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Objective and Design: This study was conducted to determine if differences in histidine decarboxylase expression and histamine levels exist between B16F10 melanoma cells and non-cancerous Melan-A melanocytes., Methods: Immunofluorescence and western blot analysis were used to detect and compare histidine decarboxylase protein levels. Enzyme-linked immunoassay was used to detect, measure, and compare histamine levels., Results: Histidine decarboxylase expression was found to be elevated in the B16F10 cells. Western blot analysis demonstrated levels of histidine decarboxylase protein expression more than twofold higher (p < 0.001) in B16F10 than in Melan-A cells. Histamine levels were 280-fold higher (p < 0.001) in B16F10 (229 ± 15 pg/mg protein) than in Melan-A (0.83 ± 0.03 pg/mg protein) cells., Conclusion: Results indicate an up-regulated histaminergic system in the B16F10 melanoma cells when compared to non-cancerous melanocytes. This supports the use of B16F10 cells as a model in which to investigate a potential role of the endogenous histaminergic system in regulating malignant cell function.

Objective: Having previously demonstrated release of histamine from mast-cell-deficient rat aorta, the objective of this study was to determine and localize histamine synthesis capability in the aorta by detecting histidine decarboxylase (HDC), the enzyme that catalyzes histamine formation., Materials and Methods: Experiments were conducted with nested reverse transcription-polymerase chain reaction (nRT-PCR) to detect HDC mRNA and with immunofluorescence and western blot analysis to detect HDC protein in rat aorta, cultured rat aortic smooth muscle (RASMC) and endothelial cells (RAEC)., Results: Gel electrophoresis of nRT-PCR products indicated HDC mRNA in liver, aorta and RASMC but not in RAEC or kidney. Sequence analysis confirmed that the band observed in RASMC was the target HDC amplicon. Immunofluorescence indicated the presence of HDC protein in RASMC and not in RAEC. Western Blot analysis revealed HDC protein (55 kDa) in liver, aorta, RASMC but not in RAEC or kidney., Conclusions: The results of this study are the first to demonstrate the presence of HDC mRNA and protein in rat aorta and more specifically in RASMC, indicative of their capability to synthesize histamine., (Copyright 2004 Birkhäuser Verlag, Basel)

Objective: The objective of this study was to investigate histamine synthesis capability of human vascular smooth muscle and endothelial cells by detecting histidine decarboxylase (HDC) mRNA., Methods: HDC catalyzes exclusively the formation of histamine in mammalian cells. Experiments utilizing nested reverse transcription-polymerase chain reaction (nRT-PCR) were conducted to detect the presence of HDC mRNA. Human aortic smooth muscle cells (HAoSMC) and human aortic endothelial cells (HAEC) were cultured and RNA was extracted and amplified using two sets of HDC-specific primers. Rat liver and kidney RNA were isolated and amplified to serve as positive and negative controls, respectively., Results: Gel electrophoresis of HAoSMC, HAEC and liver mRNA revealed bands coinciding with an expected product size of 440 base pairs. Sequence analysis revealed that the observed bands were the appropriate HDC amplicons., Conclusions: These findings are the first to indicate the presence of HDC mRNA in vascular smooth muscle cells and confirm the presence of HDC mRNA in endothelial cells which is consistent with an ability of these cell types to synthesize histamine in the vascular wall.

We previously reported that A23187 induces release of histamine from bovine intrapulmonary vein and provided pharmacological evidence against an involvement of mast cells as the source of histamine. This study was conducted to test more definitively the hypothesis that histamine is released from non-mast cell sources in blood vessels. The effects of A23187 on release of histamine were determined using rat aorta which does not contain mast cells. Aortic rings were mounted for recording of isometric tension, and following exposure to A23187 or vehicle, histamine in the bathing media was measured using enzyme immunoassay. A23187 (100 nmol/l - 10 micromol/l) induced concentration-related release of histamine from rings with endothelium. The accumulation of histamine in the bathing media induced by 10 microM A23187 reached plateau at 60 min (6.2 +/- 1.1 pmol/mg) and was markedly and significantly higher than vehicle control (0.4 +/- 0.1 pmol/mg, p < 0.05). Destruction of endothelium significantly inhibited A23187-induced histamine release (5.5 +/- 1.5 pmol/mg with endothelium, 1.1 +/- 0.3 pmol/mg without endothelium, p < 0.05). The results demonstrate that A23187 induces release of histamine from rat aorta which does not contain mast cells and that the release of histamine is largely dependent on the presence of endothelium.

