Intracellular Ca(2+) oscillations induced by over-expressed Ca(V)3.1 T-type Ca(2+) channels in NG108-15 cells.

T-type Ca(2+) channel family includes three subunits Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3 and have been shown to control burst firing and intracellular Ca(2+) concentration ([Ca(2+)](i)) in neurons. Here, we investigated whether Ca(V)3.1 channels could generate a pacemaker current and contribute to cell excitability. Ca(V)3.1 clones were over-expressed in the neuronal cell line NG108-15. Ca(V)3.1 channel expression induced repetitive action potentials, generating spontaneous membrane potential oscillations (MPOs) and concomitant [Ca(2+)](i) oscillations. These oscillations were inhibited by T-type channels antagonists and were present only if the membrane potential was around -61mV. [Ca(2+)](i) oscillations were critically dependent on Ca(2+) influx through Ca(V)3.1 channels and did not involve Ca(2+) release from the endoplasmic reticulum. The waveform and frequency of the MPOs are constrained by electrophysiological properties of the Ca(V)3.1 channels. The trigger of the oscillations was the Ca(V)3.1 window current. This current induced continuous [Ca(2+)](i) increase at -60mV that depolarized the cells and triggered MPOs. Shifting the Ca(V)3.1 window current potential range by increasing the external Ca(2+) concentration resulted in a corresponding shift of the MPOs threshold. The hyperpolarization-activated cation current (I(h)) was not required to induce MPOs, but when expressed together with Ca(V)3.1 channels, it broadened the membrane potential range over which MPOs were observed. Overall, the data demonstrate that the Ca(V)3.1 window current is critical in triggering intrinsic electrical and [Ca(2+)](i) oscillations.

Stimulation of L-type Ca2+ channels by inositol pentakis- and hexakisphosphates in rat vascular smooth muscle cells.

The electrophysiological effects of D-myo-inositol 1,3,4,5,6-pentakisphosphate (InsP5) and D-myo-inositol hexakisphosphate (InsP6), which represent the main cellular inositol polyphosphates, were studied on L-type Ca2+ channels in single myocytes of rat portal vein. Intracellular infusion of InsP5 (up to 50 micro M) or 10 micro M InsP6 had no action on Ba2+ current, whereas 50 micro M InsP6 or 10 micro M InsP5 plus 10 micro M InsP6 (InsP5,6) stimulated the inward current. The stimulatory effect of InsP5,6 was also obtained in external Ca2+-containing solution. The stimulated Ba2+ current retained the properties of L-type Ba2+ current and was oxodipine sensitive. PKC inhibitors Ro 32-0432 (up to 500 nM), GF109203X (5 micro M) or calphostin C (100 nM) abolished the InsP5,6-induced stimulation. Neither the PKA inhibitor H89 (1 micro M) nor the protein phosphatase inhibitors okadaic acid (500 nM) or cypermethrin (1 micro M) prevented or mimicked the InsP5,6-induced stimulation of Ba2+ current. However, InsP5 or InsP6 could mimic some effects of protein phosphatase inhibitor so as to extend after washing-out forskolin the stimulatory effects of the adenylyl cyclase activator on Ba2+ current. These results indicate that InsP5 and InsP6 may act as intracellular messengers in modulating L-type Ca2+ channel activity and so could be implicated in mediator-induced contractions of vascular smooth muscle cells.

Identification and function of ryanodine receptor subtype 3 in non-pregnant mouse myometrial cells.

Subtype 3 of the ryanodine receptor (RYR3) is a ubiquitous Ca2+ release channel which is predominantly expressed in smooth muscle tissues and certain regions of the brain. We show by reverse transcription-polymerase chain reaction (RT-PCR) that non-pregnant mouse myometrial cells expressed only RYR3 and therefore could be a good model for studying the role of endogenous RYR3. Expression of RYR3 was confirmed by Western blotting and immunostaining. Confocal Ca2+ measurements revealed that in 1.7 mM extracellular Ca2+, neither caffeine nor photolysis of caged Ca2+ were able to trigger any Ca2+ responses, whereas in the same cells oxytocin activated propagated Ca2+ waves. However, under conditions of increased sarcoplasmic reticulum (SR) Ca2+ loading, brought about by superfusing myometrial cells in 10 mM extracellular Ca2+, all the myometrial cells responded to caffeine and photolysis of caged Ca2+, indicating that it was possible to activate RYR3. The caffeine-induced Ca2+ responses were inhibited by intracellular application of an anti-RYR3-specific antibody. Immunodetection of RYR3 with the same antibody revealed a rather homogeneous distribution of fluorescence in confocal cell sections. In agreement with these observations, spontaneous or triggered Ca2+ sparks were not detected. In conclusion, our results suggest that under conditions of increased SR Ca2+ loading, endogenous RYR3 may contribute to the Ca2+ responses of myometrial cells.

Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels.

Heterodimeric class I phosphoinositide 3-kinase (PI3K) has been shown to be involved in the stimulation of voltage-gated Ca(2+) channels by various mediators. In this study, we bring evidences that vascular L-type Ca(2+) channels can be modulated by both tyrosine kinase-regulated class Ia and G protein-regulated class Ib PI3Ks. Purified recombinant PI3Ks increased the peak Ca(2+) channel current density when applied intracellularly. Furthermore, PI3Kalpha-, beta-, and delta-mediated stimulations of Ca(2+) channel currents were increased by preactivation by a phosphotyrosyl peptide, whereas PI3Kgamma- and beta-mediated effects were increased by Gbetagamma. In freshly isolated and cultured vascular myocytes, angiotensin II and Gbetagamma stimulated L-type Ca(2+) channel current. In contrast, platelet-derived growth factor (PDGF)-BB and the phosphotyrosyl peptide did not stimulate Ca(2+) channel current in freshly isolated cells despite the presence of endogenous PDGF receptors and PI3Kalpha and PI3Kgamma. Interestingly, when endogenous PI3Kbeta expression arose in cultured myocytes, both PDGF and phosphotyrosyl peptide stimulated Ca(2+) channels through PI3Kbeta, as revealed by the inhibitory effect of an anti-PI3Kbeta antibody. These results suggest that endogenous PI3Kbeta but not PI3Kalpha is specifically involved in PDGF receptor-induced stimulation of Ca(2+) channels and that different isoforms of PI3K regulate physiological increases of Ca(2+) influx in vascular myocytes stimulated by vasoconstrictor or growth factor.

Calcium signalling through nucleotide receptor P2X1 in rat portal vein myocytes.

1. ATP-mediated Ca2+ signalling was studied in freshly isolated rat portal vein myocytes by means of a laser confocal microscope and the patch-clamp technique. 2. In vascular myocytes held at -60 mV, ATP induced a large inward current that was supported mainly by activation of P2X1 receptors, although other P2X receptor subtypes (P2X3, P2X4 and P2X5) were revealed by reverse transcription-polymerase chain reaction. 3. Confocal Ca2+ measurements revealed that ATP-mediated Ca2+ responses started at initiation sites where spontaneous or triggered Ca2+ sparks were not detected, whereas membrane depolarizations triggered Ca2+ waves by repetitive activation of Ca2+ sparks from a single initiation site. 4. ATP-mediated Ca2+ responses depended on Ca2+ influx through non-selective cation channels that activated, in turn, Ca2+ release from the intracellular store via ryanodine receptors (RYRs). Using specific antibodies directed against the RYR subtypes, we show that ATP-mediated Ca2+ release requires, at least, RYR2, but not RYR3. 5. Our results suggest that, in vascular myocytes, Ca2+ influx through P2X1 receptors may trigger Ca2+-induced Ca2+ release at intracellular sites where RYRs are not clustered.

Phosphoinositide 3-kinase gamma mediates angiotensin II-induced stimulation of L-type calcium channels in vascular myocytes.

