Role of cGMP mechanisms in response of rat pulmonary arteries to hypoxia.

We have demonstrated previously that in response to hypoxia, isolated rat pulmonary arteries show an initial endothelium-dependent relaxation followed by an endothelium-independent transient contraction. In the presence of increased extracellular Ca2+, both of these responses were enhanced in endothelium-intact arteries. Nitro-L-arginine, a blocker of the biosynthesis of endothelium-derived relaxing factor (EDRF), abolished the initial endothelium-dependent relaxation and Ca(2+)-induced enhancement of hypoxic contraction in endothelium-intact arteries but did not alter responses in endothelium-denuded vessels. Inhibition of prostaglandin formation with indomethacin had no effect on the hypoxia-elicited responses. Preincubation with LY 83583, an inhibitor of guanylate cyclase activation, abolished the initial hypoxia-elicited relaxation and subsequent contraction. M & B 22948, a guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase inhibitor, decreased tone under O2 but not under N2, causing an apparent enhancement of the contraction to hypoxia. Thus the modulation of hypoxic responses by the endothelium is dependent on changes in EDRF production, and a decrease in smooth muscle cGMP not involving an EDRF mechanism appears to mediate the endothelium-independent hypoxic contraction observed in the isolated rat pulmonary artery.

Association of pulmonary artery photorelaxation with H2O2 metabolism by catalase.

We have examined the mechanism governing guanosine 3',5'-cyclic monophosphate (cGMP)-associated photoinduced relaxation elicited by long-wavelength ultraviolet (UV) light of endothelium-removed, isolated bovine pulmonary arteries. Hypoxia, produced by gassing of the organ bath solution with 95% N2-5% CO2, inhibited photorelaxation. Photorelaxation was also inhibited by cyanide (1 mM NaCN) but was potentiated by lactate (5 mM). Irradiation of bovine pulmonary arterial smooth muscle with UV light (or exposure to exogenous H2O2) stimulated cyanide-inhibitable oxidation of methanol to formaldehyde, suggesting that UV light increased H2O2 metabolism via catalase. The UV light-induced oxidation of methanol by pulmonary arterial smooth muscle was also inhibited by hypoxia. Consumption of O2 was detected when pulmonary arterial tissue was exposed to UV light, but cyanide failed to interfere with this effect, consistent with the photochemical reduction of O2 within vascular smooth muscle in a manner independent of mitochondrial respiration. We propose that photorelaxation is associated with the intracellular photochemical reduction of O2 to form H2O2, which elicits increases of vascular smooth muscle cGMP levels via the catalase-dependent activation of soluble guanylate cyclase. In addition, we hypothesize that the photooxidation of NAD(P)H could contribute to the generation of H2O2, since the enhancement of photorelaxation by lactate may originate from increased levels of NADH.

Inhibition of coronary artery superoxide dismutase attenuates endothelium-dependent and -independent nitrovasodilator relaxation.

Isolated bovine coronary arteries were treated with 10 mM diethyldithiocarbamate (DETCA) for 30 minutes to deplete the cytosolic ZnCu form of superoxide dismutase (SOD). This treatment completely inhibited the endothelium- and cGMP-dependent relaxation to acetylcholine (mediated via the endothelium-derived relaxing factor, which is thought to be nitric oxide) without significantly inhibiting endothelium-dependent relaxation to arachidonic acid (mediated by prostaglandins). DETCA treatment of endothelial cells cultured from the coronary arteries inhibited bradykinin-elicited release of endothelium-derived relaxing factor, which was detected by bioassay on an isolated rabbit aorta in the presence of extracellular SOD. DETCA also inhibited cGMP-associated relaxations to nitric oxide and to vasodilators thought to function via the generation of this mediator (nitroglycerin and nitroprusside), but cAMP-associated relaxations to isoproterenol and papaverine were not altered. The inhibitory effects of DETCA against the relaxation to nitroprusside and nitroglycerin were attenuated by severe hypoxia. DETCA treatment of isolated coronary arterial smooth muscle or cultured endothelial cells produced an increase of chemiluminescence elicited in the presence of lucigenin, a detector of superoxide anion generation. The addition of SOD markedly attenuated the effects of DETCA treatment on arterial relaxation and chemiluminescence. Therefore, control of cellular superoxide anion levels by endogenous SOD appears needed for the release of endothelium-derived relaxing factor and relaxation of vascular smooth muscle to nitrovasodilators mediated via cGMP in the bovine coronary artery, but SOD is not critical for other endothelium-dependent or cAMP-associated relaxant mechanisms.

