Protein kinase C modulates the release of [3H]5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels.

The present studies examined the relationship between protein kinase C (PKC) and L-type voltage-dependent calcium channels in modulating the release of neurotransmitter from K(+)-depolarized rat spinal cord synaptosomes. Activators of PKC, such as phorbol 12-myristate 13-acetate (PMA), mezerein and oleoyl acetylglycerol produced a concentration-dependent potentiation of K(+)-induced release of [3H]5-hydroxytryptamine ([3H]5-HT). Enhanced release was dependent on the concentration of both Ca2+ and K+ in the superfusion medium. Calcium-independent release of [3H]5-HT or release induced by the Ca2+ ionophore were unaffected by PKC activators. Calcium-dependent release of [3H]5-HT, evoked by K+, was enhanced under similar conditions by the L-type Ca2+ channel agonists Bay K 8644 and (+)-SDZ 202-791. Nimodipine, an L-type Ca2+ channel antagonist, while having no independent effect on K(+)-induced release of [3H]5-HT, abolished the potentiative effects of Bay K 8644 and PMA. Similarly, the PKC inhibitors, polymyxin B and staurosporine, blocked effects of both PMA and Bay K 8644 on K(+)-stimulated release of [3H]5-HT. Neither PMA nor Bay K 8644 altered the uptake of [3H]5-HT. These results suggest that PKC-dependent mechanisms utilize calcium influx, via the L-type calcium channel, to modulate release of neurotransmitter and indicate a possible functional link between PKC and L-type voltage-dependent calcium channels in the spinal cord.     

An operational model of pharmacological agonism: the effect of E/[A] curve shape on agonist dissociation constant estimation.

An operational model of pharmacological agonism has been analysed to predict the behaviour of rectangular hyperbolic and non-hyperbolic agonist-concentration effect, E/[A], curves with variation in receptor concentration, [Ro]. Irreversible antagonism is predicted to cause E/[A] curve gradient changes in non-hyperbolic cases but not in hyperbolic cases; in both cases estimation of agonist dissociation constants (KAS) is theoretically valid. 5-Hydroxytryptamine (5-HT) produced "steep' E/[A] curves in contracting the rabbit isolated aorta preparation. Irreversible antagonism by phenoxybenzamine (Pbz) produced a flattened E/[A] curve, consistent with theoretical predictions. Fitting 5-HT E/[A] curves in the presence and absence of Pbz to the model provided an estimate of KA for 5-HT which was not significantly different from the estimate obtained using Furchgott's null method. The operational model of agonism appears to account qualitatively and quantitatively for the effects of [Ro] changes on hyperbolic and non-hyperbolic E/[A] curves. Under conditions where irreversible antagonism may be used to estimate KAS, fitting the operational model directly to E/[A] data represents a valid, economical and analytically simple alternative to the conventional null method.     

Inhibition of radioligand binding to A1 adenosine receptors by Bay K8644 and nifedipine

Two dihydropyridine compounds, Bay K8644 (a calcium entry activator) and nifedipine (a calcium entry blocker), were found to inhibit the binding of [3H]phenylisopropyladenosine ([3H]PIA) to A1 adenosine receptors in rat cerebral cortex membranes with comparable potencies (IC50 10-30 microM). Scatchard analyses indicated that both Bay K8644 and nifedipine inhibited the binding of [3H]PIA by increasing the KD but without significant effect on the Bmax. When tested at 100 microM, neither Bay K8644 nor nifedipine showed a significant effect on [3H]-p-aminoclonidine ([3H]PAC; alpha 2-adrenergic receptor), [3H]dihydroalprenolol ([3H]DHA; beta-adrenergic receptor), [3H]spiperone (dopamine receptor), and [3H]nitrobenzylthioinosine [( 3H]NBMPR; nucleoside transporter) binding. In the presence of 10 mM Mg2+, the ability of 2-chloroadenosine (2-Cl-Ad, an A1 adenosine receptor agonist) to displace [3H]PIA binding was increased. Conversely, the potencies of 1,3-diethyl-8-phenylxanthine (DPX; an A1 receptor antagonist), Bay K8644 and nifedipine in inhibiting [3H]PIA binding were unchanged. It is suggested that both Bay K8644 and nifedipine may act as antagonists of adenosine A1 receptors, in addition to their well-known effects on calcium channels.     