Previous results have shown that in rat portal vein myocytes the betagamma dimer of the G(13) protein transduces the angiotensin II-induced stimulation of calcium channels and increase in intracellular Ca(2+) concentration through activation of phosphoinositide 3-kinase (PI3K). In the present work we determined which class I PI3K isoforms were involved in this regulation. Western blot analysis indicated that rat portal vein myocytes expressed only PI3Kalpha and PI3Kgamma and no other class I PI3K isoforms. In the intracellular presence of an anti-p110gamma antibody infused by the patch clamp pipette, both angiotensin II- and Gbetagamma-mediated stimulation of Ca(2+) channel current were inhibited, whereas intracellular application of an anti-p110alpha antibody had no effect. The anti-PI3Kgamma antibody also inhibited the angiotensin II- and Gbetagamma-induced production of phosphatidylinositol 3,4,5-trisphosphate. In Indo-1 loaded cells, the angiotensin II-induced increase in [Ca(2+)](i) was inhibited by intracellular application of the anti-PI3Kgamma antibody, whereas the anti-PI3Kalpha antibody had no effect. The specificity of the anti-PI3Kgamma antibody used in functional experiments was ascertained by showing that this antibody did not recognize recombinant PI3Kalpha in Western blot experiments. Moreover, anti-PI3Kgamma antibody inhibited the stimulatory effect of intracellularly infused recombinant PI3Kgamma on Ca(2+) channel current without altering the effect of recombinant PI3Kalpha. Our results show that, although both PI3Kgamma and PI3Kalpha are expressed in vascular myocytes, the angiotensin II-induced stimulation of vascular L-type calcium channel and increase of [Ca(2+)](i) involves only the PI3Kgamma isoform.

Involvement of both G protein alphas and beta gamma subunits in beta-adrenergic stimulation of vascular L-type Ca(2+) channels.

1. Previous data have shown that activation of beta(3)-adrenoceptors stimulates vascular L-type Ca(2+) channels through a G alphas-induced stimulation of the cyclic AMP/PKA pathway. The present study investigated whether beta-adrenergic stimulation also uses the G beta gamma/PI3K/PKC pathway to modulate L-type Ca(2+) channels in rat portal vein myocytes. 2. Peak Ba(2+) current (I(Ba)) measured using the whole-cell patch clamp method was maximally increased by application of 10 microm isoprenaline after blockade of beta(3)-adrenoceptors by 1 microM SR59230A. Under these conditions, the isoprenaline-induced stimulation of I(Ba) was reversed by ICI-118551 (a specific beta(2)-adrenoceptor antagonist) but not by atenolol (a specific beta(1)-adrenoceptor antagonist). The beta(2)-adrenoceptor agonist salbutamol increased I(Ba), an effect which was reversed by ICI-118551 whereas the beta(1)-adrenoceptor agonist dobutamine had no effect on I(Ba). 3. Application of PKA inhibitors (H-89 and Rp 8-Br-cyclic AMPs) or a PKC inhibitor (calphostin C) alone did not affect the beta(2)-adrenergic stimulation of I(Ba) whereas simultaneous application of both PKA and PKC inhibitors completely blocked this stimulation. 4. The beta(2)-adrenergic stimulation of L-type Ca(2+) channels was blocked by a pre-treatment with cholera toxin and by intracellular application of an anti-G alphas antibody (directed against the carboxyl terminus of G alphas). In the presence of H-89, intracellular infusion of an anti-Gss(com) antibody or a beta ARK(1) peptide as well as a pre-treatment with wortmannin (a PI3K inhibitor) blocked the beta(2)-adrenergic stimulation of I(Ba). 5. These results suggest that the beta(2)-adrenergic stimulation of vascular L-type Ca(2+) channels involves both G alphas and G beta gamma subunits which exert their stimulatory effects through PKA and PI3K/PKC pathways, respectively.

Contribution of ryanodine receptor subtype 3 to ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes.

Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca(2+) release during Ca(2+) sparks and global Ca(2+) responses, evoked by activation of voltage-gated Ca(2+) channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca(2+) loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca(2+) responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes. RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes. Confocal Ca(2+) measurements revealed that the number of cells showing spontaneous Ca(2+) sparks was strongly increased by superfusing the vascular myocytes in 10 mm Ca(2+)-containing solution. These Ca(2+) sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3. In contrast, inhibition of RYR3 reduced the global Ca(2+) responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca(2+) responses in Ca(2+)-overloaded myocytes. Ca(2+) transients evoked by photolysis of caged Ca(2+) with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca(2+)](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca(2+) loading, the RYR3 becomes activable by caffeine and local increases in [Ca(2+)](i).

