The preclinical pharmacological profile of the potent and selective leukotriene B4 antagonist CP-195543.

CP-195543 [(+)-2-(3-benzyl-4-hydroxy-chroman-7-yl)-4-trifluoromethyl-benzoic acid] is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro CP-195543 inhibited [3H]LTB4 binding to high-affinity LTB4 receptors on human neutrophils (HN) and murine spleen membranes with IC50 values of 6.8 nM (Ki = 4.9 nM) and 37.0 nM (Ki = 26.9 nM), respectively. CP-195543 inhibited human and mouse neutrophil chemotaxis mediated by LTB4 with IC50 values of 2.4 nM and 7.5 nM, respectively. Evidence of noncompetitive antagonist effects on the HN high-affinity LTB4 receptor was obtained by Scatchard analysis of [3H]LTB4 binding to and chemotaxis of HN to LTB4. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on HN indicated that CP-195543 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b up-regulation on HN was inhibited competitively by CP-195543 (pA2 = 7.66). In whole blood, CP-195543 also blocked CD11b up-regulation on HN (pA2 = 7.12) and murine neutrophils (pA2 = 7.06) with a similar potency. LTB4-mediated CD11b up-regulation on human monocytes and eosinophils in whole blood were inhibited by CP-195543 with IC50 values of 270 nM and 420 nM, respectively. CP-195543 at 10 microM failed to inhibit HN chemotaxis and CD11b up-regulation mediated through alternative (i.e., complement fragment 5a, interleukin-8, platelet-activating factor) G-protein-coupled chemotactic factor receptors. In vivo, after oral administration, CP-195543 blocked LTB4-mediated neutrophil infiltration in guinea pig and murine skin with ED50 values of 0.1 mg/kg and 2.8 mg/kg p.o., respectively. When administered in osmotic pumps, CP-195543 reduced the clinical symptoms and attendant weight loss in an IL-1-exacerbated murine model of collagen-induced arthritis with half-maximal effects associated with plasma drug levels of 0.4 to 0.5 microg/ml. Collectively these data provide evidence of the in vitro potency and in vivo efficacy of a novel LTB4 antagonist and support its clinical evaluation in a variety of inflammatory diseases in man.

trans-3-Benzyl-4-hydroxy-7-chromanylbenzoic acid derivatives as antagonists of the leukotriene B4 (LTB4) receptor.

The SAR of a series of 2-(7-chromanyl)benzoic acids has been investigated with the aim of identifying potent and selective LTB4 receptor antagonists that maintain potency in complex biological fluids. We found optimal activity in derivatives with electron-withdrawing groups in the benzoic acid ring and with an unsubstituted C-3 benzyl group on the chromanol nucleus. While compounds containing a 3-(4-phenyl)benzyl chromanol substituent were potent LTB4 receptor antagonists, the increased lipophilicity imparted by the additional phenyl substituent led to decreased potency in the presence of plasma proteins. From among the potent compounds identified, CP-195543, the 5'-trifluoromethyl 3-benzyl chromanol, was selected for development.

Differential in vivo and in vitro bronchorelaxant activities of CP-80,633, a selective phosphodiesterase 4 inhibitor.

CP-80,633, a selective phosphodiesterase (PDE) 4 inhibitor, potently reverses histamine bronchoconstriction in anesthetized guinea pigs (ED50 10 micrograms/kg) but only weakly relaxes histamine-constricted guinea pig trachea (EC50 137 microM). Using CP-80,633 as a prototype PDE4 inhibitor, we evaluated the hypothesis that bronchodilation induced by PDE4 inhibitors is not mediated by direct relaxation of airway smooth muscle. In anesthetized guinea pigs, a bronchodilatory dose of CP-80,633 did not increase plasma catecholamines, nor did propranolol pretreatment significantly alter the ability of CP-80,633 to reverse histamine bronchoconstriction. In an isolated organ system, the activity of bronchorelaxants may be attenuated by the lack of endogenous activators of adenylyl cyclase or by decreased levels of intracellular cyclic nucleotides. Pretreatment with the beta-adrenoceptor agonist, salbutamol, or the PDE3 inhibitor imazodan did not potentiate the bronchorelaxant ability of CP-80,633. Milrinone pretreatment increased the potency of CP-80,633 to relax carbachol-constricted tracheal rings, but only at concentrations where nonspecific effects have been reported. By comparing the bronchorelaxant abilities of PDE inhibitors in tracheal rings with or without epithelium, we determined that the epithelium did not serve as a barrier to drug penetration. In conclusion, CP-80,633 is a potent bronchodilator in vivo, whose activity is neither mediated by direct airway smooth muscle relaxation nor dependent upon endogenous catecholamines.