Effect of Bay K 8644 on the synthesis and metabolism of dopamine and 5-hydroxytryptamine in various brain areas of the rat

Abstract— The effects of Bay K 8644 (1,2 and 4 mg kg^?1, i.p.) on the synthesis and metabolism of dopamine and 5-hydroxytryptamine (5-HT) in rat brain after m-hydroxybenzylhydrazine administration were studied. Bay K 8644 (2 and 4 mg kg^?1, i.p.) caused an increase in the synthesis of both dopamine in the striatum and 5-HT in the midbrain and striatum, measured as the accumulation of 3,4-dihydroxyphenylalanine (dopa) and 5-hydroxytryptophan, respectively. Moreover, Bay K 8644 at the dose of 4 mg kg^?1 increased the turnover of dopamine in the striatum and of 5-HT in midbrain and striatum. These neurochemical changes were antagonized by the calcium channel antagonist nimodipine (10 mg kg^?1, i.p.). It is concluded that dihydropyridine receptors may mediate the brain region-specific changes in the dopaminergic and 5-HT-ergic neurotransmission which occur following activation of neuronal calcium channels.     

Effect of bay K 8644, a calcium channel agonist,on dog cardiac muscarinic receptors

To investigate further whether the effects of the dihydropyridine (DHP) drugs on calcium channels are related to those of these drugs on muscarinic receptors, the binding characteristics of the DHP calcium channel agonist, Bay K 8644, on muscarinic receptors and calcium channels were compared to those of the DHP calcium channel antagonists, nicardipine and nimodipine in the dog cardiac sarcolemma. Bay K 8644, nicardipine and nimodipine inhibited the specific [³H]QNB binding with K _i values of 16.7μM, 3.5μM and 15.5μM respectively. Saturation data of [³H]QNB binding in the presence of these DHP drugs showed this inhibition to be competitive. Bay K 8644, like nicardipine and nimodipine, blocked the binding of [³H]nitrendipine to the high affinity DHP binding sites, but atropine did not, indicating that the muscarinic receptors and the DHP binding sites on calcium channels are distinct. The K _i value of Bay K 8644 for the DHP binding sites was 4 nM. Nicardipine and nimodipine (K _i :0.1–0.2 nM) were at least 20 times more potent than Bay K 8644 in inhibiting [³H]nitrendipine binding. Thus, the muscarinic receptors were about 4000 times less sensitive than these high affinity DHP binding sites to Bay K 8644. These results suggest that the DHP calcium agonist Bay K 8644 binds directly to the muscarinic receptors but its interaction with the muscarinic receptors is not related to its binding to the DHP binding sites on calcium channels.     

The effect of Bay K 8644 on angiotensin II-induced contractions of rabbit aortic strips

The effect of Bay K 8644 on contractile responses to angiotensin II (AII) was investigated in rabbit aorta. Bay K 8644 alone did not elicit contraction while contractile responses to AII were enhanced at low concentrations (10(-8), 3 X 10(-8), 10(-7) M) and depressed at a higher concentration (10(-6) M). The potentiating effect was always observed and increased after treatment by phentolamine. These findings suggest that Bay K 8644 activates calcium channels operated by angiotensin receptors.     

Calcium channel activation does not increase release of endothelial-derived relaxant factors (EDRF) in rat aorta although tonic release of EDRF may modulate calcium channel activity in smooth muscle

