Bob Wilffert

VASCULAR SMOOTH-MUSCLE CONTRACTION INITIATED BY POSTSYNAPTIC ALPHA-2-ADRENOCEPTOR ACTIVATION IS INDUCED BY AN INFLUX OF EXTRACELLULAR CALCIUM

Vasoconstriction in pithed, normotensive rats elicited via stimulation of postsynaptic alpha 2-adrenoceptors by B-HT 920 was antagonized by EDTA and the calcium antagonists nifedipine, D 600 and verapamil, whereas pressor responses to the alpha 1-agonist methoxamine were unaffected. This indicates that vasoconstriction in vivo initiated via postsynaptic alpha 2-adrenoceptors requires an influx of extracellular calcium. Thus, the antihypertensive effect of calcium antagonists may be based upon a diminution of vascular tone maintained by postsynaptic alpha 2-adrenoceptors.

Ca2+ influx insensitive to organic Ca2+ entry blockers contributes to noradrenaline-induced contractions of the isolated guinea pig aorta

We determined the contribution of intracellular Ca2+ to the noradrenaline (NA, 3 X 10(-5) mmol/l)-induced contraction of the isolated guinea pig aorta. Since only about 55% of the NA-induced contraction could be attributed to intracellular Ca2+ release, we assumed that a Ca2+ influx component contributes to the NA-induced contraction. This influx component proved resistant to the organic calcium entry blockers (CEBs) nifedipine, diltiazem, flunarizine and gallopamil which, in contrast, antagonized K(+)-induced Ca2+ influx completely. Conversely, the NA-induced Ca2+ influx component could be antagonized by the inorganic cations La3+, Cd2+, Mn2+, Ni2+ and Co2+. 45Ca2+ uptake experiments also revealed that both KCl and NA induce Ca2+ influx of which only the latter one is resistant to nifedipine. It was concluded that in the guinea pig aorta NA activates a receptor-operated channel through which Ca2+ can be translocated from the extracellular space to the cytosol to contribute directly to contraction.

SGD 101-75 IS DISTINGUISHED FROM OTHER SELECTIVE ALPHA-1-ADRENOCEPTOR AGONISTS BY THE INHIBITION OF ITS PRESSOR-RESPONSES BY CALCIUM ENTRY BLOCKADE AND VASODILATATION IN PITHED RATS AND CATS

The vasopressor effects of the selective alpha 1-adrenoceptor agonist Sgd 101/75 (2-[2-methylindazol-4-imino]-imidazolidine HCl) were analyzed in pithed rats and cats. Vasodilatation by the beta 2-adrenoceptor agonist salbutamol (1 mg/kg i.v.) or by the converting enzyme inhibitor captopril (5 mg/kg i.v.) antagonized the vasoconstriction by Sgd 101/75 in pithed rats. The effect of salbutamol was abolished by restoration of the baseline diastolic pressure by infusion of vasopressin. Calcium entry blockade by nifedipine (0.1-3 mg/kg i.v.) and (-)-verapamil (0.3 and 1 mg/kg i.v.) dose dependently inhibited the rise in the diastolic pressure induced by Sgd 101/75 pithed rats. This inhibition could not be attenuated by an infusion of vasopressin. In pithed cats, nifedipine most effectively antagonized the pressor effects of Sgd 101/75. In this respect, Sgd 101/75 is different from other alpha 1-adrenoceptor agonists, which are known to elicit a vasoconstriction which is virtually insensitive to vasodilatory measures and calcium entry blockade. These findings may be explained on the basis of a further subdivision of vascular postjunctional alpha 1-adrenoceptors.

