G protein heterotrimer Galpha13beta1gamma3 couples the angiotensin AT1A receptor to increases in cytoplasmic Ca2+ in rat portal vein myocytes.

The subunit composition of angiotensin AT1 receptor-activated G protein was identified by using antisense oligonucleotide injection into the nucleus of rat portal vein myocytes. In these cells, we have previously shown that increases in the cytoplasmic calcium concentration ([Ca2+]i) induced by activation of angiotensin AT1 receptors were dependent on extracellular Ca2+ entry by L-type Ca2+ channels and subsequent Ca2+-induced Ca2+ release from the intracellular stores. The angiotensin AT1 receptor-activated increases in [Ca2+]i were selectively inhibited by injection of antisense oligonucleotides directed against the mRNAs coding for the alpha13, beta1, and gamma3 subunits. A correlating reduction in Galpha13, Gbeta1, and Ggamma3 protein expression was confirmed by immunocytochemistry. In addition, anti-alpha13 antibody and synthetic peptide corresponding to the carboxyl terminus of the Galpha13 subunit inhibited, in a concentration-dependent manner, the angiotensin AT1 receptor-mediated Ca2+ response. Reverse transcription-polymerase chain reaction analysis showed that only the angiotensin AT1A receptor was expressed in rat portal vein smooth muscle. Furthermore, injection of anti-AT1A oligonucleotides selectively inhibited the angiotensin II-induced increase in [Ca2+]i. We conclude that the receptor-activated signal leading to increases in [Ca2+]i is transduced by the heterotrimeric G13 protein composed of alpha13/beta1/gamma3 subunits and that the carboxyl terminus of the Galpha13 subunit interacts with the angiotensin AT1A receptor.

Distinct functions of Gq and G11 proteins in coupling alpha1-adrenoreceptors to Ca2+ release and Ca2+ entry in rat portal vein myocytes.

In this study, we identified the subunit composition of Gq and G11 proteins coupling alpha1-adrenoreceptors to increase in cytoplasmic Ca2+ concentration ([Ca2+]i) in rat portal vein myocytes maintained in short-term primary culture. We used intranuclear antisense oligonucleotide injection to inhibit selectively the expression of subunits of G protein. Increases in [Ca2+]i were measured in response to activation of alpha1-adrenoreceptors, angiotensin AT1 receptors, and caffeine. Antisense oligonucleotides directed against the mRNAs coding for alphaq, alpha11, beta1, beta3, gamma2, and gamma3 subunits selectively inhibited the increase in [Ca2+]i activated by alpha1-adrenoreceptors. A corresponding reduction of the expression of these G protein subunits was immunochemically confirmed. In experiments performed in Ca2+-free solution only cells injected with anti-alphaq antisense oligonucleotides displayed a reduction of the alpha1-adrenoreceptor-induced Ca2+ release. In contrast, in Ca2+-containing solution, injection of anti-alpha11 antisense oligonucleotides suppressed the alpha1-adrenoreceptor-induced stimulation of the store-operated Ca2+ influx. Agents that specifically bound Gbetagamma subunits (anti-betacom antibody and overexpression of a beta-adrenergic receptor kinase carboxyl-terminal fragment) had no effect on the alpha1-adrenoreceptor-induced signal transduction. Taken together, these results suggest that alpha1-adrenoreceptors utilize two different Galpha subunits to increase [Ca2+]i. Galphaq may activate phosphatidylinositol 4,5-bisphosphate hydrolysis and induce release of Ca2+ from intracellular stores. Galpha11 may enhance the Ca2+-activated Ca2+ influx that replenishes intracellular Ca2+ stores.

Angiotensin II-mediated activation of L-type calcium channels involves phosphatidylinositol hydrolysis-independent activation of protein kinase C in rat portal vein myocytes.