Superoxide anion inhibits cGMP-associated bovine pulmonary arterial relaxation.

We have reported evidence that endothelium-independent relaxations of isolated bovine pulmonary arteries to H2O2 and to reoxygenation with 95% O2-5% CO2 after brief exposure to N2 (5% CO2) appear to be mediated by the activation of guanylate cyclase via H2O2 metabolism through catalase. Treatment of endothelium-removed pulmonary arteries with a potential guanylate cyclase-inhibitor, LY 83583, or with the inhibitor of the Zn+2, Cu+2-superoxide dismutase (SOD) diethyldithiocarbamic acid (DETCA), antagonized guanosine 3',5'-cyclic monophosphate (cGMP)-associated relaxation to H2O2, to reoxygenation and to glyceryl trinitrate, but not the adenosine 3',5'-cyclic monophosphate-associated relaxation to isoproterenol. Superoxide anion (O2-.) levels, detected by lucigenin-elicited chemiluminescence, were enhanced by LY 83583 or DETCA treatment of pulmonary arteries at ambient PO2. Chemiluminescence produced by LY 83583 was markedly potentiated by DETCA treatment, decreased at addition of exogenous SOD, and inhibited markedly by anoxia. LY 83583, but not DETCA, stimulated cyanide-insensitive O2 consumption, consistent with redox cycling of the compound independent of mitochondrial respiration. We propose that O2-. generated on the metabolism of LY 83583, or from cellular electron donors after SOD inhibition by DETCA, inhibits cGMP-mediated relaxations of pulmonary arteries.

Methylene blue inhibits vasodilation of skeletal muscle arterioles to acetylcholine and nitric oxide via the extracellular generation of superoxide anion.

The mechanism of modulation of cyclic GMP-associated vascular responses by methylene blue, an agent employed to inhibit the activation of soluble guanylate cyclase in tissues, was investigated in the cremaster muscle microcirculation of pentobarbital-anesthetized rats. The effect of topically applied agents on the diameter of third-order arterioles (15-20 microns diameter) was determined by in vivo television microscopy. Topical application (100 microliters) of acetylcholine (0.01 microgram) or nitric oxide (0.06-6 micrograms) caused vasodilator responses that were inhibited (P less than .05, n = 6-8) 64% and 30 to 100%, respectively, by suffusion of the preparation with 5 microM methylene blue. Agents that are thought to produce activation of guanylate cyclase via cellular metabolism to nitric oxide, nitroglycerin (0.5 ng-0.5 microgram) or nitroprusside (0.5 ng-0.5 microgram), also produced vasodilation. However, methylene blue suffusion did not inhibit these responses (n = 6-9). The inhibition of vasodilation to acetylcholine or nitric oxide by methylene blue was completely prevented by suffusion of superoxide dismutase, but not affected by suffusion of catalase. Based on the current conceptualization of the mechanism of action of these vasodilator agents in isolated larger blood vessels, methylene blue appears to inhibit responses in this skeletal muscle microcirculatory preparation through the extracellular generation of superoxide anion and not via a direct interaction with guanylate cyclase.

Ascorbate activates soluble guanylate cyclase via H2O2-metabolism by catalase.