Adjunctive cabergoline therapy of Parkinson's disease: comparison with placebo and assessment of dose responses and duration of effect.

Adjunctive cabergoline or placebo, in doses up to 5 mg daily, were administered to Parkinson's disease patients with short-duration levodopa responses in a 6-month double-blind trial. The 13 patients randomized to cabergoline and completing the study had significantly improved Unified Parkinson's Disease Rating Scale (UPDRS) motor scores and timed hand-tapping test scores. Serial measurements on test days documented improved scores: (a) before the first levodopa (and cabergoline) dose of the day, (b) at the time of the peak levodopa effect, and (c) at the end of the levodopa response cycle, 5 h after test doses. Continued testing verified that these therapeutic responses were sustained for at least 48 h after the last cabergoline dose. Patients randomized to placebo failed to improve on any of these measures. In a subsequent open-label dose-escalation phase, further improvement was documented as the dosage was gradually raised to 10 mg daily. As in the double-blind phase, levodopa reduction allowed the improvement to occur in the absence of significantly increased dyskinesias. Other side effects were more substantial with higher doses, however, including two of 11 patients with hallucinations and confusion. In summary, adjunctive single-daily-dose cabergoline therapy resulted in long-lasting, dose-related improvement in parkinsonism not seen in patients receiving placebo.

Fluctuating Parkinson's disease. Treatment with the long-acting dopamine agonist cabergoline.

OBJECTIVE: Assessment of the very long-acting dopamine agonist medication cabergoline in the control of motor fluctuations in Parkinson's disease. DESIGN: Open-label trial (13 weeks). SETTING: Referral centers (Mayo Clinic, Rochester, Minn, and Scottsdale, Ariz). PATIENTS: Volunteer sample of 41 patients with idiopathic Parkinson's disease who were experiencing motor fluctuations while receiving stable doses of carbidopa and levodopa. INTERVENTION: Adjunctive oral cabergoline was incrementally administered once daily with the maintenance dose determined by the clinical response (maximum dose, 5 mg/d). MAIN OUTCOME MEASURES: Standardized serial motor examinations were performed, beginning anywhere from 30 minutes before and continuing to 6 hours after test doses of medications were administered. Scores during adjunctive cabergoline therapy were compared with the prestudy baseline scores during therapy with carbidopa and levodopa without cabergoline. RESULTS: Adjunctive cabergoline therapy significantly improved mean motor scores at the time of each standardized serial examination, from 30 minutes to 6 hours after the administration of test doses of medications. Significant motor score improvement was also measured 24 hours after the last cabergoline dose was administered, suggesting a very long-acting antiparkinsonian effect. Mean dyskinesia scores were slightly but nonsignificantly elevated. Diary card "off-time" was improved by 42%, whereas the levodopa dosage was reduced by 18%. Only three patients dropped out (7% of the total), which contrasts with much higher dropout rates owing to adverse events in previous clinical trials of other antiparkinsonian dopamine agonists. CONCLUSIONS: Cabergoline improved motor control in patients with Parkinson's disease who were experiencing clinical fluctuations. Possible advantages of this medication include an extended clinical response (persisting to 24 hours), tolerability, and ease of use (once per day administration).

A selective adenosine A1 receptor antagonist attenuates renal dysfunction during controlled hypotension with adenosine in rats.