Effects of the irreversible α-adrenoceptor antagonists phenoxybenzamine and benextramine on the effectiveness of nifedipine in inhibiting α1- and α2-adrenoceptor mediated vasoconstriction in pithed rats

SummaryIn pithed normotensive rats, i.v. injection of the selective α1-adrenoceptor agonist cirazolien produced vasoconstriction which was largely resistant to inhibition by nifedipine. On the other hand, the pressor effects of the selective α1-adrenoceptor agonists St 587 and Sgd 101/75 were much more effectively blocked by nifedipine, although not as effectively as the pressor effects to the selective α2-adrenoceptor agonist B-HT 920. The sensitivity to inhibition of vasoconstriction in pithed rats to the different agonists increased in the order cirazoline ≪ St 587<Sgd 101/75<B-HT 920. Phenoxybenzamine (3–300 μg/kg, i.v., −60 min) irreversibly antagonized the vasoconstriction to cirazoline, St 587, Sgd 101/75 and B-HT 920. After treatment of the rats with phenoxybenzamine the potency and efficacy of nifedipine in antagonizing vasoconstriction to α1-, but not to α2-adrenoceptor activation was dose-dependently enhanced. The potency of nifedipine to inhibit α1-vasoconstriction by cirazoline, St 587 and Sgd 101/75 was increased maximally to the level of efficacy at which nifedipine antagonized B-HT 920-induced vasoconstriction. The dose of phenoxybenzamine required to maximally increase the potency and efficacy of nifedipine to antagonize vasoconstriction of the α1-adrenoceptor agonists was inversely related to the level of sensitivity to blockade by nifedipine of the vasoconstriction they produced. In contrast, pretreatment of rats with the irreversible antagonist, benextramine (10 mg/kg, i.v., −100 to −60 min) did not increase the potency or efficacy of nifedipine to antagonize vasoconstriction to cirazoline, St 587, Sgd 101/75 or B-HT 920, despite irreversible blockade of α1- and α2-adrenoceptors. These data suggest that phenoxybenzamine, but not benextramine, selectively inhibits the α1-adrenoceptor mediated vasoconstrictor mechanism that is independent of influx of extracellular calcium. Moreover, the results show that the existence of receptor reserve or the number of α1-adrenoceptors activated does not determine the relative contribution of calcium influx-independent mechanisms in α1-adrenoceptor-mediated vasoconstriction.

The contractions induced in rat and guinea-pig aortic strips by the α2-adrenoceptor selective agonists B-HT 920 and UK 14,304 are mediated by α1-adrenoceptors

The alpha-adrenergic action of the selective alpha 2-adrenoceptor agonists B-HT 920 and UK 14,304 was investigated on helically cut preparations of rat and guinea-pig isolated aorta. The alpha 1-adrenoceptor selective stimulant (-)-phenylephrine was included for comparison. All agonists induced concentration-dependent contractions in both preparations. Calcium entry blockade by D 600 almost abolished the contractions evoked by B-HT 920 and UK 14,304 in rat aorta while those evoked in guinea-pig aorta were less strongly affected. Contractions elicited by (-)-phenylephrine were moderately impaired by D 600 in rat aorta whereas there was only a limited effect in guinea-pig aorta. Analysis of the prazosin and yohimbine antagonism of B-HT 920- and UK 14,304-evoked contractions showed the involvement of alpha 1-like adrenoceptors in rat and guinea-pig aorta, prazosin being approximately 1000 times more potent that yohimbine. The results show that B-HT 920 and UK 14,304 contract rat and guinea-pig aorta via alpha 1-like adrenoceptors which are not identical. It is submitted that rat and guinea-pig alpha 1-adrenoceptors activate different contractile processes.

