Preclinical enantioselective pharmacology of (R)- and (S)- ketorolac.

Many of the nonsteroidal anti-inflammatory drugs (NSAIDs) are marketed as racemic mixtures, composed of (R)- and (S)- enantiomers. Racemic NSAIDs are potent cyclooxygenase (COX) inhibitors only through the action of the (S)- enantiomers, as the (R)- enantiomers do not exhibit COX inhibition. However, the (R)- enantiomer of ketoprofen exhibits potent analgesic activity and minimal ulcerogenic potential. To extend these observations, we examined the (R)- and (S)- enantiomers of RS- ketorolac, (S)- ketorolac exhibited potent COX1 and COX2 enzyme inhibition, whereas (R)- ketorolac was > 100-fold less active on both COX subtypes. Both enantiomers did not affect norepinephrine or serotonin uptake sites, and nitric oxidase or lipoxygenase activities, nor did they demonstrate any affinity for opioid receptors (mu, delta, or kappa). In experimental models, (S)- ketorolac exhibited about 10-fold greater activity than (R)- ketorolac in the murine phenylquinone writhing model. In this model, morphine sulfate was effective at much lower doses, however, and neither (R)- nor (S)- ketorolac showed any morphine-sparing effect. In the rat gait test for analgesia in the foot paw after injection of brewers yeast suspension, neither (R)- nor (S)- ketorolac affected paw volume. However, both provoked changes in gait scores, the (S)- enantiomer being 30-fold more potent than the (R)- enantiomer. A similar reduction was observed with respect to ulcerogenic potential, measured by direct microscopic changes after test conclusion. These findings suggest that (R)- ketorolac may possess analgesic activity that is independent of COX inhibition and may be associated with reduced ulcerogenic potential compared to effects exhibited by (S)- ketorolac.

Differences in beta 3-adrenergic receptor cardiovascular regulation in conscious primates, rats and dogs.

The goal of our study was to compare the effects of two beta 3-adrenergic receptor agonists, i.e., BRL37344 (BRL) and CL316243 (CL), in conscious dogs, rats and nonhuman primates, instrumented with aortic and atrial catheters, ascending aortic flow probes and left ventricular (LV) pressure gauges. In conscious dogs, CL, 10 micrograms/kg, i.v., reduced, P < .05, mean arterial pressure by 8 +/- 4% and total peripheral resistance by 51 +/- 4% and increased, P < .05, LV dP/dt by 79 +/- 10% and heart rate by 88 +/- 8%. These responses were similar to those induced by BRL, 8.3 micrograms/kg, i.v. After autonomic blockade (beta 1/beta 2-adrenergic, cholinergic and ganglionic), CL still reduced total peripheral resistance by 49 +/- 4%, and increased LV dP/dt by 21 +/- 4%, without an effect on heart rate. In conscious rats, 10 times the dose of CL 100 micrograms/kg, i.v., reduced, P < .05, total peripheral resistance by 18 +/- 4%, without major effects on LV dP/dt and heart rate. BRL, 83 micrograms/kg i.v., reduced total peripheral resistance similarly by 31 +/- 3%, and increased heart rate (25 +/- 3%) and LV dP/dt (27 +/- 5%) more, P < .05. In conscious monkeys and baboons, CL, 100 micrograms/kg, i.v., did not induce significant effects, whereas BRL, 83 micrograms/kg, i.v., increased heart rate (36 +/- 6%), LV dP/dt (85 +/- 9%) and decreased total peripheral resistance (30 +/- 2%). After beta 1/beta 2-adrenergic receptor blockade in rats and primates, there were almost no differences between BRL and CL, i.e., in primates there were almost no significant cardiovascular effects with either compound. Both CL and BRL increased circulating free fatty acids in the dog, but not in the baboon. Thus, CL appears to be a more specific beta 3-adrenergic receptor agonist than BRL. More importantly, beta 3-adrenergic receptor stimulation was most profound in dogs, where even a direct effect on LV contractility was identified, was diminished but still significant in rats, and essentially absent in primates.

Depolarization-induced 86Rb+ efflux in CHO cells expressing a recombinant potassium channel.