We investigated whether Ca2+ channel activation by K+ or Bay K 8644 could cause release of endothelium-derived relaxant factor (EDRF) from rat aorta. Bay K 8644 (0.1-100 nM) did not relax rat aorta preparations partially contracted with phenylephrine, although acetylcholine caused large relaxations. Following partial K+-depolarization (12 or 15 mM), Bay K 8644 (10 nM-1 microM) contracted rat aorta preparations directly. Preparations were more sensitive to Bay K 8644 when stripped of the endothelium in 12 mM K+; concentration-effect curves were displaced to the left, and maximum responses were enhanced. In 15 mM K+, there was a leftward shift of the curves without change of maximal responses. However, Bay K 8644 (1 microM), did not increase the guanosine 3',5' cyclic-monophosphate (cGMP) content of rat aorta in the presence of endothelium, which is a function of EDRF release. Representatives (nifedipine, verapamil, cinnarizine) from different calcium antagonist subgroups had differential effects on contractions induced by Bay K 8644, and the effects of verapamil (1 and 10 microM) and lower concentrations of cinnarizine (1 microM) were reduced in the presence of endothelium. We propose that there is a tonic liberation of EDRF in rat aorta, which is unaffected by Ca2+ channel activation and which gives effects similar to a weak hyperpolarization of the smooth muscle cells. Consequently, Ca2+ channel activation and sensitivity to certain calcium-antagonists may be modified.     

Effect of selective agonists and antagonists on atrial adenosine receptors and their interaction with Bay K 8644 and [3H]-nitrendipine.

1. (-)-N6-phenylisopropyladenosine (R-PIA) and N6-cyclohexyladenosine (CHA), highly selective agonists at A1-adenosine receptors, 5'-N-ethyl-carboxamidoadenosine (NECA), a non-selective agonist at A1 and A2 receptors, and 2-phenylaminoadenosine (CV-1808), a selective A2 agonist, were compared in spontaneously beating and electrically driven atria. R-PIA, CHA and NECA inhibited contraction in both preparations. CV-1808 was not effective up to 500 nM. 2. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX), a new selective A1 receptor antagonist, competitively inhibited the effects of the adenosine agonists, at low concentrations (IC50 less than 1 nM). 3. CHA and NECA were able to inhibit the positive inotropic effect of Bay K 8644 both in spontaneously beating and in electrically driven atria. 4. R-PIA, CHA and NECA (agonists), 8-phenyltheophylline (PT) and DPCPX (antagonists), failed to influence [3H]-nitrendipine binding on microsomal membranes from guinea-pig atria and ventricles in a range of concentrations from 1 nM to 100 microM. 5. The data support the existence of A1 receptors in atrial tissue. No evidence for a direct interaction between adenosine analogues and Bay K 8644 was found at the level of slow calcium channels. Adenosine analogues appear to antagonize the effects of Bay K 8644 indirectly by activation of A1 receptors.     

Effects of Bay K 8644 on contraction of the human isolated bronchus and guinea-pig isolated trachea.

The effects of Bay K 8644, a dihydropyridine which increases calcium flux through the potential-operated channels were studied on the contractions induced by histamine, acetylcholine, KCl and Ca2+ on human isolated bronchial strips and the results were compared to those obtained on guinea-pig isolated tracheal spirals. Subsequently the contractant effects of Bay K 8644 in K+-enriched medium and in the presence of Ca2+ 0.03 mM were investigated. In Krebs normal calcium medium, Bay K 8644 did not significantly modify the EC50 of acetylcholine or histamine on the human bronchus, but in concentrations of 10(-7)-10(-6)M it potentiated the effects of KCl on that preparation. It did not modify the EC50 of acetylcholine, histamine or KCl on the guinea-pig trachea. In Ca2+-free Krebs medium with additional K+ (30 mM), Ca2+ concentration-response curves were displaced to the left by Bay K 8644 in the two preparations. Shifts were 0.52 +/- 0.11 and 0.72 +/- 0.16 log units respectively with Bay K 8644 10(-8) and 10(-7) M on human bronchus (n = 4) and 0.67 +/- 0.16 and 1.06 +/- 0.19 log units respectively with Bay K 8644 10(-7) and 10(-6) M on the guinea-pig trachea (n = 5). In Krebs medium with Ca2+ 0.03 mM and K+ 30 mM, Bay K 8644 (10(-8) to 10(-6) M) contracted both the human bronchus and the guinea-pig isolated trachea. This effect was competitively antagonized by nicardipine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the Ca agonist Bay K8644 on 45Ca influx and net Ca uptake into rabbit aortic smooth muscle

