Extracellular Ca2+ influx and endothelin-1-induced intracellular mitogenic cascades in rabbit internal carotid artery vascular smooth muscle cells

Endothelin-1 (ET-1) has been proven to activate two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in rabbit internal carotid artery vascular smooth muscle cells (ICA VSMCs). Ca2+ influx through these channels plays an essential role for ET-1-induced mitogenesis in ICA VSMCs. The purpose of the current study was to investigate the effects of Ca2+ influx on intracellular pathways of ET-1-induced mitogenesis in ICA VSMCs using receptor-operated Ca2+ channel blockers, SK&F 96365 and LOE 908. We focused on extracellular-signal regulated kinase 1 and 2 (ERK1/2) in this context. PD 98059, an inhibitor of mitogen-activated protein kinase kinase, abolished the ET-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenesis. ERK1/2 activation by ET-1 was partially suppressed in the absence of extracellular Ca2+. Moreover, based on the sensitivity to SK&F 96365 and LOE 908, Ca2+ influx through NSCC-1, NSCC-2 and SOCC plays essential roles in the extracellular Ca2+-dependent component of ERK1/2 activity. In addition, Ca2+ influx through these channels was also involved in the PD 98059-resistant component of ET-1-induced mitogenesis. These results indicate that (1) the ET-1-induced mitogenesis involves both ERK1/2-dependent and -independent mechanisms in ICA VSMCs (2), ERK1/2 activation by ET-1 involves a Ca2+ influx-dependent cascade as well as a Ca2+ influx-independent cascade (3), Ca2+ influx through NSCC-1, NSCC-2 and SOCC has important roles in the Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, and (4) Ca2+ influx through these channels also plays important roles in mitogenic pathways downstream of ERK1/2.     

Cl~-通道在内皮素-1引起的血管平滑肌细胞增殖中的作用

目的　研究Cl-通道在内皮素 1(endothelin 1,ET 1)引起的血管平滑肌细胞增殖中的作用 ,并探讨其可能的作用机制。方法　通过细胞计数和3H TdR参入实验 ,并结合fura 2 /AM荧光测定胞浆游离Ca2 + 浓度 ([Ca2 + ]i)等技术 ,观察了Cl-通道阻断剂对ET 1引起的 [Ca2 + ]i 变化及血管平滑肌细胞增殖的影响。结果　Cl-通道阻断剂DIDS可呈浓度依赖性地抑制 10nmol·L-1ET 1引起的血管平滑肌细胞增殖 ,其它Cl-通道阻断剂如IAA 94、NPPB、SITS、DPC和速尿均无此作用 ,DIDS也能抑制 10nmol·L-1ET 1引起的内流相 [Ca2 + ]i 升高 ,而对ET 1引起的Ca2 + 释放无影响 ;预先将细胞与 1μmol·L-1nifedipine作用后 ,3μmol·L-1DIDS对 10nmol·L-1ET 1引起的内流相 [Ca2 + ]i 升高及血管平滑肌细胞增殖不再有效 ,将细胞与 10 μmol·L-1SK&F96 36 5预孵后 ,DIDS却能进一步抑制ET 1的上述作用 ;3μmol·L-1DIDS对 30mmol·L-1KCl引起的胞浆[Ca2 + ]i升高无影响。结论　DIDS可通过阻断Cl-通道来抑制ET 1因促发Cl-通道开放经电压依赖性钙通道的Ca2 +内流及细胞增殖 ,DIDS敏感的Cl-通道可能在ET 1促发的Ca2 + 内流及血管平滑肌细胞增殖的调控上都起着重要的作用     