Effects of the Selective β1-Adrenoceptor Antagonist, Nebivolol, on Cardiovascular Parameters in the Pithed Normotensive Rat

In the pithed rat we investigated the cardiovascular properties of d,l-nebivolol and its enantiomers. We used the increase in heart rate elicited by (-)-adrenaline and (-)-noradrenaline as a model for studying beta 1-adrenoceptors. A leftward shift of the logarithmic dose-pressor response curve of (-)-adrenaline reflects beta 2-adrenoceptor-blocking properties. The blood pressure responses of methoxamine, B-HT 920 and serotonin (5-HT) were studied in order to test whether d,l-nebivolol has alpha 1-, alpha 2- and 5-HT2-receptor-blocking properties. Furthermore, the interaction of d,l-nebivolol with the peripheral sympathetic neurotransmission was investigated in pithed rats by electrical stimulation of the spinal cord. d,l-Nebivolol and d-nebivolol (threshold concentration 10(-8) mol/kg) were demonstrated to be selective beta 1-adrenoceptor antagonists. l-Nebivolol was a factor of 1,000 less potent as beta 1-adrenoceptor blocker. Up to a dose of 10(-5) mol/kg, d,l-nebivolol appeared to have neither alpha 1-, alpha 2-, beta 2-, 5-HT2-, angiotensin II-receptor antagonistic, calcium entry blocking, converting enzyme inhibiting nor direct vasodilating properties and did not interact with the sympathetic neurotransmission in the vascular wall. An explanation for an antihypertensive effect independent of beta-adrenoceptor blockade as found in spontaneously hypertensive rats and man could not be found in this model, therefore we suggest that this blood-pressure-lowering effect does not originate from conventional peripheral mechanisms.

Inhibitory dopamine receptors on sympathetic neurons innervating the cardiovascular system of the pithed rat

SummaryAdditional experimental evidence was obtained for an inhibitory function of prejunctional α2-adrenoceptors and/or dopamine receptors located on noradrenergic neurons innervating the heart and resistance vessels of the pithed normotensive rat. Mixed α2-adrenoceptor receptor agonists, differing in selectivity towards either receptor type, i.e. N,N-di-n-propyldopamine (DPDA), 2-N, N-di-n-propylamino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphthalene (DP-6,7-ADTN), B-HT 920 and B-HT 933 (azepexole) were used.In pithed normotensive rats, DPDA (30 and 100 μg/kg/min) dose-dependently inhibited the electrical stimulation-induced increase in diastolic pressure, but did not significantly affect the stimulation-evoked increase in heart rate. The inhibition exerted by DPDA was blocked by haloperidol and sulpiride (0.3 mg/kg of each), but not by yohimbine (1 mg/kg), indicating the involvement of dopamine receptors. In this respect, sulpiride and haloperidol were found approximately equipotent.DP-6,7-ADTN (10 and 30 μg/kg/min) impaired both tachycardic and vasoconstrictor responses in a dose-dependent manner. Sulpiride (0.3 mg/kg) only partially restored the DP-6,7-ADTN-depressed stimulation-evoked increase in diastolic pressure, whereas yohimbine (1 mg/kg) alone was without effect. The combination of both antagonists completely prevented the inhibition caused by DP-6,7-ADTN. On the other hand, yohimbine (1 mg/kg), but not sulpiride (0.3 mg/kg), selectively antagonized the DP-6,7-ADTN-induced inhibition of stimulation-evoked tachycardia.B-HT 920 (1, 3 and 10 μg/kg/min) very effectively reduced the increase in diastolic pressure and heart rate caused by electrical stimulation. Inhibitory dopamine as well as α2-adrenoceptors participated in the vascular effects of B-HT 920, whereas α2-adrenoceptors were only involved in the cardioinhibitory response to this agonist.B-HT 933 (0.6 and 1 mg/kg/min) dose-dependently reduced the stimulation-evoked increase in arterial pressure through selective stimulation of inhibitory α2-adrenoceptors, dopamine receptors not taking a part.The results confirm and extend the observations that in addition to α2-adrenoceptors inhibitory dopamine receptors are located on the sympathetic neurons connected with the arterial vasculature of the pithed normotensive rat. The sympathetic nerves innervating the rat heart do not contain inhibitory dopamine receptors; their activity only can be modulated by α2-adrenoceptor stimulation. In the pithed normotensive rat, activation of prejunctionally located α2-adrenoceptors more effectively inhibits the sympathetic activity directed to the heart than that to the resistance vessels.

