Robert R. Ruffolo

RECEPTOR INTERACTIONS OF IMIDAZOLINES: α‐ADRENOCEPTORS OF RAT AND RABBIT AORTAE DIFFERENTIATED BY RELATIVE POTENCIES, AFFINITIES AND EFFICACIES OF IMIDAZOLINE AGONISTS

1 Noradrenaline and a series of imidazolines were used to characterize and differentiate the postsynaptic α‐adrenoceptors of rat and rabbit aortae. 2 Dose‐response curves in each tissue revealed marked differences in the profile of agonist activity among the compounds. Based on the ED50 values for each compound, a rank order of potency of oxymetazoline > noradrenaline > tramazoline > tetrahydrozoline > clonidine was obtained in rabbit aorta and an order of noradrenaline > clonidine > tramazoline > oxymetazoline was obtained in rat aorta. Tetrahydrozoline had no agonist activity in rat aorta. 3 Dissociation constants were determined for each agonist in rat and rabbit aortae. Again, differences between the tissues were observed to the extent that the rank order of affinities for the imidazolines were exactly opposite for the two tissues. In rabbit aorta the order was, oxymetazoline > tramazoline > tetrahydrozoline > clonidine, whereas in rat aorta it was, clonidine > tetrahydrozoline > tramazoline > oxymetazoline. The extremes in tissue selectivity were observed with clonidine, which had approximately 125 fold higher affinity in rat aorta, and oxymetazoline, which had approximately 4 times higher affinity in rabbit aorta. 4 The absolute values of relative efficacies of the imidazolines studied, and their rank order, also differed between the two tissues. The relative efficacies of oxymetazoline and tramazoline were more than 15 fold greater in rabbit aorta than in rat aorta. Furthermore, tetrahydrozoline had a greater relative efficacy than clonidine in rabbit aorta while the converse was true in rat aorta. 5 Differences in the rank order of potency, affinity and relative efficacy of noradrenaline and a series of imidazolines in rat and rabbit aortae indicate that the postsynaptic α‐adrenoceptors in these tissues are different. While the postsynaptic α‐adrenoceptor of rabbit aorta is clearly of the α1‐subtype, the exact nature of the postsynaptic α‐receptor of rat aorta is not clear. The unique α‐receptor of rat aorta has properties of both α1‐ and α2‐adrenoceptors.

Myocardial protection with carvedilol.

Carvedilol is a multiple-action cardiovascular agent that is both a beta-adrenoceptor antagonist and a vasodilator and has recently been made available for the treatment of mild-to-moderate hypertension. Clinical trials are ongoing to establish the efficacy of carvedilol in angina and congestive heart failure. beta-Adrenoceptor antagonists are known to reduce myocardial work secondary to reductions in heart rate and contractility; accordingly, they have been shown to be cardioprotective in animals and in humans. Because carvedilol has beta-adrenoceptor antagonist activity, it also should provide significant cardioprotection. The additional vasodilating activity of carvedilol, which will further reduce myocardial work by decreasing afterload and myocardial wall tension, should provide more salvage of ischemic myocardium than that afforded by a pure beta-adrenoceptor antagonist, such as propranolol. We investigated the ability of carvedilol and propranolol to reduce infarct size in experimental models of acute myocardial infarction in the rat, pig, and dog. The left anterior descending coronary artery was occluded for 30 (rat) or 45 min (pig) and then reperfused for 24 h (rat) or 4 h (pig). In the dog, the left circumflex coronary artery was occluded for 60 min and reperfused for 24 h. Vehicle, carvedilol, or propranolol was administered intravenously 15 min before ischemia (and, in the rat only, repeated 4 h after ischemia). An additional group of dogs was subjected to permanent left anterior descending coronary artery occlusion for 6 h, and carvedilol or propranolol was given 15 min after occlusion. Infarct size was examined on stained tissue sections using quantitative image analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Differences in the applicability of the Easson-Stedman hypothesis to the α1- and α2-adrenergic effects of phenethylamines and imidazolines

The enantiomers of 2-(3,4, alpha-trihydroxybenzyl)imidazoline and the corresponding desoxy derivative, 2-(3,4-dihydroxybenzyl)imidazoline, were evaluated at alpha 1- and alpha 2-adrenergic receptors to test the applicability of the Easson-Stedman hypothesis to the imidazoline class of alpha-adrenergic agonists. A series of closely related phenethylamines was included for comparison. The Easson-Stedman hypothesis states that optically active adrenergic agonists possessing an asymmetric hydroxyl-substituted benzylic carbon atom will have the following relative potencies: R(-) greater than S(+) = desoxy. While the phenethylamines were found to adhere to the Easson-Stedman hypothesis at both alpha 1- and alpha 2-adrenergic receptors, the optically active imidazolines did not. These findings further support our previous observations that the phenethylamines and imidazolines may interact differently with alpha-adrenergic receptors.

