Hypotensive effect of Aspidosperma subincanum Mart. in rats and its mechanism of vasorelaxation in isolated arteries.

ETHNOPHARMACOLOGICAL RELEVANCE: Aspidosperma subincanum is a medicinal herb that is known to be useful for the treatment of cardiovascular-related illnesses. However, its effects and pharmacological mechanisms of action have not been studied. The aim of the present study was to determine the effect of an ethanol extract of Aspidosperma subincanum (EEAS) on blood pressure (in vivo) and vascular tension (in vitro) in the rat thoracic aorta. MATERIALS AND METHODS: Catheters were inserted into the right femoral vein and artery of anesthetized rats for EEAS infusion and the measurement of blood pressure, heart rate and aortic blood flow (flow probes were placed around the aorta). Moreover, the vasodilator effect of EEAS in isolated pre-contracted rat aortas was examined. RESULTS: Intravenous infusion of EEAS resulted in significant and dose-dependent hypotension, bradycardia and increased aortic blood flow. In isolated arteries, EEAS (0-27 µg/mL) induced a concentration-dependent relaxation of pre-contracted aortic rings; endothelial denudation potentiated this effect. Pre-treatment of the aortic rings with ODQ, an inhibitor of soluble guanylyl cyclase (sGC); MDL-12,330A, an inhibitor of adenylyl cyclase (AC); or CPA, a SERCA inhibitor, reduced EEAS-induced vasorelaxation. Treatment with an EEAS impaired contractions induced by phenylephrine (an adrenergic agonist) and Bay K 8644 (an L-type Ca(2+) channel activator). The blockade of K(+) channels with tetraethylammonium, clotrimazole, glibenclamide or 4-aminopyridine reduced the relaxation stimulated by EEAS. CONCLUSIONS: These findings suggest that EEAS induces hypotension associated with bradycardia. EEAS induces endothelium-independent vascular relaxation. The sGC/cGMP and AC/cAMP pathways, SERCA activation and Ca(2+) and K(+) flux across the sarcolemma, are likely involved in this relaxation.

Ent-7α-acetoxytrachyloban-18-oic acid and ent-7α-hydroxytrachyloban-18-oic acid from Xylopia langsdorfiana A. St-Hil. & Tul. modulate K(+) and Ca(2+) channels to reduce cytosolic calcium concentration on guinea pig ileum.

In this study we investigated the mechanism underlying the spasmolytic action of ent-7α-acetoxytrachyloban-18-oic acid (trachylobane-360) and ent-7α-hydroxytrachyloban-18-oic acid (trachylobane-318), diterpenes obtained from Xylopia langsdorfiana, on guinea pig ileum. Both compounds inhibited histamine-induced cumulative contractions (slope=3.5±0.9 and 4.4±0.7) that suggests a noncompetitive antagonism to histaminergic receptors. CaCl(2)-induced contractions were nonparallelly and concentration-dependently reduced by both diterpenes, indicating blockade of calcium influx through voltage-dependent calcium channels (Ca(v)). The Ca(v) participation was confirmed since both trachylobanes equipotently relaxed ileum pre-contracted with S-(-)-Bay K8644 (EC(50)=3.5±0.7×10-(5) and 1.1±0.2×10-(5)M) and KCl (EC(50)=5.5±0.3×10-(5) and 1.4±0.2×10-(5)M). K(+) channels participation was confirmed since diterpene-induced relaxation curves were significantly shifted to right in the presence of 5mM tetraethylammonium (TEA(+)) (EC(50)=0.5±0.04×10-(4) and 2.0±0.5×10-(5)M). ATP-sensitive K(+) channel (K(ATP)), voltage activated K(+) channels (K(V)), small conductance calcium-activated K(+) channels (SK(Ca)) or big conductance calcium-activated K(+) channels (BK(Ca)) did not seem to participate of trachylobane-360 spasmolytic action. However trachylobane-318 modulated positively K(ATP), K(V) and SK(Ca) (EC(50)=1.1±0.3×10-(5), 0.7±0.2×10-(5) and 0.7±0.2×10-(5)M), but not BK(Ca). A fluorescence analysis technique confirmed the decrease of cytosolic calcium concentration ([Ca(2+)](c)) induced by both trachylobanes in ileal myocytes. In conclusion, trachylobane-360 and trachylobane-318 induced spasmolytic activity by K(+) channel positive modulation and Ca(2+) channel blockade, which results in [Ca(2+)](c) reduction at cellular level leading to smooth muscle relaxation.