We examined the effects of Bay K8644 and external K+ on isolated rings of rabbit aorta. Previous evidence has suggested that the Ca channel agonist, Bay K8644, preferentially opens the potential-sensitive Ca2+ channels (PSCs) rather than receptor-operated Ca2+ channels (ROCs). Bay K8644 stimulated 45Ca influx, net Ca gain and contractile tension were measured as a function of extracellular K+ concentration. Although Bay K8644 induced little tension at 5 mM K+ (PSS), Ca2+ influx was stimulated from a resting value of 12 mumol X kg-1 X min-1 (0.02 pmol X cm-2 X s-1) to 17 mumol X kg-1 X min-1 while the total Ca content increased by 95 mumol X kg-1. This net Ca gain was reversed by 50 mM caffeine. As the external K+ was increased from 5 to 80 mM, the Bay K8644 stimulation of influx and contraction first increased and then decreased. The data are explained in terms of a calcium entry model consisting of membrane Ca2+ channels in series with a subplasmalemmal barrier consisting of superficial sarcoplasmic reticulum (SR).     

Adenosine: A natural modulator of L-type calcium channels in atrial myocardium?

The mechanism of action of adenosine at the level of atrial myocardium has been a matter of debate. Electrophysiological studies showed that adenosine increases K+ efflux which may reduce Ca2+ influx, indirectly, by shortening the myocardial action potential. Recently some authors proposed that adenosine also depresses Ca2+ influx by a direct action on the L calcium channel, but, this effect being lower than that on voltage-dependent K+ channels, it was considered of minor importance. The effect of adenosine and its stable analogues was studied in the presence of the dihydropyridine Bay K 8644, a highly specific L-type calcium channel agonist, on isolated guinea-pig atria. The inotropic effect of the calcium channel activator was found to be antagonized by adenosine A1-receptor agonists. Binding studies showed that the effect on Bay K 8644 was not due to the interaction between adenosine analogues and dihydropyridines at the level of a common receptor site on L-type Ca2+ channels. Inhibitors of K+ channels did not antagonize the effect of adenosine analogues against Bay K 8644. Experimental conditions aimed to unmask an effect on slow Ca2+ currents (i.e. K+ depolarized paced atria), further supported that adenosine analogues may act in atria as negative modulators on L-type Ca2+ channels. Finally, the use of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a highly specific A1-receptor antagonist, demonstrated that the antagonism of Bay K 8644 by adenosine analogues is strictly dependent on A1 receptors. The above data support the possibility of a dual signal transduction pathway to ion channels (K+ and Ca2+) linked to A1 receptors in atrial myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antagonism between (-)-N6-phenylisopropyladenosine and the calcium channel facilitator Bay K 8644, on guinea-pig isolated atria.

Antagonism between (-)-N6-phenylisopropyladenosine (PIA) and the dihydropyridine calcium channel facilitator Bay K 8644 was investigated in guinea-pig spontaneously beating or electrically driven isolated atria, taken from normal and from reserpine-treated animals. PIA (3-100 nM) produced a dose-dependent decrease in contractile tension and frequency in spontaneously beating atria being more effective in reserpinized preparations. Bay K 8644 (5-200 nM) produced an increase in contractile tension in both normal and reserpinized atria. In electrically driven left atria the positive inotropic effect of Bay K 8644 was similar to that in spontaneously beating preparations. The positive chronotropic effect of Bay K 8644 was slight and variable. PIA produced a rightward parallel shift of the concentration-response curves for the positive inotropic effects of Bay K 8644 in all experimental conditions. In spontaneously beating atria from normal guinea-pigs, the Schild regression plot was linear and its slope near to unity; pA2 of PIA 8.63 +/- 0.05 (IC50 2.35 +/- 0.25 nM). In electrically driven atria the antagonism by PIA of the effects of Bay K 8644 was apparently competitive, and the IC50 of PIA was 18.6 +/- 0.4 nM. PIA antagonized the positive chronotropic effect of Bay K 8644 in spontaneously beating preparations, both from normal and from reserpine-treated animals. Carbachol did not modify the positive inotropic effects of Bay K 8644. These data indicate that PIA may interact with Bay K 8644 at the level of the slow calcium channels, and may decrease the transmembrane calcium flux into the cell.     