Antihypertensive activity of the non-peptide angiotensin II receptor antagonist, SK&F 108566, in rats and dogs

SummaryThe antihypertensive activity of the nonpeptide angiotensin II receptor antagonist, SK&F 108566 (E) - α - [[2 - butyl -1 - [(4 - carboxyphenyl)methyl] -1H -imidazol-5-yl]methylene]-2-thiophene propanoic acid, was examined in rats and dogs. SK&F 108566 produced dose-dependent decreases in blood pressure in renin-dependent hypertensive rats. At 10 mg/kg intraduodenally, mean arterial blood pressure fell from between 150–160 mm Hg to approximately 124 mm Hg. A sustained infusion of SK&F 108566 at 25 μg/min intraduodenally normalized blood pressure during 3 days of infusion and for 18 h following cessation of the infusion. Evaluation of the systemic hemodynamic effects of SK&F 108566 in chronically instrumented renin-dependent hypertensive rats demonstrated that the antihypertensive effects of SK&F 108566 were accompanied by a significant increase in cardiac output with little change in stroke volume. In dogs made acutely hypertensive by an intravenous infusion of angiotensin 1, SK&F 108566 resulted in dose-dependent decreases in blood pressure. The antihypertensive activity of SK&F 108566 at 10 mg/kg p.o. was maintained for between 13–15 h, a similar duration of action as observed with enalapril (1 mg/kg, p.o.). Administration of DuP 753 (losartan) intravenously caused a small and short-lived fall in blood pressure in the angiotensin I-infused hypertensive dog. However, the active metabolite of losartan, EXP 3174, resulted in a response of longer duration. In dogs made hypertensive by placement of an ameroid constrictor on the left renal artery, SK&F 108566 (10 mg/kg, p.o.) or enalapril (1 mg/ kg, p.o.) resulted in antihypertensive responses of at least 12 h duration. The data indicate that SK&F 108566 is a long-acting antihypertensive agent in the rat and dog.

Properties of 8,9-dichloro-2,3,4,5-tetrahydro-1H- 2-benzazepine, an inhibitor of norepinephrine N-methyltransferase

Abstract LY134046, 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine hydrochloride, was a potent inhibitor of norepinephrine N -methyltransferase (NMT) from rat brain or rabbit adrenal glands in vitro . The inhibition was competitive with respect to the methyl-accepting substrate, (-)-norepinephrine, the K i for LY134046 being 2.4 × 10 −8 M. LY134046 inhibited the NMT activity in rat brain stem and hypothalamus in vivo at doses of 10–40 mg/kg, i.p., and lowered the epinephrine (but not norepinephrine or dopamine) concentration in these brain regions. The epinephrine reduction produced by a single 40 mg/kg, i.p. dose of LY134046 persisted at 24 hr and daily injections of 10–40 mg/kg doses for 5 days produced cumulative reductions in epinephrine concentration. LY134046 was similar to SK&F 64139 (7,8-dichloro-1,2,3,4-tetrahydroisoquinoline hydrochloride), a structurally related compound, as an inhibitor of NMT in vitro and in vivo , but the two compounds differed in their relative abilities to block α 2 receptors. SK&F 64139 was 20-to 50-fold more potent than LY134046 in antagonizing [ 3 H]clonidine binding to rat brain membranes and phenylephrine-induced contractions of rat aortic strips, but it was only about twice as potent as LY134046 in inhibiting NMT activity. LY134046 seems to be more selective than other currently known inhibitors of NMT and may be useful for pharmacologic intervention in the function of epinephrine-forming neurons in brain.

Comparison between carvedilol and captopril in rats with partial ablation-induced chronic renal failure

