Endothelin-induced Ca2 Research Articles

Possible mechanism of endothelin-induced Ca2+ mobility in A7r5 cultured vascular smooth muscle cells.

We studied the mechanism by which endothelin-1 (ET-1) affects the mobility of intracellular free Ca2+ ([Ca2+]i) in cultured A7r5 aortic smooth muscle cells. ET-1 at 10(-9) to 10(-7) M increased [Ca2+]i in Ca2+-containing buffer and Ca2+-free buffer. Pretreatment with ET-1 inhibited thapsigargin- and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca2+]i increases in Ca2+-free buffer. Pretreatment with thapsigargin and CCCP partially abolished the [Ca2+]i increase induced by ET-1. The ET-1-induced Ca2+ signal was partially suppressed by the ETA receptor antagonist BQ123 and the ETB receptor antagonist BQ788 and nifedipine. Pretreatment of cells with the phospholipase C inhibitor U73122 reduced the ET-1-induced [Ca2+]i increase. These results suggest that the ET-1-induced [Ca2+]i increase in A7r5 smooth muscle cells initially activates the ETA receptor, leading to Ca2+ influx and increased internal Ca2+ release from endoplasmic reticulum and mitochondrial Ca2+ stores. The ETB receptor and L-type Ca2+ channel are involved in maintaining further extracellular Ca2+ influx. ET-1-induced intracellular Ca2+ release was also modulated by phospholipase C-coupled events.

Vascular biology of endothelin signal transduction.

1. Endothelins regulate cell function by interacting with two classes of cell surface receptors, ETA and ETB receptors. Both receptor types are members of the heptahelical transmembrane-spanning receptor superfamily and couple via G-proteins to multiple intracellular effectors. 2. Many of the cellular responses induced by endothelins are mediated by changes in cytoplasmic Ca2+ concentration. Stimulation of inositol 1,4,5-trisphosphate (IP3) formation promotes release of Ca2+ from intracellular stores via IP3-sensitive Ca2+ channels. This mechanism accounts for the initial transient peak of the Ca2+ elevation. The entry of Ca2+ across the plasma membrane through multiple types of Ca2+ channels is responsible for the sustained phase of Ca2+ elevation and, together, both mechanisms regulate cell function. 3. Endothelin-mediated Ca2+ signals vary markedly in duration, spatial organization and temporal pattern. The elevations in Ca2+ are sustained, transient or oscillatory and occur either globally or are localized to discrete spatial domains. These different Ca2+ signals, which are dependent on the availability of specific ion channels, control distinct cellular functions. Ryanodine-sensitive Ca2+ release channels may be important in determining the organization of the Ca2+ signal. 4. Endothelin-induced Ca2+ elevations near the plasma membrane stimulate the opening of Ca(2+)-dependent K+ and Cl- channels. These channels are key regulators of membrane potential and, consequently, regulate the activity of voltage-dependent Ca2+ influx pathways. 5. Endothelin regulates the growth and differentiation of cells. It markedly potentiates the mitogenic response of other growth factors, an effect that involves activation of the mitogen-activated protein kinase cascade and induction of early response genes. 6. Finally, the vascular actions of endothelin are influenced by the relative expression of specific ion channels, the spatial and temporal pattern of the Ca2+ signal and the cellular composition of the vascular wall.

Opioids inhibit endothelin-mediated DNA synthesis, phosphoinositide turnover, and Ca2+ mobilization in rat C6 glioma cells

Opioid agonists inhibit DNA synthesis in C6 rat glioma cells that express opioid receptors, induced by desipramine (DMI). This inhibition was not observed in cells that were not treated with DMI, and thus did not express opioid-binding sites. Endothelin, a known mitogen, increased thymidine incorporation dose dependently (up to 1.7-fold) in DMI-treated C6 cells. This increase was reversed by an anti-idiotypic antibody to opioid receptors, Ab2AOR, which has opioid agonist properties. The opioid antagonist naltrexone blocked the inhibition caused by Ab2AOR. Endothelin also stimulated phosphoinositide (PI) turnover and this effect was inhibited by morphine (50%) or by Ab2AOR (72%) in DMI-treated but not in DMI-untreated C6 cells. These actions of morphine and Ab2AOR were reversed by naltrexone. The inhibition of PI turnover and of thymidine incorporation by Ab2AOR or morphine was insensitive to pertussis toxin (PTX). Since PI turnover is known to induce Ca2+ mobilization, it was of interest to examine the effects of the applied opioids on intracellular Ca2+ concentrations. Endothelin increased the concentration of cytosolic free Ca2+ in the cells while Ab2AOR, morphine, and beta-endorphin reversed the endothelin-induced Ca2+ mobilization in DMI-treated but not in DMI-untreated C6 cells. The effect of these agonists was also blocked by naltrexone. The results indicate that glial cells can be a target of an opioid receptor-mediated antimitogenic action and that an abatement in PI turnover and Ca2+ mobilization may be associated with this mechanism.