Inhibitory effect of calcium antagonist drugs on vasoconstriction induced by vascular alpha2-adrenoceptor stimulation

A survey is given of the mechanisms of the antihypertensive effect of calcium entry blockers. The main background of the antihypertensive/hypotensive action is dilatation of precapillary arterioles (resistance vessels that cause a reduction in total peripheral resistance and, hence, a decrease in blood pressure). The vascular relaxation is caused by an inhibition of the transmembranous calcium influx and, probably less so, by interference with calmoduline. Calcium entry blockers significantly reduce the vasoconstriction induced by the excitation of vascular postsynaptic alpha 2 adrenoceptors. The inhibitory effect of calcium entry blockers is reversed by the calcium entry promoter Bay k 8644. The vasoconstriction induced by alpha 1-adrenoceptor stimulation is less generally influenced by calcium entry blockers than the alpha 2 effects. The interference with alpha 2-adrenoceptor-induced vasoconstriction may contribute to the vasodilator action of the calcium entry blockers, especially in hypertensive patients who show a hyperreactivity to pressor responses toward catecholamines.

HEMODYNAMIC CHARACTERIZATION OF PINACIDIL IN RATS - COMPARISON WITH HYDRALAZINE

Pinacidil (N-cyano-N-4-pyridyl-N-1,2,2-trimethylpropylguanidine monohydrate; P1134) is a new vasodilator drug with a direct relaxant effect on vascular smooth muscle. Its hemodynamic properties, in comparison with those of hydralazine, were studied in conscious normotensive and spontaneously hypertensive rats; anesthetized normotensive rats; pithed normotensive rats; pithed normotensive rats subjected to electrical stimulation of the spinal cord. Radioligand binding studies on rat cerebral membranes were carried out to study a possible affinity for pinacidil towards alpha 1- and alpha 2-adrenoceptors, respectively. The observations made in conscious and anesthetized rats suggest that both pinacidil and hydralazine are predominantly arterial vasodilators. In conscious animals reflex tachycardia was elicited by both drugs. Neither pinacidil nor hydralazine possessed substantial affinity for alpha 1- or alpha 2-adrenoceptors, as concluded from radioligand binding studies. Pinacidil interferes with the pressor response to postsynaptic alpha 2-adrenoceptor stimulation in pithed rats, possibly reflecting weak calcium antagonistic activity of the drug. Pinacidil did not interfere with the electrically induced release of noradrenaline from presynaptic sites. All results suggest that pinacidil is a direct-acting arteriolar dilator, which on a molar base is somewhat more potent than hydralazine.

R 56865 Differentiates between Contractile Agents with Respect to the Nifedipine-Sensitive Component in the Isolated Rat Aorta

The interaction of the benzothiazolamine R 56865 with the nifedipine-sensitive component of the serotonin (5-HT)-, angiotensin II (AII)- and arginine-vasopressin (AVP)-induced contractions was studied in the isolated rat aorta. Nifedipine caused concentration-dependently (10(-9)-10(-6) mol/l) a slight rightward shift accompanied by a limited depression of the maximum of the concentration-response curves for 5-HT-, AII- and AVP-induced contractions. R 56865 (10(-5) mol/l) antagonized the contraction elicited by 5-HT and AII in a similar manner as nifedipine. The effect of R 56865 on 5-HT- and AII-induced contractions was no longer observed after pretreatment with nifedipine. The AVP-induced contraction was not affected by R 56865 (10(-5) mol/l). As shown previously, R 56865 is a weak inhibitor of potential-operated channels but inactive on Ca2+ channels activated by NA. In conclusion, R 56865 does not only differentiate between depolarization and receptor-stimulation, but also between the activation of Ca2+ channels by different types of receptors. We propose that R 56865 may interact with Ca2+ channels at a site which plays a role in their activation.