O2-sensitivity of beta adrenergic responsiveness in isolated bovine and porcine coronary arteries

The relation between pO2 and beta adrenergic responsiveness was studied in isolated bovine and porcine coronary artery rings. Isoproterenol elicited a concentration-dependent relaxation of bovine and porcine coronary artery rings precontracted with KCI (2 X 10(-2) M) or histamine (10(-6) M); beta adrenergic responsiveness was significantly lower in K+-depolarized coronary arteries. A decrease of bath pO2 from 95 to 40% significantly reduced beta adrenergic responsiveness in both coronary preparations precontracted with either KCI or histamine. Similarly, exogenous arachidonic acid (3 X 10(-6) to 3 X 10(-5) M) depressed isoproterenol-induced relaxations in both tissues. Indomethacin (5 X 10(-6) M) augmented beta adrenergic responsiveness in the presence of 95% O2 and prevented the inhibitory effects of the decrease in bath pO2 and arachidonic acid in both preparations. The experimental data suggest that the demonstrated O2-sensitivity of beta adrenergic responsiveness is mediated by vascular prostaglandin synthesis in isolated large coronary arteries.     

Effects of inhibitors of arachidonic acid metabolism and calcium entry on responses to acetylcholine, potassium and norepinephrine in the isolated canine saphenous vein

The present study was designed to determine whether interference with the metabolism of arachidonic acid or the entry of extracellular calcium affects the responses of the canine saphenous vein to acetylcholine, potassium or norepinephrine. Rings of canine saphenous vein, with or without endothelium, were suspended in organ chambers filled with physiological salt solution and set at their optimal length for isometric tension recording. Removal of the endothelium, confirmed by the absence of the characteristic relaxation induced by thrombin in intact rings, did not affect concentration-response curves to acetylcholine or norepinephrine. The cyclooxygenase inhibitor, indomethacin, augmented the response to acetylcholine. This effect was comparable in rings with and without endothelium and in rings pretreated with phentolamine. Indomethacin did not alter the response to norepinephrine but augmented that to potassium. Similar results were obtained with the cyclooxygenase inhibitors, acetylsalicylic acid and meclofenamate. The antioxidant and lipoxygenase inhibitor nordihydroguaiaretic acid and the cyclooxygenase/lipoxygenase inhibitor phenidone blocked the ability of indomethacin to augment acetylcholine- and potassium-induced contractions. Arachidonic acid-induced contractions were not blocked by indomethacin but were inhibited by nordihydroguaiaretic acid, phenidone and the calcium entry blockers diltiazem and nimodipine. Diltiazem and nimodipine inhibited responses to potassium and acetylcholine without affecting those to norepinephrine. The augmentation by indomethacin of potassium- and acetylcholine-evoked contractions was inhibited by diltiazem and nimodipine. In rings of femoral artery denuded of endothelium, indomethacin had no effect on the responses to acetylcholine, norepinephrine or potassium. These results suggest that, in the canine saphenous vein but not in the femoral artery, activation of the lipoxygenase pathway for the metabolism of arachidonic acid augments preferentially contractions which depend upon the entry of extracellular calcium.     

Inhibitory effect of quinacrine on myocardial reactive hyperemia in the dog

We investigated the effect of quinacrine, a phospholipase A2 inhibitor, on myocardial reactive hyperemic response after 5-, 10- and 20-sec coronary occlusions and adenosine-induced coronary vasodilatation in perfused dog hearts in situ. Continuous infusions of quinacrine (30-300 micrograms/min) into the left coronary artery significantly reduced both reactive hyperemic response and increase in coronary blood flow produced by intracoronary injections of adenosine (1-30 micrograms) without significant effect on basal coronary blood flow, perfusion pressure and heart rate. Neither acetylcholine- nor verapamil-induced increases in coronary blood flow were modified by quinacrine. The reactive hyperemic response and the vasodilatory effect of adenosine were both reduced by intracoronary infusions of theophylline (100-1000 micrograms/min). The inhibitory effect of theophylline on the reactive hyperemic response was less prominent than that of quinacrine, whereas the vasodilatory effect of adenosine was reduced to the same extent by both drugs. Intracoronary infusions of indomethacin (a cyclooxygenase inhibitor), phenidone (an inhibitor of both cyclooxygenase and lipoxygenase) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) did not affect the reactive hyperemic response. The present results support the hypothesis that adenosine may be a mediator of myocardial reactive hyperemia and suggest that other factors, possibly phospholipase products, may also contribute to this phenomenon. We were unable to define the mechanism involved; however, it can be excluded that cyclooxygenase and lipoxygenase products of arachidonic acid contribute to myocardial reactive hyperemia.     