Quercetin antagonism of Bay K 8644 effects on rat tail artery L-type Ca(2+) channels.

The functional interaction between two L-type Ca(2+) channel activators, quercetin and (S)-(-)-methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)pyridine-5-carboxylate (Bay K 8644), has been investigated in vascular smooth muscle cells. L-type Ca(2+) currents [I(Ca(L))] were recorded in freshly isolated rat tail main artery myocytes using the whole-cell patch-clamp method. Bay K 8644 increased I(Ca(L)) in a concentration-dependent manner with a pEC(50) value of 8.25. Pre-incubation of myocytes with concentrations of quercetin per se ineffective as an L-type Ca(2+) channel activator (0.1 and 0.3 microM) inhibited significantly the maximal response evoked by Bay K 8644, but left unaltered its potency. Quercetin (0.1 microM) prevented the hyperpolarizing shift of the steady-state inactivation curve induced by 0.1 microM Bay K 8644 and its stimulation of I(Ca(L)) tail current intensity without modifying Bay K 8644-induced effects on I(Ca(L)) activation, inactivation, deactivation kinetics as well as on use-dependence and recovery from inactivation. Quercetin at nutritionally meaningful concentrations, limited the responsiveness of vascular L-type Ca(2+) channels to the pharmacological stimulation operated by Bay K 8644. These data contribute to a better understanding of quercetin effects on experimental in vivo cardioprotection.

Influence of naloxone on catecholamine release evoked by nicotinic receptor stimulation in the isolated rat adrenal gland.

The present study was designed to investigate the effect of naloxone, a well known opioid antagonist, on the secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane-depolarization in the isolated perfused rat adrenal glands, and to establish its mechanism of action. Naloxone (10(-6) approximately 10(-5) M), perfused into an adrenal vein for 60 min, produced dose- and time-dependent inhibition of CA secretory responses evoked by ACh (5.32 x 10(-3) M), high K+ (5.6 x 10(-2) M), DMPP (10(-4) M) and McN-A-343 (10(-4) M). Naloxone itself also failed to affect the basal CA output. In adrenal glands loaded with naloxone (3 x 10(-6) M), the CA secretory responses evoked by Bay-K-8644, an activator of L-type Ca2+ channels, and cyclopiazonic acid, an inhibitor of cytoplasmic Ca(2+)-ATPase, were also inhibited. In the presence of met-enkephalin (5 x 10(-6) M), a well known opioid agonist, the CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly inhibited. Taken together, these results suggest that naloxone greatly inhibits the CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as that by membrane depolarization. It seems that these inhibitory effects of naloxone does not involve opioid receptors, but might be mediated by blocking both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of Ca2+ into the cytoplasmic calcium store, which are at least partly relevant to the direct interaction with the nicotinic receptor itself.

Cortisol rapidly suppresses intracellular calcium and voltage-gated calcium channel activity in prolactin cells of the tilapia (Oreochromis mossambicus).

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.

L-type Ca2+ channels activation and contraction elicited by myricetin on vascular smooth muscles.