We hypothesized that substance P and capsaicin would cause the release of prostaglandin E2 (PGE2) from intrapulmonary bronchi isolated from Sprague-Dawley rats. Substance P (1 microM) caused the release of PGE2, measured with enzyme immunoassay, from the isolated airway segments; PGE2 release was inhibited by the neurokinin (NK)1-receptor antagonist, RP-67580, by inhibition of cyclooxygenase with meclofenamate, and by removal of the epithelium. The release of PGE2 caused by capsaicin (1 microM) was similar in magnitude to that caused by substance P. The capsaicin-induced release of PGE2 was inhibited by desensitization of sensory nerves with capsaicin and by RP-67580, meclofenamate, and epithelial denudation. We conclude that activation of NK1 receptors on epithelium causes release of PGE2, which most likely represents the ultimate mediator of airway smooth muscle relaxation, produced by exogenous neuropeptides and by activation of the sensory nerve inhibitory system. Epithelial damage, such as that seen in asthmatic airways, would disrupt this protective system in the lungs, which could contribute to the development of airway disease.

Previous studies in our laboratory and others suggested that activation of 5-HT2 receptors mediates 5-hydroxytryptamine (5-HT)-induced contraction of airway smooth muscle and that this response is dependent in part on endogenous acetylcholine (ACh). The purpose of the present study was to confirm a role for 5-HT2 receptors and endogenous ACh in 5-HT-induced contraction of rat bronchi. In this study, we examined the effects of 5-HT2 receptor antagonists (ketanserin and LY53857), acetylcholinesterase inhibitors (physostigmine and neostigmine), and a muscarinic receptor alkylating agent [propylbenzilylcholine mustard (PBCM)] on contractile responses evoked by 5-HT and the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT). Concentration-response curves generated in isolated rat intrapulmonary bronchi in response to 5-HT and alpha-Me-5-HT were superimposable. Inhibition of acetylcholinesterase by physostigmine or neostigmine potentiated contractile responses elicited by 5-HT and alpha-Me-5-HT. Alkylation of muscarinic receptors with PBCM decreased maximal responses elicited by 5-HT or alpha-Me-5-HT in a concentration-dependent manner. Maximum contraction attained with exogenous ACh was decreased by PBCM in a concentration-dependent manner and, at the highest concentration evaluated, ACh-induced contractions were abolished. 5-Hydroxytryptamine-induced contraction was inhibited competitively by low concentrations of the 5-HT2-receptor selective antagonist, ketanserin; higher concentrations abolished contractile responses to the amine. The inhibition of 5-HT-induced contractile responses by another 5-HT2-receptor selective antagonist, LY53857, was non-competitive in nature. Together, the results suggest that 5-HT contracts rat airways directly by activating 5-HT2 receptors located on airway smooth muscle and indirectly by activation of 5-HT2 receptors on parasympathetic nerve endings to cause release of ACh. The potential physiological implication of these findings is that 5-HT released in inflammatory conditions such as asthma may play a role in causing bronchoconstriction by releasing ACh or by augmenting release of ACh from activated cholinergic nerves.