Endothelium-dependent effects of carteolol

Experiments were designed to study the effect of the beta adrenergic antagonist, carteolol, on the endothelium-dependent responsiveness of isolated arteries. Rings of canine coronary arteries were suspended in organ chambers for isometric tension recording; carteolol inhibited the relaxation to isoproterenol and abolished the difference in responsiveness to the beta adrenergic agonist between rings with and without endothelium. Carteolol did not cause endothelium-dependent relaxations of femoral or coronary arteries. In bioassay experiments, carteolol augmented the basal release of relaxing factors from the endothelium of the femoral artery; this effect was prevented by indomethacin. In rings of femoral arteries, carteolol increased the endothelium-dependent relaxations induced by the alpha-2 adrenergic agonist UK 14,304; this was not affected by indomethacin but prevented by propranolol. Carteolol did not modify the endothelium-dependent relaxations to acetylcholine, adenosine diphosphate, bradykinin, thrombin and the Ca+-ionophore A23187. Carteolol inhibited the endothelium-dependent hypoxic contraction of the canine coronary artery. It did not affect endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. These experiments suggest that carteolol facilitates the abluminal release of endothelium-dependent relaxing factor caused by alpha-2 adrenergic activation, and causes the intraluminal release of vasodilator prostaglandins. The compound prevents the endothelium-dependent contractions which are not mediated by products of cyclooxygenase. These actions may contribute to the vasodilatator properties of carteolol in the intact organism.     

Effects of adenosine triphosphate and prostaglandins on vascular adrenergic transmission.

The effects of prostaglandins (PGs) on the adrenergic neuroeffector transmission and their relationship to the action of ATP were investigated. This was in view of the putative negative feedback mediated by ATP or a related purine compound, and the reported stimulation of PG synthesis by adenine nucleotides. The central ear artery and saphenous vein of the rabbit were isolated and their contractile responses to adrenergic nerve stimulation monitored. These responses were markedly reduced by PGE1 and PGE2 and significantly augmented by indomethacin and aspirin, suggesting the occurrence of the PGE-mediated negative feedback. PGF2a facilitated the response of the vein but was without affect on the artery, while arachidonic acid was facilitatory on the former and inhibitory on the latter. Possibly a PGF2a-like substance is formed in the presence of arachidonic acid and, in the vein, masks the effect of any PGE. ATP depressed the arterial and venous contractile response to adrenergic nerve stimulation. This inhibition was not significantly affected by indomethacin or aspirin. It was enhanced in the artery and diminished in the vein by arachidonic acid, but only to the extent of algebraic sum of the effects of ATP and the acid. It seems possible that the purine- and PGE-mediated feedback mechanisms are independent and parallel pathways.     

Calcium-independent phospholipase A(2)-derived arachidonic acid is essential for endothelium-dependent relaxation by acetylcholine