Adenosine infusion produces sustained, reversible, controlled hypotension in humans, but markedly compromises renal function. Since adenosine inhibits renal perfusion and filtration via stimulation of intrarenal A1 receptors, we hypothesized that antagonism of A1-subtype receptors with the selective adenosine A1-receptor antagonist (+-)N6-endonorbornan-2-yl-9-methyladenine (N-0861) would attenuate adenosine-induced renal dysfunction while still allowing induction and maintenance of hypotension. Systemic and renal hemodynamic and excretory responses were measured in clearance studies conducted in six groups of anesthetized rats treated with: vehicle+saline, vehicle+adenosine, N-0861 (10 or 30 mumol/kg intravenously [i.v.])+saline, or N-0861 (10 or 30 mumol/kg i.v.),+adenosine. The A1-receptor antagonist had little effect on the magnitude, maintenance, or reversibility of controlled hypotension produced by adenosine infusion. Oliguria, hypofiltration, and electrolyte retention induced by adenosine infusion were attenuated in rats pretreated with N-0861. Adenosine A1-receptor antagonism also reduced the rebound hyperfiltration and hyperperfusion associated with cessation of adenosine infusion. N-0861 alone had only a modest effect on renal or systemic hemodynamics, but produced significant dose-related diuresis/natriuresis. These results suggest that coadministration of, or pretreatment with, a selective adenosine A1-receptor antagonist may attenuate the undesirable renal effects of adenosine while allowing maintenance of controlled hypotension.

Discrimination of A1 versus A2 receptor subtype selectivity of adenosine receptor agonists in vivo.

Previous attempts to discern and quantify the selectivity of agonists for A1 versus A2 adenosine receptors in vivo have been confounded by the activation of baroreceptor reflexes and/or simultaneous expression of responses to both A1 and A2 receptor activation. In anesthetized, vagotomized rats with isolated in situ constant-flow perfused hindquarters (HQ), bradycardic responses to i.v. agonist injections measured A1 receptor activation and HQ vasodilation elicited by i.a. agonist injections measured the stimulation of A2 receptors. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) produced A2 receptor-mediated HQ vasodilation at doses 8- and 4-fold lower (-log ED50 values, 7.3 +/- 0.04 mol and 8.7 +/- 0.06 mol, respectively) than those required to evoke A1 receptor-mediated bradycardia (-log ED50 values, 6.4 +/- 0.01 mol and 8.1 +/- 0.07 mol, respectively). N6-cyclopentyladenosine (CPA) was approximately 8-fold selective for A1 receptors (-log ED50 values, A1, 8.5 +/- 0.05 mol; A2, 7.6 +/- 0.16 mol). 2-(Phenylamino)adenosine (CV-1808) and 2[2(4-fluorophenyl)ethoxy]adenosine (FPEA) were at least 125- and 200-fold more potent agonists at A2 receptors (-log ED50 values, 7.7 +/- 0.10 mol and 8.0 +/- 0.24 mol, respectively) than at A1 receptors (-log ED50 values, 5.6 +/- 0.08 mol and 5.7 +/- 0.01 mol, respectively). These studies demonstrated that stimulation of A1 and A2 receptors may be discriminated in vivo and that such responses are selective, reproducible, dose-dependent and quantifiable.(ABSTRACT TRUNCATED AT 250 WORDS)

Multicenter study of cabergoline, a long-acting dopamine receptor agonist, in Parkinson's disease patients with fluctuating responses to levodopa/carbidopa.

We administered cabergoline, a potent, once-a-day dopamine against, to 61 patients with advanced Parkinson's disease (PD) and response fluctuations--"wearing-off" and "on-off" phenomena. The patients were on stable doses of levodopa/carbidopa. During the first 5 weeks, patients were randomized to allow equal numbers to end titration at each of five daily doses of cabergoline from 0.5 to 2.5 mg. We evaluated the patients using the Unified Parkinson's Disease Rating Scale (UPDRS) and diaries of motor performance. This part of the study was double-blinded. After 5 weeks, the mean Activities of Daily Living (ADL) score on the UPDRS decreased by 22% (p < 0.0001), the mean Motor Score in the "off" period decreased by 14% (p < 0.0001), and the mean Motor Score in the "on" period decreased by 22% (p < 0.0001). The mean percent "off" time decreased by 9.0%. Twenty-three patients (38%) achieved at least a 25% improvement in the combined ADL and motor examination of the UPDRS. Four patients dropped out because of adverse effects. We treated 37 patients, including some patients who had experienced a transient 25% improvement, for an additional 8 weeks in an open manner until they achieved a 25% improvement or reached a maximum of 5 mg/d. The other patients were continued in a double-blind manner on the dose of cabergoline they had achieved at the end of week 5. At the end of 13 weeks, the group of 37 patients achieved additional significant improvements in mean ADL and mean motor scores in the "on" and "off" periods. The percent time "off" decreased by 31%.(ABSTRACT TRUNCATED AT 250 WORDS)

