (PASMCs), but not in mesenteric artery SMCs. Inhibition of mitochondrial ROS generation with rotenone, myxothiazol, or glutathione peroxidase-1 overexpression prevented hypoxia-induced increases in total PKC and PKCepsilon activity in pulmonary arteries. The inhibitory effects of rotenone were reversed by exogenous hydrogen peroxide. A PKCepsilon translocation peptide inhibitor or PKCepsilon gene deletion decreased hypoxic increase in [Ca(2+)](i) in PASMCs, whereas the conventional PKC inhibitor GO6976 had no effect. These data suggest that acute hypoxia may specifically increase mitochondrial ROS generation, which subsequently activates PKC, particularly PKCepsilon, contributing to hypoxia-induced increase in [Ca(2+)](i) and contraction in PASMCs.     

N(omega)-nitro-L-arginine decreases resting cytosolic [Ca2+] and enhances heat stress-induced increase in cytosolic [Ca2+] in human colon carcinoma T84 cells

N(omega)-nitro-L-arginine (LNNA) inhibits the synthesis of heat shock proteins in animals and cultured cells exposed to heat stress. Heat shock protein synthesis is known to be Ca2+-dependent. In this study, we have characterized the effect of LNNA on [Ca2+]i before and after heat stress in human colon carcinoma T84 cells. In untreated cells incubated in the presence of external Ca2+, the resting [Ca2+]i was 201+/-3 nM. If these cells were exposed to 45 degrees C for 10 min, [Ca2+]i increased by 50+/-2%. Preincubation with LNNA (100 microM) without subsequent heating led to a decrease in [Ca2+]i in a LNNA concentration-dependent manner. Preincubation with LNNA followed by heating increased [Ca2+]i to levels 88+/-5% greater than cells heated without LNNA pretreatment. Incubating cells in medium without external Ca2+ (no heating, no LNNA treatment) lowered resting [Ca2+]i to 115+/-2 nM and greatly reduced the increase in [Ca2+]i observed if cells were heated in the presence of Ca2+, indicating that external Ca2+ plays an important role in the maintenance of [Ca2+]i in T84 cells. With external Ca2+ absent, LNNA pretreatment further reduced [Ca2+]i in unheated cells, and heating failed to enhance [Ca2+]i. We determined (with external Ca2+ present) that the heat-stress induced increase in [Ca2+]i in T84 cells was blocked by dichlorobenzamil, a Na+/Ca2+ exchanger inhibitor, suggesting that the exchanger mediates Ca2+ entry. The median inhibitory concentration (IC50) in cells not treated with LNNA was 0.970+/-0.028 microM. With LNNA pretreatment, the IC50 was 5.099+/-0.107 microM. Heat stress of T84 cells did not affect the binding affinity of the Na+/Ca2+ exchanger for external Ca2+, but it increased the maximal velocity of the exchanger. In unheated cells, preincubation with LNNA decreased the binding affinity of the exchanger for Ca2+, but after heat treatment, both the binding affinity and maximal velocity of the exchanger increased. Our data are consistent with the idea that LNNA affects the activity of the Na+/Ca2+ exchanger. We also determined there are intracellular Ca2+ pools in T84 cells sensitive to thapsigargin, monensin, and ionomycin. Treatment with TMB-8, a blocker of Ca2+ sequestration and mobilization, or ionomycin inhibited the LNNA-induced decrease in [Ca2+]i observed in the absence of external Ca2+, suggesting that LNNA promotes Ca2+ sequestration.     

Endothelin isopeptides evoke Ca2+ signaling and oscillations of cytosolic free [Ca2+] in human mesangial cells

Isopeptides of the newly discovered peptide family, endothelins (ET), caused a concentration-dependent increase in intracellular free [Ca2+] ([Ca2+]i) in human glomerular mesangial cells. ET isopeptides and sarafotoxin S6b caused transient and sustained [Ca2+]i waveforms which resulted from mobilization of intracellular Ca2+ stores and from Ca2+ influx through a dihydropyridine-insensitive Ca2+ channel. Ca2+ signaling evoked by ET isopeptides underwent a marked adaptive, desensitization response. Although activation of protein kinase C attenuated ET-induced Ca2+ signaling, desensitization by ET isopeptides was independent of protein kinase C. High concentrations of ET-1 and ET-2 also caused oscillations of [Ca2+]i that partially depended on extracellular Ca2+. These results suggest that an increase in [Ca2+]i constitutes a common pathway of signal transduction for the ET peptide family.     