This study was initiated to test the hypothesis that histamine can act as an endothelium-derived contracting factor in bovine isolated intrapulmonary vein. The effects of calcium ionophore, calcimycin (A23187), on isometric tension were compared in unstimulated rings of intrapulmonary vein with and without endothelium. A23187 (0.1-10 microM) induced concentration-related contraction when endothelium was present. Destruction of endothelium markedly inhibited A23187-induced contraction. Methylene blue, hemoglobin or NG-methyl-L-arginine significantly enhanced A23187-induced contraction only in venous rings with endothelium consistent with attenuation of the contraction by the concomitant release of endothelium-derived relaxing factor (nitric oxide) [EDRF(NO)]. Histamine H1 receptor antagonists inhibited, and iproniazid enhanced, contraction elicited by A23187. A23187 induced release of greater amounts of histamine from venous rings with than without endothelium. A23187-induced contraction was not mimicked by the mast cell activator, compound 48/80, and was not inhibited by preexposure to compound 48/80 or in the presence of cromolyn or doxantrazole. A23187-induced contraction was not inhibited by pretreatment with indomethacin, phentolamine, lipoxygenase inhibitors or superoxide dismutase. The results indicate that A23187 induces endothelium-dependent contraction in bovine intrapulmonary vein and support histamine as one major mediator involved. The association of destruction of endothelium with an inhibition of both A23187-induced contraction and histamine release is consistent with the endothelium as a source for histamine which can exert a local vasoconstrictor effect in bovine intrapulmonary vein.

Estrogens have been postulated to play an important role in modulation of vascular responses to endogenous reactive substances. The effects of chronic in vivo treatment with 17 beta-estradiol on relaxant responses to acetylcholine were investigated in the rat aorta isolated from prepubertal female rats. The selectivity of effects of 17 beta-estradiol on acetylcholine-induced relaxation was evaluated using histamine, another endothelium-dependent relaxant in the rat aorta. 17 beta-Estradiol significantly enhanced endothelium-dependent relaxation induced by acetylcholine, but did not alter the vascular responses to acetylcholine in endothelium-denuded aortic rings isolated from prepubertal female rats. In contrast, 17 beta-estradiol did not change endothelium-dependent relaxation induced by histamine in endothelium-intact aortic rings. The results of the present study demonstrate that 17 beta-estradiol selectively enhances acetylcholine-induced endothelium-dependent relaxation in the rat aorta.

5-Hydroxytryptamine (5-HT) potentiated contractions of isolated rat bronchi evoked by electrical field stimulation (EFS). The degree of potentiation caused by 5-HT was dependent upon concentration of the amine present in the tissue bath. The effects of antagonists selective for different subtypes of the 5-HT receptor on potentiation of EFS-induced contractions by 5-HT were examined. Propranolol, a nonselective beta-adrenoceptor antagonist which can act as a 5-HT1 receptor antagonist, did not inhibit the effect of 5-HT on EFS-induced contractile responses. Similarly, 5-HT3 receptor antagonism with MDL 72222 or ICS 205-930, did not inhibit the facilitatory effects of 5-HT. However, ketanserin, mianserin and spiperone, 5-HT2 receptor antagonists, abolished the effects of 5-HT on EFS-induced responses. These latter results suggested that the potentiation was dependent upon activation of 5-HT2 receptors thus additional experiments were conducted using the 5-HT2 receptor agonist, alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT). alpha-Me-5-HT caused a concentration-dependent potentiation of EFS-induced contractile responses comparable to that observed with 5-HT. Concentrations of alpha-Me-5-HT that significantly potentiated EFS-induced contraction were essentially without effect on airway smooth muscle contraction elicited by exogenous acetylcholine. These results are consistent with a role for 5-HT2 receptor activation in mediating the facilitatory effects of 5-HT on cholinergic nerve-mediated responses in airways.