N-0861 selectively antagonizes adenosine A1 receptors in vivo.

Experiments were performed to determine the antagonistic actions of (+/-)N6-endonorbornan-2-yl-9-methyladenine (N-0861) at A1 and A2 adenosine receptors in vivo, and to evaluate the pharmacodynamics of the observed responses. The selectivity of antagonism of A1 vs. A2 receptors by N-0861 was evaluated by generating dose-response curves to adenosine-induced bradycardia (A1 effect), and vasodilation in the in situ constant-flow perfused rat hindquarter vasculature (A2 effect). N-0861, at doses greater than or equal to 1 mumol/kg + 0.04 mumol/kg per min, i.v. produced dose-related rightward shifts of the A1 dose-response curve, but had no effect on the A2 dose-response curve at doses as high as 100 mumol/kg, i.v. In contrast, the non-selective A1/A2 adenosine receptor antagonist 8-phenyltheophylline antagonized both A1 and A2 receptor-mediated responses to adenosine. The minimum effective i.v. dose and the duration of action of N-0861 were determined by evoking bradycardic responses to i.v. adenosine (A1 effect) in anesthetized, vagotomized, beta-blocked rats before and after single bolus doses of vehicle or N-0861 (0.3, 0.6, 1.0, 3.0 or 10.0 mumol/kg). The lowest i.v. dose of N-0861 to antagonize A1 receptor-mediated bradycardia was 0.3 mumol/kg i.v.; the duration of effect ranged from 1 min (following 0.3-1 mumol/kg) to approximately 2.5 h (following 10 mumol/kg). N-0861 is a selective (by greater than or equal to 333-fold) antagonist of adenosine A1 receptors.

Cardiovascular and renal actions of AHR-16303B, an antagonist of 5-HT2 receptors and calcium channels, in hypertensive and normotensive rats.

The cardiovascular and renal responses to AHR-16303B, a novel antagonist of 5-hydroxytryptamine (5-HT2) receptors and calcium channels, were examined in spontaneously hypertensive (SHR) and normotensive rats (NTR) and compared with verapamil and ritanserin. In SHR, AHR-16303B (10-300 mg/kg orally, p.o.) produced dose-related reductions in mean arterial blood pressure (MABP), accompanied by modest isokaliuretic diuresis and unchanged heart rate (HR). In NTR, 10-30 mg/kg p.o. AHR-16303B had no effect on MABP or renal excretory function; 100 and 300 mg/kg reduced MABP but had only transient effects on HR; 100 mg/kg produced antidiuresis in NTR. Both strains of rats tolerated doses of AHR-16303B as high as 300 mg/kg. In both SHR and NTR, verapamil (10-100 mg/kg p.o.) produced dose-related reductions in MABP, antinatriuresis at 60 and 100 mg/kg, and variable effects on HR. Oral ritanserin had no effect on MABP of SHR or NTR at 3 or 10 mg/kg. AHR-16303B is unique in that it simultaneously antagonizes 5-HT2 receptors and produces safe and effective reduction of elevated BP without altering HR or triggering renal compensatory antidiuresis. At effective 5-HT2/calcium antagonistic doses, AHR-16303B has no effect on cardiorenal homeostasis in normotensive animals.

Protective effect of p-chlorophenylalanine in glycerol-induced acute renal failure in rats.