Cells expressing a recombinant human voltage-activated potassium channel (K-channel), Kv1.5, have been used in a functional assay that measures depolarization-stimulated 86Rb+ efflux as an indicator of K-channel function. Neither untransfected nor vector-transfected cells display measurable 86Rb+ efflux under depolarizing conditions. The depolarization-induced 86Rb+ efflux is blocked by standard K-channel blockers quinine, 4-aminopyridine and 3,4-diaminopyridine, but not by tetraethylammonium, quinidine, glibenclamide, or several peptide toxins. The pharmacological profile of the recombinant system reflects that reported for the channel in its native state. In such a system with no observable endogenous background, analysis of recombinant K-channel subtypes allows rapid assessment of pharmacological agents with isoform selectivity and specificity. Inclusion of compounds of unknown activity in an assay such as this could identify agents capable of modulating specific K-channel isoforms. Development of this high through-put assay system for the study of specific isoforms is a critical step in the identification and development of drugs that affect the desired target tissues with predictable pharmacology and minimal side effects due to nonselective K-channel interaction.

Identification and characterization of angiotensin II receptors in rat epididymal adipocyte membranes.

To better understand the role of the mitogenic vasoactive peptide angiotensin II (AII) in growth and differentiation, we have investigated the existence of membrane receptors for this peptide in rat adipocytes. Following isolation of epididymal fat cells, membrane protein was removed and incubated with varying concentrations of 125I-AII with or without submicromolar concentrations of unlabeled AII. Binding of AII was highly specific, rapid, and reversible. Scatchard analysis indicated that adipocyte membranes contain a high-affinity AII receptor with a Kd of 0.90 nmol/L and a binding site concentration (Bmax) of 53.7 fmol/mg protein. Additional pharmacologic analyses resulted in a rank order potency for peptide agonists and antagonists similar to that reported for the vascular receptor. Determination of subtype specificity with selective organic compounds indicated that the epididymal adipocyte receptor was displaced at low concentrations of DuP753, a selective AT1 subtype antagonist. These studies have successfully identified and characterized a high-affinity membrane receptor for AII in fat cells, further establishing adipose tissue as a peripheral site containing regulatory components of the local renin-angiotensin system.

Transforming growth factor alpha and atrial natriuretic peptide in white adipose tissue depots in rats.

To detect the presence in adipose tissue of peptides known to affect tissue growth and to investigate potential regional differences, epididymal and perirenal adipose tissue depots from male Sprague-Dawley rats were separated into adipocyte and stroma-vascular fractions by collagenase digestion, sequential centrifugation and filtration. Identity and integrity of the fractions were demonstrated by light and electron microscopy, while dose-response curves for angiotensin-converting enzyme (ACE) were performed, revealing maintained functional capacity of the stroma-vascular fraction. ACE, atrial natriuretic peptide (ANP), and transforming growth factor-alpha (TGF-alpha) concentrations were significantly greater in epididymal than perirenal stroma-vascular tissue. Adipocyte fractions from both depots contained significant concentrations of ANP and TGF-alpha. There was no detectable ACE in the adipocyte fractions, indicating that no contaminating stromal-vascular cells were present in these fractions. These data show significant concentrations of peptides with effects on growth in subfractions of adipose tissue and demonstrate regional differences in concentrations between fat depots.

Relationship of cardiac hemodynamic and biochemical adaptations to mortality during long-term aortic constriction.

To determine the biochemical and hemodynamic responses to aortic ligation, and to assess the survival rate after the induction of hypertension, 90 normotensive rats were subjected to surgical constriction of the abdominal aorta. Mortality, left ventricular hemodynamics, myocardial biochemical assays, and plasma renin assays were determined 1 week, 1 month, 3 months, or 1 year later. Mortality was greatest between 1 week and 3 months after aortic ligation, during which plasma renin activity was significantly elevated. The rate of left ventricular pressure rise, contractile index, and myocardial alpha-adrenoceptor number were increased at 1 month, but were comparatively depressed at 3 months after the operation, suggesting that the heart was in failure at this time. At 1 year after ligation, hemodynamic and biochemical parameters continued toward normalization. Our data suggest that, in this rodent model, cardiac pump failure occurs through a combination of time-dependent, pressure-induced mechanical adaptations and myocardial biochemical changes that involve both the renin-angiotensin and sympathetic nervous systems. The observed relationship between mortality, myocardial hemodynamics, and biochemical parameters may be used for additional basic research investigations concerning the early periods of cardiac failure.