Characterization of Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery

This study attempted to characterize Ca2+ channels involved in endothelin-1-induced contraction of rabbit basilar artery using whole-cell patch-clamp and measurement of intracellular free Ca2+ concentration. Endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in addition to the voltage-operated Ca2+ channel (VOCC). These channels can be discriminated using Ca2+ channel blockers, SK&F 96365 and LOE 908. Tension study was conducted to clarify the Ca2+ channels involved in endothelin-1-induced contraction of basilar artery. Endothelin-1-induced basilar artery contraction is fully dependent on extracellular Ca2+ influx. Based on sensitivity to nifedipine, an L-type VOCC blocker, VOCCs have a minor role in endothelin-1-induced contraction. Both LOE 908 and SK&F 96365 inhibit endothelin-1-induced contraction in a concentration-dependent manner, and their combination abolished it. The median inhibitory concentrations of these blockers for endothelin-1-induced contraction correlated well with those of the endothelin-1-induced [Ca2+]i responses. Thus, the inhibitory action of these blockers on endothelin-1-induced contraction may be mediated by blockade of NSCC-1, NSCC-2, and the SOCC. Extracellular Ca2+ influx through NSCC-1, NSCC-2, and SOCC may be essential for endothelin-1-induced basilar artery contraction.     

高糖增强兔血管平滑肌细胞对内皮素—1的增殖反应性

目的;观察高糖对内皮素－１促兔主动脉血管平滑肌细胞增殖的影响。方法：ＶＳＭＣ分别培养于含正常葡萄糖,高糖或高渗的培养基中,「＾３Ｈ」胸腺嘧啶掺入法检测ＤＮＡ合成速率,蛋白质印迹法检测磷酸化ｐ４４／４２ ＭＡＰＫ的表达。结果：在１０＾－１２至１０＾－８ｍｏｌ．Ｌ＾－１浓度范围内,ＥＴ－１浓度依赖方式增加ＶＳＭＣ的「＾３Ｈ」胸腺嘧啶掺入及磷酸化ｐ４４／ｐ４２ＭＡＰＫ的表达。     

高糖增强兔血管平滑肌细胞对内皮素-1的增殖反应性(英文)

观察高糖对内皮素－１（ＥＴ－１）促兔主动脉血管平滑肌细胞（ＶＳＭＣ）增殖的影响．方法： ＶＳＭＣ分别培养于含正常葡萄糖、高糖或高渗（５．５，２５，葡萄糖 ５．５＋甘露醇 １９．５ ｍｍｏｌ·Ｌ－１）的培养基中［３Ｈ］胸腺嘧啶掺入法检测ＤＮＡ合成速率，蛋白质印迹法检测磷酸化 ｐ４４／４２ ＭＡＰＫ的表达．结果：在 １０至 １０－８ｍｏｌ·Ｌ－１浓度范围内， ＥＴ－１以浓度依赖方式增加 ＶＳＭＣ的［３Ｈ］胸腺嘧啶掺入及磷酸化ｐ４４／４２ ＭＡＰＫ的表达，从 １０－１１到 １０－８ｍｏｌ·Ｌ－１，培养于高糖的 ＶＳＭＣ对相同浓度 ＥＴ－１的增殖反应性高于正常糖或高渗培养条件下的ＶＳＭＣ（Ｐ＜ ０．０５，或 Ｐ＜ ０．０１），而在后两种条件下，ＶＳＭＣ对ＥＴ－１的增殖反应无显著差别．同样，在高糖条件下，ＥＴ－１诱导的ＶＳＭＣ磷酸化ｐ４４／４２ＭＡＰＫ的表达较正常糖和高渗ＶＳＭＣ增加 ６０％－６５％结论：高糖增强ＶＳＭＣ对ＥＴ－１的增殖反应性，可能与磷酸化的 ｐ４４／４２ ＭＡＰＫ高表达有关     

Ca2+ entry channels in rat thoracic aortic smooth muscle cells activated by endothelin-1.