The action of angiotensin II (ANG II) was studied in single myocytes from rat portal vein, in which the cytoplasmic Ca++ concentration was estimated by emission from fluorescent dyes and the Ca++ channel current was measured with the whole-cell mode of the patch-clamp technique. ANG II stimulated Ca++ channel current through L-type Ca++ channels and initiated a slow and small increase in the cytoplasmic Ca++ concentration in cells in which intracellular Ca++ stores had been depleted by pretreatment with ryanodine and caffeine. Both Ca++ channel current stimulation and Ca++ responses were selectively inhibited by losartan, indicating activation of angiotensin AT1 receptors. Activation of Ca++ channels by ANG II was insensitive to treatment with pertussis toxin and cholera toxin. Intracellular applications of anti-G alpha q/alpha 11 and anti-phosphatidylinositol antibodies had no effect on the ANG II-induced stimulation of Ca++ channel current, indicating that phosphatidylinositol-specific phospholipase C was not involved in this signaling pathway. Down-regulation of protein kinase C and application of an inhibitor of protein kinase C blocked the ANG II-induced effects. Tricyclodecan-9-yl xanthogenate (an inhibitor of non-phosphatidylinositol-specific phospholipases C and phospholipases D) but not propranolol (an inhibitor of phospholipase D-derived diacylglycerol formation) suppressed the ANG II-induced effects. These data suggest that phosphatidylcholine-specific phospholipase C is involved in the ANG II signaling pathway leading to stimulation of L-type Ca++ channels by protein kinase C.

Ca2+ sparks and Ca2+ waves activate different Ca(2+)-dependent ion channels in single myocytes from rat portal vein.

Ca2+ release from intracellular stores was examined with the use of a confocal microscope in single, voltage-clamped myocytes from rat portal vein loaded with both Fluo-3 and Fura-red. Spontaneous local increases in [Ca2+]i from the sarcoplasmic reticulum, termed Ca2+ sparks, were observed in about 30% of the quiescent cells tested. Ca2+ sparks could be evoked by low concentrations of caffeine (1 mM) or ryanodine (1 microM). Both spontaneous and caffeine-evoked Ca2+ sparks were insensitive to blockers of voltage-dependent Ca2+ channels. Caffeine (10 mM) triggered propagating Ca2+ waves of large amplitude which started from the same site than spontaneous Ca2+ sparks in 73% of the cells, as expected if Ca2+ sparks were the elementary events that could account for the initiation of Ca2+ waves. Spontaneous Ca2+ sparks activated both Ca(2+)-dependent K+ and non-selective cation currents, whereas Ca2+ waves were able to evoke Ca(2+)-dependent chloride current. These results suggest that both inward cation current and outward K+ current activated by Ca2+ sparks may exert a key role in controlling the basal activity of vascular myocytes.

Angiotensin II-activated Ca2+ entry-induced release of Ca2+ from intracellular stores in rat portal vein myocytes.

1. The action of angiotensin II (AII) was studied in single myocytes from rat portal vein in which the cytoplasmic Ca2+ concentration was estimated by emission from dyes Fura-2 or Indo-1 and the Ca2+ channel current was measured with the whole-cell mode of the patch-clamp technique. 2. Most of the AII-evoked increases in [Ca2+]i were reduced by about 60% after pretreatment with ryanodine and caffeine to deplete intracellular Ca2+ stores. However, in some cells the AII-induced Ca2+ responses were of small amplitude and resembled those obtained in the presence of ryanodine and caffeine. Both types of Ca2+ responses induced by AII were selectively inhibited by losartan, suggesting that the AII effects resulted from activation of the angiotensin AT1 receptors. 3. The concentration-response curve to AII had an EC50 value close to 1 nM for the increase in [Ca2+]i obtained after depletion of intracellular Ca2+ stores. This value was increased to around 18 nM in experiments where the intracellular Ca2+ stores were not depleted. 4. AII-evoked Ca2+ responses were abolished in the absence of external Ca2+ and in the presence of 1 microM oxodipine to block L-type Ca2+ channels. 5. Intracellular applications of the InsP3 receptor antagonist, heparin or an anti-PdtIns antibody did not modify AII-induced Ca2+ responses. 6. Our results show that AII releases Ca2+ from intracellular stores without involving InsP3 but through a Ca2+ release mechanism activated by Ca2+ influx through L-type Ca2+ channels.

Activation of calcium sparks by angiotensin II in vascular myocytes.

