Lala Rakotoarisoa

Relation between α1‐adrenoceptor subtypes and noradrenaline‐induced contraction in rat portal vein smooth muscle

1 In vascular smooth muscle, α1‐adrenoceptors have been classified recently into two or three subtypes. We examined which α1‐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 (α‐ethyl‐3,4,5‐trimethoxy‐α‐(3‐((2‐(2‐methoxyphenoxy)ethyl)‐amino)‐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 μm 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 μm CEC induced a slight rightward displacement of the concentration‐response curve but the maximal contraction was not significantly affected suggesting that the CEC‐sensitive α1‐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 μm 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 μm 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 α1‐adrenoceptor subtypes in rat portal vein smooth muscle, which show high‐ and low‐affinities respectively for each of prazosin, WB4101 and HV723 and correspond to α1H‐ and α1L‐adrenoceptor subtypes. According to recent α1‐adrenoceptor subclassifications, the α1H‐adrenoceptor subtype which is sensitive to inactivation by CEC may correspond to the α1B‐adrenoceptor subtype. The contraction induced by noradrenaline seems to be predominantly mediated through the α1L‐adrenoceptor subtypes which may include the α1N‐adrenoceptor subtype, as recently proposed.

Rat vena cava α1B-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.

Effect of dihydropyridines on calcium channels in isolated smooth muscle cells from rat vena cava

1 . Whole‐cell patch‐clamp method was applied to single smooth muscle cells freshly isolated from the rat inferior vena cava. 2 . Depolarizing pulses, applied from a holding potential of − 90 mV, activated both Na+ and Ca2+ channels. The fast Na+ current was inhibited by nanomolar concentrations of tetrodotoxin (TTX). The slow Ba2+ current (measured in 5 mm Ba2+ solution) was inhibited by Cd2+ and modulated by dihydropyridine derivatives. When the cells were held at a holding potential of −80 mV, racemic Bay K 8644 increased the Ba2+ current (ED50 = 10 nm) while racemic isradipine inhibited the current (IC50 = 21 nm). 3 . The voltage‐dependency of isradipine blockade was assessed by determining the steady‐state availability of the Ca2+ channels. From the shift of the inactivation curve in the presence of isradipine, we calculated a dissociation constant of 1.11 nm for inactivated Ca2+ channels. Scatchard plots of the specific binding of (+)‐[3H]‐isradipine obtained in intact strips incubated in 5.6 mm or 135 mm K+ solutions confirmed the voltage‐dependency of isradipine binding. 4 . Specific binding of (+)‐[3H]‐isradipine was completely displaced by unlabelled (±)‐isradipine, with an IC50 of 15.1 nm. This value is similar to the IC50 for inhibition of the Ba2+ current (21 nm) in cells maintained at a holding potential of −80 mV. 5 . Bay K 8644 had no effects on the Ba2+ current kinetics during a depolarizing test pulse. The steady‐state inactivation‐activation curves of Ba2+ current were not significantly shifted along the voltage axis. 6 . The present data suggest the existence of two distinct dihydropyridine binding sites which can be bound preferentially by agonist or antagonist derivatives.

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 μm) or 10 μm InsP6 had no action on Ba2+ current, whereas 50 μm InsP6 or 10 μm InsP5 plus 10 μm 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 μm) or calphostin C (100 nm) abolished the InsP5,6‐induced stimulation. Neither the PKA inhibitor H89 (1 μm) nor the protein phosphatase inhibitors okadaic acid (500 nm) or cypermethrin (1 μm) 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.

(+)−[3H]‐PN 200–110 binding to cell membranes and intact strips of portal vein smooth muscle: characterization and modulation by membrane potential and divalent cations

1 Specific binding of the calcium‐antagonist dihydropyridine derivative, (+)−[3H]‐PN 200–110 (isradipine), to cell membranes of equine portal vein smooth muscle was compared with binding to intact strips isolated from rat portal veins. 2 Specific binding to vascular smooth muscle membranes was of high affinity, saturable and reversible. The dissociation constant obtained from association and dissociation kinetics of (+)−[3H]‐PN 200–110 was similar to that obtained from equilibrium binding and competition experiments. 3 Specific binding of (+)−[3H]‐PN 200–110 was completely displaced by unlabelled dihydropyridines. Among other calcium antagonists, D888 and (+)−cis‐diltiazem partially inhibited the binding at 25°C. At 37°C, only (+)−cis‐diltiazem stimulated the binding. LaCl3, CdCl2, NiCl2, CoCl2 had inhibitory effects, whereas KCl and NaCl had no effect. 4 When intact strips of portal vein were incubated in high external potassium concentrations for 30 min, the Kd was lowered to 0.04 ± 0.01 nm from the control value of 0.14 + 0.02 nm (n = 5), thereby indicating that (+)−[3H]‐PN 200–110 bound to voltage‐dependent calcium channels, with a higher affinity, in the depolarized state. 5 When external Ca2+ was removed or substituted with Ba2+ or Sr2+, Kd values increased suggesting that the dihydropyridine binding to intact strips was modulated by binding of Ca2+ ions to voltage‐dependent calcium channels.