The contraction of the rat aorta induced by endothelin-1 (ET-1) requires entry of extracellular Ca2+, but involvement of voltage-operated Ca2+ channel is minor. Using whole-cell recordings of patch-clamp and monitoring of the intracellular free Ca2+ concentration ([Ca2+]i), we characterized Ca2+ entry channels in A7r5 cells activated by ET-1. ET-1 activates three types of voltage-independent Ca2+ entry channels: two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC). Furthermore, it was found that these channels can be pharmacologically discriminated using Ca2+ channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is resistant to SK&F 96365, but sensitive to LOE 908, whereas NSCC-2 is sensitive to both SK&F 96365 and LOE 908. SOCC is sensitive to SK&F 96365, but resistant to LOE 908. Using these channel blockers, we analyzed Ca2+ entry channels involved in the ET-1-induced contractions of rat thoracic aorta and increases in [Ca2+]i of single smooth muscle cells. The responses to lower concentrations of ET-1 (< or = 0.1 nM) were abolished by either SK&F 96365 or LOE 908 alone. In contrast, the responses to higher concentrations of ET-1 (> or = 1 nM) were suppressed by SK&F 96365 or LOE 908 to about 10% and 35% of controls, respectively, and abolished by combined treatment with SK&F 96365 and LOE 908. These results show that the responses of rat aorta to lower concentrations of ET-1 involve only one Ca2+ channel that is sensitive to SK&F 96365 and LOE 908 (NSCC-2), whereas those to higher concentrations of ET-1 involve NSCC-1, NSCC-2 and SOCC, contributing 10%, 55% and 35%, respectively, to total Ca2+ entry.     

The Na(+)/Ca(2+) exchanger inhibitor KB-R7943 activates large-conductance Ca(2+)-activated K(+) channels in endothelial and vascular smooth muscle cells

The effect of the selective inhibitor of Na(+)/Ca(2+) exchanger (NCX), KB-R7943, on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in cultured human umbilical vein endothelial cells (HUVECs) and freshly isolated mouse aortic smooth muscle cells (MASMCs). In voltage-clamped cells, KB-R7943 reversibly activated BK(Ca) currents in HUVECs and MASMCs. The EC(50) of KB-R7943 for BK(Ca) current activation in HUVECs was determined to be 6.78+/-0.7 microM. In inside-out and outside-out patches, KB-R7943 markedly increased BK(Ca) channel activity and slightly decreased single channel current amplitudes. In inside-out patches, KB-R7943 shifted the relationship between [Ca(2+)](i) and open probability (P(o)) to the left; the [Ca(2+)](i) required to evoke half-maximal activation changed from 1220+/-68 nM (in the absence of KB-R7943) to 620+/-199 nM (in the presence of 10 microM KB-R7943). In addition, KB-R7943 shifted the relationship between membrane potential and P(o) to the left; the membrane potential to evoke half-maximal activation changed from 76.86+/-1.09 mV (in the absence of KB-R7943) to 49.62+/-2.55 mV (in the presence of 10 microM KB-R7943). In conclusion, KB-R7943 was found to act as a potent BK(Ca) channel activator, which increases the sensitivity of BK(Ca) channels to cytosolic free Ca(2+) and membrane potential, and thereby BK(Ca) channel activity. These results should be considered when KB-R7943 is used as NCX blocker.     

Agonist-stimulated Ca(2+) transport in mesenteric vascular smooth muscle cells of vitamin D(3)-induced calcium overload rats

Altered intracellular Ca(2+) homeostasis and accumulated Ca(2+) deposition in arterial walls contribute to the natural arterial aging and aging-related vascular pathologies. To gain further insight into internal relationship between these two factors, a vitamin D(3)-induced vascular Ca(2+) overload rat model was employed. Mesenteric vascular smooth muscle cells (VSMCs) were isolated from both vitamin D(3) and Wistar control rats and were maintained in primary culture for 24 h. Cytosolic and nuclear Ca(2+) ([Ca(2+)](i), [Ca(2+)](n)) in VSMCs were compared between vitamin D(3) and Wistar groups using laser scanning confocal microscopy and Ca(2+)-sensitive-dye Fluo-3. Cytosolic and nuclear Ca(2+) were evaluated under both resting and agonist-stimulated conditions including the voltage-dependent Ca(2+) channel openers BayK8644 and KCl, the inositol-1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) release channel activator IP(3), the ryanodine-sensitive Ca(2+) release channel activator trichloromethane, the sarcoplasmic reticulum Ca(2+) -ATPase inhibitor cyclopiazonic acid, and angiotensin II. Although the levels of [Ca(2+)](n) and [Ca(2+)](i) were comparable between vitamin D(3) and Wistar groups under the resting condition, the increase of [Ca(2+)](n) and [Ca(2+)](i) elicited by various agonists was significantly enhanced in VSMCs from the vitamin D(3) group compared with those from the Wistar group, suggesting abnormality of membrane Ca(2+) gating and intracellular Ca(2+) release under Ca(2+) overload condition. In conclusion, our study indicated that vitamin D(3)-induced vascular Ca(2+) overload may directly interrupt cytosolic and nuclear Ca(2+) homeostasis.     