1 The effect of the novel β‐adrenoceptor antagonist and vasodilator, carvedilol (SK&F 105517, ∼ 70 mg kg−1 daily in the food), and captopril (∼ 38 mg kg−1 daily in the drinking fluid) on the progression of chronic renal failure in rats was studied. 2 Six weeks following partial renal ablation, the urinary protein excretion of the carvediol‐ (60 ± 21 mg day−1) and captopril‐treated (35 ± 9mgday−1) animals was less than 50% that of control rats (133 ± 27 mg d−1). 3 Serum creatinine (Scr) and urea nitrogen (SUN) concentrations of the carvedilol‐(Scr, 0.63 ± 0.09 mg dl−1; SUN, 11.3 ± 1.2 mg dl−1) and captopril‐treated (Scr, 0.82 ± 0.05 mg dl−1; SUN, 14.1 ± 1.5 mg dl−1) animals were also significantly (P < 0.05) lower than that observed in control animals (Scr, 1.4 ± 0.3 mg dl−1; SUN, 19.2 ± 3.9 mg dl−1), indicating that glomerular filtration rate was improved by both drugs. Plasma renin activity was significantly (P < 0.05) higher in captopril‐treated rats (24.7 ± 4.6 ng angiotensin I ml−1 h−1) than in either carvedilol‐treated (7.9 ± 1.4 ng angiotensin I ml−1 h−1) or control animals (7.4 ± 1.0 ng angiotensin I ml−1 h−1). 4 Histological examination of the kidneys demonstrated a significantly reduced glomerular hypertrophy and glomerulosclerosis in those animals receiving carvedilol or captopril compared to controls. 5 Serum carvedilol concentration measured every 6 h for 24 h was variable and ranged on average from 57 ± 13 ng ml−1 at 16 h 00 min to 121 ± 31 ng ml−1 at 03 h 00 min. These data indicate that the rats probably had 24 h systemic exposure to carvedilol. 6 The present study indicates that carvedilol is effective in attenuating the progression of chronic renal failure in rats.

Systemic hemodynamic effects of dopamine, (±)-dobutamine and the (+)- and (−)-enantiomers of dobutamine in anesthetized normotensive rats

The hemodynamic effects of dopamine, (+/-)-dobutamine (racemic mixture) and the (+)- and (-)-enantiomers of dobutamine were evaluated in anesthetized normotensive rats. Dopamine and (+/-)-dobutamine produced hemodynamic effects in anesthetized rat that were qualitatively similar to those reported for these compounds in man. The increase in cardiac output produced by dopamine and (+/-)-dobutamine was due mainly to an increase in stroke volume, with heart rate being only minimally affected. Dopamine produced a large increase in mean arterial pressure and slightly increased total peripheral vascular resistance, whereas (+/-)-dobutamine only modestly increased blood pressure and significantly reduced total peripheral resistance. The (-)-enantiomer of dobutamine, which possesses mainly alpha 1-adrenoceptor agonist activity, produced marked increases in cardiac output, stroke volume, total peripheral resistance and mean arterial pressure, but did not significantly increase heart rate. In contrast, (+)-dobutamine, which possesses predominantly beta 1-and beta 2-adrenoceptor agonist activity, elicited only a modest increase in cardiac output which was due entirely to increased heart rate since stroke volume was not increased. Total peripheral vascular resistance and mean arterial blood pressure were both reduced by (+)-dobutamine, characteristic of a beta-adrenoceptor agonist. The increase in cardiac output and blood pressure produced by (+/-)-dobutamine, but not the positive chronotropic effect, were significantly inhibited by alpha 1-adrenoceptor blockade with prazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

Existence of spare alpha 1-adrenoreceptors, but not alpha 2-adrenoreceptors, for respective vasopressor effects of cirazoline and B-HT 933 in the pithed rat.

Summary: The existence of a receptor reserve (spare receptors) was investigated for postsynaptic vascular α1-and α2-adrenoceptors in the pithed rat by evaluating the effects of progressive inactivation of α1- and α2-adrenoceptor pools by the irreversible antagonist phenoxybenzamine on the pressor responses of cirazoline and B-HT 933. Dose-response curves for the α1-adrenoceptor-mediated vasoconstrictor effects of cirazoline were shifted in a rightward direction with no depression of the maximum response by lower doses of phenoxybenzamine (0.1-0.2 mg/kg, i.v.). Progressively higher doses of phenoxybenzamine (>1 mg/kg, i.v.) produced further rightward shifts in the dose-response curves of cirazoline, but also depressed the maximum response. In contrast, all doses of phenoxybenazmine that inhibited the α2-adrenoceptor-mediated pressor effects of B-HT 933 produced a reduction in the maximum response. These results are highly suggestive of the existence of a receptor reserve in the pithed rat for the postsynaptic vascular α1-adrenoceptor-mediated effects of cirazoline, but not for the postsynaptic vascular α2-adrenoceptor-mediated effects of B-HT 933. Confirmation of the existence of a receptor reserve for only the postsynaptic vascular α1-adrenoceptor-mediated effects of cirazoline came from further analysis of the antagonism, by phenoxybenzamine, of cirazoline and B-HT 933 dose-response curves. The maximum pressor response that could be elicited by the α1-adrenoceptor agonist cirazoline was a hyperbolic function of the size of the α1-adrenoceptor pool, the latter being progressively decreased by phenoxybenzamine treatment. Such a hyperbolic relationship is indicative of a receptor reserve. In marked contrast, the maximum pressor response that could be evoked by the α2-adrenoceptor agonist B-HT 933 was a linear function of the size of the intact α2-adrenoceptor pool, characteristic of a lack of spare receptors. Further analysis showed that the occupancy-response relationship is fivefold more favorable for the α1-adrenoceptor-mediated pressor effects of cirazoline than for the α2-adrenoceptor-mediated pressor effects of B-HT 933, indicating that any given maximum pressor response in the pithed rat may be obtained with one-fifth as many α1-adrenoceptors being activated by cirazoline than α2-adrenoceptors being stimulated by B-HT 933. The large receptor reserve for the postsynaptic vascular α1-adrenoceptor-mediated pressor effects of cirazoline, but not for the postsynaptic vascular α2-adrenoceptor-mediated pressor effects of B-HT 933, must be considered when assessing possible differential effects of noncompetitive antagonists on α1- or α2-adrenoceptor-mediated pressor effects in the pithed rat.