Effects of hindlimb suspension on cytosolic Ca2+ and [3H]ryanodine binding in cardiac myocytes.

Effects of a 14-day hindlimb suspension were examined on [3H]ryanodine binding to rat ventricular microsomes and on cytosolic Ca2+ concentration ([Ca2+]i) and voltage-dependent Ca2+ channels in isolated ventricular myocytes. In suspended rats, the amplitude of the twitch [Ca2+]i transient was increased without significant modifications of the basal [Ca2+]i and sarcoplasmic reticulum content. Because cell capacitance, L-type Ca2+-current density, and Ca2+-channel gating were not significantly modified after suspension, the increase in [Ca2+]i was expected to reside in a change in ryanodine receptors. Scatchard analysis of [3H]ryanodine binding revealed that suspension enhanced binding by increasing the affinity of the receptors for [3H]ryanodine without affecting the maximal binding capacity. Both Ca2+-release channel activity and [3H]ryanodine binding are modulated by Ca2+. However, the Ca2+ sensitivity of [3H]ryanodine binding remained unchanged after suspension. Taken together, these results suggest that the increase in twitch [Ca2+]i transients after suspension may result from a change in the intrinsic properties of the ryanodine receptors but not from a change in the expression level of these receptors.

Inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channel-dependent Ca2+ signalling in rat portal vein myocytes.

Ca2+ signalling events were analyzed in single myocytes from rat portal vein by using a laser confocal microscope combined with the patch-clamp technique. Increase in inositol 1,4,5-trisphosphate (InsP3) concentration was obtained by photorelease from a caged precursor or intracellular dialysis of 3F-InsP3. Low InsP3 concentrations activated either small elevations of [Ca2+]i or localized Ca2+ transients whereas high InsP3 concentrations activated either homogeneous Ca2+ responses or propagated Ca2+ waves. The InsP3-evoked localized Ca2+ transients had spatio-temporal properties characteristic of Ca2+ sparks. In addition, compounds that blocked Ca2+ sparks and Ca2+ responses activated by Ca2+ jumps reduced the global InsP3-activated Ca2+ responses and suppressed the Ca2+ transients. In contrast, Ca2+ responses evoked by flash-photolytic Ca2+ jumps or caffeine were not affected by heparin (an InsP3 receptor antagonist). These results suggest that the absence of elementary Ca2+ events evoked by InsP3 may be related to the lack of clustered InsP3 receptor units in these cells, as confirmed by immunocytochemistry. Cooperativity between InsP3- and ryanodine-sensitive Ca2+ channels may represent a novel mechanism to amplify Ca2+ release from the same intracellular store and give rise to propagated Ca2+ waves.

L-type and Ca2+ release channel-dependent hierarchical Ca2+ signalling in rat portal vein myocytes.

Ca2+ signalling events and whole-cell Ca2+ currents were analyzed in single myocytes from rat portal vein by using a laser scanning confocal microscope combined with the patch-clamp technique. In myocytes in which the intracellular Ca2+ store was depleted or Ca2+ release channels were blocked by 10 microM ryanodine, inward Ca2+ currents induced slow and sustained elevations of [Ca2+]i. These Ca2+ responses were suppressed by 1 microM oxodipine and by depolarizations to +120 mV, a potential close to the reversal potential for Ca2+ ions, suggesting that they reflected Ca2+ influx through L-type Ca2+ channels. With functioning intracellular Ca2+ stores, flash photolysis of caged Ca2+ gave rise to a small increase in [Ca2+]i with superimposed Ca2+ sparks, reflecting the opening of clustered Ca2+ release channels. Brief Ca2+ currents in the voltage range from -30 to +10 mV triggered Ca2+ sparks or macrosparks that did not propagate in the entire line-scan image. Increasing the duration of Ca2+ current for 100 ms or more allowed the trigger of propagating Ca2+ waves which originated from the same initiation sites as the caffeine-activated response. Both Ca2+ sparks and initiation sites of Ca2+ waves activated by Ca2+ currents were observed in the vicinity of areas that excluded the Ca2+ probes, reflecting infoldings of the plasma membrane close to the sarcoplasmic reticulum, as revealed by fluorescent markers. The hierarchy of Ca2+ signalling events, from submicroscopic fundamental events to elementary events (sparks) and propagated waves, provides an integrated mechanism to regulate vascular tone.