The effects of myricetin (3,3',4',5,5',7-hesahydroxyflavone), a natural flavonoid found in edible plants, were studied on vascular smooth muscle L-type Ca(2+) channels by comparing its mechanical, radioligand binding, and electrophysiological properties to those of the Ca(2+) channel agonist (S)-(-)-Bay K 8644. In rat aorta rings, both myricetin and (S)-(-)-Bay K 8644 induced contractile responses, which were dependent upon prior exposure to K(+). At 15 mM K(+) (K15) the pEC(50) values for myricetin and (S)-(-)-Bay K 8644 were 4.43+/-0.03 and 7.92+/-0.13, respectively. Furthermore, the maximum tension response to myricetin was not significantly different from that elicited by either (S)-(-)-Bay K 8644 or K60. The Ca(2+) channel blockers nifedipine, verapamil and diltiazem antagonised and fully reverted myricetin-, (S)-(-)-Bay K 8644- as well as K60-induced contractions. Both myricetin and (S)-(-)-Bay K 8644 potentiated rat aorta ring responses to K(+), shifting the K(+) concentration-response curve to the left. (S)-(-)-Bay K 8644, but not myricetin, inhibited in a concentration-dependent manner (+)-[(3)H]PN200-110 binding in porcine aortic membranes. Electrophysiological recordings from single rat tail artery myocytes, under amphotericin B-perforated as well as conventional methods, showed that both myricetin and (S)-(-)-Bay K 8644 increased L-type Ba(2+) current (I(Ba(L))) and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarising direction, without, however, modifying the threshold potential. Furthermore, (S)-(-)-Bay K 8644 accelerated both activation and inactivation kinetics of I(Ba(L)) while myricetin slowed down the activation kinetics. Finally, both (S)-(-)-Bay K 8644 and myricetin slowed down deactivation kinetics of I(Ba(L)). These results suggest that myricetin induces vasoconstriction by activating L-type Ca(2+) channel with similar efficacy but a site of action different to that of (S)-(-)-Bay K 8644.

Inhibitory effects of D2 agonists by striatal injection on excessive release of dopamine and hyperactivity induced by Bay K 8644 in rats.

We investigated by means of behavioral and neurochemical studies the effects of either D(1) or D(2) agonist on excessive dopamine release and hyperactivity induced by the microinjection of Bay K 8644, and an L-type Ca(2+) channel stimulant, into the rat caudate putamen under a novel environmental condition. Hyperactivity (locomotor activity and rearing counts) and significant increases in extracellular dopamine levels induced by Bay K 8644 were concomitantly observed. D(1) agonist, SKF81297, administered into the caudate putamen did not block Bay K 8644-induced hyperactivity measured by monitoring both animal activity and increases in extracellular dopamine levels detected by microdialysis. Pretreatment with the D(2) agonists, bromocriptine, talipexole and pramipexole, into the caudate putamen significantly blocked Bay K 8644-induced hyperactivity for 45 min after Bay K 8644 administration, although the single administration of these agonists significantly potentiated locomotor activity and rearing behavior. Furthermore, these agonists significantly suppressed Bay K 8644-induced extracellular dopamine levels. Our results indicate that these D(2) agonists (1) act on postsynaptic neuronal D(2) receptors under conditions of normal or low dopamine release in the caudate putamen, and (2) act on presynaptic D(2) receptors (autoreceptors) when excessive levels of dopamine are released or hyperdopamine neuronal activity is induced. Consequently, the effect of D(2) agonists in the clinical treatment of Parkinson's disease may be due to stimulation of postsynaptic D(2) receptors rather than presynaptic autoreceptors.

Effects of tetrandrine on smooth muscle contraction induced by mediators in pulmonary hypertension.

AIM: In attempt to characterize tetrandrine on pulmonary hypertension, biological activities induced by a range of mediators implicated in the pathogenesis of pulmonary hypertension were investigated. METHODS: Pulmonary artery rings and tracheal segments were contracted with couples of bioactive substances in which a series experiments including effects of tetrandrine on calcium agonist, endothelin, thromboxane A2, angiotensin II, neuropeptide Y, histamine, 5-methyl furmethide were performed, the influences of tetrandrine in the concentration of 1 to 30 micromol/L were investigated. RESULTS: Tetrandrine inhibited calcium agonist BayK8644, endothelin-1 and thromboxane A2 mimetic U46619, angiotensin II- and neuropeptide Y-induced contractile responses with depression of the maximal contraction of pulmonary artery rings in a varying extent. Tetrandrine inhibited leukotriene E4-induced concentration-response curve in a competitive antagonist manner with a pKB of (5.29+/-0.11) without any influence leukotriene C4, leukotriene D4, histamine, and 5-methyl furmethide induced contractile responses of guinea pig trachea. CONCLUSION: Tetrandrine may produce multiple pharmacological effects against calcium channel antagonist, U46619, endothelin-1,angiotension II, and neuropeptide Y induced vasoconstriction in rat pulmonary arteries in varying extent and inhibition of leukotriene E4 rather than C4, D4, histamine, and 5-methyl furmethide induced contractile responses on rat tracheal segments. These pharmacological characteristics are considered to contribute to its antihypertensive action during pulmonary hypertension.

