Biochemical and Biophysical Research Communications
Endothelin-sensitive intracellular Ca2+ store overlaps with caffeine-sensitive one in rat aortic smooth muscle cells in primary culture
Summary
We made use of quin2 microfluorometry to determine the effects of endothelin (ET) on cytosolic free Ca2+ concentrations ([Ca2+]i) in rat aortic smooth muscle cells in primary culture. In Ca2+-containing medium, ET induced a rapid and sustained elevation of [Ca2+]i. In the latter component, in particular, the elevation of [Ca2+]i was inhibited by diltiazem. In Ca2+-free medium, ET induced a rapid and transient [Ca2+]i elevation, which was not inhibited by diltiazem. When the caffeine-sensitive intracellular Ca2+ store was practically depleted by repeated treatment with caffeine in Ca2+-free media, ET did not elevate [Ca2+]i. Thus, it was suggested that ET induces [Ca2+]i elevation not only by extracellular Ca2+-dependent mechanisms but also by releasing Ca2+ from the intracellular store, and that the ET-sensitive Ca2+ store may overlap with the caffeine-sensitive one, in cultured vascular smooth muscle cells.
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Cited by (74)
Role of nonselective cation channels as Ca<sup>2+</sup> entry pathway in endothelin-1-induced contraction and their suppression by nitric oxide
1998, European Journal of PharmacologyThe present study was carried out to clarify the role of nonselective cation channels as a Ca2+ entry pathway in the contraction and the increase in [Ca2+]i induced by endothelin-1 in endothelium-denuded rat thoracic aorta rings, and their suppression by nitric oxide (NO). In Ca2+-free medium, the endothelin-1-induced contraction was suppressed to about 20% of control values, although the increase in [Ca2+]i became negligible. The contraction and the increase in [Ca2+]i monitored by fura 2 fluorescence were unaffected by a blocker of L-type voltage-operated Ca2+ channels nifedipine. A blocker of nonselective cation channels 1-[β-[3-(4-methoxyphenyl)propoxyl]-4-methoxyphenethyl]-1H-imidazole·HCl(SK&F 96365) suppressed the endothelin-1-induced contraction and increase in [Ca2+]i to the level similar to that after removal of extracellular Ca2+. SK&F 96365 had no further effect on the endothelin-1-induced contraction in the absence of extracellular Ca2+. The endothelin-1-induced contraction and increase in [Ca2+]i were abolished by a donor of NO sodium nitroprusside. The effects of another NO donor 3-morpholinosydnonimine (SIN-1) were also tested and yielded essentially similar results to those for sodium nitroprusside on the endothelin-1-induced contraction. Furthermore, the inhibitory effects of sodium nitroprusside could be blocked with a guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ) at 30 μM. These findings suggest that Ca2+ entry through nonselective cation channels but not voltage-operated Ca2+ channels plays a critical role in the endothelin-1-induced increase in [Ca2+]i and the resulting contraction and that inhibition by NO of the endothelin-1-induced contraction is mainly the result of blockade of Ca2+ entry through these channels.
Endothelin regulation of vascular tonus
1996, General Pharmacology- 1.
1. Following the identification of endothelin-1 (ET-1) in 1988, an increasing body of work has accumulated on this endothelium-derived vasoconstrictor peptide.
- 2.
2. ET-1 may regulate the regional vascular tonus.
- 3.
3. ET-1 may directly act on the underlying smooth muscle to increase the force in a paracrine manner. ET-1 may also act on the endothelium to release relaxing factors in an autocrine manner. In either case, both ETA and ETB receptors may be involved.
- 1.
Endothelin-1 and vasopressin activate Ca<sup>2+</sup>-permeable non-selective cation channels in aortic smooth muscle cells: Mechanism of receptor-mediated Ca<sup>2+</sup> influx
1996, Journal of Molecular and Cellular CardiologyThe effects of vasopressin and endothelin-1 on cultured aortic smooth muscle cell lines (A7r5) were investigated by measurements of intracellular calcium [Ca2+]iand the patch-clamp techniques. Vasopressin and endothelin-1 (100 nm) evoked an initial peak followed by a smaller sustained rise of [Ca2+]iin the presence of extracellular calcium [Ca2+]o. In the absence of [Ca2+]o, only the initial peak of [Ca2+]iwas observed. Therefore, the initial peak of [Ca2+]iwas mainly due to calcium release from the storage sites, whereas the later sustained rise of [Ca2+]iwas due to the calcium entry from outside. The sustained rise of [Ca2+]iwas unaffected by nifedipine (10μm) significantly, but was completely abolished by La3+(1 mm). Under current clamp conditions with K+-internal solution, vasopressin and endothelin-1 (100 nm) produced hyperpolarization, then followed by depolarization. Under voltage clamp conditions at a holding potential of −40 mV, both vasopressin and endothelin-1 first activated the outward current, then followed by a long-lasting inward current with a high noise level. The first outward current was abolished by charybdotoxin (100 nm), Cs+in the patch pipette and high EGTA (10 mm) in the pipette, suggesting that it was a Ca2+-sensitive K+current (IK.Ca). The inward current was still elicited with the patch pipette containing Cs+-internal solution, and reversed at about 0 mV. The reversal potential was not significantly altered by the replacement of [Cl−]ior [Cl−]o, proposing that the inward current is a cation selective channel (IN.S.). The inward current was also observed even when extracellular cations are Ca2+. La3+(1 mm), Cd2+(1 mm) completely abolished the vasopressin-induced IN.S., however, nifedipine (10μm) failed to inhibit it significantly. Single channel activities were recorded in the cell-attached configurations when vasopressin or endothelin-1 was applied to the bathing solution. The unitary conductance of the channels was approximately 20 pS with 140 mmNa+, Cs+, or K+in the pipette, but was 15 pS with 110 mmCa2+in the pipette. Permeabilities sequence calculated from the reversal potentials was Na+≊Cs+≊K+>Ca+. These results provide evidence that calcium entry and membrane depolarization elicited by vasopressin or endothelin-1 are mediated by a receptor-mediated Ca2+-permeable non-selective cation channel in aortic smooth muscle cells.