The role of calcium-independent phospholipase A(2) (iPLA(2))-produced arachidonic acid (AA) in acetylcholine (ACh)-mediated, endothelium-dependent vascular relaxation was investigated. ACh-induced relaxation of phenylephrine-constricted isolated rat mesenteric resistance arteries was attenuated following pretreatment with (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL; 1 microM; p < 0.01), a highly selective suicide substrate inhibitor of iPLA(2). Following BEL, the ACh relaxation could be completely restored following pretreatment with picomolar quantities of the cell-permeant methyl ester analog of AA (arachidonic acid methyl ester, AA-Me). Higher amounts of AA-Me (1 microM) had a direct endothelium-dependent relaxing action, which was inhibited by the nitric-oxide synthase inhibitor (N(omega)-nitro-L-arginine; 100 microM), independent of ACh, and unaffected by BEL. Neither the ACh relaxation restoring action nor the direct relaxing action of AA-Me was affected by preincubation with inhibitors of the lipoxygenase (esculetin, 10 microM) or cytochrome P450 monooxygenase (17-octadecynoic acid; 10 microM) pathways; and both actions of AA-Me were enhanced following preincubation with the cyclooxygenase inhibitor indomethacin (10 microM; p < 0.05). The results of the present study indicate that iPLA(2)-produced AA plays an essential role in ACh-mediated endothelium-dependent relaxation in rat mesenteric resistance arteries.     

Role of reactive oxygen in bile salt stimulation of colonic epithelial proliferation.

Our previous studies had suggested a link between bile salt stimulation of colonic epithelial proliferation and the release and oxygenation of arachidonate via the lipoxygenase pathway. In the present study, we examined the role of reactive oxygen versus end products of arachidonate metabolism via the cyclooxygenase and lipoxygenase pathways in bile salt stimulation of rat colonic epithelial proliferation. Intracolonic instillation of 5 mM deoxycholate increased mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA. Responses to deoxycholate were abolished by the superoxide dismutase mimetic CuII (3,5 diisopropylsalicylic acid)2 (CuDIPS), and by phenidone or esculetin, which inhibit both lipoxygenase and cyclooxygenase activities. By contrast, indomethacin potentiated the response. Phenidone and esculetin suppressed deoxycholate-induced increases in prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 5, 12, and 15-hydroxyeicosatetraenoic acid (HETE), whereas CuDIPS had no effect. Indomethacin suppressed only PGE2. Deoxycholate (0.5-5 mM) increased superoxide dismutase sensitive chemiluminescence 2-10-fold and stimulated superoxide production as measured by cytochrome c reduction in colonic mucosal scrapings or crypt epithelium. Bile salt-induced increases in chemiluminescence were abolished by CuDIPS, phenidone, and esculetin, but not by indomethacin. Intracolonic generation of reactive oxygen by xanthine-xanthine oxidase increased colonic mucosal ornithine decarboxylase activity and [3H]thymidine incorporation into DNA approximately twofold. These effects were abolished by superoxide dismutase. The findings support a key role for reactive oxygen, rather than more distal products of either the lipoxygenase or cyclooxygenase pathways, in the stimulation of colonic mucosal proliferation by bile salts.     

Ouabain inhibits endothelium-dependent relaxations to arachidonic acid in canine coronary arteries

Experiments were designed to analyze the effects of ouabain on the actions of exogenous arachidonic acid on endothelial and vascular smooth muscle cells. Rings or strips were prepared from left circumflex canine coronary arteries and suspended for isometric tension recording in organ chambers filled with oxygenated modified Krebs-Ringer-bicarbonate solution. During contractions evoked by prostaglandin F2 alpha, arachidonic acid caused relaxations both in the presence and the absence of endothelium. However, removal of the endothelium reduced its inhibitory action. Indomethacin prevented the relaxations in rings without endothelium, but did not affect the response to high doses (10(-6) to 10(-5) M) of arachidonic acid in preparations with endothelium. The inhibitor of lipoxygenase, nordihydroguaiaretic acid, had no effect on the inhibitory responses to arachidonic acid in rings with or without endothelium. Ouabain abolished both the endothelium-dependent and the direct relaxations to arachidonic acid. Endothelium-dependent relaxations in response to oleic acid, elaidic acid, adenosine diphosphate and thrombin were not affected by ouabain. In the presence of indomethacin, coronary artery strips without endothelium were relaxed by arachidonic acid only when layered (intimal surface against intimal surface) with a longitudinal strip with endothelium. In layered preparations, treatment of the intact longitudinal strip with ouabain before layering prevented the relaxation, whereas pretreatment of the strip without endothelium had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation by arachidonic acid of the effect of vasoconstrictor stimuli in the canine kidney

