Human postjunctional alpha-1 and alpha-2 adrenoceptors: differential distribution in arteries of the limbs

Experiments were performed in order to characterize the post-junctional alpha adrenoceptors that mediate contraction in arteries of human limbs. Blood vessels were obtained from patients undergoing amputation of an extremity for reasons other than vascular disease. Proximal (dorsalis pedis and arcuate arteries of the foot, superficial palmer arch of the hand) and distal (digital arteries of the foot and hand) blood vessels were studied from each limb. The blood vessels were removed within 60 min of amputation and were suspended for isometric tension recording in modified Krebs-Ringer bicarbonate solution. In proximal and distal arteries, alpha-1 adrenergic blockade with prazosin produced a nonparallel shift in the concentration-effect curve to high compared to low concentrations of the agonist. In contrast, alpha-2 adrenergic blockade with rauwolscine was more effective against responses evoked by low concentrations of norepinephrine. This suggests that the alpha-2 adrenergic component of the response to norepinephrine is a low-maximum effect compared to the alpha-1 adrenergic component. Prazosin was less potent and rauwolscine more potent in distal arteries, compared to proximal arteries which might indicate an increased alpha-2 adrenergic response in distal arteries. The selective alpha-1 adrenergic agonist, phenylephrine, produced similar responses in proximal and distal arteries. However, the selective alpha-2 adrenergic agonist, B-HT 920, caused greater contractile responses in distal arteries compared to proximal arteries. The results suggest that alpha-1 and alpha-2 adrenoceptors are present on the vascular smooth muscle of arteries of human limbs, and that alpha-2 adrenoceptors are more prominent on distal arteries. This may be related to an increased contribution of the distal arteries to thermoregulation.     

Postjunctional alpha-1 and alpha-2 adrenoceptors in human skin arteries. An in vitro study

Experiments were performed to characterize the postjunctional alpha adrenoceptors that mediate adrenergic constriction in human skin arteries. Abdominal s.c. arteries were obtained from patients who died 3 to 12 hr before, and vascular segments 2 mm in length and 600 to 1050 microns in external diameter were prepared for isometric tension recording. On application of norepinephrine, phenylephrine (alpha-1 adrenergic agonist) or clonidine (alpha-2 adrenergic agonist) the arteries contracted in a dose-dependent manner and, in terms of the mean EC50 values, the order of potencies was clonidine greater than norepinephrine greater than phenylephrine. With regard to their ability to induce maximal contraction, the order was norepinephrine = phenylephrine greater than clonidine. In the presence of phentolamine (nonspecific alpha adrenergic antagonist) or yohimbine (selective alpha-2 adrenergic antagonist) the control curve for norepinephrine was displaced to the right in a parallel way. Prazosin (selective alpha-1 adrenergic antagonist) depressed both the slope and maximal response of the control curve for norepinephrine but the shift was not significant. Prazosin and yohimbine produced a parallel rightward shift in the control curve for phenylephrine and clonidine, respectively. These results suggest that skin arteries of humans have a mixed population of postjunctional alpha-1 and alpha-2 adrenoceptors and that alpha-2 adrenoceptors are more prominent. They also suggest that the alpha-2 adrenergic component of the response to norepinephrine is a low-maximum effect compared to the alpha-1 adrenergic component. This could be of significance in regulating skin blood flow and thermoregulatory function.     

Naloxone-induced bradycardia in pithed rats: evidence for an interaction with the peripheral sympathetic nervous system and alpha-2 adrenoceptors.