Ca(2+) influx through nonselective cation channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells

Ca(2+) channels activated by endothelin-1 (ET-1) in C6 glioma cells (C6 cells) were characterized using whole-cell patch-clamps and by monitoring the intracellular free Ca(2+) concentration ([Ca(2+)](i)), when administering Ca(2+) channel blockers such as LOE 908 and SK&F 96365. Using this methodology, the Ca(2+) channels involved in ET-1-induced mitogenesis were identified.The patch-clamp study and [Ca(2+)](i) monitoring showed that 10 nM ET-1 activated two types of Ca(2+)-permeable nonselective cation channels (NSCC); one was sensitive to LOE 908 but resistant to SK&F 96365 (NSCC-1) and the other was sensitive to both LOE 908 and SK&F 96365 (NSCC-2). Conversely, 0.1 nM ET-1 activated only NSCC-1.ET-1-induced mitogenesis in a concentration-dependent manner, with the maximum effect arising at concentrations > or =10 nM. LOE 908 completely suppressed the 10 nM ET-1-induced mitogenesis, whereas SK&F 96365 only partially suppressed it. The IC(50) values of these blockers for the ET-1-induced mitogenesis were similar to those for the 10 nM ET-1-induced increase in [Ca(2+)](i). In contrast, LOE 908 completely suppressed 0.1 nM ET-1-induced mitogenesis, whereas SK&F 96365 did not affect it.Collectively, these results demonstrate that the sustained increase in [Ca(2+)](i), via NSCC-1 and NSCC-2, may be essential for ET-1-induced mitogenesis in C6 cells. Moreover, the sensitivity of NSCC-1 to ET-1 is higher than that of NSCC-2 to ET-1.     

Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.     

Direct block of Ca2+ channels by calmidazolium in cultured vascular smooth muscle cells.

We investigated the action of calmidazolium (CMZ), an inhibitor of calmodulin (CaM), on the L-type Ca2+ currents (ICa(L)) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), by using the whole-cell voltage-clamp method. All experiments were conducted at room temperature (24-25 degrees C). The peak IBa (Ca2+ channel current with 5 mM Ba2+ as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of -60 mV. To elevate intracellular free Ca2+ concentration ([Ca]i) to pCa 6.5, the pipette solution contained a Ca2+-EGTA buffer (pCa 6.5) to allow equilibration with the cells. Bath application of 1 microM CMZ reduced the peak amplitude of IBa to 36.7+/-4.9% (n = 8); maximal effect occurred within 7-8 min. Peak IBa continued to decrease even after washing out the CMZ. Recovery of IBa was not observed even after 10 min of washout. Even in presence of an peptide inhibitor of CaM-dependent protein kinase-II (5.2 microM) in the pipette solution, CMZ inhibited IBa to 27.8 +/-5.3% (n = 7). To exclude the possibility that other Ca2+/ CaM-dependent kinases and phosphatases may regulate Ca2+ channel activity, we examined the effect of CMZ on IBa when [Ca]i was reduced by use of Ca2+/EGTA-buffered pipette solutions. At pCa approximately equal to 10 (10 mM EGTA and only contaminant Ca2+), CMZ inhibited IBa to 33.4+/-5.9% (n = 14) with a median inhibitory concentration (IC50) value of 0.29 microM. The activation curve (pCa approximately equal to 10) was shifted in the positive direction by 6.3 mV; the inactivation curve was shifted in the negative direction by 5.0 mV. CMZ decreased IBa progressively during repetitive step depolarizations. CMZ did not slow the rate of recovery from inactivation. In conclusion, CMZ inhibits Ca2+ channel current in a use-dependent manner. This inhibition is independent of CaMK-II and other Ca2+/CaM-dependent pathways. Therefore it is likely due to direct blockade of Ca2+ channels by CMZ. CMZ may reduce the outer surface charge and block the open state of the Ca2+ channels.     