The effect of arachidonic acid on the renal vasoconstrictor response elicited by sympathetic nerve stimulation, norepinephrine and angiotensin II in pentobarbital-anesthetized dogs, with and without pretreatment with the cyclooxygenase inhibitors, sodium meclofenamate or indomethacin was investigated. Stimulation of renal nerves at 2 to 8 Hz or injections into the renal artery of either norepinephrine or angiotensin II (0.06-0.5 micrograms) produced vasoconstriction and decreased blood flow to the kidney in frequency- and dose-related manners, respectively. In animals that were untreated with the cyclooxygenase inhibitors, renal arterial infusion of arachidonic acid at 15 micrograms kg-1 min-1 increased blood flow to the kidney and attenuated the vasoconstrictor effect of renal nerve stimulation, injected norepinephrine and angiotensin II. However, in dogs pretreated with either sodium meclofenamate or indomethacin (5 mg/kg), infusion of arachidonic acid failed to alter the renal blood flow and the vasoconstrictor response elicited by both adrenergic stimuli and by angiotensin II. In contrast, pretreatment of animals with the cyclooxygenase inhibitor, sodium meclofenamate, did not prevent either prostaglandin E2 or prostacyclin (4 ng kg-1 min-1) from increasing renal blood flow and inhibiting the renal vasoconstrictor response produced by either adrenergic stimuli or by angiotensin II. These data suggest that arachidonic acid produces renal vasodilation and attenuates the vasoconstrictor effect of the adrenergic stimuli and angiotensin II through its transformation by cyclooxygenase into one or more produced, presumably prostaglandin E2 and/or prostacyclin.     

Role of platelets and the arachidonic acid pathway in the regulation of neutrophil oxidase activity.

The intercellular mechanisms involved in platelet-mediated regulation of neutrophil function remain incompletely understood. This study investigated the role of the arachidonic acid pathway in the modulation of chemoattractant-induced production of oxygen metabolites, measured as luminol-amplified chemiluminescence (CL). We demonstrate that platelets dose-dependently inhibit the CL response in neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Incubation with eicosatetrayonic acid (ETYA), a combined cyclooxygenase and lipooxygenase inhibitor, dramatically decreased the fMLP-induced CL response in neutrophils, an effect that was further enhanced in the presence of platelets. The separate effects of eicosatriyonic acid (ETI) and indomethacin, specific inhibitors of lipoxygenase and cyclooxygenase, respectively, were significantly lower compared to the action of ETYA. On the contrary, impediment of arachidonic acid release with the phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone (ATK) markedly increased the production of oxygen radicals triggered by fMLP. The addition of exogenous arachidonic acid clearly decreased the fMLP-induced CL response in neutrophils, which further strengthens a downregulating effect of arachidonic acid on oxidase activity. This inhibitory action of arachidonic acid, however, was reversed upon co-incubation with platelets. In conclusion, this study suggests that an accumulation of arachidonic acid, following chemotactic peptide stimulation, turns off neutrophil oxidase activity. Furthermore, platelets may support the synthesis of reactive arachidonic acid metabolites, which modulate oxygen radical production in neutrophils.     

Role of platelets and the arachidonic acid pathway in the regulation of neutrophil oxidase activity