Interaction of Dihydropyridine Calcium Channel Agonists and Antagonists with Adenosine Receptors

Abstract: We have confirmed our previous ( Fredholm et al. 1986a ) finding that the dihydropyridine calcium channel agonist Bay K 8644 can displace [³H]-R-PIA from its binding site, the adenosine A₁-receptor. Bay K 8644 had an apparent K_i of 5.2 × 10^-6 M. The effect was shared by the two dihydropyridine calcium channel antagonists nifedipine and felodipine (K_i 4.2 and 8.7 × 10^-6 M, respectively). By contrast, two non-dihydropyridine calcium channel antagonists, verapamil and diltiazem, did not affect binding. Bay K 8644 displaced [³H]-R-PIA from its binding sites in a solubilized preparation. [³H]-XAC, a novel, potent A₁-receptor antagonist ligand, was also displaced by the dihydropyridine compounds with a similar or slightly higher potency as the displacement of R-PIA. This suggests a direct interaction with the adenosine receptor rather than an effect on regulatory GTP-binding proteins. However, at 1 μmol/l neither Bay K 8644 nor nifedipine significantly attenuated cyclic AMP accumulation in rat hippocampi or the R-PIA-mediated adenylate cyclase inhibition. The results show that dihydropyridine compounds that act as agonists or antagonists on L-type calcium channels can also affect adenosine receptors. The potency of the compounds for this effect is much lower than their potency as calcium channel agonists or antagonists. The results may therefore be of more experimental than clinical significance.     

吉非罗齐拮抗Bay K 8644增加大鼠主动脉平滑肌细胞L-型电压依赖性钙电流

目的 观察吉非罗齐拮抗Bay K 8644对离体大鼠主动脉的收缩效果,并进一步研究其机制。方法 采用离体血管张力记录法记录大鼠主动脉血管环的张力变化,膜片钳电生理学记录大鼠主动脉平滑肌细胞L-型电压依赖性钙电流。结果 Bay K 8644可以引起大鼠主动脉血管环收缩,最大收缩幅度为(2.13±0.42)g,提前孵育吉非罗齐可以对Bay K 8644的收缩幅度起到一定程度的抑制作用,使其收缩幅度降至(1.02±0.36)g。提前孵育L-NAME和去内皮均没有对吉非罗齐的抑制效果产生明显影响。Bay K 8644可以使得大鼠主动脉平滑肌细胞L-型钙电流增大127.62%,而吉非罗齐的干预,可以使其增幅降低到79.48%。结论 吉非罗齐可以一定程度上抑制Bay K 8644引起的大鼠主动脉收缩,其效果不受一氧化氮合酶抑制剂L-NAME及去内皮的影响。吉非罗齐可以部分拮抗Bay K 8644引起的大鼠离体主动脉平滑肌细胞L-型电压依赖性钙电流的增大。     

Dihydropyridine Bay K 8644 activates T lymphocyte calcium-permeable channels

The effects of the dihydropyridine calcium channel agonist Bay K 8644 on indo-1-loaded Jurkat human leukemia T lymphocytes was assessed by flow cytometry. Bay K 8644 from 10(-9) to 10(-4) M caused a dose-dependent rise in the intracellular free Ca concentration, an effect that was not mimicked by the dihydropyridine Ca antagonist nifedipine. Single channel recordings by the extracellular patch-clamp technique indicated that Bay K 8644 activated an 8-pS, barium-permeable channel that opened as bursts of brief events. The channel appeared to be identical to the previously described voltage-insensitive, messenger-mediated, calcium-permeable channel involved in T cell activation. The predominant effect of Bay K 8644 on these channels was to increase the probability of channel reopening, apparently without a major effect on mean channel open-time. The results suggest that the dihydropyridine Ca agonist Bay K 8644 interacts with both voltage-gated and receptor-operated Ca channels and also suggest potential strategies for development of a new class of immunomodulatory drugs.     