Characterization of serotonin receptors in isolated rat intramyocardial coronary artery.

The purpose of the present study was to characterize the receptor subtypes that mediate serotonin (5-HT)-induced contraction in isolated rat intramyocardial coronary artery. In coronary artery with and without endothelium, only 5-HT and alpha-methylserotonin maleate (5-HT2 agonist) elicited equipotent concentration-dependent contractions. The EC50 values for 5-HT and alpha-methylserotonin maleate in endothelium-intact arteries were 4.7 x 10(-7) and 4.5 x 10(-7) M, respectively, whereas in endothelium-denuded arteries they were 2.8 x 10(-7) and 1.9 x 10(-7) M, respectively. The other subtype agonists, such as (+/-)-8-hydroxy-dipropylaminotetralin hydrobromide (5-HT1A agonist), 1-(3-chlorophenyl)piperazine dihydrochloride and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo(1,2-a)quinoxaline (5-HT1B) and 2-methyl-serotonin maleate (5-HT3), only elicited a small percentage of the maximum contraction to 5-HT. In prostaglandin F2 alpha-precontracted coronary arteries with intact endothelium or denuded of endothelium, the addition of 5-HT resulted in a further increase in tension. No relaxation was observed with 5-HT up to 1 x 10(-5) M. The contraction induced by 5-HT in artery both with and without endothelium was inhibited by ketanserin (5-HT2 antagonist) but not by l-propranolol (5-HT1 antagonist) nor by 3-tropanyl-indole-3,5-dichlorobenzoate (5-HT3 antagonist). Ketanserin, the selective 5-HT2 antagonist, effectively antagonized 5-HT-induced contraction by shifting the 5-HT response curve to the right without inhibiting the maximal response in both endothelium-intact and -denuded arteries.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of experimental obesity and subsequent weight reduction upon circulating atrial natriuretic peptide.

The effect of obesity and weight reduction upon circulating concentrations of atrial natriuretic peptide was assessed in an experimental model of the disease. Obese rats weighing in excess of 750 g were compared with formerly obese animals subjected to a 15-week period of caloric restriction resulting in a 40% reduction in body weight. Mean adipocyte size was significantly reduced with weight loss, as was estimated body fat. Mean arterial blood pressure remained normotensive for both groups, but a significant reduction in heart rate was associated with weight reduction. Circulating atrial natriuretic peptide was significantly elevated in the lean rats, which also exhibited decreased plasma renin activity and a negative sodium balance. Analysis of heart to body weight ratios implied that an obesity-associated, volume-induced cardiac hypertrophy remained even after the normalization of body fat. These results suggest that the diuresis and natriuresis accompanying weight reduction may be facilitated by atrial natriuretic peptide, which was elevated in part due to a persistent left ventricular hypertrophy following the transition from the obese to lean condition.

Cardiovascular effects of intermittent drinking: assessment of a novel animal model of human alcoholism.

The development of a novel model of human alcoholism has involved the presentation of a 30% alcohol solution to Sprague-Dawley rats via a syringe-feeding needle apparatus. With twice daily intermittent drinking, rats consumed an equivalent of 7-8 g/kg body weight of alcohol, which represented 25% of total daily caloric intake. Alcohol was absorbed rapidly, as significant circulating concentrations were observed within 15 min of gavage, eventually peaking at approximately 200 mg% 1 h later. Hemodynamic recordings in the conscious state after a 10-week drinking program indicated a normotensive blood pressure at peak blood alcohol levels, yet a hypertensive response 24 h after the final drink at a time when blood alcohol was not detected. Alcoholic rats continued to gain weight in parallel with controls fed ad libitum throughout the study, and changes in cardiac size and indices of contractility were not affected by 10 weeks of intermittent drinking. Additionally, no histological evidence of cardiac muscle damage was observed in alcoholic animals. Our animal model closely resembles the clinical situation in terms of the pattern of alcohol consumption, circulating concentrations of alcohol and the percentage of caloric intake in the form of alcohol. The hemodynamic changes observed support the hypothesis that alcoholic hypertension may be a manifestation of withdrawal, as opposed to any direct pressor effect of alcohol itself.