Earlier experiments performed in this laboratory have demonstrated that naloxone infusion (1 mg/kg/min i.v.) into conscious rats results in a bradycardia that has a peripheral component, is dependent on a certain level of sympathetic activity and is sensitive to alpha adrenoceptor blockade (5 mg/kg of phentolamine i.v.). The main objective of this investigation was to examine the underlying mechanism(s) responsible for the peripherally mediated naloxone-induced bradycardia, and to test the hypothesis that naloxone interacts with peripheral inhibitory alpha adrenoceptors associated with depression of peripheral sympathetic activity. Naloxone infusion (1 mg/kg/min i.v.) in pithed rats, in the absence of sympathetic nerve activation, resulted in a bradycardia that could not be blocked by 1 mg/kg (i.v.) of atropine, 5 mg/kg (i.v.) of phentolamine, 0.1 mg/kg (i.v.) of prazosin or 0.5 mg/kg (i.v.) of rauwolscine. Isoproterenol or norepinephrine-induced tachycardia was not blocked by naloxone infusion, suggesting that naloxone does not antagonize the postjunctional activation of cardiac adrenoceptors to cause bradycardia. In the presence of sympathetic nerve activity, naloxone depresses neurogenic tachycardia. This effect was blocked completely by 5 mg/kg (i.v.) of phentolamine or 0.5 mg/kg (i.v.) of rauwolscine, but not 0.1 mg/kg (i.v.) of prazosin or 1 mg/kg (i.v.) of atropine. The results of this investigation suggest that the naloxone-induced bradycardia in pithed rats is mediated postjunctionally and prejunctionally, and that this prejunctional effect is dependent on sympathetic nerve activity and inhibitory alpha-2 adrenoceptors. Furthermore, these results confirm results obtained from conscious rats in an earlier investigation.     

Calcium utilization coupled to stimulation of postjunctional alpha-1 and alpha-2 adrenoceptors in isolated human resistance arteries.

Human s.c. resistance arteries (internal diameters 158-353 microns) were mounted in a microvascular myograph, and experiments were designed to examine the calcium pools utilized by selective stimulation of alpha-1 and alpha-2 adrenoceptors. In a concentration-dependent manner, phenylephrine and B-HT 933 evoked contractions mediated by alpha-1 and alpha-2 adrenoceptors, respectively, both in calcium-containing and in calcium-free saline. With respect to the maximum response to potassium in calcium-containing saline, the maximum responses to phenylephrine and B-HT 933 were 96 +/- 6 and 85 +/- 8%, respectively, in calcium-containing saline, and 79 +/- 4 and 14 +/- 2%, respectively, in calcium-free saline. A qualitatively similar difference in maximum responses to alpha-1 vs. alpha-2 adrenoceptor stimulation in calcium-free saline was demonstrated for norepinephrine in the presence of antagonists selective for the two alpha adrenoceptor subtypes. The maximum relaxation in calcium-containing saline produced by the calcium antagonist nitrendipine was 52 +/- 3% in vessels precontracted with phenylephrine, but 80 +/- 5% in vessels precontracted with B-HT 933. A quantitative difference in receptor reserves was demonstrated between alpha-1 and alpha-2 adrenoceptors; 90% of the maximum response was obtained at 34 +/- 5 and 57 +/- 8% receptor occupation, respectively. These data suggest that compared to responses mediated by stimulation of postjunctional alpha-1 adrenoceptors, stimulation of postjunctional alpha-2 adrenoceptors relies heavily on calcium influx. Stimulation of postjunctional alpha-2 adrenoceptors is, however, also coupled to intracellular release of calcium in isolated human s.c. resistance arteries.     

Evidence for central alpha-2 adrenoceptors, not imidazoline binding sites, mediating the ethanol-attenuating properties of alpha-2 adrenoceptor antagonists.