The intercellular mechanisms involved in platelet-mediated regulation of neutrophil function remain incompletely understood. This study investigated the role of the arachidonic acid pathway in the modulation of chemoattractant-induced production of oxygen metabolites, measured as luminol-amplified chemiluminescence (CL). We demonstrate that platelets dose-dependently inhibit the CL response in neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). Incubation with eicosatetrayonic acid (ETYA), a combined cyclooxygenase and lipooxygenase inhibitor, dramatically decreased the fMLP-induced CL response in neutrophils, an effect that was further enhanced in the presence of platelets. The separate effects of eicosatriyonic acid (ETI ) and indomethacin, specific inhibitors of lipoxygenase and cyclooxygenase, respectively, were significantly lower compared to the action of ETYA. On the contrary, impediment of arachidonic acid release with the phospholipase A 2 inhibitor arachidonyl trifluoromethyl ketone (ATK ) markedly increased the production of oxygen radicals triggered by fMLP. The addition of exogenous arachidonic acid clearly decreased the fMLP-induced CL response in neutrophils, which further strengthens a downregulating effect of arachidonic acid on oxidase activity. This inhibitory action of arachidonic acid, however, was reversed upon co-incubation with platelets. In conclusion, this study suggests that an accumulation of arachidonic acid, following chemotactic peptide stimulation, turns off neutrophil oxidase activity. Furthermore, platelets may support the synthesis of reactive arachidonic acid metabolites, which modulate oxygen radical production in neutrophils.     

Flavonoid potentiation of contractile responses in rat blood vessels.

Certain bioflavonoids and phenolic compounds have long been known to enhance catecholamine responses, in vivo and in vitro. In the present studies the flavone, baicalein, potentiated nerve-stimulated contractions in vitro in rat tail and femoral artery isometric ring preparations. Inhibition of catecholamine reuptake with cocaine or catecholamine metabolism with tropolone and parglyine (monoamine oxidase and catecholamine-O-methyl transferase inhibitors, respectively) did not alter baicalein's ability to potentiate contractile responses to nerve stimulation. Baicalein (10(-5) M), the prototype flavone, also increased sensitivity to exogenous norepinephrine, serotonin, arginine vasopressin and to the noncatecholamine alpha-1 and alpha-2 adrenergic agonists, cirazoline and tramazoline. Structure-function studies indicated that flavone potentiation required three contiguous A or B ring hydroxylations. Several nonflavone phenol derivatives with three contiguous hydroxyls also potentiated nerve stimulation responses. As baicalein is a potent lipoxygenase inhibitor, comparisons were made between potentiating ability and lipoxygenase inhibitory activity in a series of flavonoids. There was no direct correlation between inhibition of 12-hydroxy-5,8,10,14-eicosatetraenoic acid levels in thrombin stimulated human platelets and potentiation of contractile responses in the femoral artery. Additionally, the specific substrate analog lipoxygenase inhibitor, 5,8,11-eicosatriynoic acid, and the cyclooxygenase inhibitor, ibuprofen, were nonpotentiating. Ibuprofen pretreatment did not alter the potentiating action of baicalein. It is concluded that flavonoids with three contiguous hydroxyls on either the A or B ring increase in vitro vascular responsiveness via a post-synaptic process, independent of cyclooxygenase, lipoxygenase, monoamine oxidase or catecholamine-O-methyl transferase activity.     

Role of cyclooxygenase products of arachidonic acid metabolism in Ascaris antigen-induced bronchoconstriction in sensitized dogs

The pharmacologic modulation of Ascaris allergen-induced bronchoconstriction was investigated in beagle dogs sensitized by experimental infection with Ascaris suum ova. Ascaris-induced increases in total lung resistance (RL) and falls in dynamic lung compliance (Cdyn) were unaffected by pretreatment with an antihistamine (pyrilamine) given alone but were significantly attenuated (P less than .05) by the cyclooxygenase enzyme inhibitor, indomethacin. The combination of pyrilamine and indomethacin also produced a significant (P less than .01) inhibition of Ascaris-induced bronchoconstriction, greater than that produced by indomethacin given alone but the difference between the two treatment groups was not statistically significant. The combination of an antihistamine, the cyclooxygenase inhibitor indomethacin and the leukotriene synthesis inhibitor, U-60,257, almost completely abolished Ascaris-induced bronchoconstriction (91% inhibition of Cdyn changes; 93% inhibition of RL changes). The inhibition was significantly greater than that produced by cyclooxygenase inhibition alone. The leukotriene synthesis inhibitor piriprost (U-60,257) given alone or in combination with pyrilamine produced no inhibition of Ascaris-induced changes in RL or Cdyn. The leukotriene antagonist FPL55712 or the thromboxane synthase inhibitor U-63,557A also showed little or no activity in this model, whereas the thromboxane receptor antagonist AH23848 produced a marked inhibition of Ascaris-induced bronchoconstriction. We conclude that Ascaris-induced bronchoconstriction is mediated primarily by cyclooxygenase products of arachidonic acid metabolism. The role of histamine and lipoxygenase products can only be revealed during an effective cyclooxygenase blockade.     

Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate

The objective of this study was to examine the relationship between responses of bovine intrapulmonary artery and vein to arachidonic acid and cyclic nucleotide levels in order to better understand the mechanism of relaxation elicited by arachidonic acid and acetylcholine. Arachidonic acid relaxed phenylephrine-precontracted arterial rings and elevated both cyclic GMP and cyclic AMP levels in arteries with intact endothelium. In contrast, endothelium-damaged arterial rings contracted to arachidonic acid without demonstrating significant changes in cyclic nucleotide levels. Indomethacin partially inhibited endothelium-dependent relaxation and abolished cyclic AMP accumulation whereas methylene blue, a guanylate cyclase inhibitor, partially inhibited relaxation and abolished cyclic GMP accumulation in response to arachidonic acid. All vessel responses were blocked by a combination of the two inhibitors. Prostaglandin (PG) I2 relaxed arterial rings and elevated cyclic AMP levels whereas PGE2 and PGF2 alpha caused contraction, suggesting that the indomethacin-sensitive component of arachidonic acid-elicited relaxation is due to PGI2 formation and cyclic AMP accumulation. The methylene blue-sensitive component is attributed to an endothelium-dependent but cyclooxygenase-independent generation of a substance causing cyclic GMP accumulation. Intrapulmonary veins contracted to arachidonic acid with no changes in cyclic nucleotide levels and PGI2 was without effect. Homogenates of intrapulmonary artery and vein formed 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from [14C]arachidonic acid, which was inhibited by indomethacin. Thus, bovine intrapulmonary vein may not possess receptors for PGI2. The failure of endothelium-intact vein to relax to acetylcholine may be related to the lack of a relaxant effect by arachidonic acid, perhaps attributed to the absence of generation of an endothelium-derived relaxing factor.     

Nonadrenergic effects of isoproterenol in dogs with hypoxic pulmonary vasoconstriction. Possible role of prostaglandins

To determine whether the pulmonary vasodilation produced by isoproterenol is mediated solely by its beta adrenergic effects, we studied the hemodynamic responses to isoproterenol in three groups of dogs with pulmonary vasoconstriction produced by continuous ventilation with 10% oxygen: (a) hypoxia alone, (b) hypoxia and propranolol 0.3 mg/kg i.v. bolus followed by an infusion of 5 micrograms/kg per min, and (c) hypoxia after pretreatment with an inhibitor of cyclooxygenase, either indomethacin or meclofenamate 5 mg/kg s.c. twice daily for 2 d prior to study. All groups had similar values for mean pulmonary artery pressure (PAPm) and pulmonary vascular resistance (PVR) during room air and hypoxic ventilation. Isoproterenol in doses of 0.0025, 0.005, and 0.05 micrograms/kg per min produced a dose-related decline in PAPm and PVR during hypoxia in group 1. Despite beta-blockade with propranolol (group 2), isoproterenol at all three doses significantly reduced PAPm and PVR. The responses to isoproterenol were comparable in the presence or absence of propranolol; at 0.05 micrograms/kg per min the effects of isoproterenol were blunted, but not abolished, by propranolol. Similar results were observed even when five times the dose of propranolol was given. Isoproterenol at all three doses had no effect, however, on PAPm and PVR in the cyclooxygenase inhibitor-pretreated group. These data suggest that the pulmonary vasodilator effects of isoproterenol are not mediated solely by pulmonary vascular beta adrenergic receptors, and that vasodilator prostaglandins may play a role in the responses to this drug.     