Effect of a 14-day hindlimb suspension on cytosolic Ca2+ concentration in rat portal vein myocytes.

Effects of a 14-day hindlimb suspension were examined on increases in cytosolic Ca2+ concentration ([Ca2+]i) evoked by vasoactive compounds and on Ca2+ channels in rat portal vein myocytes. The maximal increases in [Ca2+]i elicited by caffeine, norepinephrine, and angiotensin II were reduced by 30-50% in suspended rats, and complete recovery was obtained 4 days after suspension removal. In contrast, voltage-gated Ca2+ channels were unaffected by hindlimb suspension. Using both confocal microscopy and the patch-clamp technique, we measured local increases in [Ca2+]i which corresponded to activation of a small number of ryanodine-sensitive Ca(2+)-release channels (Ca2+ sparks) and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-gated Ca2+ channels. After hindlimb suspension, these local Ca2+ events, as well as the Ca2+ sensitivity of ryanodine-sensitive Ca2+ release channels, remained unchanged. In contrast, the propagated Ca2+ responses (Ca2+ waves) were significantly reduced in parallel with a noticeable inhibition of [3H]ryanodine binding to vascular membranes. Taken together, these results suggest that inhibition of the vasoconstrictor-induced increases in [Ca2+]i during long-term suspension may be related to a reduction of the number of functional ryanodine-sensitive and Ins(1,4,5)P3-gated channels in the sarcoplasmic reticulum of rat portal vein myocytes.

Angiotensin II receptor subtypes and contractile responses in portal vein smooth muscle.

The selective biphenylimidazole and tetrahydroimidazopyridine antagonists exemplified by losartan (DuP 753) and PD 123319 have been shown to bind selectively to angiotensin AT1 and AT2 receptor subtypes, respectively. To characterize which subtypes of angiotensin II receptors are expressed in mammalian portal vein smooth muscle, we performed, using both membrane and strip preparations, [3H]angiotensin II binding experiments and then contraction experiments to investigate the functional relevance of these binding sites. Specific binding of [3H]angiotensin II was of high affinity, saturable and reversible. Specific binding of [3H]angiotensin II was completely displaced by angiotensin II and the peptide antagonist [Sar1,Ile8]angiotensin II. The inhibition of [3H]angiotensin II binding by losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)- methyl]imidazole, potassium salt) and DuP 532 (2-n-propyl-4-pentafluoroethyl-1-[(2'-(1H-tetrazol-5-yl)biph enyl-4-yl)- methyl]imidazole-5-carboxylic acid) was biphasic and LIGAND curve-fitting analysis revealed two populations of specific binding sites. One subpopulation represented 75% of the total binding and showed high affinity for angiotensin II, losartan and DuP 532, but low affinity for the peptide angiotensin AT2 receptor antagonist CGP 42112A (N-alpha-nicotinoyl-Tyr-Lys-[N-alpha-CBZ-Arg]-His-Pro-Ile-OH) and thus appeared identical to the cloned angiotensin AT1 receptor subtype. The remaining 25% of the sites showed nearly 1000-fold lower affinity for losartan, 6500-fold lower affinity for DuP 532 and high affinity for PD 123319 (S-1-[[4-(dimethylamino)-3-methylphenyl]methyl]-5-diphenylacetyl- 4,5,6,7-tetrahydro-1H-imidazo-[4,5-c] pyridine-6-carboxylic acid, difluoroacetate monohydrate) and CGP 42112A, with values of Ki in the same range (nM) as those found for losartan and DuP 532 at angiotensin AT1 binding sites. These sites appear to be angiotensin AT2 receptors. Only the angiotensin AT1 receptor subtype interacted with G-proteins, as indicated by the 80% inhibition of [3H]angiotensin II binding in the presence of guanosine 5'-O-(3-thiophosphate) or fluoroaluminates. Although the angiotensin II-induced contraction was completely inhibited by losartan with a pA2 value of 8.8, PD 123319 reduced the angiotensin II-induced contraction by 20-25%, indicating that both angiotensin AT1 and AT2 receptor subtypes are functional in portal vein smooth muscle.