The role of central adrenoceptors in the ethanol-attenuating effects of alpha-2 adrenoceptor blockers was investigated in mice; the centrally active antagonist atipamezole was compared with L 659,066, which penetrates the brain poorly. L 659,066 (1-10 mg/kg) had no effect on the hypothermia induced by ethanol (2 g/kg) or ethanol ataxia (2.4 g/kg), whereas atipamezole (1 mg/kg) significantly attenuated both ethanol-induced hypothermia and ataxia. Atipamezole (1-3 mg/kg) significantly attenuated the ethanol-induced reduction in exploratory head-dipping in a holeboard test whereas L 659,066 was only effective at a dose of 1 mg/kg, higher doses (3 and 10 mg/kg) and a lower dose (0.3 mg/kg) were ineffective. Atipamezole was without effect on ethanol's locomotor stimulant effect in the holeboard but L 659,066 attenuated this effect at doses less than 3 mg/kg Many alpha-2 adrenoceptor ligands also have affinity for nonadrenergic imidazoline-binding sites. The role these sites may play in attenuating ethanol's effects was investigated by comparing RX 821002 (methoxy idazoxan), which has little or no affinity for imidazoline-binding sites with atipamezole. Both atipamezo 1 e (1 mg/kg) and RX 821002 (0.06-0.2 mg/kg) significantly attenuated ethanol-induced hypothermia, ataxia and reduction in head-dipping, but were without effect on ethanol-induced locomotor stimulation. These results suggest that nonadrenergic imidazoline-binding sites are not implicated in the ethanol-attenuating properties of alpha-2 adrenoceptor antagonists.     

Classification of bovine aortic alpha-1 adrenoceptors by the criteria of ligand affinity and molecular mass

[3H]Prazosin bound to a single class of alpha-1 adrenoceptors in bovine aortic membranes with a Kd of 25 pM. Digitonin solubilized 30% of the receptors as assayed by specific [3H] prazosin binding. The rank order potency of displacing ligands was the same for both membrane-bound and soluble alpha-1 adrenoceptors [prazosin greater than phentolamine greater than yohimbine and (-)-epinephrine = (-)-norepinephrine much greater than (+)-norepinephrine]. Prazosin had a significantly lower affinity for the soluble receptor, whereas the other adrenergic ligands had the same affinity for both forms of the receptor. The alpha-1 adrenoceptor was affinity-labeled with 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6,7- dimethoxyquinazoline and analyzed by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the receptor binding subunit in both bovine aorta and rat liver was found to be 86,000 MW. It is concluded from this study that bovine aortic alpha-1 adrenoceptors have pharmacologic and biochemical characteristics similar to those in other tissues.     

Ontogeny of renal alpha-1 and alpha-2 adrenoceptors in the spontaneously hypertensive rat

Renal alpha-1 and alpha-2 adrenoceptors were characterized during the development of the spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto rats through Scatchard analysis of [3H]prazosin and [3H]yohimbine binding to kidney membrane preparations in an attempt to correlate biochemical changes with the reported functional changes occurring in hypertension development in the SHR. Renal alpha-1 and alpha-2 receptor density was higher in SHR than in Wistar-Kyoto rats at all ages tested. In contrast to Wistar-Kyoto rats, in which the number of alpha-1 and alpha-2 receptors remained relatively constant with age, the number of renal alpha-1 adrenoceptors in the SHR was lowest at the 4th week of age (61 fmol/mg) increasing transiently at 5 weeks and then again at 8 week reaching a plateau at that time. The maximum number of binding sites for [3H]yohimbine binding in the SHR was also age dependent. The number of renal alpha-2 adrenoceptors in the SHR was lowest at 4 weeks of age (125 fmol/mg) increasing to 220 fmol/mg at 5 weeks of age and to 260 fmol/mg at 8 weeks of age. Adult levels (308 fmol/mg) were reached by 18 weeks of age. Unlike receptor densities, affinity constants were not significantly altered during postnatal development. The changes in alpha-1 and alpha-2 adrenoceptors in the kidneys of SHR may suggest an important developmental role which is not yet understood.     

Postjunctional alpha-1 and alpha-2 adrenoceptors in the coronaries of the perfused guinea-pig heart

Prazosin and yohimbine were used to differentiate postjunctional alpha adrenoceptors in the coronaries of the perfused guinea-pig heart. Two postjunctional alpha adrenoceptor subtypes were distinguished by the affinities of the receptor for yohimbine and prazosin. The pA2 for yohimbine were 8.74 against alpha-methylnorepinephrine and 8.98 against BHT-920, and the pA2 for prazosin was 9.84 against phenylephrine. Yohimbine was not very active against the alpha-1 selective agonist as was prazosin against the alpha-2 selective agonists. Alpha-1 and alpha-2 postjunctional adrenoceptors mediate vasoconstriction in the whole coronary bed of the perfused guinea-pig heart.     