Contraction in smooth muscle is triggered by an increase in cytoplasmic free calcium ([Ca2+]i) which depends on both Ca2+ influx through L-type Ca2+ channels and Ca2+ release from the sarcoplasmic reticulum (SR). Two mechanisms have been shown to be involved in SR Ca2+ release, one is stimulated by Ca2+ and involved ryanodine-sensitive Ca2+-release channels; the other is stimulated by an increase in inositol 1,4,5-trisphosphate (InsP3) generation induced by various mediators and involved InsP3-sensitive Ca2+ release channels. Here, we examined the effects of angiotensin II on [Ca2+]i in single rat portal vein myocytes using both the whole cell patch-clamp method and a laser scanning confocal microscope. Elementary Ca2+ release events (Ca2+ sparks) were obtained spontaneously or in response to L-type Ca2+ channel current activation, and resulted from activation of ryanodine-sensitive Ca2+-release channels in the SR. We show that angiotensin AT1 receptors stimulate Ca2+ sparks through activation of L-type Ca2+ channels without involving InsP3-induced Ca2+ release. This novel transduction pathway may be a common mechanism for vasoconstrictors which do not stimulate generation of chemical second messengers.

Beta adrenergic receptor activation attenuates the generation of inositol phosphates in the pregnant rat myometrium. Correlation with inhibition of Ca++ influx, a cAMP-independent mechanism.

In the pregnant rat myometrium, an averaged 30% of inositol phosphate accumulation induced by carbachol and oxytocin was inhibited by oxodipine indicating that a part of receptor-mediated generation of inositol phosphates depended on Ca++ influx through voltage-gated Ca++ channels. In fura-2-loaded cells, carbachol and oxytocin caused a two-phase [Ca++]i response, made up of a transient [Ca++]i peak of about 700 nM followed by a sustained phase of about 120 nM. Oxodipine reduced the [Ca++]i peak by 40% and the plateau phase by 50%, pointing to a contribution of Ca++ influx in both the [Ca++]i peak and sustained phase. Isoproterenol reduced inositol phosphate response to carbachol and oxytocin to an amount equivalent to that elicited by oxodipine. No additional reduction could be obtained in a combination of isoproterenol and oxodipine. Isoproterenol decreased by 40% the [Ca++]i peak and by 70% the [Ca++]i plateau phase. Differently from isoproterenol, forskolin did not affect inositol phosphate accumulation induced by oxytocin and failed to attenuate the [Ca++]i peak. The inhibitory effect of isoproterenol on both inositol phosphate accumulation and [Ca++]i increase induced by oxytocin was abolished by pertussis toxin. These data suggest that beta adrenergic receptor activation is linked via a cAMP-independent, pertussis toxin-sensitive process to an activation of K+ channels, as revealed by use of selective K+ channel antagonists, with the consequent closure of voltage-gated Ca++ channels, resulting in the inhibition of the Ca(++)-associated generation of inositol phosphates.

Effects of phospholipase C inhibitors on Ca2+ channel stimulation and Ca2+ release from intracellular stores evoked by alpha 1A- and alpha 2A-adrenoceptors in rat portal vein myocytes.

The ability of phospholipase C inhibitors to inhibit Ca2+ channel stimulation and Ca2+ release from intracellular stores evoked by norepinephrine in single rat portal vein myocytes was investigated in the aim of identifying the type of phospholipase C involved in the transduction pathways activated by alpha 1A- and alpha 2A-adrenoceptors. U73122 (an inhibitor of phosphatidylinositol-phospholipase C) inhibited in a concentration-dependent manner the release of Ca2+ from the intracellular stores induced by activation of alpha 1A-adrenoceptors and related to inositol phosphate production whereas U73343 was ineffective. Both compounds had no effect on the release of Ca2+ induced by caffeine. However, U73122 and U73343 inhibited the L-type Ca2+ channel. D609 (an inhibitor of phosphatidylcholine-phospholipase C) had no direct inhibitory effects on the L-type Ca2+ channel but it inhibited concentration dependently the alpha 2A-adrenoceptor-induced stimulation of Ca2+ channels, which had been shown to be independent of phosphatidylinositol hydrolysis. Therefore, these results suggest that alpha 2A-adrenoceptors activate a phosphatidylcholine-phospholipase C in vascular myocytes. However, D609 had other sites of action as it blocked norepinephrine- and caffeine-induced Ca2+ release from the intracellular stores.

A Gi1-2-protein is required for alpha 2A-adrenoceptor-induced stimulation of voltage-dependent Ca2+ channels in rat portal vein myocytes.

In rat portal vein myocytes, alpha 2A-adrenoceptors activate voltage-dependent Ca2+ channels via a transduction pathway requiring protein kinase C activation mediated by a pertussis-toxin-sensitive G-protein. As revealed by the use of antibodies directed against the different alpha-subunits expressed in portal vein myocytes, we show that the clonidine-induced stimulation of voltage-dependent Ca2+ channels is mainly mediated by a Gi1-2-protein.