This study examined the effects of chlorpheniramine, citalopram and fluoxetine on 5-hydroxytryptamine (5-HT)-induced contraction and 5-HT uptake in rat thoracic aortic rings in vitro. Chlorpheniramine and citalopram markedly potentiated 5-HT-induced contraction. Potentiation by fluoxetine was less pronounced. Chlorpheniramine (0.01-1 microM) and citalopram (0.1-1 microM) induced concentration-dependent parallel shifts to the left of the 5-HT concentration-response curves. The potentiation by chlorpheniramine was selective as chlorpheniramine (1 microM) did not potentiate phenylephrine-induced contraction. The potentiation did not depend upon the presence of endothelium, and was not related to H1 receptor antagonism as diphenhydramine and pyrilamine (1 microM) did not similarly enhance 5-HT-induced contractions. Whereas cocaine (1-10 microM) similarly potentiated 5-HT-induced contraction, imipramine (1-10 microM) inhibited, rather than enhanced, contraction elicited by 5-HT. In the presence of 10 microM cocaine, maximally effective concentrations of chlorpheniramine (1 microM) or citalopram (100 nM) did not induce any additional potentiation of 5-HT-induced contraction. Cooling (4 degrees C) markedly inhibited uptake of [3H]5-HT in rings with and without endothelium. Although less marked, imipramine (10 microM), cocaine (1 microM), chlorpheniramine (1 microM) and citalopram (100 nM) inhibited [3H]5-HT uptake in endothelium-intact and endothelium-denuded rings. Fluoxetine also inhibited [3H]5-HT uptake, but the inhibition was only statistically significant in endothelium-intact rings. The monoamine oxidase (MAO) inhibitor, pargyline (10-100 microM), did not significantly affect 5-HT-induced contraction. The results demonstrate that chlorpheniramine, citalopram and to a lesser extent, fluoxetine potentiate 5-HT-induced contraction in rat aorta in which neuronal 5-HT uptake is negligible. The data are consistent with inhibition of non-neuronal 5-HT uptake as at least one mechanism responsible for potentiation of 5-HT-induced contraction in rat aorta by chlorpheniramine, citalopram and fluoxetine.

The organic nitrates and related nitrovasodilators are relaxants of vascular and airway smooth muscle. Very little information is currently available regarding the influence of nitrates and related nitrovasodilators on pulmonary autacoid release. This study examined the influence of glyceryl trinitrate, isosorbide dinitrate and sodium nitroprusside on histamine release from bovine lung mince. Spontaneous histamine release from bovine lung mince was not altered by 0.1 nM to 1 microM glyceryl trinitrate, isosorbide dinitrate or sodium nitroprusside. Glyceryl trinitrate, isosorbide dinitrate and sodium nitroprusside produced a concentration-dependent decrease in A23187 (10 microM) stimulated histamine release. Glyceryl trinitrate also inhibited histamine liberation following the addition of compound 48/80. Further studies indicated that the inhibitory action of glyceryl trinitrate was reversed by coincubation with the guanylate cyclase inhibitor, methylene blue (10 microM). These findings indicate that glyceryl trinitrate, sodium nitroprusside and isosorbide dinitrate inhibit non-immunologically stimulated pulmonary histamine release and suggest that alterations in guanylate cyclase activity may influence pulmonary histamine release.

Sex hormones have been postulated to play an important role in the modulation of vascular responsiveness to endogenous vasoactive substances. This study was designed to establish the effects of chronic treatment with estrogen in vivo on subsequent contractile responsiveness of aortic rings to angiotensin II or norepinephrine in vitro. Concentration-response curves for angiotensin II were compared in aortic rings with or without endothelium isolated from ovariectomized rats (275-299 g) pretreated with 17 beta-estradiol (approximately 1 mg/kg per day) or placebo for 14 days and from normal prepubertal female rats (125-149 g) pretreated with 17 beta-estradiol (approximately 5 mg/kg per day) or placebo for 14 days. Whether or not endothelium was present, angiotensin II-induced contraction was significantly depressed in rings from 17 beta-estradiol-treated ovariectomized or prepubertal rats when compared to controls, and the pattern of the effects of 17 beta-estradiol-treatment on the concentration-response curves for angiotensin II was similar in the two models. In contrast to angiotensin II-induced contraction, norepinephrine-induced contraction was significantly enhanced in aortic rings with or without endothelium from ovariectomized rats pretreated with 17 beta-estradiol (approximately 1 mg/kg per day, 14 days) and from normal prepubertal female rats pretreated with 17 beta-estradiol (approximately 5 mg/kg per day, 14 days) when compared to controls. The results demonstrate that chronic treatment with 17 beta-estradiol selectively reduced and enhanced contractile responsiveness of aortic rings to angiotensin II and norepinephrine, respectively. Further, the results indicate that the normal prepubertal female rat is an efficient model to investigate modulation by estrogen of aortic responsiveness to vasoactive agents in vitro.