Presynaptic alpha-2 adrenoceptor activation and coupling of the receptor-presynaptic effector system in the perfused rat heart: affinity and efficacy of phenethylamines and imidazoline derivatives

The right sympathetic nerves of perfused rat hearts were stimulated in the presence of inhibitors of neuronal and extraneuronal uptake and propranolol. The inhibition by alpha adrenoceptor agonists of stimulation-evoked (10 pulses, 0.1 Hz) [3H]norepinephrine (NE) overflow into the perfusate was taken as a parameter of presynaptic adrenoceptor activation. Under the present conditions, autoinhibition of NE release is not activated by endogenous NE as evident from ineffectiveness of adrenoceptor antagonists in facilitating evoked [3H]NE overflow. The potency (EC50, -log10), affinity (agonist-presynaptic receptor dissociation constant KA, -log10) and relative efficacies (RE) were determined for phenethylamines (NE or alpha-methylepinephrine) and for imidazoline derivatives. NE (-log EC50, 7.76) was 0.88 log units more potent than alpha-methylepinephrine (-log EC50, 6.88) and about the same difference was observed for the -log KA values (5.92 vs. 4.75). RE were similar (NE, 100%; alpha methylepinephrine, 98%) and 22- to 50-fold higher than efficacies of imidazoline derivatives. Hydroxylations in positions 3 and 4 of the phenyl moiety of phenylaminoimidazoline (-log EC50, less than 5; -log KA, less than 5; RE, less than 1%) resulted in a marked increase in potency (-log EC50, 8.32) of the resulting dihydroxyphenylaminoimidazoline due to a high affinity (-log KA, 8.22) at a low efficacy (2% of NE). In contrast, hydroxylation in positions 3 and 4 of the phenyl ring of tolazoline (no agonist activity under the present conditions; antagonist affinity constant from the literature, 6.4-6.6) produced dihydroxytolazoline, a moderately potent agonist (-log EC50, 7.25) with an efficacy of 3.5% at an affinity (-log KA, 6.92) not much different from that of tolazine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Variation in sensitivity of alpha adrenoceptor-mediated contraction of the vascular smooth muscle of rabbit elastic and muscular arteries is related to receptor affinity

Norepinephrine sensitivity (pD2) and agonist dissociation constant (pKA) have been determined in the following 12 rabbit arteries: thoracic and abdominal aorta, basilar, ear, common, external and internal iliac, ovarian, large and medium pulmonary, renal and superior mesenteric. They were determined in the presence of beta adrenoceptor blockade and uptake 1 and uptake 2 inhibition to prevent compromising additional actions of norepinephrine and intrinsic processes that influence its concentration at the site of action. In the superior mesenteric artery, determinations were made after endothelial inactivation and in the presence of indomethacin, because in this vessel blockade by these procedures influences norepinephrine sensitivity. In 12 arteries a positive correlation was found between norepinephrine pD2 and pKA (r = 0.74, P less than .01). The slope of the regression line did not differ from unity. Norepinephrine pD2 did not correlate with receptor reserve in these arteries when assessed as antilog pD2-pKA. In three arteries, the ear and common and external iliac, a large receptor reserve was found. If these were excluded from the series, the following correlation would be found: r = 0.9; P less than .001. Here again the slope of the regression line did not differ from unity. The pD2 and pKA were determined for the more selective alpha-1 adrenoceptor agonist, phenylephrine in six of these arteries, and similar results were obtained. The KB for prazosin in this series did not correlate with norepinephrine KA (r = 0.45, P greater than .05), and the slope (0.17) was not significantly different from zero. The pD2 for histamine, determined after H2 receptor blockade, does not differ in the arteries.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of alpha-1 and alpha-2 adrenoceptors in the sympathetic control of the proximal urethra