Magnesium sulfate inhibits the oxytocin-induced production of inositol 1,4,5-trisphosphate in cultured human myometrial cells.

OBJECTIVE: The purpose of this study was to determine the effects of magnesium sulfate on inositol trisphosphate production and the mechanism of these effects. STUDY DESIGN: Myometrium was obtained at the time of cesarean delivery from women before labor at term. Inositol trisphosphate was measured in the primary myometrial cell cultures after stimulation with oxytocin, sodium fluoride, or Bay K 8644 with or without preincubation with magnesium sulfate or nifedipine. Experiments were performed in either calcium-containing or calcium-free medium that contained egtazic acid and after preincubation with the intracellular calcium chelator BAPTA-acetoxymethylester. Inositol trisphosphate production was measured by radioreceptor assay. In separate experiments, changes in intracellular calcium concentrations ([Ca(2+)](i)) were measured with the use of Fura-2 and spectrophotofluorometry. RESULTS: Oxytocin, sodium fluoride, and Bay K 8644 increased inositol trisphosphate production 2- to 4-fold. Preincubation with magnesium sulfate (3 x 10(-3) mol/L) for > or = 5 minutes decreased oxytocin-, sodium fluoride-, and Bay K 8644-induced inositol trisphosphate production in either calcium-containing or calcium-free media. Preincubation with BAPTA-acetoxymethylester decreased oxytocin-stimulated inositol trisphosphate production by 78% in calcium-containing media and completely prevented the oxytocin response in calcium-free media. Magnesium sulfate decreased inositol trisphosphate production in calcium-containing media but had no additional effect in calcium-free media. Oxytocin and Bay K 8644 increased [Ca(2+)](i) in either calcium-containing or calcium-free media, and magnesium sulfate reduced this in both cases. CONCLUSION: Magnesium sulfate appears to inhibit phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activity and subsequent calcium release in cultured myometrial cells by a direct effect on phospholipase C.

Inhibitory effects of 1,4-DHP antagonists on synaptic GABA release modulated by BAY-K 8644 in mechanically dissociated rat substantia innominata.

The effects of dihydropyridine (1,4-DHP) agonist and antagonists on miniature inhibitory postsynaptic currents (mIPSCs) were investigated in mechanically dissociated rat substantia innominata neurons attached to native GABAergic presynaptic nerve terminals, namely 'synaptic bouton preparation', using nystatin perforated patch recording mode under voltage-clamp conditions. BAY-K 8644 (BAY-K), an L-type Ca(2+) channel agonist, reversibly and concentration dependently facilitated the GABAergic mIPSC frequency without altering the distribution of current amplitudes. Removal of extracellular Ca(2+) completely suppressed the facilitatory effect of BAY-K on mIPSC frequency. The facilitatory effect of BAY-K on mIPSC frequency was maintained even in the presence of selective N-, P- and Q-type Ca(2+) channel antagonists, such as 3 x 10(-6) M omega-conotoxin-GVIA (omega-CgTX-GVIA), 3 x 10(-8) M omega-agatoxin-IVA (omega-AgTX-IVA) and 3 x 10(-6)M omega-conotoxin-MVIIC (omega-CmTX-MVIIC). However, nicardipine (3 x 10(-6) M) and nimodipine (3 x 10(-6) M), 1,4-DHP antagonists, significantly inhibited the mIPSC frequency enhanced by BAY-K by 37 +/- 5 and 42 +/- 6%, respectively. These results suggest the possible existence of L-type Ca(2+) channels in GABAergic presynaptic nerve terminals.

Inhibitory effects of melatonin on free intracellular calcium in mouse brain cells.