Effects of several calcium channels modulators on the [3H]noradrenaline release and 45Ca influx in the rat vas deferens.

1. The effects of nifedipine (1 microM), CdCl2 (0.1 mM) and the Bay K 8644 enantiomers (1 microM) on [3H]noradrenaline release and 45Ca uptake in epididymal and prostatic rat vas deferens were investigated. 2. Nifedipine, CdCl2 and Bay K 8644 optical isomers did not affect the basal tritium release. However, the [3H]noradrenaline release evoked by high potassium (50 mM) from both portions of rat vas deferens was markedly inhibited by CdCl2, scarcely affected by nifedipine and not modified by Bay K 8644 enantiomers. 3. (-)-Bay K 8644 increased the basal and potassium (50 mM) induced 45Ca uptake whereas (+)-Bay K 8644, nifedipine and CdCl2 did not alter the basal 45Ca uptake. However, they strongly inhibited the uptake induced by potassium in both portions of rat vas deferens. 4. These results suggest that the calcium channels (mainly L type) are involved on the contractions in rat vas deferens epididymal and prostatic halves; these channels differ from those present in sympathetic nerve terminals (likely of N Type) which modulates the NA release. 5. This study also shows that Bay K 8644 optical isomers possess opposite effects on the L channels of bisected rat vas deferens smooth muscle.     

Effects of Bay K 8644 on 45Ca uptake and efflux and on contraction in the rabbit aorta.

Contractions induced by K+, noradrenaline and 11,9-epoxymethano prostaglandin H2 (11,9-epoxymethano PGH2) were accompanied by a large, moderate and negligible stimulation of 45Ca uptake in rabbit aortic rings, respectively. Bay K 8644, 14 and 56 nM, enhanced both the contraction and the 45Ca uptake stimulated by all 3 agonists. In the absence of agonists, Bay K 8644 (14 and 56 nM) caused a small contraction and increase in 45Ca uptake. 45Ca efflux was increased by noradrenaline, and Bay K 8644 augmented this. In Ca-free solution, contractions induced by noradrenaline or 11,9-epoxymethano PGH2 were not augmented by Bay K 8644. Nifedipine (0.1 microM) antagonized 45Ca uptake stimulated by K+ or noradrenaline. Nifedipine also reduced the stimulant effect of Bay K 8644 on 45Ca uptake in the presence of all three agonists. It is concluded that, in the rabbit aorta, Bay K 8644 enhances the opening of Ca channels both during depolarization and in the presence of receptor-specific agonists and is also able to open Ca channels under basal conditions. Bay K 8644 appears not to reduce Ca efflux or enhance Ca release from intracellular stores.     

The effects of the dihydropyridine Bay K 8644 in guinea-pig isolated trachealis.

In trachea bathed by Krebs solution containing indomethacin 0.8 mumol l-1, Bay K 8644 (0.01-1 mumol l-1) evoked mild spasm. Peak tension was achieved after 10 min and was generally less than 20% of an acetylcholine (ACh) maximum. The effect of Bay K 8644 was not potentiated by addition of 2.5 mmol l-1 potassium chloride (KCl) to the Krebs solution. Bay K 8644 (1 mumol l-1) caused a small potentiation of KCl and tetraethylammonium (TEA). In contrast it did not modify the actions of ACh or histamine. Bay K 8644 (1 mumol l-1) caused a small potentiation of the effect of calcium chloride (CaCl2) tested in trachea bathed by a K+-rich, Ca2+-free, MOPS-buffered physiological salt solution. Organic inhibitors of calcium influx such as nifedipine (0.1 mumol l-1), verapamil (1 mumol l-1) or diltiazem (10 mumol l-1) each caused marked depression of concentration-effect curves to KCl. Bay K 8644 (0.01-1 mumol l-1) provided concentration-dependent protection against this effect in all three cases. Estimation of calcium influx by the lanthanum technique revealed that Bay K 8644 (1 mumol l-1) was able to promote the cellular influx of Ca2+. Intracellular electrophysiological recording showed that Bay K 8644 (1 mumol l-1) caused no change in the resting membrane potential of trachealis cells and no change in the properties of the spontaneous electrical slow waves. However, Bay K 8644 was able to delay the slow wave suppression evoked by 1 mumol l-1 nifedipine. The ability of Bay K 8644 to promote Ca2+ influx and its ability to protect against the effects of several structurally-unrelated inhibitors of Ca2+ influx are consistent with Bay K 8644 acting as an agonist at the dihydropyridine receptor associated with the voltage-operated Ca2+ channel (VOC) of trachealis muscle. By this action it potentiates those spasmogens (KCl, TEA) which act by permitting Ca2+ influx through VOCs. In contrast it has no effect on those spasmogens (ACh, histamine) which principally act to liberate Ca2+ from intracellular sites of sequestration.     