Leukotriene biosynthesis by canine and human coronary arteries

Canine and human epicardial coronary arteries were tested for their ability to metabolize exogenous arachidonic acid to lipoxygenase products. Unextracted medium from incubations of canine or human arteries with arachidonic acid and the calcium ionophore, A23187, contained a substance which exhibited a leukotriene (LT)-like smooth muscle contracting activity when tested on the superfused guinea pig lung parenchymal strip bioassay. This activity could be blocked by the LT antagonist, FPL-55712. Compounds present in these media were purified by high-performance liquid chromatography and identified as LTC4, LTD4 and LTE4 by liquid scintillation counting, bioassay and radioimmunoassay. In addition, coronary artery rings converted synthetic [3H]LTC4 to [3H]LTD4 with a half-life of 44 +/- 8 min. LTD4 metabolism to LTE4 was also demonstrated. The metabolism of [3H]LTC4 was abolished by incubation of the arterial rings with a gamma-glutamyl transpeptidase inhibitor, serineborate. Production of monohydroxyeicosatetraenoic acids (5-, 12- and 15-HETE) which have been isolated previously from vascular tissue incubations was confirmed. Production of HETE was inhibited by nordihydroguaiaretic acid and unaffected by indomethacin. These findings indicate that coronary vessels can metabolize exogenous arachidonic acid by the lipoxygenase pathway. In addition to HETE, the vessels were shown to synthesize LTC4, LTD4 and LTE4, compounds which possess potent biological actions on the coronary circulation.     

Contribution of M2 alpha and M2 beta muscarinic receptors to the action of cholinergic stimuli on prostaglandin synthesis and mechanical function in the isolated rabbit heart

This study was performed to determine the subtype of M2 muscarinic receptor that is involved in the action of cholinergic agents on prostaglandin (PG) synthesis as well as on the mechanical function of the isolated rabbit heart perfused at a constant flow rate with Krebs-Henseleit buffer. The increase in PG output elicited by acetylcholine (ACh) or arecaidine propargyl ester (APE), a selective M2 agonist was attenuated by both 11-[2-[(diethylamino)methyl]-1-piperidinyl]acetyl-5,11-dihydro-6H- pyrido-[2,3-b][1,4]-benzodiazepine-6-one (AF-DX 116), an M2 alpha antagonist, and hexahydro-sila-difenidol (HHSiD), an M2 beta antagonist. The coronary vasodilating effect of ACh and APE was inhibited by HHSiD, but not by AF-DX 116, whereas the vasoconstrictor effect was blocked by AF-DX 116, but not by HHSiD. The decrease in heart rate produced by ACh or APE was blocked by AF-DX 116, but not by HHSiD; however, the decrease in developed tension produced by the cholinergic stimuli was abolished by all these muscarinic receptor antagonists. The increase in PG output or changes in the mechanical parameters of the heart produced by ACh or APE were not altered by adrenergic receptor antagonists, phentolamine and propranolol, or by the nicotinic receptor antagonist, hexamethonium. The effect of isoproterenol or exogenous arachidonic acid to enhance PG output was not altered by these M2 receptor antagonists; however, the cyclooxygenase inhibitor indomethacin abolished the output of PG elicited by these agents or by ACh or APE. These data indicate that the effect of cholinergic stimuli to promote cardiac PG synthesis and decrease developed tension is mediated through the activation of both M2 alpha and M2 beta subtypes of muscarinic receptors. The cholinergically induced vasodilating component of the coronary response is mediated through the activation of M2 beta, whereas the coronary vasoconstriction and the decrease in heart rate is mediated through the activation of M2 alpha muscarinic receptors.     