We studied the morphological and contractile characteristics of rat thoracic aortic segments perfused for 3 or 6 days under pulsatile conditions. Light microscopic examination of the segments revealed the presence of an unchanged tunica media. However, the intimal surface was mostly devoid of endothelial cells. The perfused aortic segments showed a dramatic increase in spontaneous tone when compared to fresh and sham-treated aortic segments. Maximum responses to potassium and norepinephrine were reduced after 3 days of perfusion (20-40% reduction), while maximum responses to 5-hydroxytryptamine and angiotensin II were not significantly different. After 6 days of perfusion, maximum responses to all agonist were reduced (50-60%). Sensitivity to norepinephrine was not affected by the treatment, while sensitivity to 5-hydroxytryptamine was reduced. The perfused aortic segments relaxed well in response to isoproterenol. Our system provides a useful experimental model for short-term studies of hypertension- and atherosclerosis-related vascular changes. Further refinement and characterization could improve the performance of the system for longer-term studies.

Histamine has been reported to cause endothelium-dependent relaxation of vascular smooth muscle and vasodilation. This study was undertaken to examine the inhibitory effects of histamine on cylindrical segments of extrapulmonary arteries isolated from male Sprague Dawley rats. In arterial segments precontracted with phenylephrine (10 microM), histamine (0.1-100 microM) elicited concentration-dependent relaxation responses. Removal of the endothelium or pretreatment with methylene blue (10 microM) abolished relaxation responses to low concentrations of histamine and markedly inhibited those caused by histamine at concentrations greater than 1 microM. Incubation of endothelium-intact arterial segments with pyrilamine (1 microM) caused a significant rightward shift of the histamine concentration-response curves. Treatment of the segments with cimetidine (100 microM) or indomethacin (10 microM) only minimally antagonized histamine-induced relaxation in arteries with endothelium. Residual relaxation responses observed in arteries stripped of endothelium were unaffected by pretreatment with cimetidine, indomethacin, or pyrilamine. The results suggest that the inhibitory effect of histamine in rat pulmonary arteries is mediated predominantly by activation of H1-receptors on the endothelium and the subsequent release of endothelium-derived relaxing factor(s).

Arterial contractile responses were studied in experimentally uremic rats. The ligation of 75% of the terminal branches of the left renal artery, followed by contralateral nephrectomy induced a chronic renal failure, as evidenced by significantly higher BUN concentrations and an elevation in blood pressure. Uremic rats exhibited elevations in total and HDL cholesterol, increased triglyceride levels, and significantly lowered HDL/total cholesterol ratios compared to control groups: 1) untreated control, 2) experimental control receiving renal infarction without contralateral nephrectomy, and 3) two kidney, one clip (2K1C) model of hypertension. Plasma total cholesterol levels in uremic rats increased by 50% in the first week following final surgery and plateaued at approximately 100% above control levels during postoperative weeks 3 through 10. Plasma transaminase levels were similar among all of the groups of rats studied indicating no effects of surgery on hepatic function. The contractile responses of aortic rings from uremic rats to submaximal concentrations of serotonin and clonidine were significantly increased and decreased, respectively, whereas the contractile response to norepinephrine was not altered. The enhanced sensitivity to serotonin of aortic rings from uremic rats was abolished following disruption of the endothelium. Neither the alteration of aortic contractile properties nor changes in plasma BUN and cholesterol concentrations were observed in hypertensive 2K1C rats. However, there was a tendency for decreased acetylcholine-induced relaxation in aortic rings of uremic and 2K1C rats. No significant histological evidence of atherosclerosis was found in aortic tissues of uremic rats during the interval of study. These results indicate that hypercholesterolemia, hypertriglyceridemia, and altered vascular sensitivity to serotonin and clonidine are likely to be primary changes in the uremic rat and are not secondary to the development of blood pressure elevation or overt evidence of structural change in the vessels of this model.