血小板源生长因子-AA对高血压血管平滑肌细胞钙信号的影响

目的 :明确自发性高血压大鼠血管平滑肌细胞 (SHR VSMC)增殖与血小板源生长因子 AA(PDGF AA)、PDGF α受体表达的关系及钙信号在其中的作用。方法 :在培养的血管平滑肌细胞模型中 ,采用免疫印迹 (Westernblot)、3 H TdR及3 H Leu掺入、荧光探针标记测定单细胞内钙浓度等方法 ,观察不同来源大鼠 (SHR/WKY)VSMC ,PDGF AA、PDGF α受体和PDGF β受体表达的差异性以及在PDGF AA刺激下 ,VSMC增殖肥大反应、胞内 [Ca2 ]i变化和钙离子阻断剂 (nimodipine)对其的影响。 结果 :与WKY VSMC相比SHR VSMC中PDGF AA、PDGF α受体蛋白表达明显增加 ,而PDGF β受体蛋白表达在SHR VSMC与WKY VSMC无明显变化。在PDGF AA刺激下 ,增殖细胞核抗原 (PCNA)、3 H掺入率及胞内 [Ca2 ]i浓度在SHR VSMC明显增强 ;钙离子阻断剂 (nimodipine)明显抑制PCNA表达及3 H掺入 ,胞内 [Ca2 ]i浓度明显下降。结论 :自发性高血压大鼠VSMCPDGF A链及其α受体的自发性增高 ,可能是导致SHR VSMC异常增殖、肥大 ,从而触发血管反应性和血管构型变化的重要原因之一 ;细胞膜钙通道在调控VSMC的钙内流时起主要作用     

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.     

Hypoxia activates NADPH oxidase to increase [ROS]i and [Ca2+]i through the mitochondrial ROS-PKCepsilon signaling axis in pulmonary artery smooth muscle cells

The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22(phox), p47(phox), and p67(phox) were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47(phox) protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47(phox) gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca(2+) ([ROS](i) and [Ca(2+)](i)), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCepsilon translocation peptide inhibitor, and PKCepsilon gene deletion, but not the conventional PKC inhibitor GO6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and [ROS](i) in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H(2)O(2) removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H(2)O(2) increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS](i) through the mitochondrial ROS-PKCepsilon signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS](i) and [Ca(2+)](i) as well as contraction in PASMCs.     

Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

Stimulus-response coupling in mammalian ciliated cells. Demonstration of two mechanisms of control for cytosolic [Ca2+]

Changes of cytosolic [Ca2+] have been proposed to couple stimulation of ciliary movement, however, quantitative measurements of fluctuations of intracellular free [Ca2+] associated with stimulation of ciliated cells have not been investigated. In primary cultures of rabbit oviductal ciliated cells, the stimulation of ciliary activity produced by micromolar concentrations of adenosine triphosphate (ATP) and prostaglandin F2 alpha (PGF2 alpha) was associated with a transient increase of intracellular [Ca2+]. Whereas the increase of cytosolic [Ca2+] and beat frequency produced by ATP were inhibited by the Ca-channel blocker LaCl3, the rise of cytosolic [Ca2+] and frequency of ciliary beat produced by PGF2 alpha was not affected by LaCl3. These results are the first direct demonstration that fluctuations of cytosolic [Ca2+] are associated with increased ciliary beat frequency in mammalian epithelial cells. The present findings suggest two different calcium-dependent mechanisms for stimulus-coupling in ciliary epithelium: ATP acting via purinergic receptor coupled to transmembrane influx of Ca2+, and PGF2 alpha acting via receptor-mediated release of intracellular sequestered Ca.     