1. Studies were conducted to test the hypothesis that glycerol-induced acute renal failure in rats may be mediated by 5-hydroxytryptamine. 2. The tryptophan hydroxylase inhibitor, p-chlorophenylalanine, or saline was administered for 4 days before performance of 24 h clearance studies after glycerol administration. 3. p-Chlorophenylalanine significantly reduced the nephrotoxic effect of glycerol, as evidenced by improved glomerular filtration rate, urine volume excretion and absolute sodium excretion, and reduced plasma creatinine concentration. 4. These results suggest that glycerol-induced acute renal failure may be mediated by the renovascular and/or tubular actions of 5-hydroxytryptamine.

Effect of bopindolol on renin secretion and renal excretory function in rats.

We investigated the effects of the new beta-adrenoceptor antagonist, bopindolol, on stimulated renin secretion and on renal function in conscious rats. Intravenous doses of 10, 31.6, and 100 micrograms/kg of bopindolol antagonized the rise in plasma renin activity (PRA) induced by the administration of isoproterenol (10 micrograms/kg, s.c.). Peak PRA, evident at 15 min after isoproterenol, reached 27 +/- 3 ng/ml/h with bopindolol (100 micrograms/kg) pretreatment vs. 105 +/- 7 ng/ml/h in saline controls. Antagonism by bopindolol (100 micrograms/kg, i.v.) of isoproterenol-stimulated rise in PRA persisted through 12 h. Within this dosage range, bopindolol itself had no significant effect on PRA. Significant antagonism of beta-adrenoceptor-induced hypotension resulted with 31.6 and 100 micrograms/kg of i.v. bopindolol. Orally administered bopindolol produced significant antagonism of isoproterenol-induced increase in PRA at a dose of 316 micrograms/kg; however, oral doses of 31.6-316 micrograms/kg of bopindolol failed to significantly alter furosemide-stimulated increase in PRA. Bopindolol alone had little effect on renal excretory function but caused a slight attenuation of furosemide-induced diuresis and electrolyte excretion. Plasma electrolytes and osmolarity were unaffected by bopindolol alone or in conjunction with furosemide. These results indicate that bopindolol (a) is a potent, long-acting, orally active, antagonist of beta-adrenoceptor-stimulated renin secretion in the rat, (b) is ineffective in antagonizing furosemide-induced rise in PRA, (c) does not elevate basal levels of PRA by virtue of its intrinsic sympathomimetic activity, and (d) does not cause dramatic alteration of renal excretory function at effective beta-blocking doses.

Cardiovascular and renal actions of calcium channel blocker chemical subgroups: a search for renal specificity.

The diuretic and natriuretic responses to structurally distinct classes of Ca2+ channel blockers have been compared, to determine whether any agent provoked K+-sparing natriuresis, and to assess the relation of such responses with drug effects on blood pressure. Conscious normotensive Sprague-Dawley rats received vehicle or one of the following drugs in an oral saline load (40 mL kg-1) nifedipine, nimodipine, nitrendipine, prenylamine, cinnarizine, flunarizine, diltiazem, verapamil, hydrochlorothiazide, amiloride, or hydralazine, at doses from 0.316 to 100 mg kg-1. Urine was collected for 6h. Blood pressure was monitored directly in parallel studies. Diltiazem (31.6, 100 mg kg-1) and flunarizine (100 mg.kg-1) enhanced urine and electrolyte excretion in spite of marked hypotension; diltiazem was the only drug to produce dose-related renal responses. In contrast, equihypotensive doses of hydralazine and nifedipine produced overt urine and electrolyte retention. Nitrendipine and prenylamine (0.316 mg kg-1 each) produced slight diuresis or natriuresis without altering blood pressure; higher doses had no effect. The 31.6 mg kg-1 doses of verapamil, nitrendipine, and nimodipine markedly reduced blood pressure, but neither enhanced nor limited urine and electrolyte excretion. Cinnarizine failed to produce any cardiovascular or renal effects. Diuretic responses evoked by the Ca2+ channel blockers were not class-specific, showed no tendency towards sparing K+, were generally weaker than those produced by low doses of amiloride or hydrochlorothiazide, and were dissociable from drug-induced changes in blood pressure.