In the pithed rat, postjunctional alpha-1 and alpha-2 adrenoceptors mediate increases in proximal urethral perfusion pressure (UPP). The present study examined the role of alpha-1 and alpha-2 adrenoceptors in sympathetic control of the isolated in situ proximal urethra of the rat. An endogenous alpha adrenergic constriction was demonstrated in the proximal urethra by eliciting dose-related and phentolamine-sensitive increases in UPP after the systemic administration of tyramine. Transient dose-related and hexamethonium-sensitive increases in UPP were also elicited by ganglionic stimulation with 1,1-dimethyl-4-phenylpiperazinium (DMPP). Based on the relative sensitivity of the DMPP response to standard autonomic blockers, it was determined that constriction is the predominant autonomic response in the proximal urethra and this response is mediated by alpha adrenoceptor mechanisms. Prazosin, a selective alpha-1 adrenoceptor antagonist, substantially reduced (72%) the steady-state increases in UPP elicited by discrete low frequency electrical stimulation of the spinal hypogastric outflow. In contrast, selective alpha-2 adrenoceptor blockade with rauwolscine significantly increased (28%) the UPP response to hypogastric stimulation. Prazosin also abolished the increase in urethral tone elicited by tyramine, whereas rauwolscine had no effect on the tyramine. A different response profile was observed for prazosin and rauwolscine when the steady-state increase in UPP was evoked by simultaneously stimulating the midthoracic and hypogastric spinal outflow. Under these conditions of sympathoadrenal activation, UPP was reduced by both prazosin and rauwolscine (63 and 50%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiovascular responses to the stimulation of alpha-1 and alpha-2 adrenoceptors in the conscious dog

Hemodynamic responses to the selective stimulation of alpha-1 and alpha-2 adrenoceptors were examined in chronically instrumented, conscious dogs. Norepinephrine (0.02-0.1 micrograms/kg/min), a mixed alpha-1/alpha-2 adrenoceptor agonist, phenylephrine (0.2-1.0 micrograms/kg/min), a selective alpha-adrenoceptor agonist and B-HT 920 (0.5-2.0 micrograms/kg/min), a selective alpha-2 adrenoceptor agonist, were infused i.v. after ganglionic (hexamethonium, 30 mg/kg i.v.), beta adrenoceptor (propranolol, 1, mg/kg i.v.) and muscarinic receptor (atropine methylbromide, 0.1 mg/kg i.v.) antagonism. Each of the alpha adrenoceptor agonists increased mean arterial pressure and total peripheral resistance but had no significant effect on cardiac output, stroke volume or heart rate. Equipressor doses of the alpha adrenoceptor agonists caused similar increases in left ventricular systolic and end-diastolic pressure, but there were no significant changes in left ventricular dP/dt or heart rate with any of the alpha adrenoceptor agonists. Selective antagonism of alpha-1 adrenoceptors with prazosin (1 mg/kg i.v.) abolished the pressor and vasoconstrictor responses to phenylephrine but had a lesser effect on the response to B-HT 920. Antagonism of alpha-2 adrenoceptors with rauwolscine (0.1 mg/kg i.v.) caused a significantly greater attenuation of the pressor and vasoconstrictor responses to B-HT 920 than to phenylephrine. The responses to norepinephrine were significantly attenuated by antagonism of either alpha-1 or alpha-2 adrenoceptors. Thus, in the conscious dog with reflex pathways blocked, selective stimulation of either postsynaptic alpha-1 or alpha-2 adrenoceptors increases arterial pressure and total peripheral resistance but does not significantly change heart rate, left ventricular dP/dt, stroke volume or cardiac output.     

Vascular alpha-1 antagonistic and agonistic effects of beta adrenoceptor agonists in rabbit common carotid arteries.