This study was conducted to compare the influence of endothelium on mechanical responses of bovine intrapulmonary artery and vein to acetylcholine (ACh) and A23187 and to determine if a relationship exists between the responses and cyclic GMP (cGMP) accumulation. ACh and A23187 induced relaxation in artery and A23187 induced relaxation in vein. The relaxant responses in both vessels were abolished by rubbing the intimal surfaces, indicating that the relaxant responses depended upon the presence of a functionally intact endothelium. cGMP accumulation was temporally associated with both ACh- and A23187-induced endothelium-dependent relaxation. Both the relaxant responses and the accompanying cGMP accumulations were abolished or reduced markedly by intimal rubbing or pretreatment with methylene blue. Atropine abolished relaxation and cGMP accumulation induced by ACh in artery, but not relaxation and cGMP accumulation induced by A23187. Whereas indomethacin did not affect either ACh- or A23187-induced relaxation in artery, it slightly, but significantly, reduced A23187-induced relaxation in vein. In contrast to its effect in artery, ACh only induced contractile responses in vein and did not alter cGMP levels, whether or not functional venous endothelium was present. However, ACh did relax veins when arterial endothelium was present in crossover experiments using a modified sandwich technique. At concentrations which induced endothelium-dependent relaxation in artery, ACh similarly induced no, or only minimal, contraction in both artery and vein in which endothelium was functionally destroyed. These findings demonstrate that bovine intrapulmonary artery and vein exhibit endothelium-dependent relaxation in response to A23187, and similar to ACh-induced relaxation in the artery, A23187-induced relaxation is associated closely with accumulation of cGMP.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to determine time course relationship between cyclic GMP accumulation and relaxation in bovine coronary artery and evaluate the effects of recently identified inhibitors, methylene blue and methemoglobin, on these relationships. Arterial strips were suspended in specially mounted tissue baths which permitted continuous recording of isometric tension until rapid freeze-clamping for subsequent determination of cyclic GMP levels by radioimmunoassay. Relaxation and cyclic GMP levels were measured in submaximally contracted strips at zero time (untreated) or 5-sec to 5-min intervals after exposure to 0.5 microliter of nitric oxide, 1 microM glyceryl trinitrate, 1 microM sodium nitroprusside of 1 mM sodium nitrite in the absence or presence of 10 mM methylene blue or 1 microM methemoglobin. Cyclic GMP accumulation preceded onset of relaxation elicited by nitric oxide and glyceryl trinitrate and temporally correlated with relaxation induced by sodium nitroprusside and sodium nitrite. Methylene blue simultaneously inhibited cyclic GMP accumulation and relaxation induced by all four relaxants. In contrast to methylene blue, methemoglobin abolished cyclic GMP accumulation and relaxation elicited by nitric oxide without altering responses to glyceryl trinitrate, sodium nitroprusside and sodium nitrite. These findings are consistent with and strongly support an involvement of cyclic GMP formation in vascular smooth muscle relaxation elicited by nitrogen oxide-containing vasodilators.

The mechanism whereby nitroglycerin relaxes vascular smooth muscle remains uncertain. A current hypothesis suggests that nitroglycerin reacts with critical cellular sulfhydryl groups to form an intermediate, which activates guanylate cyclase, resulting in cGMP accumulation and relaxation. This study investigated further the potential involvement of sulfhydryls in nitroglycerin-induced vascular smooth muscle relaxation by evaluating effects of a variety of sulfhydryl alkylating and reducing agents on responses to nitroglycerin and other relaxants in bovine coronary arterial strips submaximally contracted using 30 mM K. Whereas 10(-4) M 5,5'-dithiobis-(2-nitrobenzoic acid), 10(-5) MN-ethylmaleimide, and 10(-4) MN-naphthylmaleimide did not affect nitroglycerin-induced relaxation, 10(-4) MN-ethylmaleimide and 10(-4) M ethacrynic acid significantly inhibited relaxation induced by nitroglycerin. Both ethacrynic acid and N-ethylmaleimide at 10(-4) M also inhibited relaxation induced by sodium nitroprusside. N-ethylmaleimide, but not ethacrynic acid, inhibited relaxation induced by isoproterenol and forskolin. Ethacrynic acid significantly reduced both relaxation and cGMP elevation induced by both 10(-7) M nitroglycerin and 10(-7) M sodium nitroprusside. Ethacrynic acid, but not N-ethylmaleimide, significantly reduced relaxation induced by 8-Br-cGMP. Pretreatment with the sulfhydryl-containing agents N-acetylcysteine, 2-mercaptoethanol, or dithiothreitol, at 10(-3) M did not affect nitroglycerin-induced relaxation in nontolerant arteries. Similarly, N-acetylcysteine and dithiothreitol did not alter the depressed responses to nitroglycerin in arteries in which tolerance to nitroglycerin was induced in vitro. A slight but statistically significant reversal of nitroglycerin-tolerance occurred after treatment of tolerant arteries with 2-mercaptoethanol.(ABSTRACT TRUNCATED AT 250 WORDS)