Regulation of endothelin-induced Ca2+ mobilization in smooth muscle cells by protein kinase C

We have investigated the role of protein kinase C (PKC) in regulating vascular smooth muscle cell responses to endothelin (ET). During the initial phase of the response, ET stimulated rapid formation of diacylglycerol due to rapid and transient activation of phosphatidyl inositol-specific phospholipase C and to rapid and prolonged activation of phospholipase D. Concurrently, ET stimulated translocation of PKC activity that reached a peak at 1 min and remained elevated for at least 20 min. Activation of PKC produced early inhibitory effects. Treatment of cells with phorbol 12-myristate 13-acetate (PMA) 5 min before stimulation with ET inhibited total inositol phosphate formation by > 50%. Because each inositol phosphate isomer was equally affected, the target appears to be either phospholipase C or some upstream component of the receptor coupling mechanism. Activation of PKC was important for sustained response to ET. Treatment of cells with staurosporine significantly reduced sustained elevation of cytosolic free Ca2+ concentration ([Ca2+]i) normally seen with ET. We had previously shown that sustained elevation of [Ca2+]i initiated by ET was due to continued activity of L-type Ca2+ channels. Our current data suggest that PKC is important in this response. For example, staurosporine inhibited both ET-induced 45Ca2+ and Mn2+ entry occurring 10 min after stimulation of influx mechanisms by the agonist. Similarly, pretreatment of cells for 18 h with phorbol dibutyrate depleted the cells of PKC and blocked the sustained activity of Ca2+ entry mechanisms stimulated by ET. Finally, PMA initiated a slowly developing, sustained elevation of [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)

Volatile Anesthetics and Agonist-induced Contractions in Porcine Coronary Artery Smooth Muscle and Ca2+ Mobilization in Cultured Immortalized Vascular Smooth Muscle Cells

These experiments addressed four specific questions. Do isoflurane and halothane (0.5-3.0% in the gas phase) inhibit contractions evoked in isolated porcine coronary artery rings (without endothelium) by the specific Ca2+ mobilizing agonists serotonin, endothelin-1, and F-? Are contractions evoked by phorbol-activated protein kinase C inhibited by the anesthetics? In a well-characterized vascular smooth muscle cell culture model (A7r5 and A10), do the anesthetics attenuate serotonin- and endothelin-induced Ca2+ mobilization? Do the anesthetics inhibit intracellular Ca2+ mobilization via facilitated cAMP formation? Tension was measured in rings suspended in organ chambers. Apparent intracellular Ca2+ was estimated in cells using indo-1 and flow cytometry. Cyclic AMP was measured by radioimmunoassay. At the anesthetic concentrations examined, isoflurane attenuated contractions evoked by serotonin and F- but not those induced by endothelin-1 or phorbol dibutyrate. In cells, isoflurane 2% attenuated 3 x 10(-5) M serotonin-induced Ca2+ mobilization by about 26%, whereas Ca2+ responses evoked by endothelin 10(-8) M were more resistant to anesthetic inhibitory effect. Halothane attenuated contractions in rings evoked by serotonin, endothelin, and F- but lacked effect on phorbol-induced responses. In cells, halothane 2% inhibited Ca2+ mobilization induced by serotonin by about 43% and that induced by endothelin by about 31%. Neither anesthetic facilitated cAMP formation. Isoflurane and halothane variably attenuated contractions evoked by Ca2+ mobilizing agonists--by a cellular action beyond the receptor level--but did not inhibit phorbol activated protein kinase C. Serotonin- and endothelin-induced Ca2+ mobilization was inhibited by isoflurane and halothane--but the mechanism does not depend upon increased cAMP.

Regulation of endothelin-mediated calcium mobilization in vascular smooth muscle cells by isoproterenol

We have investigated the effect of isoproterenol on endothelin-induced Ca2+ mobilization in A10 vascular smooth muscle cells. Endothelin (ET) stimulates a rapid and sustained elevation of intracellular Ca2+ mediated by production of inositol phosphates, release of intracellular Ca2+, and activation of a plasmalemmal Ca2+ influx pathway. This influx pathway appears to be a L-type channel because it is inhibited by nicardipine and activated by BAY K 8644. Depolarization of the cells, by elevating extracellular K+, activated a pharmacologically similar channel and produced a similar change in intracellular Ca2+ concentration. Preincubation of cells with isoproterenol reduced the peak Ca2+ response to endothelin and blocked the sustained elevation. However, isoproterenol did not alter K(+)-induced Ca2+ entry. Thus it appears that ET-induced entry is mediated by intracellular signals and not by depolarization. With the use of cells incubated in Ca2(+)-free medium containing 1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, isoproterenol was shown to inhibit Ca2+ release from intracellular pools by 36 +/- 3%. Furthermore, isoproterenol pretreatment or addition of adenosine 3',5'-cyclic monophosphate (cAMP) to saponin-permeabilized cells inhibited inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-induced Ca2+ release from intracellular sites. Similar effects were seen with forskolin. Propranolol reversed the inhibitory effects of isoproterenol. Isoproterenol pretreatment also inhibited the rapid formation of Ins(1,4,5)P3 and [2-3H]inositol 1,3,4,5-tetrakisphosphate stimulated by endothelin and reduced the sustained formation of these compounds. Finally, isoproterenol and forskolin led to a greater than 10-fold increase in intracellular cAMP levels. This stimulation of adenylate cyclase by isoproterenol was completely blocked by propranolol. It appears then that the beta-agonist isoproterenol interacts with a beta-adrenergic receptor, elevates cAMP, and thereby alters endothelin-induced Ca2+ mobilization. Inhibition of Ins(1,4,5)P3 formation, reduction in the responsiveness of the Ins(1,4,5)P3 intracellular receptor, and perhaps inhibition of ET-induced Ca2+ entry appear to be involved.