Influence of spaceflight, hindlimb suspension, and venous occlusion on alpha 1-adrenoceptors in rat vena cava.

Effects of hindlimb suspension, spaceflight, and venous occlusion were examined in isolated strips from rat vena cava by using both [3H]prazosin-binding and contraction responses evoked by norepinephrine. Sensitivity to norepinephrine was decreased without modification of the maximal contractile response. Furthermore, the high K(+)-induced contractions were not affected, suggesting that there was no interference with voltage-dependent Ca2+ channels. The sensitivity of the norepinephrine-induced contraction to prazosin was decreased, and Scatchard analysis of [3H]prazosin binding indicated an increase in the dissociation constant without variation in maximal binding capacity. A similar increase in the dissociation constant was obtained in control rats after pretreatment with 3 microM norepinephrine or 0.1 microM phorbol 12,13-dibutyrate to desensitize the protein kinase C. This effect was completely abolished in the presence of GF-109203X, a selective inhibitor of protein kinase C. Taken together, these data indicate that altered gravity conditions induce a desensitization of alpha 1B-adrenoceptors depending on increased protein kinase C activity. This effect can be mimicked by venous occlusion and may be responsible for reduced contractile responses to norepinephrine.

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)

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.

Relation between alpha 1-adrenoceptor subtypes and noradrenaline-induced contraction in rat portal vein smooth muscle.

1. In vascular smooth muscle, alpha 1-adrenoceptors have been classified recently into two or three subtypes. We examined which alpha 1-adrenoceptor subtypes are involved in the noradrenaline-induced contraction of rat portal vein smooth muscle. 2. Binding studies with [3H]-prazosin in membranes from equine portal vein smooth muscle revealed the presence of two distinct affinity binding sites. The high-affinity site for [3H]-prazosin was also identified in intact strips of rat portal vein. Prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)- propyl) benzene-acetonitrile fumarate), WB4101 (2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane), 5-methylurapidil, phentolamine and yohimbine antagonized [3H]-prazosin binding at both types of sites. Pretreatment with 50 microM chloroethylclonidine (CEC) eliminated the high-affinity sites for prazosin but had no effect on the low-affinity sites. 3. Noradrenaline produced a concentration-dependent contraction in the rat portal vein. Pretreatment with 50 microM CEC induced a slight rightward displacement of the concentration-response curve but the maximal contraction was not significantly affected suggesting that the CEC-sensitive alpha 1-adrenoceptors played a minor role in the noradrenaline-induced contraction. Prazosin, WB4101 and HV723 produced a concentration-dependent inhibition of noradrenaline-induced contractions. The inhibition curves were little affected by CEC-pretreatment and yielded a relative order of potency of WB4101 > prazosin > HV723. 4. In the presence of 0.1 microM isradipine to block voltage-dependent Ca2+ channels, the noradrenaline-induced contraction is due to release of Ca2+ ions from agonist-sensitive intracellular Ca2+ stores. Under these conditions, the noradrenaline-induced contraction was not significantly affected by pretreatment with 50 microM CEC but was inhibited by the antagonists mentioned above with affinities different from those in the absence of isradipine. The rank order of potency became HV723 > WB4101 > prazosin.5. The present results indicate the existence of two distinct o1-adrenoceptor subtypes in rat portal vein smooth muscle, which show high- and low-affinities respectively for each of prazosin, WB4101 andHV723 and correspond to alphalH- and alphalL-adrenoceptor subtypes. According to recent alpha1-adrenoceptor subclassifications, the alpha l H-adrenoceptor subtype which is sensitive to inactivation by CEC may correspond to the alpha1B-adrenoceptor subtype. The contraction induced by noradrenaline seems to be predominantly mediated through the alphalL-adrenoceptor subtypes which may include the alpha1N-adrenoceptor subtype, as recently proposed.

Rat vena cava alpha 1B-adrenoceptors: characterization by [3H]prazosin binding and contraction experiments.