Involvement of dopamine receptor subtypes in dopaminergic modulation of aldosterone secretion in rats.

The postulation that dopamine (DA) may tonically inhibit aldosterone (ALDO) secretion has arisen from the finding that metoclopramide, a non-selective DA receptor antagonist with prominent non-dopaminergic actions, stimulates ALDO secretion. Experiments were performed to determine: (a.) the ability of several non-specific and subtype-specific DA receptor antagonists to stimulate ALDO secretion, (b.) the subtype DA receptor involved in regulating ALDO secretion, and (c.) if ALDO responses were associated with changes in plasma Na+(pNa), K+(pK), or osmolality (pOsm). Blood samples were withdrawn from carotid arterial catheters in conscious, fasted male Sprague-Dawley rats before and following intra-arterial administration of lactated Ringer's placebo, furosemide (10 mg/kg), or one of several DA receptor antagonists. Furosemide stimulated ALDO, decreased pK, and left pNa and pOsm unchanged. The non-selective DA receptor antagonists metoclopramide (0.2, 0.6 mg/kg), rs-sulpiride (0.2 mg/kg), and haloperidol (0.1 mg/kg), and the DA-2 receptor antagonists domperidone (0.1 mg/kg) and s-sulpiride (0.1 mg/kg) each stimulated ALDO, and left pNa, pK, and pOsm unchanged. Conversely, the DA-1 receptor antagonists SCH 23390 (0.03, 0.1 mg/kg) and r-sulpiride (0.1 mg/kg) failed to stimulate ALDO, and left pNa, pK, and pOsm unaltered. These studies suggest that ALDO secretion in rats is modulated by a mechanism involving DA-2, but not DA-1 subtype receptors, and that the ALDO responses to DA receptor antagonism are independent of changes in pNa, pK, and pOsm.

The effect of tranylcypromine on levels of 6-keto-PGF1 alpha and other prostaglandins in brain and mesentery of the mouse.

Tranylcypromine (TCP), which can inhibit prostacyclin (PGI2) synthesis in vitro, has been shown to facilitate platelet aggregation in damaged cerebral arterioles of the mouse when given intraperitoneally (50 mg/kg) one hour before inducing aggregation. The same dose has no effect on platelet aggregation in damaged mesenteric arterioles. The present experiments used HPLC and GC/MS to analyze PG levels and show that 5 or 50 mg/kg TCP, given intraperitoneally one hour before sacrificing the mouse, moderately reduces the level of 6-keto-PGF1 alpha, the stable metabolite of PGI2, in incubated brain homogenates. This finding supports the hypothesis that TCP's enhancement of platelet aggregation in the brain was affected by a reduction in PGI2 levels. When 500 micrograms/ml TCP was added to the incubate of brain homogenate from mice given 50 mg/kg, PGE2 levels were reduced as well as the levels of 6-keto-PGF1 alpha. In incubated mesentery, the level of 6-keto-PGF1 alpha was also reduced by treating mice with 50 mg/kg TCP. The latter result failed to support the hypothesis that levels of mesenteric 6-keto-PGF1 alpha would be unaltered by TCP in parallel with the inability of TCP to alter platelet aggregation in mesenteric arterioles. Thus our data fails to support an overall hypothesis relating TCP action on platelet aggregation to its inhibitory effect on PGI2 synthesis. At the same time the data do not rule out such a relationship for mouse brain.

Cyclooxygenase products of arachidonic acid metabolism in cat cerebral cortex after experimental concussive brain injury.