Endothelium-dependent basilar and aortic vascular responses in normotensive and coarctation hypertensive rats.

The responsiveness of acetylcholine (ACh), nitroglycerin (NG) and norepinephrine (NE) (aorta only) in both basilar arteries (BA) and thoracic aortic (TA) rings from coarctation hypertensive rats (CHR) were studied and compared to their sham-operated normotensive control rats (SNR). The effects of these agents were also evaluated in TA or BA with and without endothelium from naive normotensive rats (NNR). Blood pressure (BP) and plasma renin activity (PRA) of CHR were significantly higher than their time-matched SNR. Endothelium removal from TA of NNR significantly enhanced NE and NG sensitivity and reduced the maximum ACh relaxation. Removal of BA endothelium of NNR abolished ACh-induced relaxation but had no effect on NG-induced relaxation. In BA from CHR at any stage of hypertension studied, the sensitivity and maximum relaxation induced by ACh or NG were not significantly different than their respective time-matched SNR. ACh sensitivity of TA did not change in 1 Day CHR but decreased in 4 and 14 Day CHR. NG sensitivity increased, did not change and decreased in 1, 4 and 14 Day CHR, respectively. NE sensitivity increased in all stages of hypertension. These data suggest that in coarctation-induced hypertension there is a complex progression of events in TA which is modulated by different mechanisms as evidenced by the changes in the effects of NE, ACh and NG at various stages of hypertension. The results also suggest that the vascular endothelium of TA but not of BA may provide an acute protective mechanism to counteract the imbalance created by the increased sensitivity of smooth muscle cells to contractile agonists in the early stage of hypertension. However, persistent hypertension appears to override this mechanism.

Skin metabolism and transdermal absorption of viprostol, a synthetic PGE2 analog, in the rat: effect of vehicle.

The effects of three formulations on the transdermal absorption and antihypertensive activity of 14C-viprostol were investigated in the spontaneously hypertensive rat. Single doses of 14C-viprostol were administered topically to rats in three formulations: silicone oil, petrolatum base, and triethyl citrate (TEC). Mean arterial blood pressure (MABP) and blood concentrations of radioactivity were measured over 24 h. Metabolic profiles in the skin were determined by HPLC; in vitro skin metabolism was also investigated. Following topical dosing with 14C-viprostol in petrolatum and silicone oil, substantial systemic concentrations of radioactivity and decreases in MABP were observed. In contrast, administration of 14C-viprostol in TEC led to negligible blood concentrations of radioactivity and the lowering of MABP was diminished. Metabolic profiles in skin at the application site from rats dosed with viprostol in petrolatum and silicone oil indicated rapid hydrolysis of the viprostol methyl ester to the active free acid, CL 115,129. When TEC was used as the dosing vehicle, the conversion of viprostol to the free acid appeared to be slower. TEC (an ester itself) was also found to reduce the rate of hydrolysis of viprostol in rat skin 10,000-g supernatants.

Effect of gamma-aminobutyric acid (GABA) on vasodilation in resistance-sized arteries isolated from the monkey, rabbit, and rat.

The effect of gamma-aminobutyric acid (GABA) and papaverine on cerebral arteries of rat, rabbit, and monkey and the small mesenteric arteries of the rat were studied in vitro with a microvessel apparatus. GABA (1 x 10(-7) to 1 x 10(-3) M) did not affect the basal tension of arteries of rats at rest. In PGF2 alpha-contracted monkey basilar artery and middle cerebral artery and rat basilar artery, cumulative addition of GABA (1 x 10(-7) to 1 x 10(-3) M) did not produce any relaxation. Also in K+-contracted rat basilar artery and small mesenteric artery, cumulative additions of GABA, muscimol, or bicuculline did not result in relaxation. In K+-contracted rabbit basilar artery, GABA did not produce relaxation. However, the addition of papaverine (1 x 10(-7) to 1 x 10(-4) M) in either PGF2 alpha- or K+-contracted arteries, produced a concentration-dependent relaxation in all arteries tested. These results suggest that the failure of GABA or muscimol to induce relaxation is not due to a defect of the arterial smooth muscle relaxant mechanism, but rather is due to the inability of GABA or muscimol to directly relax the artery in this in vitro preparation. Therefore, the hypotensive effect of GABA seen in the rat is probably not due to direct vasodilation of mesenteric or cerebral arteries. These findings lend further support to the idea that GABA mediates its hypotensive effect through its action as an inhibitory neurotransmitter, as previously suggested by others.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of varying concentrations of linoleic acid on alpha-adrenoceptor responses in spontaneously hypertensive rats.