Conditions necessary for the activation by ascorbic acid of soluble guanylate cyclase purified from bovine lung have been examined. Ascorbic acid (0.1-10 mM) did not directly activate the enzyme, nonetheless, pronounced activation by ascorbate (3-10 mM) was observed in incubation mixtures containing 1 microM bovine liver catalase. Superoxide dismutase (SOD) and mannitol did not affect the catalase-dependent activation of guanylate cyclase elicited by ascorbate, suggesting that superoxide anion and hydroxyl radical were not mediating the activation of the enzyme. However, SOD enhanced the relatively low level activation of the enzyme elicited by catalase in the absence of added ascorbate. Pronounced inhibition (both with and without added ascorbate) was observed of catalase-dependent activation of guanylate cyclase by either ethanol (100 mM) or a fungal catalase preparation. Neither ethanol nor fungal catalase inhibited activation of guanylate cyclase by S-nitrosyl-N-acetyl-penicillamine (SNAP), a source of the nitric oxide free radical. These observations indicate that autoxidation of ascorbic acid or thiols present with the guanylate cyclase preparation leads to generation of H2O2, and its metabolism by bovine liver catalase mediates the concomitant activation of guanylate cyclase. The mechanism of activation appears to be associated with the presence of Compound I of catalase and to be inhibited by superoxide anion.

Antagonism of acetylcholine-mediated relaxation of rabbit pulmonary arteries by phorbol myristate acetate.

We studied the effect of 4-phorbol 12-myristate 13-acetate (PMA) on endothelium-dependent relaxation of rabbit isolated lobar pulmonary arteries contracted with phenylephrine. After full development of relaxation in response to 1.0 microM acetylcholine, addition of 10.0 nM PMA reversed relaxation rapidly and completely. This effect of PMA on acetylcholine-induced relaxation was unchanged when catalase was included in the medium. In separate groups of experiments, lobar pulmonary arteries were preincubated with 10.0 nM PMA for 20 min and then challenged with acetylcholine without washout of PMA. In these experiments PMA did not block completely the relaxation in response to addition of 1.0 microM acetylcholine. Log-concentration response curves for 0.01 to 31.6 microM acetylcholine vs. relaxation as percentage of phenylephrine-induced tone exhibited rightward shifts and depression of maxima after pretreatment with PMA. The EC50 for acetylcholine-induced relaxation was increased from 57 +/- 9.0 to 377 +/- 6.30 nM (values are mean +/- S.E.). Relaxation of lobar pulmonary arteries induced by 10.0 nM A23187 was much less sensitive to treatment with PMA than relaxation induced by acetylcholine. At least for the low concentration of PMA used here, these data are consistent with the following: 1) PMA blocks acetylcholine-induced relaxation by disrupting the link between acetylcholine receptor occupancy and the formation or release of the endothelium-derived relaxing factor, 2) PMA does not directly block the relaxant action of endothelium-derived relaxing factor on vascular smooth muscle and 3) the effect of PMA on acetylcholine-induced relaxation is not mediated by hydrogen peroxide or derived oxygen radicals.

Evidence for the role of endothelium-derived relaxing factor in acetylcholine-induced vasodilatation in the intact lung.