Previous studies have suggested that following experimental fluid percussion brain injury, increased prostaglandin (PG) synthesis, with its concomitant production of oxygen free radicals, causes functional and morphological abnormalities of the cerebral arterioles. The purpose of this study was to chemically determine if PGs are altered following this injury. To facilitate interpretation of neurochemical measurements the cats were ventilated, blood pressure was measured, and a cranial window, for microscopic observation of pial arteriolar diameter was inserted. PG levels were determined in quick-frozen cortical tissue removed from control and 3 groups of injured cats at 1.5, 8,0, and 60 min after injury. Analysis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was performed by HPLC and GC/MS. The control levels of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were 216 +/- 44, 210 +/- 48, and 48 +/- 12 ng/g wet weight, respectively. Following injury, produced by a 22 ms increase in intracranial pressure, the pial arterioles dilated irreversibly and a transient hypertensive response occurred, thereby producing hyperemia. During the maximum hyperemic response, the total PGs were 75% of control. At 8 min after injury, when blood pressure returned to control level, the PGs were 158% of control and PGs fell to 111% of control at 60 min. These experiments supported our previous studies implicating increased PG synthesis in te genesis of the physiologic and morphologic sequelae of experimental concussive brain injury.

Prostaglandin levels in isolated brain microvessels and in normal and norepinephrine-stimulated cat brain homogenates.

The levels of PGD2, PGE2, PGF2 alpha and 6-keto-PGF1 alpha (6KF1 alpha) produced from endogenous arachidonic acid (AA) were quantitated in cat cerebral cortical homogenates and microvessels isolated from cat cerebral cortex using gas chromatography/mass spectrometry (GC/MS). There was a six-fold enrichment of 6KF1 alpha levels in isolated microvessels, compared to homogenates, suggesting that 6KF1 alpha is of vascular, rather than neuronal origin. In order to further understand any possible role that norepinephrine (NE) might have on modulation of PG synthesis, we studied the effects of 0.5 mM NE on PG synthesis from endogenous AA and from 3H-PGG2, the endoperoxide precursor of PGs. In cat cortical homogenates NE induced a 74% increase in PGD2 and PGF2 alpha, a 62% increase in PGE2, and a 36% increase in 6KF1 alpha, as measured by GC/MS. NE caused a twofold increase in the conversion of 3H-PGG2 to 3H-PGF2 alpha, with a concomitant decrease in 3H-PGE2 and 3H-6KF1 alpha formation, and no change in 3H-PGD2 synthesis. NE had no effect on the total conversion of 3H-PGG2 to 3H-PGs, nor on the breakdown of 3H-PGG2 in the absence of brain tissue. We conclude that NE stimulates extravascular synthesis of PGD2, PGE2 and PGF2 alpha by stimulation of the prostaglandin synthetase complex, in addition to NE's stimulatory effect on the conversion of PGG2 to PGF2 alpha, and that the lack of effect on NE on 6KF1 alpha synthesis reflects either a failure to achieve an adequate concentration at the vascular tissue, or an absence of the mechanism whereby NE stimulates PG synthetase.

Effect of oral aspirin dose on platelet aggregation and vascular prostacyclin (PGI2) synthesis in humans and rabbits.

Large doses of oral aspirin inhibit platelet aggregation and vascular synthesis of the antiaggregatory vasodilator prostaglandin I2 (PGI2) by irreversibly acetylating the cyclooxygenase enzyme. In order to determine if one can achieve selective inactivation of platelet cyclooxygenase using oral doses of aspirin, we studied human and rabbit platelet aggregation and rabbit aortic synthesis of PGI2 before and 3 hr after various doses of aspirin. In rabbits, lower doses of aspirin produced a major inhibition of platelet aggregation and a minor inhibition of PGI2 synthesis, while higher doses of aspirin inhibited both platelet aggregation and vascular PGI2 synthesis. In humans, we found that a dose equivalent to approximately 1/4 of one 300 mg aspirin tablet consistently produced a major inhibition of cyclooxygenase-dependent platelet aggregation in a pattern similar to the inhibition of rabbit platelet aggregation where the majority of rabbit PGI2 synthetic capacity was not inhibited. In another rabbit study, we found that it takes the vasculature over 24 hr to return to control PGI2 synthetic capacity following a single, high dose of oral aspirin. In conclusion, we speculate that approximately 1/4 of an aspirin tablet, which inhibits a major portion of cyclooxygenase-dependent human platelet aggregation, may not inhibit a major portion of vascular cyclooxygenase-dependent PGI2 synthesis and may be more efficacious as an antithrombotic agent in man than are higher doses of aspirin.