The effect of increased intake of linoleic acid on the alpha-adrenergic system was assessed by safflower oil supplementation to spontaneously hypertensive rats. Linoleic acid-enriched intake at 5%, 15% and 30% by weight of total food intake for 12 wk was associated with a reduction in resting arterial blood pressure, while heart rate and heart to body weight ratios were similar to control group values. A dose-response analysis to norepinephrine bitartrate administered intravenously indicated a significant reduction in the vascular reactivity to this alpha-adrenergic agonist in all groups given linoleic acid. Direct assessment of alpha-adrenoceptor number (Bmax) and affinity (KD) in cardiac sarcolemma with [3H]-prazosin indicated that receptor binding properties were not affected by linoleic acid intake. Our results suggest that short-term linoleic acid supplementation in the established hypertensive state may lower blood pressure through effects upon alpha-adrenergic reactivity in vascular tissue, without associated effects in cardiac tissue.

Relaxant actions of viprostol and prostaglandin E2 on tracheal muscle and their effects on calcium influx.

The relaxant effect of viprostol was studied in monkey and guinea-pig tracheal muscle rings in vitro and compared to that of prostaglandin E2 (PGE2), isoproterenol (ISO) and verapamil (guinea-pig trachea only). Viprostol, PGE2, ISO and verapamil produced a concentration-dependent relaxation of carbachol-contracted tracheal preparations. The rank order of potency in monkey trachea was viprostol = ISO greater than PGE2, while in guinea-pig treachea it was ISO greater than viprostol greater than PGE2 greater than verapamil. The relaxant effect of viprostol or PGE2 was not antagonized by propranolol, suggesting that beta-adrenoceptors are not involved. Epithelium removal did not affect the bronchorelaxant effects of viprostol, PGE2 or ISO. In K+-rich, Ca++-free Krebs solution, preincubation with an IC30 of verapamil antagonized CaCl2-induced contractions while an IC30 of viprostol, PGE2 or ISO did not. Preincubation with an IC90 of viprostol, PGE2 or ISO produced 0.5, 0.5 and 1.0 log unit shifts to the right of the CaCl2 concentration response curves, respectively. At this concentration, viprostol did not reduce the maximum effect of CaCl2, but PGE2 and ISO reduced it approximately 20%. However, preincubation with an IC90 of verapamil completely abolished the CaCl2 contraction. In conclusion, viprostol is a potent bronchodilator whose effect does not depend on the epithelium, beta-adrenoceptors or antagonism of Ca++ influx. Whether the bronchodilator effect of viprostol is via intracellular sequestration of calcium as that of PGE2 remains to be studied.

Development of an animal model for investigating disparate myocardial effects of obesity and hypertension.

An experimental model for investigating the disparate effects of obesity and hypertension on the heart was developed by ligation of the aorta of male Sprague-Dawley rats made obese through ad libitum feeding. Experimental obesity was associated with an increased body fat and cardiac muscle mass, yet a normotensive systemic arterial pressure. Aortic ligation produced an elevated mean arterial pressure and resting heart rate, whereas body weight was similar to that of normotensive lean control rats. Obesity and hypertension together were associated with a significantly increased percent body fat, mean arterial pressure, and left ventricular mass compared with lean controls, whereas pressure and left ventricular weight were greater than those observed in rats with only obesity or hypertension. Cardiac adaptations corrected for body weight indicated that left ventricular weight increased as a function of body weight and body fat, but hypertension produced left ventricular adaptations independent of these variables. These initial studies indicate an additional contribution of hypertension to the left ventricular adaptations of obesity, and this model could therefore be used in future investigations concerning the cardiovascular effects of the simultaneous occurrence of these separate diseases.

