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.

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.

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.

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.

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.

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.

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.

High-affinity binding sites for [3H]saxitoxin are associated with voltage-dependent sodium channels in portal vein smooth muscle.

Saturable, high-affinity binding sites for [3H]saxitoxin were identified in equine portal vein smooth muscle membranes. These sites had a dissociation constant of 0.29 nM and a maximal binding capacity of 115 fmol.mg-1 of protein. A similar dissociation constant was obtained with cells prepared from rat portal vein. Specific binding of [3H]saxitoxin was completely displaced by unlabelled saxitoxin and tetrodotoxin, with inhibition constants of 0.42 and 2.10 nM, respectively. Tetrodotoxin blocked the fast Na+ current in single cells of rat portal vein in a concentration-dependent manner, with an IC50 of 3.15 nM. These results suggest that the high-affinity binding sites for tetrodotoxin and saxitoxin may be associated with voltage-dependent Na+ channels in vascular myocytes.

Selective modulation by membrane potential of desmethoxyverapamil binding to calcium channels in rat portal vein.

(-)-[3H]Desmethoxyverapamil (D888) binds saturably to intact strips from rat portal vein bathed in physiological solution with a Kd value of 363 pM and a maximal binding capacity value of 15.6 fmol.mg-1 wet weight. Unlabeled dihydropyridines, phenylalkylamines and benzothiazepines inhibited (-)-[3H]D888 specific binding in a concentration-dependent manner. Scatchard analyses and dissociation kinetics of (-)-[3H]D888 binding revealed the existence of mutual allosteric interactions between (+)-isradipine, (+)-cis diltiazem and (-)-D888 binding sites in portal vein strips. When voltage-dependent Ca++ channels transported Ca++, Ba++, Sr++ or Na+ the binding capacity of (-)-[3H]D888 remained unchanged. In contrast, both depolarization (induced by elevation of external K+) and hyperpolarization (in the presence of cromakalim) induced a gradual decrease in (-)-D888 binding capacity. These observations suggest that membrane potential variation would change the conformational state of Ca++ channels, in such a way that it would be less favorable for access of (-)-[3H]D888 to the binding site. This would provide an experimental argument in favor of the "guarded receptor hypothesis" according to which membrane potential modulates ligand affinity by alteration of the amount of time during which the receptor binding site is available to (-)-[3H]D888.

Guanine nucleotide-binding proteins modulate desmethoxyverapamil binding to calcium channels in vascular smooth muscle.

Specific binding of the Ca++ antagonist desmethoxyverapamil, (-)-[3H]D888, to cell membranes of equine portal vein smooth muscle was inhibited in a concentration-dependent manner by guanosine 5'-O-(gamma-thio)triphosphate and ATP but was little affected by guanosine 5'-O-(beta-thio)diphosphate, noradrenaline or phorbol 12-myristate 13-acetate ester. Inhibition constants for GTP and ATP were in the range of 0.1 to 0.3 mM. From Scatchard plots and dissociation kinetic experiments, it is proposed that D888 high affinity binding sites are transferred into low affinity sites. In intact strips of rat portal vein bathed in physiological solution, both noradrenalin and a combination of aluminum chloride and sodium fluoride inhibited (-)-[3H]D888 binding, whereas guanosine 5'-O-(gamma-thio)triphosphate was without effect. In strips pretreated with 1 microM prazosin or 10 micrograms/ml pertussis toxin (PTX) for 6 h, noradrenalin had no effect on specific (-)-[3H]D888 binding. In addition, inhibition of (-)-[3H]D888 binding in the presence of 3 microM noradrenalin was reversed in a concentration-dependent manner by prazosin but not by propranolol. Noradrenalin-induced contractions were inhibited in a concentration-dependent manner by D888. In strips preincubated with 10 micrograms/ml PTX for 6 h, the concentration-response curve was shifted to the left, indicating that removal of the PTX sensitive transduction pathway increased D888 affinity for its specific binding sites. These results show that (-)-[3H]D888 binding to Ca++ channels is changed by GTP analogs in a way that suggests that a PTX-sensitive guanine nucleotide-binding protein may directly interact with Ca++ channels in response to activation of alpha 1 adrenoceptors.

Identification and properties of voltage-sensitive sodium channels in smooth muscle cells from pregnant rat myometrium.

Saturable high and low affinity binding sites for [3H]saxitoxin were identified in myometrial membranes of pregnant rats, with dissociation constants of 0.53 and 27 nM, respectively. The maximal binding capacity of the low affinity binding sites was about 10 times higher than that of the high affinity binding sites. The dissociation constants obtained from association and dissociation kinetics of [3H]saxitoxin were similar to those obtained from equilibrium binding. Saxitoxin and tetrodotoxin specifically displaced [3H]saxitoxin binding at both types of sites. Isradipine (1-10 microM) and amiloride (50-100 microM) were without effect on the binding of [3H]saxitoxin. At high concentrations (10-100 microM), veratridine induced a partial inhibition of [3H]saxitoxin binding. In dispersed myometrial cells, [3H]saxitoxin binding revealed the presence of both high and low affinity binding sites, with KD values similar to those obtained in myometrial membranes. Sodium currents were studied in both freshly dispersed and cultured myometrial cells in the presence of veratridine (100 microM), using the whole-cell patch-clamp technique. Steady state inactivation curves indicated that sodium channels were available at negative membrane potentials (between -110 and -40 mV). Isradipine (1-10 microM) and amiloride (50-100 microM) were without effect on the sodium current. Applications of saxitoxin or tetrodotoxin inhibited the amplitude of the sodium current in a concentration-dependent manner. The concentrations of saxitoxin and tetrodotoxin producing half-maximal inhibition were 1.4 and 8.8 nM, respectively. Although the IC50 values for saxitoxin and tetrodotoxin found from electrophysiological experiments are not identical to the equilibrium dissociation constants for the high and low affinity sites found from binding experiments, these results suggested that binding of the neurotoxins to the high affinity sites may be involved in their inhibitory effects on sodium channels. Furthermore, low affinity binding sites may be associated with a non-functional subtype of sodium channels in myometrial cells.