Modulation of Ca2+ channels by alpha 1A- and alpha 2A-adrenoceptors in vascular myocytes: involvement of different transduction pathways.

Using subtype-selective agonists and antagonists, and antibodies directed against phosphatidylinositol and G-proteins, it has been shown in single myocytes of rat portal vein that both alpha 1A- and alpha 2A-adrenoceptors modulate Ca2+ channels through two distinct transduction pathways. alpha 1A-adrenoceptors couple with a Gq/G11 protein to activate a phospholipase C (PLC) which hydrolyses phosphatidylinositol to generate inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 releases intracellular stored Ca2+ as evidenced by microspectrofluorimetry with Fura-2. The large and transient increase in [Ca2+]i activates chloride channels leading to a membrane depolarization that opens voltage-gated Ca2+ channels. In addition, DAG activates transiently protein kinase C (PKC) which increases the opening probability of Ca2+ channels through a phosphorylation-dependent process. alpha 2A-adrenoceptors do not induce Ca2+ release from intracellular stores but promote sustained Ca2+ influx through voltage-gated Ca2+ channels. The coupling involves a Gi-protein and activation of PKC by DAG. These two transduction pathways may be involved in the physiological action of noradrenaline in vascular smooth muscles.

Electrophysiological and radioligand binding studies of elgodipine and derivatives in portal vein myocytes.

The effects of a novel dihydropyridine, elgodipine, and of three derivatives have been studied on the calcium channel currents of isolated cells from rat portal vein by the patch-clamp technique, and on specific (+)-[3H]isradipine binding to vascular membranes. Elgodipine inhibited both T- and L-type calcium channels in a concentration-dependent manner. Half-inhibitions of T- and L-type calcium channel current were obtained at concentrations of 32 and 2.3 nM, respectively. Currents activated repetitively were similarly inhibited than those after a rest period, indicating absence of use-dependent inhibition by elgodipine. When cells were held at depolarized membrane potentials at which T- or L-type calcium channels were inactivated, the inhibitory effects of elgodipine were enhanced on both calcium channel currents, indicating that the elgodipine-induced inhibition was voltage-dependent. The elgodipine concentration which blocked the inactivated calcium channels were 5 to 7 times lower than those which blocked the resting calcium channels. The inhibition constant for elgodipine obtained from the displacement of (+)-[3H]isradipine binding to the L-type calcium channels in vascular membranes was identical to the dissociation constant calculated from electrophysiological data on inactivated calcium channels. At concentrations that completely inhibited calcium channels, elgodipine had no effect on chloride and potassium channels, and did not interfere with the intracellular calcium stores.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 2-adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes.

The presence of functional alpha 2-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca2+]i) was estimated using emission from the dye Fura-2. Activation of alpha 2-adrenoceptors by clonidine (an alpha 2-adrenoceptor agonist) or noradrenaline (a non-selective alpha-adrenoceptor agonist), both in the presence of 0.1 microM prazosin to block alpha 1-adrenoceptors, caused a slow and sustained increase in [Ca2+]i which was inhibited by 0.1 microM rauwolscine (an alpha 2-adrenoceptor antagonist). A similar Ca2+ response was obtained with oxymetazoline (a selective alpha 2A-adrenoceptor agonist) suggesting that the increase in [Ca2+]i resulted from activation of the alpha 2A-adrenoceptor subtype. The increase in [Ca2+]i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The alpha 2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 microM prazosin. Taken together, these results indicate that alpha 2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Whole-cell patch-clamp experiments showed that alpha 2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Both alpha 1A- and alpha 2A-adrenoreceptor subtypes stimulate voltage-operated L-type calcium channels in rat portal vein myocytes. Evidence for two distinct transduction pathways.