Endothelin-1-induced signaling pathways in vascular smooth muscle cells

Endothelin-1 (ET-1), a vasoactive peptide, is believed to contribute to the pathogenesis of vascular abnormalities such as hypertension, atherosclerosis, hypertrophy and restenosis. ET-1 elicits its biological effects through the activation of two receptor subtypes, ET-A and ET-B that belong to a large family of transmembrane guanine nucleotide-binding protein-coupled receptors (GPCRs). ET-1 receptor activation results in the stimulation of several signaling pathways including mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB). An intermediary role of Ca(2+)/calmodulin-dependent protein kinases (CaMK), protein kinase C (PKC) as well as receptor and non-receptor protein tyrosine kinases in triggering the activation of MAPK and PI3-K/PKB signaling in response to ET-1 has been suggested. Activation of these pathways by ET-1 is intimately linked with the regulation of cellular hypertrophy, growth, proliferation and cell survival. Here we provide an overview of these signaling pathways in vascular smooth muscle cells (VSMCs) with an emphasis on their potential role in vascular pathophysiology.     

TRPC1 Ca(2+)-permeable channels in animal cells

The full-length transient receptor (TRPC)1 polypeptide is composed of about 790 amino acids, and several splice variants are known. The predicted structure and topology is of an integral membrane protein composed of six transmembrane domains, and a cytoplasmic C- and N-terminal domain. The N-terminal domain includes three ankyrin repeat motifs. Antibodies which recognise TRPC1 have been developed, but it has been difficult to obtain antibodies which have high affinity and specificity for TRPC1. This has made studies of the cellular functions of TRPC1 somewhat difficult. The TRPC1 protein is widely expressed in different types of animal cells, and within a given cell is found at the plasma membrane and at intracellular sites. TRPC1 interacts with calmodulin, caveolin-1, the InsP3 receptor, Homer, phospholipase C and several other proteins. Investigations of the biological roles and mechanisms of action of TRPC1 have employed ectopic (over-expression or heterologous expression) of the polypeptide in addition to studies of endogenous TRPC1. Both approaches have encountered difficulties. TRPC1 forms heterotetramers with other TRPC polypeptides resulting in cation channels which are non-selective. TRPC1 may be: a component of the pore of store-operated Ca2+ channels (SOCs); a subsidiary protein in the pathway of activation of SOCs; activated by interaction with InsP3R; and/or activated by stretch. Further experiments are required to resolve the exact roles and mechanisms of activation of TRPC1. Cation entry through the TRPC1 channel is feed-back inhibited by Ca2+ through interaction with calmodulin, and is inhibited by Gd3+, La3+, SKF96365 and 2-APB, and by antibodies targeted to the external mouth of the TRPC1 pore. Activation of TRPC1 leads to the entry to the cytoplasmic space of substantial amounts of Na+ as well as Ca2+. A requirement for TRPC1 is implicated in numerous downstream cellular pathways. The most clearly described roles are in the regulation of growth cone turning in neurons. It is concluded that TRPC1 is a most interesting protein because of the apparent wide variety of its roles and functions and the challenges posed to those attempting to elucidate its primary intracellular functions and mechanisms of action.     

Receptor-mediated mitogenic effect of thromboxane A2 in vascular smooth muscle cells

The effects of thromboxane A2 (TXA2) on the proliferation of vascular smooth muscles cells (VSMC) were examined using primary cultures of VSMC from rat aorta. U46619, a stable TXA2 mimetic, stimulated DNA synthesis of VSMC only in the presence of insulin. The effect was concentration-dependent with a half-maximal effect obtained at approximately 1 x 10(-8) M. The mitogenic effect of U46619 was larger than that of endothelin, another mitogen derived from endothelium. Among several TXA2/PGH2 analogs, the proliferative activity was detected only in the agonists, and not in the antagonists or in the metabolite of TXA2. A series of TXA2/PHG2 receptor antagonists completely suppressed the U46619-stimulated DNA synthesis as well as the [3H]SQ29,548 binding to the TXA2/PGH2 receptors in VSMC. The rank order of binding affinities to the receptors among the respective antagonists correlated well with the potencies for suppression of the proliferative effects of U46619. The mitogenic effects of U46619 were also attenuated by the presence of calcium antagonists. U46619 caused activation of phospholipase C with the production of inositol trisphosphate, leading to increases in the intracellular free Ca2+ concentration as measured with the fluorescent indicator fura-2. These results suggest that TXA2 induces mitogenic effects on VSMC through binding to its specific receptors. This effect of TXA2 on the proliferation of VSMC may be related to the development of atherosclerosis.     