AIM: To study the effects of melatonin (Mel) on cortical intrasynaptosomal calcium concentration in old mice and on [Ca2+]i elevation induced by Bay-K-8644, KCl, and sodium l-glutamate in isolated brain cells of neonatal mouse, and to determine the antiaging mechanism of Mel. METHODS: [Ca2+]i was measured in an RF-5000 recording spectrofluorophotometer by preloading the synaptosomes or cells with Fura 2-AM. RESULTS: Long term of administrating Mel inhibited the overload of [Ca2+]i in old mouse cerebral cortex. The [Ca2+]i in both high (20 mg.L-1) and low dose (1 mg.L-1) of Mel groups was reduced from (434 +/- 32) nmol. L-1 (the older control group) to (330 +/- 41) and (313 +/- 56) nmol.L-1, respectively, P < 0.01. Mel 0.01, 0.1, 1, and 3 mumol.L-1 remarkably reduced [Ca2+]i elevations in isolated newborn mouse brain cells induced by Bay-K-8644, KCl, and Glu. CONCLUSIONS: The inhibitory effect of Mel on neuronal [Ca2+]i overload is involved in its antiaging effect.

Effects of excitatory amino acids and nimodipine on calcium currents in cultured rat cortical neurons.

AIM: To study the effect of excitatory amino acid (EAA) and calcium channel blocker on neuronal calcium channels. METHODS: With path-clamp technique (whole-cell recording), the effects of Bay-K-8644, cesium glutamate, potassium aspartate, and nimodipine (Nim) on calcium currents (ICa) in cultured cortical neurons of neonatal rats were studied. RESULTS: ICa was raised obviously by Bay-K-8644 and glutamate. ICa was raised concentration-dependently by aspartate (0.5, 5, 50 mmol.L-1), with increasing rates 15% +/- 3%, 37% +/- 3%, and 53% +/- 6%, respectively. The inhibition of ICa was obvious while adding Nim in the bath solution. With Nim 10 mumol.L-1, the inhibitory rate was 46% +/- 4%. CONCLUSION: EAA had increasing effects on neuronal calcium currents and Nim inhibited Ca2+ influx in neurons.

Bethanechol activates a post-receptor negative feedback mechanism in rabbit urinary bladder smooth muscle.

PURPOSE: Recent studies using vascular and gut smooth muscles indicate that contractile receptor agonists may activate post-receptor down-regulatory mechanisms causing a temporary reduction in the strength of subsequent contractions. Our data indicate a similar mechanism exists in detrusor smooth muscle of the urinary bladder. MATERIALS AND METHODS: Each isolated strip of female rabbit detrusor was placed in a tissue bath, secured to an isometric force transducer, and length-adjusted until depolarization with 110 mM KCl produced a maximum contraction (S0). Subsequent contractions were normalized to S0 (S/S0) or to a first stimulus with 30 mM KCl or caffeine (S/S1). Tissues were pretreated with the muscarinic receptor agonist, bethanechol (BE), then stimulated with KCl, caffeine, or Bay k 8644 to identify potential post-receptor down-regulation. RESULTS: Contractions induced by 30 mM KCl had three phases labeled fast peak (FP), slow peak (SP) and steady-state (SS). In tissues exposed for 30 min. to a maximum BE concentration then washed for 5 min., the KCl-induced FP and SP, but not SS, responses were reduced by approximately 40%. Smaller reductions in peak KCl-induced contractions occurred in tissues pretreated for a shorter duration or with a 100-fold lower BE concentration. This down-regulation induced by bethanechol pretreatment was reversible, lasting approximately 1-2 h. Not only were KCl-induced contractions reduced by BE pretreatment, but also those produced by the intracellular Ca(2+)-mobilizer, caffeine, and the L-type Ca2+ channel agonist, Bay k 8644. CONCLUSIONS: Pretreatment of isolated strips of rabbit detrusor with a muscarinic receptor agonist produced short-term down-regulation of KCl-induced peak contractions that may have involved inhibition of both influx of extracellular Ca2+ and release of intracellular Ca2+. Reductions in the degree of this novel modulatory response during disease conditions and aging could enhance contractile activity, possibly causing detrusor instability.

Effect of praeruptorin C on spontaneous [Ca2+]i transients in cultured myocardial cells of neonatal rats.