Previously, we have shown that myocytes from rat portal vein express alpha 1A-adrenoreceptors that couple with a Gq/G11-protein to stimulate phosphoinositide turnover and release of calcium from intracellular stores. The purpose of this study was to investigate the contribution of both alpha 1- and alpha 2-adrenoreceptor subtypes in inducing stimulation of voltage-operated calcium channels. Norepinephrine (a nonselective alpha-adrenoreceptor agonist), phenylephrine (an alpha 1-adrenoreceptor agonist), clonidine, and oxymetazoline (alpha 2-adrenoreceptor agonists) stimulated the calcium channel current by a similar extent. Using subtype-selective antagonists we showed that both alpha 1A- and alpha 2A-adrenoreceptors modulated voltage-operated calcium channels through two distinct transduction pathways. alpha 1A-Adrenoreceptors coupled with a pertussis toxin-insensitive G-protein whereas alpha 2A-adrenoreceptors coupled with a pertussis toxin-sensitive G-protein. Portal vein myocytes expressed G-proteins that were recognized by anti-alpha q/alpha 11, -alpha i(1-2), and -alpha i(3) antibodies. As internal applications of anti-phosphatidylinositol and anti-alpha q/alpha 11 antibodies had no effect on the alpha 2A-adrenoreceptor-induced enhancement of calcium channel current, these findings suggest that phosphatidylinositol hydrolysis and Gq/G11-protein are not involved in the alpha 2A-adrenoreceptor-induced coupling process. A protein kinase C inhibitor, GF 109203X, and a long term (24 h) treatment with phorbol dibutyrate to decrease the activity of protein kinase C blocked the alpha 1A- and alpha 2A-adrenoreceptor-induced stimulation of calcium channels as well as that stimulation induced by phorbol dibutyrate. Moreover, activation of alpha 2A-adrenoreceptors did not induce a significant calcium release from intracellular stores. These data suggest that two distinct G-proteins, probably Gq/G11 and Gi, coupled to alpha 1A- and alpha 2A-adrenoreceptors regulate calcium influx through voltage-operated calcium channels by two different transduction pathways leading to activation of protein kinase C.

Inhibition of L-type Ca2+ channels in portal vein myocytes by the enantiomers of oxodipine.

We studied the effects of the enantiomers of the dihydropyridine derivative, 4-(2,3 methylenedioxyphenyl)-1,4-dihydro-2,6-dimethyl-3 carboxyethyl-5-carboxymethyl-pyridine (oxodipine), on voltage-dependent Ca2+ channels of rat portal vein myocytes by combining electrophysiological techniques and binding studies. (+)- and (-)-oxodipine depressed the L-type Ca2+ current in a concentration-dependent manner, with similar IC50 values (around 10 nM) but had no appreciable effect on the intracellular Ca2+ stores. The steady-state inactivation curve for the Ca2+ current was shifted along the voltage axis to negative membrane potentials indicating that the block of the Ca2+ current by oxodipine enantiomers increased with depolarization. The voltage-dependent inhibitory property of oxodipine was related to an increase in [3H](+)-4-(benzo-2-oxa-1,3-diazol-4-yl)-1,4-dihydro-2,6-dimethy lpy ridine- 3,5-dicarboxylic acid 3-isopropyl, 5-methyl ester (isradipine) binding affinity without change in binding capacity. In normally polarized intact strips, interactions of (+)- and (-)-oxodipine with [3H](+)-isradipine binding indicated a stimulation of the radioligand binding at low concentrations of (-)-oxodipine while the (+) enantiomer seemed to act as a competitive ligand. Depolarization of intact strips with 135 mM K(+)-solutions increased the apparent affinity of the enantiomers of oxodipine, and abolished the stimulating effect of (-)-oxodipine on the binding of [3H](+)-isradipine. Inhibition of Ca2+ current was increased in the simultaneous presence of 1 nM of (+)- and (-)-oxodipine when compared to the inhibitions induced by 2 nM of each enantiomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxytocin mobilizes calcium from a unique heparin-sensitive and thapsigargin-sensitive store in single myometrial cells from pregnant rats.

Intracellular free Ca2+ concentration ([Ca2+]i) was monitored using the fluorescence from the dye Fura-2-AM in single myometrial cells from pregnant rats. Oxytocin and acetylcholine applied to the cell evoked an initial peak in [Ca2+]i followed by a smaller sustained rise which was rapidly terminated upon removal of acetylcholine or persisted after oxytocin removal. A Ca2+ channel blocker (oxodipine) and external Ca2+ removal decreased both the transient and sustained rises in [Ca2+]i suggesting that Ca2+ influx through L-type Ca2+ channels participated in the global Ca2+ response induced by oxytocin. However, the initial peak in [Ca2+]i produced by oxytocin was mainly due to Ca2+ store release: it was abolished by inclusion of heparin [which blocks inositol 1,4,5-trisphosphate (InsP3) receptors] in the pipette (whole-cell recording mode of patch-clamp) and external application of thapsigargin (which blocks sarcoplasmic reticulum Ca(2+)-ATPases). In contrast, the transient Ca2+ response induced by oxytocin was unaffected by ryanodine. Moreover, caffeine failed to induce a rise in [Ca2+]i but reduced the oxytocin-induced transient Ca2+ response. The later sustained rise in [Ca2+]i produced by oxytocin was due to the entry of Ca2+ into the cell as it was suppressed in external Ca(2+)-free solution. The Ca2+ entry pathway is permeable to Mn2+ ions, in contrast to that described in various vascular and visceral smooth muscle cells. Oxytocin-induced Ca2+ release is blocked by the oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT. The prolonged increase in [Ca2+]i after oxytocin removal is rapidly terminated by addition of the oxytocin antagonist suggesting that oxytocin dissociation from its receptor is very slow.(ABSTRACT TRUNCATED AT 250 WORDS)