Studies on the effects of viprostol in isolated small blood vessels and thoracic aorta of the rat.

1. The effects of viprostol, prostaglandin E2 (PGE2) and nitroglycerin were studied in basilar artery, small mesenteric artery and the vein parallel to it as well as thoracic aorta of the rat. 2. In KCl-contracted basilar artery, viprostol produced a concentration-related biphasic response, contraction at concentrations less than 3 X 10(-6) M and relaxation at concentrations greater than 3 X 10(-6) M. PGE2 produced a concentration-related contraction while nitroglycerin produced a concentration-related relaxation. 3. In KCl-contracted small mesenteric artery, viprostol produced a biphasic response which was similar to that in the basilar artery. PGE2 produced a contraction and nitroglycerin produced relaxation in a concentration-dependent manner. 4. In KCl-contracted small mesenteric vein, in contrast to basilar and mesenteric artery, viprostol produced only a concentration-related relaxation in the range of 1 X 10(-6) to 1 X 10(-4) M. PGE2 produced a contraction and nitroglycerin produced a concentration-related relaxation. 5. In KCl-contracted thoracic aorta, PGE2 produced a biphasic response, relaxation at concentrations less than 3 X 10(-7) M and a concentration-related contraction at concentrations greater than 3 X 10(-7) M. Viprostol only produced a concentration-related contraction at concentrations greater than 1 X 10(-6) M, which was significantly less in magnitude than the contraction produced by PGE2. Nitroglycerin produced a concentration-related relaxation as seen in the small vessels. 6. In conclusion, the present data demonstrate that viprostol is a vasorelaxant agent which effectively dilates KCl-contracted basilar, small mesenteric artery and vein, but not the thoracic aorta of rat. The potent antihypertensive action of viprostol is probably due to its relaxant effect on the small arteries and veins but not on the large conduit artery.

Antilipolytic activity of viprostol, a transdermally active antihypertensive PGE2 analog.

Viprostol, a novel prostaglandin E2 congener, was assessed for in vitro antilipolytic activity in the spontaneously obese rat. In isolated epididymal adipocytes, viprostol exhibited a dose-dependent inhibition of catecholamine-stimulated lipolysis at concentrations ranging from 10 microM to 1 mM, but was ineffective at lower concentrations. Additionally, viprostol exhibited approximately 50% of the antilipolytic activity of naturally-occurring PGE1 and PGE2 at similar concentrations, but was as potent as PGF2 alpha. At 10 microM, viprostol inhibited maximum catecholamine-stimulated lipolysis by approximately 35% of the total, hormone-stimulated glycerol release. The results of these experiments indicate that viprostol exhibits antilipolytic activity in vitro, but is less potent than the naturally-occurring PGE's to which it is most closely related structurally.

Antiarrhythmic agents inhibit the in vitro formation of thromboxane (TX) in platelets from spontaneously hypertensive rats (SHR).

The occurrence of early life threatening arrhythmias in the ischemic myocardium has been associated with local generation of thromboxane (TX). Antiarrhythmic agents are typically classified according to the fundamental mechanism involved in restoring normal rhythm but identifying those agents also capable of suppressing TX formation offers a means of improving the rationale of antiarrhythmic therapy. Accordingly, representative antiarrhythmic agents from classes I-IV were evaluated in the present study for their ability to suppress TX formation from intact rat platelets, in vitro. Agents from class I, II and IV achieved significant reductions in TX levels as compared to a reference TX inhibitor, dazoxiben. The IC-50 value for dazoxiben was 1.5 X 10(-6) M. Nicardipine and flecainide, being the most active of the antiarrhythmic agents tested, had IC-50's of 5 X 10(-6) and 1 X 10(-5) M respectively. Verapamil and propranolol had values of 1 X 10(-5) and 5 X 10(-5) M respectively; labetalol and quinidine were 5 X 10(-5) M, and phenytoin and diltiazem were approximately 1 X 10(-4) M. These data suggest a subsidiary antiarrhythmic property of these particular agents as related to their ability to suppress TX generation in the ischemic myocardium and implies these agents may be preferred in the treatment of early life threatening arrhythmias resulting as an initial response to myocardial ischemia.