Effect of Ca2+ Agonist Bay K 8644 in human placental arteries

• 1.1. Bay K 8644 (0.1 μM) induced weak contractions in human placental artery segments that were increased in the presence of 7.5 mM K⁺. K⁺ and serotonin (5-HT) induced contractions that were enhanced by preincubation of segments with Bay K 8644. These enhancements were reduced by nifedipine (0.1 μM) and diltiazem (1 μM).
• 2.2. Bay K 8644 induced a ⁴⁵Ca²⁺ uptake increase which was potentiated by depolarization with K⁺ (less than 30 mM) and antagonized by nifedipine. K⁺ (15 and 30 mM) and 5-HT (1 μM) induced ⁴⁵Ca²⁺ uptake that was enhanced by Bay K 8644.
• 3.3. These results suggest that Bay K 8644: (1) is unable to activate the quiescent potential-operated Ca²⁺ channels (POCs) of these arteries, and (2) activates receptor (5-HT)-operated Ca²⁺ channels or facilitates Ca²⁺ influx through POCs activated by 5-HT.

     

Possible role of Ca2+ channels in the vasodilating effect of 5beta-dihydrotestosterone in rat aorta.

It has previously been shown that the androgen, 5beta-dihydrotestosterone (17beta-hydroxy-5beta-androstan-3-one, 5beta-DHT), is able to produce an endothelium-independent vasodilating effect in rat aorta. The present study analyzed the mechanisms underlying the above vasodilator effect of 5beta-dihydrotestosterone, with particular emphasis on verifying a possible interaction with GABA(A) receptors, beta-adrenoceptors and Ca2+ channels. Rat aortic rings without endothelium were isometrically recorded. 5Beta-dihydrotestosterone produced a concentration-dependent relaxation on the contractions induced by noradrenaline (NA; 0.3 microM) or K+ (KCl; 60 mM), with the latter being more sensitive to 5beta-dihydrotestosterone-induced relaxation than the former; the concentration-response curves showed that 5beta-dihydrotestosterone is significantly more potent than 17beta-estradiol(1,3,5(10)-estratrien-3,17beta-diol) to induce vasodilatation. The vasodilating effect of 5beta-dihydrotestosterone on noradrenaline-induced contraction was resistant to blockade by the GABA(A) receptor antagonists, picrotoxin or bicuculline, and the beta-adrenoceptor antagonist, propranolol, a finding that excludes an interaction of the steroid with GABA(A) receptors and beta-adrenoceptors. Interestingly, the contractions evoked by calcium in depolarized tissues were substantially inhibited by 5beta-dihydrotestosterone, implying that this steroid could be an endogenous calcium channel blocker; consistent with this finding, 5beta-dihydrotestosterone was able to relax tissues precontracted with the calcium channel opener, Bay K 8644. Moreover, although the rings precontracted with noradrenaline and potassium were almost equipotently relaxed by 5beta-dihydrotestosterone. Nifedipine was more potent than 5beta-dihydrotestosterone to block the potassium-induced contraction, but the steroid was more effective than nifedipine to prevent noradrenaline-induced contraction. The above results suggest that 5beta-dihydrotestosterone causes relaxation of rat aorta by acting directly on the membrane of smooth muscle cells; this non-genomic action may be explained in terms of a blockade of voltage- and receptor-dependent calcium channels, a mechanism that restricts the availability of extracellular calcium in the contractile machinery.