Interactions of three inotropic agents, ASL-7022, dobutamine and dopamine, with α- and β-adrenoceptors in vitro

SummaryThree inotropic agents, ASL-7022, dobutamine and dopamine, were evaluated for their α-and β-adrenoceptor mediated effects in vitro in a variety of isolated organs and in radioligand binding studies. All compounds were α1-adrenoreceptor agonists in rat and guinea pig aortae, but the rank orders of potency were exactly opposite in these two tissues. Only the rank potency order of dobutamine>ASL-7022>dopamine obtained in rat aorta was consistent with the results obtained in radioligand binding studies to α1-adrenoreceptors in rat cerebral cortex and to previous results obtained in vivo in the pithed rat. The results obtained in guinea pig aorta did not parallel the radioligand binding studies in rat brain or our previous results in pithed rat, and suggests that species differences exist between postsynaptic vascular α1-adrenoreceptors in rat and guinea pig aorta, consistent with previous conclusions. ASL-7022 was found to be a potent α2-adrenoreceptor agonist in field-stimulated guinea pig ileum, and was approximately 10-fold more potent than dobutamine in this respects, which was also confirmed by radioligand binding studies to α2-adrenoreceptors in rat cerebral cortex. The β1-adrenoreceptor mediated effects of these compounds were evaluated in guinea pig atria, where the rank order of potency was dobutamine>ASL-7022>dopamine. An identical rank order of affinity was established for these compounds by displacement of 3H-dihydroalprenolol from β1-adrenoreceptors in rat cerebral cortex. The β1-adrenoreceptor mediated effects of dobutamine and ASL-7022 in guinea pig atria were completely direct in nature and not secondary to the release of endogenous catecholamines. In contrast, a major component of the β1-adrenoreceptor mediated tachycardia produced by dopamine in guinea pig atria was indirect in nature as evidenced by the marked attenuation in potency that occurred following catecholamine depletion with reserpine. All three compounds elicited β2-adrenoreceptor mediated inhibition of tone in rat uterus, with the rank order of potency being ASL-7022>dobutamine>dopamine. Again, this rank order of β2-adrenoreceptor potency was also reflected in β2-adrenoreceptor affinity as assessed by displacement of 3H-dihydroalprenolol from β2-adrenoreceptors in rat cerebellum. Based on these results, it may be concluded that for α-adrenoceptors, dobutamine is a selective β2-adrenoreceptor agonist, ASL-7022 is a selective α2-adrenoreceptor agonist, and dopamine is a nonselective α-adrenoceptor agonist. For β-adrenoceptor mediated effect, ASL-7022 is a selective α2 agonist, while dobutamine and dopamine are nonselective β-adrenoceptor agonists. It is likely that the complex inotropic and hemodynamic activities of ASL-7022, dobutamine and dopamine result from the sum of their individual effects at the α-and β-adrenoceptor subtypes.

Computerized graphic methods for determining dissociation constants of agonists, partial agonists, and competitive antagonists in isolated smooth muscle preparations

Computer programs have been written that calculate dissociation constants for agonists, partial agonists, and competitive antagonists from data generated in isolated smooth muscle preparations. The data are analyzed according to standard procedures and are presented in graphical form with the aid of an inexpensive digital plotter. The digital plotter coupled with a computer markedly decreases the time required to perform these fundamental analyses and also reduces the chance for error and experimenter bias.