The vascular effects of acetylcholine were evaluated in blood-free lungs of rabbits perfused in situ at a constant flow rate of 15 ml/min with Kreb's-bicarbonate solution containing 3% dextran. Infusion of 1 microM acetylcholine normally elicited vasoconstriction. However, acetylcholine (3.0 nM-1.0 microM) produced concentration-dependent vasodilatation when administered to lungs in which pulmonary artery pressure (i.e., perfusion pressure) was elevated and cyclooxygenase activity was blocked, respectively, by infusing the endoperoxide analog U46619 (2.0-10.0 nM) and 30.0 microM indomethacin. Three additional groups of lungs treated with indomethacin and preconstricted with U46619 were challenged twice with 1.0 microM acetylcholine. Two of the groups were equilibrated with an infusion of either 20.0 microM quinacrine or 10.0 microM ferrous hemoglobin before and during their second challenges with acetylcholine and a control group received no treatment between trials. Neither quinacrine nor hemoglobin altered base-line pulmonary artery pressure, but both agents reversed the effect of acetylcholine from vasodilatation to vasoconstriction. This study agrees with previous reports of a pulmonary vasoconstrictor action of acetylcholine dependent on an intact cyclooxygenase pathway, but also provides new evidence for a vasodilator action of acetylcholine in the intact lungs of rabbits. Quinacrine and hemoglobin are known to antagonize the endothelium-dependent relaxation of vascular smooth muscle elicited by acetylcholine; therefore, this study provides indirect evidence supporting a role for the endothelium-derived relaxing factor in the regulation of pulmonary vascular tone.

Endothelial cells as mediators of vasodilation of arteries.

A brief review is first presented of findings during the past few years by the authors and by others on the nonprostaglandin endothelium-dependent relaxation of isolated arteries by a large number of vasoactive agents. Among these agents are acetylcholine (ACh); the calcium ionophore A23187; ATP and ADP; substance P; bradykinin (canine, human, and porcine arteries); histamine, acting via an H1-receptor (rat arteries); thrombin (canine arteries); serotonin (canine coronary artery); and norepinephrine, acting via an alpha2-receptor (canine coronary artery). The endothelium-derived relaxing factor (EDRF) released by ACh and other agents has not yet been identified. Our original hypothesis that arachidonic acid is the precursor of EDRF is not supported by the finding that other unsaturated fatty acids in addition to arachidonic acid, and even stearic acid, elicited nonprostaglandin endothelium-dependent relaxations. Methylene blue and hemoglobin (but not methemoglobin) rapidly inhibited relaxation of rabbit aorta by ACh or A23187, suggesting that our proposal that EDRF is a labile free radical may be correct. The endothelium-dependent relaxation by each of these agents was shown to be preceded by an endothelium-dependent increase in cyclic GMP in the smooth muscle--a finding consistent with the hypothesis that EDRF stimulates guanylate cyclase in the muscle, leading to an increase in cyclic GMP that somehow activates relaxation. Some questions relating to the potential physiological important of endothelium-dependent relaxations are discussed.

Role of endothelial cells in relaxation of isolated arteries by bradykinin.

Bradykinin elicits relaxation of isolated transverse rings of canine coronary, celiac, superior mesenteric, renal, splenic, pulmonary, gastric, and femoral arteries. After endothelial cells of the vessel wall are removed by rubbing of the intimal surface, canine arteries fail to relax upon addition of bradykinin. The endothelium-dependent relaxation of canine arteries remains intact after treatment with cyclooxygenase inhibitors (indomethacin and flurbiprofen), and this argues against mediation by prostaglandins. When they are stimulated with bradykinin, endothelial cells of canine arteries appear to release a substance mediating vascular smooth muscle relaxation. In contrast, preparations of arteries of cats (superior mesenteric) and rabbits (superior mesenteric and celiac) may be rubbed on the intimal surface without a consistent loss of sensitivity to the relaxing effects of bradykinin. In addition, relaxation of the cat and rabbit arteries is completely blocked by cyclooxygenase inhibitors. Preliminary studies indicate that bradykinin relaxes human arteries in an endothelium-dependent manner and that this effect is not mediated by prostaglandins. We have previously reported that arteries of all species tested require the presence of endothelial cells for relaxation in response to acetylcholine and we have also demonstrated, using the rabbit aorta, that this effect is mediated by the release of an uncharacterized substance from these cells that relaxes vascular smooth muscle. We conclude that bradykinin relaxes canine and human arteries via a similar mechanism but that it relaxes cat and rabbit arteries by stimulating release of prostaglandins from as yet undefined cell types.