AIMS: To study the effects of praeruptorin C (Pra-C) on [Ca2+]i transients in cultured neonatal myocardiocytes. METHOD: Using Ca(2+)-sensitive fluorescent indicator, Fura 2-AM, spontaneous cytosolic Ca2+ transients were measured in cultured myocardial cells of neonatal rats. RESULTS: Pra-C 10, 30 mumol.L-1 caused a decrease in the peak of Ca2+ transients. Pra-C 30 mumol.L-1 and 10-30 mumol.L-1 inhibited partly the stimulatory effects of CaCl2 4.8 mmol.L-1 and Bay k 8644 100 nmol.L-1 on peak Ca2+ transients, respectively. Pra-C did not cause any marked change in the basal [Ca2+]i level between beats. Pra-C inhibited the reduced [Ca2+]i transients after inhibition of sarcoplasmic reticulum Ca2+ release in ryanodine pretreated cells. CONCLUSIONS: Pra-C inferred with the Ca2+ influx responsible for excitation-contraction coupling in myocardiocytes.

Effect of the Ca(2+)-channel agonist Bay K 8644 on the contractile responses in human placental veins.

1. The aim of the present study was to analyse, in segments of human placental veins, the effect of the Ca2+ channel agonist Bay K 8644 (0.1 microM) and Ca2+ channel antagonists nifedipine (0.1 microM) and diltiazem (1 microM) on vascular contractility and 45Ca2+ uptake. 2. The Ca2+ channel agonist Bay K 8644 (0.1 microM) caused small concentration dependent contractions that were increased by a moderate membrane depolarization with 7.5 mM K+. This increase was reversed by nifedipine and diltiazem. Ca2+ addition to segments previously depolarized with 75 mM K+ and exposed to a Ca(2+)-free medium caused contractile responses that were increased by 0.1 microM Bay K 8644; such an increase was blocked by 0.1 microM nifedipine and 1 microM diltiazem. 3. K+ and 5-HT induced concentration dependent contractile responses which were increased by Bay K 8644 (0.1 microM). Both 0.1 microM nifedipine and 1 microM diltiazem inhibited the increasing effect of Bay K 8644. Bay K 8644 (30 nM and 0.1 microM) caused an enhancement in 45Ca2+ accumulation over the basal value, that was increased by membrane depolarization with K+ (7.5, 15 and 30 nM) and inhibited by nifedipine (0.1 microM). K+ (15 and 30, but not 7.5 mM) and 5-HT (1 microM) induced 45Ca2+ uptake over the basal level that was increased by Bay K 8644 (0.1 microM). Such an increase was antagonized by nifedipine (0.1 microM). 4. These data indicate that: (1) a small depolarization with K+ is needed for Bay K 8644 to be able to produce consistent contractile responses, suggesting that voltage gated Ca2+ channels (VGCCs) are not activated in a basal situation in placental veins; (2) the increase of 5-HT contraction by Bay K 8644 may be produced by either the capability of this amine to depolarize the membrane of smooth muscle cells and subsequent facilitation of Ca2+ influx through VGCCs or direct activation by Bay K 8644 of receptor (5-HT) operated Ca2+ channels (ROCs), and (3) the increasing effect of Bay K 8644 appears to be due to a Ca2+ entry activation through VGCCs.

A tyrosine kinase regulates alpha-adrenoceptor-stimulated contraction and phospholipase D activation in the rat aorta.

Since previous studies had indicated a role for tyrosine kinases in alpha 2-adrenoceptor-induced contractile responses in other blood vessels, as well as in the activation of phospholipase D, we examined the sensitivity of these responses in rat aorta to the tyrosine kinase inhibitor genistein. Contractions induced by both noradrenaline and the alpha 2-adrenoceptor-selective agonist UK14304 (5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline) were fully inhibited by genistein, with the latter responses being more sensitive. Contractions induced by high K+ buffer were also inhibited, but to a lesser extent. Both agonists caused a stimulation of phospholipase D activity, which could be blocked by pretreatment with pertussis toxin, indicating involvement of either Gi or Go. Genistein completely inhibited the agonist-induced phospholipase D activity and also substantially reduced the basal level of phospholipase D activity. Pretreatment with either the alpha 1-adrenoceptor antagonist prazosin or the alpha 2-adrenoceptor antagonist rauwolscine was also effective in eliminating the agonist-induced increase of phospholipase D. These results indicate that a tyrosine kinase-regulated phospholipase D plays a critical role in alpha-adrenoceptor-induced contractions of the rat aorta and that stimulation of both alpha 1- and alpha 2-adrenoceptors is essential to allow phospholipase activation.