Chloride and monovalent ion-selective cation currents activated by oxytocin in pregnant rat myometrial cells.

OBJECTIVE: The purpose of our study was to characterize the membrane mechanisms responsible for oxytocin-induced depolarization in single cells from pregnant rat myometrium. STUDY DESIGN: Membrane currents were recorded with the whole-cell mode of the standard patch-clamp technique. Intracellular calcium concentration was monitored with the fluorescence from Fura 2 added to the pipette solution. RESULTS: We found that oxytocin predominantly activates potassium, chloride, and cation conductances. Chloride and cation currents were evoked by an increase in the intracellular calcium concentration dependent on calcium release from the heparin-sensitive intracellular stores. Chloride and cation current showed different calcium dependences so that they could be activated separately. CONCLUSION: Stimulation of oxytocin receptors induces opening of calcium-activated chloride and cation channels, leading to depolarization of the myometrial cells. This depolarization opens, in turn, voltage-dependent calcium channels.

Effects of oxodipine and elgodipine on (+)-[3H]-isradipine binding to cardiac and vascular membranes: cardiovascular selectivity.

We studied the effects of six dihydropyridines on the specific binding of (+)-[3H]-isradipine to vascular (portal vein) and cardiac isolated membranes to achieve the relative cardiovascular selectivity of these compounds. Elgodipine, (+)-oxodipine and nifedipine had a significantly higher affinity for the vascular L-type calcium channel than for the cardiac calcium channel while nicardipine showed opposite properties. The other dihydropyridines (nitrendipine and (+)-isradipine) had similar affinities for the cardiac and vascular calcium channels. As the membrane potential of isolated membranes is about 0 mV, these results suggest that the differences in binding of these dihydropyridines to L-type calcium channels in vascular and cardiac cells may be attributed to differences in the molecular structure of these calcium channels.

Activation of alpha-1A adrenoceptors mobilizes calcium from the intracellular stores in myocytes from rat portal vein.

Intracellular free Ca++ concentration ([Ca++]i) was monitored using the fluorescence from the dye fura-2-acetoxymethylester in single myocytes from rat portal vein. In the presence of oxodipine (a L-type Ca++ channel inhibitor), norepinephrine (10 microM) evoked transient increases in [Ca++]i which were related to release of Ca++ from intracellular stores. The alpha-1 adrenoceptors mediating intracellular Ca++ release and inositol phosphate accumulation were identified by using subtype-selective agonists and antagonists. Pretreatment with chloroethylclonidine had little effect on the norepinephrine-induced increase in [Ca++]i and inositol phosphate accumulation. In contrast, prazosin, 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane and alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amino )- propyl)benzeneacetonitrile fumarate produced a concentration-dependent inhibition of both intracellular Ca++ release and inositol phosphate accumulation. The rank of potency was prazosin > 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane > alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amino - propyl) benzeneacetonitrile fumarate. Methoxamine was as effective as norepinephrine but was less potent as shown by the rightward shift of the concentration-response curves. These results indicate that myocytes from rat portal vein express alpha-1A adrenoceptors whose activation stimulates phosphoinositide turnover and release of Ca++ from intracellular stores. The alpha-1A adrenoceptor stimulation of [Ca++]i and subsequent activation of Ca(++)-activated Cl- current was insensitive to intracellular applications of pertussis toxin, but concentration-dependently blocked by intracellular dialysis with a pipette solution containing anti-alpha q/alpha 11 antibody (whole cell recording mode).(ABSTRACT TRUNCATED AT 400 WORDS)