We examined which subtypes of alpha 1-adrenoceptors are expressed in rat vena cava by using both functional and [3H]prazosin binding experiments. Pretreatment with chloroethylclonidine inactivated about 80% of the specific [3H]prazosin binding sites and reduced the maximal noradrenaline-induced contraction to the same extent. Competition with subtype-selective agonists and antagonists showed primarily the alpha 1B-adrenoceptor subtype in vena cava. The number of alpha 1-adrenoceptors estimated with [3H]prazosin binding and the maximal noradrenaline-induced contraction were dose-dependently inhibited by phenoxybenzamine, indicating the absence of receptor reserve for noradrenaline in vena cava. As the noradrenaline-induced contraction was largely inhibited in Ca(2+)-free solution, these results suggest that alpha 1B-adrenoceptors can be mainly linked to Ca2+ influx in rat vena cava.

Dual effect of thrombin on voltage-dependent Ca2+ channels of portal vein smooth muscle cells.

Thrombin induces a number of physiological responses in several types of cells. To determine the action of thrombin in the vein, the electrophysiological and mechanical effects of thrombin were studied in rat portal vein smooth muscle cells. Ca2+ channel currents were recorded using the whole-cell patch-clamp technique. Thrombin had both inhibitory and stimulatory effects on the Ca2+ channel current. The inhibitory effect was reversed on washout of thrombin, whereas the stimulatory effect was maintained after thrombin was removed. Thrombin (1 unit/ml) produced a reversible decrease of 27.3 +/- 3.3% (n = 12) in the current amplitude and a sustained increase of 71.2 +/- 12.9% (n = 20). The thrombin-induced inhibition of Ca2+ channel current was blocked by the thrombin inhibitor hirudin and by the protease inhibitor leupeptin. The stimulatory effect of thrombin was inhibited by hirudin, by intracellular application of guanosine 5'-O-(beta-thio)diphosphate, and by antiphophatidylinositide antibodies but not by pertussis toxin. The thrombin-induced enhancement of the Ca2+ channel current amplitude was not observed when the current was previously stimulated by phorbol 12,13-dibutyrate. This suggests that the inhibitory effect of thrombin was related to its proteolytic activity and that the stimulatory effect involved activation of a pertussis toxin-insensitive GTP-binding protein, phosphatidylinositide hydrolysis, and protein kinase C activation. Both thrombin effects occurred in the same concentration range (0.001-10 units/ml). The thrombin-induced contraction of portal vein strips was completely inhibited by isradipine, and thrombin did not produce an increase in cytosolic [Ca2+], measured by indo-1 fluorescence in cells clamped at -50 mV, sufficient to activate Ca(2+)-dependent chloride current.(ABSTRACT TRUNCATED AT 250 WORDS)

Noradrenaline-activated heparin-sensitive Ca2+ entry after depletion of intracellular Ca2+ store in portal vein smooth muscle cells.

Single portal vein smooth muscle cells were voltage-clamped using the whole cell patch-clamp technique. Intracellular free Ca2+ concentration ([Ca2+]i) was simultaneously monitored using the fluorescence from the dye indo-1. Noradrenaline (NA, 10 microM) evoked a transient increase in [Ca2+]i, due to inositol 1,4,5-trisphosphate (InP3)-induced Ca2+ release, followed by a sustained increase in [Ca2+]i, caused by extracellular Ca2+ entry. This phase was maintained as long as NA was present and was never observed when the agonist was only briefly applied (3 s). Neither ryanodine, nor caffeine produced the Ca2+ influx whereas the complete depletion of intracellular Ca2+ pool in the absence of external Ca2+ allowed, when Ca2+ was readmitted, the activation of the Ca2+ entry which was used to replenish Ca2+ store. During NA stimulation, the Ca2+ entry was activated even if the Ca2+ pool had not been totally emptied. The Ca2+ entry pathway involved was blocked by Ni2+ and Mn2+, was not permeable to these ions, and was more sensitive to heparin than the InsP3-induced Ca2+ release. Thus, the complete depletion of Ca2+ store activates a Ca2+ influx which is modulated by NA such as in its presence, a partial depletion is enough to induce Ca2+ entry.