Endothelin-1 and angiotensin II act as progression but not competence growth factors in vascular smooth muscle cells
1996, European Journal of PharmacologyThe direct effects of endothelin-1 and angiotensin II on cell cycle progression were investigated in rat aorta smooth muscle cells in primary culture. The phase of the cell cycle was determined by an immunocytochemical analysis of cell cycle-specific nuclear antigens. The primary cultured cells were synchronized in the G0 phase (100%) by serum deprivation for 24 h. Endothelin-1 (0.1 μM) or angiotensin II (1 μM) had no effect on the cell cycle of G0 cells, whereas platelet-derived growth factor (PDGF) stimulated the entry of the G0 cells into the G1 phase (100%) without a further progression to the S and M phases. Endothelin-1 or angiotensin II stimulated the progression of the PDGF-pretreated G1 cells to the S and M phases. Fura-2 microfluorometry revealed that, between the G0 and G1 cells, there were no differences in the extent and time course of cytosolic Ca2+ elevations induced by endothelin-1 or angiotensin II, which suggested that endothelin-1 and angiotensin II receptors and their signaling pathways regulating cytosolic Ca2+ remained intact in these cell phases. We thus conclude that endothelin-1 and angiotensin II require the prior transition induced by a competence growth factor such as PDGF to exert their mitogenic effects. These results suggest the important role of endothelin-1 and angiotensin II in atherosclerosis as promoters (progression growth factors), but not as initiators.
Effect of endothelin-1 and its combination with adenosine on myocardial contractility and myocardial energy metabolism in vivo
1995, Journal of Molecular and Cellular CardiologyContradictory results have been reported about the isotropic effects of the vasoconstrictive peptide endothelia-1 (ET-1). In contrast to in vitro experiments in vivo studies could not demonstrate a positive inotropy of ET-1. It may be possible, that the direct positive isotropic effect of ET-1 observed in in vitro studies is counterbalanced in vivo by an indirect negative inotropy due to its coronar-constrictive effect.
This study examined the hemodynamic and isotropic effects of 2500 ng ET-1/kg without and after pretreatment with the vasodilating nucleoside adenosine (0.5. 2.0, 5.0 mg ADO/kg/min). Data were compared with NaCl controls in open-chest rats during and after a 7-min infusion. Besides measurements in the intact circulation isovolumic measurements were carried out for quantification of myocardial contractility independently of peripheral vascular effects. We further examined the effect of ET-1 and its combination with 2.0 mg ADO/kg/min on myocardial high-energy phosphates (ATP. AMP. ADP, creatine phosphate). ET-1 causes a strong and longlasting vasoconstriction (+ 186% v preinfusion values), which is dose-dependently antagonized in part by ADO (+ 109%, + 1361%. + 60%). While the maximum of the isovolumic LVSP (peak LVSP) and the corresponding dP/dtmax (peak dP/dtmax) were unchanged with sole ET-1 (peak LVSP: + 5%, peak dP/dtmax −2%). these indexes of myocardial contractility were increased after pretreatment with ADO (peak LVSP: + 11%, + 13%, + 4%; peak dP/dtmax: +9%, + 20%, + 10%) indicating a positive isotropic effect of ET-1, ET-1 causes a reduction of the high energy-phosphates (ATP: −19%, P<0.01; creatine phosphate: −23%, P<0.05; v controls) that can be prevented by ADO (ATP: −7%, creatine phosphate: −5%, both N.S.).
The vasoconstrictive-induced ischaemia of ET-1 counteracts the direct positive isotropic effect of the peptide. ADO antagonizes the vasoconstriction, normalizes the energy metabolism and unmasks the positive inotropy of ET-1.
Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells
1995, Cell CalciumRegulation of the increase in inositol 1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP3 formation and caused an initial transient peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 μM ) for 30 min blocked the ET-1-induced IP3 formation and Ca2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-IogEC50) of ET-1-induced IP3 formation and increase in [Ca2+]i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the ET-1-induced IN formation and Ca2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.