Quercetin as a novel activator of L-type Ca(2+) channels in rat tail artery smooth muscle cells

1. The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage-dependent Ca(2+) channels of smooth muscle cells freshly isolated from the rat tail artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. 2. Quercetin increased L-type Ca(2+) current [I(Ca(L))] in a concentration- (pEC(50)=5.09+/-0.05) and voltage-dependent manner and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction, without, however, modifying the threshold and the equilibrium potential for Ca(2+). 3. Quercetin-induced I(Ca(L)) stimulation was reversible upon wash-out. T-type Ca(2+) current was not affected by quercetin. Quercetin shifted the voltage dependence of the steady-state inactivation and activation curves to more negative potentials by about 5.5 and 7.5 mV respectively, in the mid-potential of the curves as well as increasing the slope of activation. Quercetin slowed both the activation and the deactivation kinetics of the I(Ca(L)). The inactivation time course was also slowed but only at voltages higher than 10 mV. Moreover quercetin slowed the rate of recovery from inactivation. 4. These results prove quercetin to be a naturally-occurring L-type Ca(2+) channel activator.     

Effects of extracellular Ca2+ influx on endothelin-1-induced intracellular mitogenic cascades in C6 glioma cells

We have recently shown that endothelin-1 activates two types of Ca2+-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. These channels can be distinguished by their sensitivity to blockers of the receptor-operated Ca2+ channel, 1-[b-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365, whereas NSCC-2 is sensitive to both LOE 908 and SK&F 96365. Moreover, extracellular Ca2+ influx through these channels plays an essential role in endothelin-1-induced mitogenesis in C6 glioma cells. The purpose of the present study was to investigate the effects of extracellular Ca2+ influx on intracellular pathways of endothelin-1-induced mitogenic responses in C6 glioma cells. We focused on extracellular signal-regulated kinase 1 and 2 (ERK1/2) in this context. An inhibitor of mitogen-activated protein kinase, 2-[2-amino-3-methoxyphenyl]-4H-1-benzopyran-4-one (PD 98059), abolished the endothelin-1-induced increase in ERK1/2 activity, but only partially suppressed the mitogenic response. ERK1/2 activation by endothelin-1 was partially suppressed in the absence of extracellular Ca2+. On the basis of the sensitivity to LOE 908 and SK&F 96365, Ca2+ influx through NSCC-1 and NSCC-2 plays an essential role in the extracellular Ca2+-dependent component of ERK1/2 activity. In contrast, Ca2+ influx through NSCC-2 is involved in the ERK1/2-independent component of endothelin-1-induced mitogenesis. These results indicate that (1) the endothelin-1-induced mitogenic response involves both ERK1/2-dependent and -independent mechanisms, (2) ERK1/2 activation by endothelin-1 involves an extracellular Ca2+ influx-dependent cascade as well as an extracellular Ca2+ influx-independent cascade, (3) because endothelin-1-induced mitogenesis is completely dependent on extracellular Ca2+ influx, extracellular Ca2+ influx also plays an important role in mitogenic pathways downstream of ERK1/2, (4) extracellular Ca2+ influx through NSCC-1 and NSCC-2 has an important role in the extracellular Ca2+ influx-dependent component of ERK1/2-dependent mitogenesis, (5) extracellular Ca2+ influx through NSCC-2 has an important role in ERK1/2-independent mitogenesis, and (6) Ca2+ influx through each Ca2+ channel may play a distinct role in intracellular mitogenic cascades.     