Modulation of 3H]dihydropyridine binding by activation of protein kinase C in vascular smooth muscle

The influence of noradrenaline and protein kinase C modulators on (+)-[3H]isradipine binding to voltage-dependent calcium channels has been studied in membranes of equine portal vein smooth muscle and intact strips isolated from rat portal vein. Specific (+)-[3H]isradipine binding to intact strips was increased by noradrenaline, a combination of aluminium and fluoride, and phorbol esters. The increase in isradipine binding induced by noradrenaline was inhibited by 1 microM prazosin while that induced by phorbol esters was inhibited by H7 (a protein kinase C inhibitor). In strips pretreated 6 h with 10 micrograms.ml-1 pertussis toxin, the noradrenaline-induced increase in isradipine binding was unchanged. In contrast, isradipine binding to membranes was unaffected by noradrenaline or GTP-gamma-S. Only phorbol esters had a stimulatory effect on isradipine binding when membranes were incubated in a medium containing 10 microM ATP and 5 mM Mg2+. Scatchard plot analysis reveals that the stimulation of isradipine binding by both noradrenaline and phorbol esters appears to result from a decrease in KD rather than an effect on the maximal binding capacity. Contractions evoked by noradrenaline were concentration-dependently depressed by isradipine. About 30% of the response was resistant to inhibition, while KCl-induced contractions were completely blocked. However, noradrenaline-induced contractions were more sensitive to isradipine inhibition than were KCl-induced contractions. These results suggest that activation of protein kinase C modulates isradipine binding to voltage-dependent Ca2+ channels independently of a separate modulation by membrane depolarization.

Interactions of spironolactone with (+)-[3H]-isradipine and (−)-[3H]-desmethoxyverapamil binding sites in vascular smooth muscle

Spironolactone partially inhibited the specific binding of (+)‐[3H]‐isradipine and (−)‐[3H]‐desmethoxyverapamil to vascular smooth muscle membranes. It is suggested that spironolactone interacts at a binding site of the calcium channel complex and allosterically modulates ligand binding at receptor sites in the channel.

Angiotensin II-induced delayed stimulation of phospholipase C γ1 requires activation of both phosphatidiylinositol 3-kinase γ and tyrosine kinase in vascular myocytes

In vascular smooth muscles, angiotensin II (AII) has been reported to activate phospholipase C (PLC) and phosphatidylinositol 3‐kinase (PI3K). We investigated the time‐dependent effects of AII on both phosphatidylinositol 3,4,5‐trisphosphate (PtdInsP3) and inositol phosphates (InsPs) accumulation in permeabilized microsomes from rat portal vein smooth muscle in comparison with those of noradrenaline (NA). AII stimulated an early production of PtdInsP3 (within 30 s) followed by a delayed production of InsPs (within 3‐5 min), in contrast to NA which activated only a fast production of InsPs. The use of pharmacological inhibitors and antibodies raised against the PI3K and PLC isoforms expressed in portal vein smooth muscle showed that AII specifically activated PI3Kδ and that this isoform was involved in the AII‐induced stimulation of InsPs accumulation. NA‐induced InsPs accumulation depended on PLCβ1 activation whereas AII‐induced InsPs accumulation depended on PLCγ1 activation. AII‐induced PLCδ1 activation required both tyrosine kinase and PI3Kδ since genistein and tyrphostin B48 (inhibitors of tyrosine kinase), LY294002 and wortmannin (inhibitors of PI3K) and anti‐PI3Kδ antibody abolished AII‐induced stimulation of InsPs accumulation. Increased tyrosine phosphorylation of PLCβ1 was only detected for long‐lasting applications of AII and was suppressed by genistein. These data indicate that activation of both PI3Kβ and tyrosine kinase is a prerequisite for AII‐induced stimulation of PLCβ1 in vascular smooth muscle and suggest that the sequential activation of the three enzymes may be responsible for the slow and long‐lasting contraction induced by AII.

Desmethoxyverapamil-sensitive calcium channels in rat portal vein smooth muscle

The whole-cell patch clamp technique was used to analyze the properties of the phenylalkylamine-sensitive calcium channels in smooth muscle cells isolated from the portal vein. (-)-D888 dose dependently inhibited the calcium current elicited from a holding potential of -40 mV (IC50 = 1.3 nM) in a frequency-dependent manner. No voltage dependence of the inhibition was noted. Independent high- and low-affinity binding sites for (-)-[3H]D888 were identified. Calcium entry blockers such as (-)-D888, d-cis-diltiazem and nicardipine completely or partially antagonized the (-)-[3H]D888 binding at both types of sites. The properties of this cross-inhibition suggest that phenylalkylamines and d-cis-diltiazem bind at common sites in vascular smooth muscles whereas dihydropyridines bind at distinct sites which are allosterically coupled to the phenylalkylamine sites. As the IC50 for (-)-D888 found from electrophysiological experiments is not identical to the equilibrium dissociation constants for the high- and low-affinity sites found from binding data (0.47 and 50 nM, respectively), it is suggested that binding of (-)-D888 to both high- and low-affinity sites may be involved in the inhibitory effect of (-)-D888 on calcium channels. Furthermore, these two different binding sites may correspond to two different subtypes of phenylalkylamine-sensitive calcium channels in smooth muscle cells.

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

Summary— 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.