The stainless-steel cannula-inserting method was used to observe vascular effects of mixed and selective beta adrenoceptor agonists, isoproterenol, procaterol and denopamine, on isolated, perfused rabbit common carotid arteries. In phenylephrine-preconstricted preparations, the three beta agonists induced a dose-dependent vasodilation which was not suppressed by treatment with beta antagonists, atenolol, a selective beta-1 antagonist and ICI 118551, a selective beta-2 antagonist. On the other hand, in prostaglandin F2 alpha-preconstricted preparations, these agonists produced no vasodilation and revealed weak vasoconstrictions which were readily suppressed by bunazosin, a selective alpha-1 antagonist. Moreover, these agonists caused a shift of the dose-response curve for phenylephrine to the right in a parallel fashion in non-preconstricted preparations. The relative pA2 values for isoproterenol, procaterol and denopamine calculated from the displacement curve were 7.47, 7.59 and 8.17, respectively. Thus, it is concluded that 1) there are little functional beta adrenoceptors in the rabbit common carotid arteries, 2) beta adrenoceptor agonists have both antagonistic and agonistic properties for alpha-1 adrenoceptor activation, 3) denopamine possesses a higher potency as an alpha-1 antagonist and 4) beta agonists generally act as vasodilators in rabbit cerebral circulation.     

Binding of [3H]prazosin to porcine aortic membranes: interaction of calcium antagonists with vascular alpha-1 adrenoceptors

The characteristics of [3H]prazosin binding and the interaction of Ca antagonists with alpha-1 adrenoceptors in the porcine aortic membranes were investigated. The binding characteristics of [3H]prazosin, namely, the kinetics and affinity of binding, saturability, competition by adrenergic agonists and antagonists, stereoselectivity and the localization of binding sites, indicated that [3H]prazosin binds specifically to the alpha-1 adrenoceptors in the sarcolemma of porcine aortic smooth muscle cells. In the inhibition study by several Ca antagonists, the specific binding of [3H]prazosin to aortic membranes was inhibited by verapamil (Ki = 0.66 microM), D600 (Ki = 0.86 microM), nicardipine (Ki = 2.3 microM) and d-cis diltiazem (Ki = 9.8 microM). Nifedipine and nitrendipine, potent dihydropyridine Ca antagonists, only partially inhibited the [3H]prazosin binding, up to 10(-4) M. l-Cis and dl-trans diltiazem, the less potent stereoisomers as Ca channel blockers compared with the d-cis form, showed a similar and greater potency as a competitor to alpha-1 adrenoceptors, respectively. These observations indicate that verapamil, D600, nicardipine and diltiazem interact with vascular alpha-1 adrenoceptors and that the potency of these compounds as a competitor to alpha-1 adrenoceptors does not parallel their potency as Ca channel blockers.     

Nifedipine and alpha adrenoceptors in rat aorta. I. Role of extracellular calcium in alpha-1 and alpha-2 adrenoceptor-mediated contraction

The ability of nifedipine to inhibit contractions induced by non-selective and selective alpha adrenoceptor agonists and by KCl depolarization was studied in the rat aorta. The presence of alpha-2 adrenoceptors which mediate vasoconstriction was demonstrated. Furthermore, this response was highly dependent on extracellular calcium. Alpha-1 adrenoceptors were also present, but this response was dependent primarily upon intracellular calcium stores. Contractions induced by maximally effective concentrations of agonists were separated into fast and slow components of the response. Nifedipine effectively inhibited the slow component of contractions induced by norepinephrine, phenylephrine and clonidine. The slow component of the response induced by these agonists is therefore due to influx of extracellular calcium. The fast component was resistant to nifedipine treatment and is therefore assumed to be due to release of intracellular calcium. Nifedipine was equally potent at inhibiting the slow component of both alpha-1 and alpha-2 adrenoceptor-stimulated contractions and contractions due to KCl depolarization. This suggests that the calcium influx pathways activated by these two types of stimuli 1) may be the same or 2) they have similar affinities for nifedipine or 3) there is a common site of action of nifedipine on both calcium entry pathways.     