Platelet-derived growth factor and angiotensin II cause increases in cytosolic free calcium by different mechanisms in vascular smooth muscle cells

Platelet-derived growth factor (PDGF) and angiotensin II (AII) are thought to mediate their biological effects in vascular smooth muscle cells (VSMCs) by causing alterations in cytosolic free calcium ([ Ca2+]i). In this study we examine the pathways by which PDGF and AII alter [Ca2+]i in VSMCs. Addition of PDGF resulted in a rapid, transient, concentration-dependent increase in [Ca2+]i; this rise in [Ca2+]i was blocked completely by preincubation of cells with ethylene glycol-bis (beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) or CoCl2, by the voltage-sensitive Ca2+-channel antagonists verapamil or nifedipine, by 12-O-tetradecanoylphorbol-13-acetate (TPA), or by pertussis toxin. AII also caused an increase in [Ca2+]i; however, AII-stimulated alterations in [Ca2+]i displayed different kinetics compared with those caused by PDGF. Pretreatment of cells with 8-(diethylamine)-octyl-3,4,5-trimethyoxybenzoate hydrochloride (TMB-8), almost totally inhibited AII-induced increases in [Ca2+]i. EGTA or CoCl2 only slightly diminished AII-stimulated increases in [Ca2+]i. Nifedipine, verapamil, TPA, and pertussis toxin pretreatment were without effect on AII-induced increases in [Ca2+]i. PDGF and AII both stimulated increases in total inositol phosphate accumulation, although the one-half maximal concentration (ED50) for alterations in [Ca2+]i and phosphoinisitide hydrolysis differed by a factor of 10 for PDGF (3 X 10(-10) M for Ca2+ vs. 2.5 X 10(-9) M for phosphoinositide hydrolysis), but they were essentially identical for AII (7.5 X 10(-9) M for Ca2+ vs. 5.0 X 10(-9) M for phosphoinositide hydrolysis). PDGF stimulated mitogenesis (as measured by [3H]-thymidine incorporation into DNA) in VSMCs with an ED50 similar to that for PDGF-induced alterations in phosphoinositide hydrolysis. PDGF-stimulated mitogenesis was blocked by pretreatment of cells with voltage-sensitive Ca2+ channel blockers, TPA, or pertussis toxin. These results suggest that PDGF and AII cause alterations in [Ca2+]i in VSMCs by at least quantitatively distinct mechanisms. PDGF binding activates a pertussis-toxin-sensitive Ca2+ influx into cells via voltage-sensitive Ca2+ channels (blocked by EGTA, verapamil, and nifedipine), as well as stimulating phosphoinositide hydrolysis leading to release of Ca2+ from intracellular stores. AII-induced alterations in [Ca2+]i are mainly the result of phosphoinositide hydrolysis and consequent entry of Ca2+ into the cytoplasm from intracellular stores. Our data also suggest that changes in [Ca2+]i caused by PDGF are required for PDGF-stimulated mitogenesis.     

Mechanisms of nordihydroguaiaretic acid-induced [Ca2+]i increases in MDCK cells

The effect of nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells has been investigated. NDGA (10-100 microM) increased [Ca2+]i concentration-dependently. The [Ca2+]i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca2+ removal partly inhibited the Ca2+ signals induced by 25-100 microM NDGA and abolished that induced by 10 microM NDGA. In Ca(2+)-free medium, pretreatment with 0.1 mM NDGA for 12 min abolished the [Ca2+]i increase induced by the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) and the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (1 microM). However, 0.1 mM NDGA still increased [Ca2+]i after Ca2+ stores had been depleted by pretreating with 2 microM CCCP, 1 microM thapsigargin and 0.1 mM cyclopiazonic acid. NDGA (50 microM) activated Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength, which was almost abolished by 50 microM La3+. This implies NDGA induced Ca2+ influx mainly via a La(3+)-sensitive pathway. Consistently, 50 microM La3+ pretreatment inhibited 0.1 mM NDGA-induced [Ca2+]i increase. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.1 mM NDGA in Ca(2+)-free medium, suggesting NDGA activated capacitative Ca2+ entry. Pretreatment with 0.1 mM NDGA for 200 s prior to Ca2+ did not alter 1 microM thapsigargin-induced capacitative Ca2+ entry. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 0.1 mM NDGA-induced Ca2+ release by 65%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This suggests NDGA-induced Ca2+ release was independent of inositol 1,4,5-trisphosphate (IP3), but was modulated by phospholipase A2.