Regulation of endothelin-1 production in cultured rat vascular smooth muscle cells

Endothelin-1 (ET-1) is secreted from all rat vascular smooth muscle cells (VSMCs) examined, in addition to endothelial cells (ECs). An average secretion rate of ET-1 from rat VSMCs was determined to be 10% that excreted from ECs. We examined the effects of 22 substances on ET-1 secretion from VSMCs and compared them with those from ECs. Transforming growth factor-beta1 (TGF-beta), acidic and basic fibroblast growth factors, epidermal growth factor, angiotensin II, and adrenaline stimulated ET-1 secretion from VSMCs, whereas forskolin, thrombin, and platelet-derived growth factor-BB reduced it. Only TGF-beta and phorbol ester elicited consistent effects on ET-1 secretion from VSMCs and ECs. Regulation of ET-1 and adrenomedullin secretion from VSMCs was distinctly different. These data suggest that ET- 1 production in VSMCs is regulated by a mechanism separate from that in ECs and from adrenomedullin production in VSMCs. Chromatographic analysis showed immunoreactive ET-1 secreted from VSMCs was mainly composed of big ET- 1, whereas approximately 90% of that from ECs was ET-1. By TGF-beta stimulation of VSMCs, the ratio of big ET-1 to ET-1 was further increased. Because big ET-1 is converted into ET-1 only on the surface of the ECs in the culture system, big ET-1 secreted from the VSMCs may function as a mediator transmitting a signal from VSMCs to ECs in vivo.     

Characterization of voltage-independent Ca2+ channels activated by endothelin-1

To clarify Ca2+ entry channels involved in the endothelin-1 (ET-1)-induced increase in the intracellular concentration ([Ca2+]i), we performed whole-cell recordings of patch-clamp techniques and monitoring of [Ca2+]i with Ca2+ indicators fura-2 and fluo-3 in A7r5 cells (a cell line derived from rat thoracic aortic smooth muscle cells). With whole-cell recordings, lower concentrations (< or = 1 nM) of ET-1 activated a Ca(2+)-permeable nonselective cation channel (designated NSCC-1). In contrast, higher concentrations (> or = 1 nM) of ET-1 activated two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and store-operated Ca2+ channel (SOCC). Importantly, we found that these Ca2+ channels can be pharmacologically discriminated using blockers of the so-called receptor operated Ca2+ influx such as SK&F 96365 and LOE 908. That is, NSCC-1 is resistant to SK&F 96365 but sensitive to LOE 908; NSCC-2 is sensitive to both SK&F 96365 and LOE 908; SOCC is sensitive to SK&F 96365 but resistant to LOE 908. Using these blockers, we analyzed the ET-1-induced increase in [Ca2+]i. The increase in [Ca2+]i induced by lower concentrations of ET-1 was resistant to SK&F 96365 but sensitive to LOE 908. In contrast, the increase in [Ca2+]i induced by higher concentrations of ET-1 was partially suppressed to approximately 30% of controls by either SK&F 96365 or LOE 908 alone, and it was abolished by their combination. These results show that the increase in [Ca2+]i induced by lower concentrations (< or = 1 nM) of ET-1 results from Ca2+ influx through NSCC-1, whereas the increase in [Ca2+]i induced by higher concentrations (> or = 10 nM) of ET-1 results from Ca2+ influx through NSCC-1, NSCC-2 and SOCC.     

Thromboxane A2-induced phosphatidylcholine hydrolysis in porcine vascular smooth muscle cells.

The effect of 9,11-epithio-11,12-methanothromboxane A2 (STA2), a thromboxane A2 receptor agonist, on phosphatidylcholine hydrolysis was examined in porcine vascular smooth muscle cells. Although STA2 stimulated diacylglycerol production in a concentration-dependent manner, it only caused a slight accumulation of [3H]phosphatidylethanol in the presence of 0.5% ethanol, reflecting its weak stimulation of phosphatidylcholine-specific phospholipase D. STA2-induced diacylglycerol production was potently and concentration dependently inhibited by potassium tricyclo-[5.2.1.0(2.6)]-decyl-(9[8])-xanthogenate (D609), an inhibitor of phosphatidylcholine-specific phospholipase C. These results suggest that the thromboxane A2 receptor in vascular smooth muscles is functionally coupled to phosphatidylcholine-specific phospholipase C to yield diacylglycerol.