Functional effects of activation of alpha-1 adrenoceptors by dexmedetomidine: in vivo and in vitro studies.

Dexmedetomidine, an alpha-2 adrenoceptor (AR) agonist, produces a biphasic hypnotic response in rats. Since central alpha-1 AR stimulation may reverse the hypnotic response produced by central alpha-2 AR stimulation, we have investigated, in both in vivo and in vitro models, the functional effects of dexmedetomidine on alpha-1 AR. For in vivo studies, stainless steel cannulas were inserted stereotaxically into the lateral ventricle of halothane-anesthetized rats to facilitate i.c.v. drug administration. Four to 7 days later, the alpha-1 AR antagonist prazosin (1 mg/kg-1) or saline was administered i.p. 15 min before i.c.v. injections of dexmedetomidine (10-333 micrograms) and the sleep-time (duration of loss of righting reflex) was assessed. The sleep-time increased, in a linear fashion, up to 33 micrograms; above this dose, there was a decrease in sleep-time. Pretreatment with prazosin prevented the decrease in sleep-time which was seen at higher doses. For in vitro studies, binding parameters of dexmedetomidine and its anesthetically inert L-isomer were determined from competition binding curves using [125I]2-[beta-(4-hydroxy-3-[125I]iodo- phenyl)ethylaminomethyl]-tetralone as the radiolabeled ligand and membranes prepared from HeLa cell lines stably expressing either alpha-1B or alpha-1C AR subtypes. Dexmedetomidine bound with equal affinity to both the alpha-1B (1178 +/- 63 nM) and the alpha-1C (1344 +/- 230 nM) isoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enantiomers of dobutamine increase the force of contraction via beta adrenoceptors, but antagonize competitively the positive inotropic effect mediated by alpha-1 adrenoceptors in the rabbit ventricular myocardium

Experiments were carried out to characterize the pharmacological properties of enantiomers and racemic mixture of dobutamine to modulate the myocardial contractility through alpha and beta adrenoceptors in the rabbit papillary muscle. Dobutamine caused the concentration-dependent positive inotropic effect: the rank order of potency was R-(+)- greater than (+/-) - greater than S-(-)-dobutamine. The positive inotropic effect of (+)-, (-)- and (+/-)-dobutamine was antagonized by a beta adrenoceptor antagonist, (+/-)-bupranolol in a competitive manner, but was not affected by an alpha-1 adrenoceptor antagonist, prazosin. The concentration-response curve for (-)-phenylephrine mediated by alpha adrenoceptors in the presence of 10(-6) M (+/-)-bupranolol was shifted by enantiomers of dobutamine to the right in a concentration-dependent manner. Thus, enantiomers of dobutamine antagonized the positive inotropic effect of (-)-phenylephrine in a competitive manner, and pA2 values [negative logarithm of the dissociation constant (KB)] for (+)- and (-)-dobutamine were 6.67 and 5.99, respectively. The specific binding of [3H]prazosin to membrane fractions of rabbit ventricular myocardium was displaced by dobutamine with a high potency: the -log Ki values for (+)- and (-)-dobutamine were 6.43 and 5.97, respectively, which correspond well with pA2 values of these compounds for functional modification. These findings indicate that enantiomers of dobutamine elicit the positive inotropic effect through activation of beta adrenoceptors, whereas both enantiomers behave as the competitive antagonist of myocardial alpha adrenoceptors mediating the positive inotropic effect in the isolated rabbit papillary muscle.     