Possible mechanisms of 15-hydroperoxyarachidonic acid-induced contraction of the canine basilar artery in vitro

Pharmacological studies of the contraction induced by 15-hydroperoxyarachidonic acid (15-HPAA) were done in the isolated canine basilar artery. The maximal contractile force produced by 15-HPAA was 1.5 times that of serotonin and was equivalent to that of prostaglandin (PG) F2 alpha or PGA1. At concentrations in which reductions of [14C]-6-keto-PGF1 alpha (the breakdown product of PGI2) occurred, both 15-HPAA and tranylcypromine contracted the artery, whereas indomethacin consistently relaxed it. In the case of indomethacin, as the drug inhibited the synthesis of [14C]-PGE2, F2 alpha and thromboxane(TX) B2 (the breakdown product of TXA2) as well, it has been considered that the balances between PGI2 and other vasoconstrictive PGs or TX may regulate the tone of the artery. Furthermore, it was shown that 15-HPAA enhanced the synthesis of lipoxygenase products from [14C]arachidonic acid. Experiments were done, therefore, to know whether these enhanced lipoxygenase products participated in the manifestation of contraction induced by 15-HPAA or not. As a result, although indomethacin did not affect the contraction, significant reductions of the contraction were shown by eicosatetraynoic acid. These results suggest that enhanced synthesis of lipoxygenase products may be involved in eliciting contractile responses of 15-HPAA.     

Age-related decrease in beta adrenergic receptor-mediated vascular smooth muscle relaxation

Beta adrenergic receptor-mediated vascular smooth muscle relaxation decreases with increasing age. We have examined the mechanism responsible for this phenomenon using rat mesenteric arteries from young (5-6 weeks) and older (10-12 months) rats. The beta adrenergic agonist isoproterenol produced a dose-dependent relaxation of serotonin-constricted mesenteric artery rings from young rats, whereas the maximal ability of isoproterenol to relax arterial rings from the older rats was found to be reduced markedly (92.7 vs. 27.6%, P less than .0001). The relaxation responses caused by acetylcholine and nitroglycerin, which appear to act independently of cyclic AMP (cAMP), are similar in the two groups. The loss in responsiveness of the mesenteric artery to isoproterenol was not explained by a change in beta receptor number in the vessels (29 +/- 4 in young rats vs. 31 +/- 7 fmol/mg of protein in the older rats). The maximal stimulation of cAMP accumulation by isoproterenol was lower in the older vessels; forskolin activated cAMP accumulation equally in the two groups. However, the vessels from the older rats were less sensitive to forskolin-induced vascular relaxation. Also, the ability of dibutyryl cAMP to promote vascular relaxation was diminished in the older vessels. These data suggest that the diminished cAMP accumulation in older vessels in response to isoproterenol might not necessarily in itself explain completely the reduced physiological response and that an additional defect in the beta adrenergic-mediated relaxation in the vascular smooth muscle of older rats may lie at the level of cAMP-dependent protein kinase activation or more distally.     

Vasorelaxing effects of prostaglandin I2 on the canine basilar and coronary arteries

The effects of prostaglandin (PG) I2 on canine basilar and coronary arteries were studied. PGI2 caused a relaxation from the basal level more effectively in the endothelium-intact preparations of the coronary artery than in those of the basilar artery. The PGI2-induced relaxation in the basilar artery was enhanced by removal of the endothelium, and by treatment with indomethacin (10(-6) M), aspirin (5 X 10(-5) M), both cyclooxygenase inhibitors, OKY-046 (3 X 10(-5) M) and RS-5186 (10(-6) M), both thromboxane (TX) A2 synthetase inhibitors and ONO-3708 (10(-8) M), a TXA2 antagonist. The enhancing effects of removal of the endothelium and treatment with indomethacin and aspirin on the PGI2-induced relaxation were greater than those of treatment with OKY-046, RS-5186 and ONO-3708. The PGI2-induced relaxation in the coronary artery was not affected by removal of the endothelium, treatment with indomethacin (10(-6) M) or methylene blue (10(-6) M). In the endothelium-removed preparations precontracted with a TXA2 agonist, PGI2-induced relaxation was less in the basilar artery than in the coronary artery. The present experiments suggest that endothelium-derived factors (TXA2 and other cyclooxygenase products) counteract the vasorelaxing effect of PGI2 in the canine basilar artery, but not in the coronary artery.