Vasorelaxant effect of the analgesic clonixin on rat aorta.

1. A novel vasorelaxant effect of clonixinate of L-lysine (Clx), analgesic and anti-inflammatory, was studied in rat aortic rings. 2. Clx completely relaxed aortic rings contracted by KCl 70 mM and together with its analog flunixin exhibited lesser potency but equal efficacy than verapamil. In comparison, indomethacin, which is a more potent cyclo-oxygenase inhibitor relaxed only about 40% of the maximal contraction of aortic rings. 3. Furthermore, Clx antagonized Ca2+ dependent aortic contraction and BAY K-8644 induced aortic contraction suggesting its calcium antagonist character. 4. From these results it can be concluded that the hypotensive effect seen in rats in vivo after Clx i.v. injection arises because of vasodilatory effect of Clx and gives further support to the proposal that the pharmacological mechanism of action of Clx should be calcium antagonism.

Mechanism of the action of angiotensin-converting enzyme inhibitors on agonist-induced Ca2+ influx.

To evaluate the direct effects of the angiotensin-converting enzyme (ACE) inhibitors, captopril, enalaprilat, enalapril (a prodrug without therapeutically significant ACE inhibitory effect) and ramiprilat, on cellular calcium metabolism, the cytosolic free calcium concentration was measured in cultured rat vascular smooth muscle cells using the fluorescent dye, fura-2. Preincubation with captopril, enalaprilat, enalapril, or ramiprilat for 40 min significantly reduced the angiotensin II-induced transplasma membrane calcium influx but did not influence the angiotension II-induced calcium release from internal stores. Captopril and ramiprilat also inhibited arginine vasopressin, but not the thapsigargin-, norepinephrine-, or the BayK 8644-induced changes in cytosolic calcium. Phorbol 12-myristate 13-acetate pretreatment for 30 s caused an increase in the angiotensin II-induced rise in cytosolic calcium. Although both captopril and verapamil reduced responses to angiotensin II to similar extents, only verapamil blocked the ability of phorbol 12-myristate 13-acetate to enhance responses to angiotensin II. It is concluded that ACE inhibitors modulate the effects of some but not all agonist-induced transplasma membrane calcium influx.

Pinacidil attenuates positive inotropic but not chronotropic responses to norepinephrine in isolated dog atrial and ventricular preparations.

We investigated whether pinacidil, a K+ATP channel opener like acetylcholine and adenosine, attenuated the positive chronotropic and inotropic responses to norepinephrine in isolated, blood-perfused dog atrial and ventricular preparations. Pinacidil (0.01-0.3 mumol) decreased atrial and ventricular contractile force to a much greater extent than sinus rate in a dose-related manner. Pinacidil dose-dependently attenuated increases in atrial and ventricular forces induced by norepinephrine but not increases in sinus rate. Pinacidil similarly attenuated the positive atrial and ventricular inotropic responses to Bay k 8644 and CaCl2. The pinacidil doses producing a fifty percent decrease (ED50) of the atrial and ventricular contractile force were not significantly different from the respective pinacidil doses producing a fifty percent inhibition (ID50) of the positive inotropic responses to norepinephrine, Bay k 8644 and CaCl2. Ouabain (5 and 15 nmol) did not affect the decreases in atrial and ventricular contractile force in response to pinacidil. These results suggest that the K+ATP-channel activator pinacidil, unlike acetylcholine or adenosine, functionally attenuates increases in ventricular and atrial contractile force in the responses to norepinephrine and other cardiotonics due to shortening of the action potential duration induced by K+ATP-channel activation in the dog heart.

The endothelin ETA receptor antagonist, BQ-123, normalizes the response of SHR aorta to Ca2+ channel activator.

Hypertension is associated with a hypersensitive response to the Ca2+ channel activator, Bay K 8644. We investigated the effect of the endothelin ETA receptor antagonist, BQ-123, on the contractions induced by Bay K 8644 in aorta from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats. BQ-123 (1 microM) decreased the sensitivity to Bay K 8644 of aorta of SHR down to that of WKY. This observation is consistent with a role for endothelin in hypertension.