Alkylation of alpha-1 receptors with a chemically reactive analog of prazosin reveals low affinity sites for norepinephrine in rabbit aorta

The effect of a newly synthesized irreversible blocker of the alpha-1 receptor [1-(4-amino-6,7-dimethoxy-2-quinazolnyl)-4-(2-bicyclo[2,2,2] octa-2,5-dienylcarbonyl)-piperazine; SZL-49] has been evaluated in contractile studies in rabbit aorta and binding studies in aorta and brain. SZL-49 produced long lasting inhibition of norepinephrine-induced contractions which was apparent 21 hr after drug washout. The inhibition, which was dose and time dependent, was characterized by progressive shift to the right in the norepinephrine dose-response curve. The ED50 for norepinephrine was shifted from 10(-7) M to 8 X 10(-7), 3 X 10(-6), 1 X 10(-5) and 5 X 10(-4) M after incubation (30 min) and washout of increasing concentrations of SZL-49. Surprisingly, SZL-49, irrespective of the dose or incubation time, did not decrease the maximal response of aortic rings to norepinephrine. This resulted in a norepinephrine dose-response curve after SZL-treatment that is parallel to the control. SZL-49 had no effect on the spasmogenic actions of histamine, serotonin, KCl or CaCl2. In contrast to the inhibitory pattern seen with SZL-49, incubation with 10(-7) M phenoxybenzamine shifted the norepinephrine dose-response curve to the right in a nonparallel manner and significantly depressed the maximal response obtainable with norepinephrine. Incubation with 10(-6) M phenoxybenzamine for 30 min virtually abolished the response to norepinephrine. Phenoxybenzamine (10(-7) M) was without effect on aortic rings treated with a maximally effective dose of SZL-49. Prazosin weakly antagonized the contractile actions of norepinephrine observed after SZL-49 treatment, whereas yohimbine was without effect on these norepinephrine-induced contractions. In control binding studies [3H]prazosin bound to two classes of sites in both aorta and brain preparations. Affinities and densities for these sites were K1 = 67.5 pM, K2 = 309 pM; R1 = 38.2 fmol/mg, R2 = 46.47 fmol/mg in aorta and K1 = 29.6 pM, K2 = 182 pM; R1 = 6.6 fmol/mg and R2 = 30.4 fmol/mg in brain. Treatment with increasing amounts of SZL-49 (10(-10) to 10(-8) M) progressively reduced the number of [3H]prazosin sites without altering the affinity of the sites remaining. At 10(-7) M, SZL-49 eliminated completely all specific [3H]prazosin binding. Our results indicate that the site mediating norepinephrine contraction after treatment with SZL-49 does not possess the characteristics of an alpha-1 receptor and supports the hypothesis that a low affinity site for norepinephrine and prazosin exists in vascular smooth muscle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evaluation of the role of alpha-1 and alpha-2 adrenoceptors in the maintenance of neurogenic tone to the hindlimb vasculature

The location of two distinct subtypes of alpha adrenoceptors on blood vessels is now well established. The present study was designed to assess the relative contribution of alpha-1 and alpha-2 adrenoceptors to the maintenance of sympathetic vasoconstrictor tone to the canine hindlimb. Bilateral hindlimb perfusion was carried out at controlled flow rates where one of the hindlimbs was neurally intact and the other limb was denervated surgically. Vascular resistances in the hindlimbs were evaluated by pressure-flow curves which were constructed by recording perfusion pressures at various flow levels. Administration of alpha-1 adrenoceptor antagonist, prazosin (50 micrograms/kg), which selectively inhibited the hindlimb vasoconstrictor effect of phenylephrine but not of B-HT 933, produced a significant decrease in the hindlimb vascular resistance in the innervated limb. This was reflected in a decrease in perfusion pressure and a downward shift of the pressure-flow curve of 30% from control. No changes in resistance occurred in the denervated limb. The alpha-2 adrenoceptor antagonist, yohimbine (100 micrograms/kg), which selectively antagonized vasoconstriction to B-HT 933 but not to phenylephrine, further decreased by 27% hindlimb vascular resistance in the innervated limb when given to the same group of prazosin-treated animals. The magnitude of the decrease in vascular resistance produced by yohimbine (27%) was similar to that caused by prazosin (30%). The vascular resistance in the innervated limb was now similar to that in the denervated limb after prazosin and yohimbine. Phentolamine, when administered after prazosin and yohimbine, did not produce any changes in the hindlimb vascular resistance in these dogs.(ABSTRACT TRUNCATED AT 250 WORDS)