The objective of this study was to determine the direct actions of nitroprusside and nitroglycerin on the pulmonary vascular bed in the intactchest dog. These widely used nitrogen oxide-containing vasodilator agents decreased pulmonary arterial pressure and increased cardiac output without altering left atrial pressure. Reductions in pulmonary arterial pressure and pulmonary vascular resistance were small under resting conditions, but were enhanced when pulmonary vascular tone was elevated by infusion of a stable prostaglandin analog that increases pulmonary vascular resistance by constricting intrapulmonary veins and upstream segments. In studies in which pulmonary blood flow to the left lower lobe was maintained constant, nitroprusside and nitroglycerin caused small but significant reductions in lobar arterial and small-vein pressures without significantly affecting left atrial pressure. With constant blood flow, lobar vascular pressures that were reduced in response to the vasodilators were more greatly reduced when lobar vascular resistance was increased by infusion of the prostaglandin analog or serotonin. However, when lobar vascular pressures were elevated by passive obstruction of lobar venous outflow, vasodilator responses to nitroprusside and nitroglycerin were not enhanced. These data suggest that nitroprusside and nitroglycerin decrease pulmonary vascular resistance by dilating intrapulmonary veins and upstream segments. These responses were minimal under control conditions but were enhanced when vascular tone was increased. This vasodilator action is independent of passive factors such as changes in pulmonary blood flow or left atrial pressure and is not secondary to an effect of these agents on the systemic circulation. Pulmonary vasodilator responses to nitroprusside and nitroglycerin were, however, found to be dependent on the existing level of vasomotor tone in the pulmonary vascular bed.

Pulmonary vascular responses to the endoperoxide PGH2, an endoperoxide analog, primary prostaglandins (PGE2, PGF2 alpha, and PGD2), arachidonic acid, and PGI2 were compared in the intact chest of the cat and dog. The primary prostaglandins as well as PGH2 and a stable endoperoxide analog all increase lobar arterial pressure in the dog and cat when injected into the lobar artery. Since pulmonary blood flow was held constant with a pump and left atrial pressure was unchanged, the increases in lobar arterial pressure indicate an increase in pulmonary vascular resistance. The endoperoxide analog and PGF2 alpha were very potent pressor agents in the pulmonary vascular bed. The primary prostaglandins and the endoperoxide analog had significant contractile activity in isolated segments of bovine intrapulmonary artery and vein and in canine intrapulmonary vein. The endoperoxide analog. PGF2 alpha, PGD2, and arachidonic acid, in addition to increasing pulmonary vascular resistance, increased bronchomotor tone. In contrast to the effects of the primary prostaglandins and the endoperoxide analog, PGI2 had vasodilator activity in the pulmonary vascular bed and this activity was enhanced when pulmonary vascular tone was elevated. The prostaglandin precursor arachidonic acid produced both increases and decreases in pulmonary vascular resistance, depending on dose and rate of administration, and these responses were blocked by indomethacin. These data indicate that the prostaglandins have marked but diverse activity in the pulmonary vascular bed.

Development of tachyphylaxis to angiotensin-induced contraction was compared in rings of rat aorta with and without functional endothelium. Rat aorta with functional endothelium rapidly developed marked tachyphylaxis to contraction induced by angiotensin II or angiotensin III. Destruction of endothelium significantly depressed, but did not prevent, development of tachyphylaxis to contractile responses induced by repeated exposure to angiotensin II or angiotensin III. The findings suggest that two mechanisms are involved in the development of tachyphylaxis to angiotensin in rat aorta, an endothelium-dependent mechanism and an endothelium-independent mechanism.

The effects of prostaglandins A2, A1, F2 alpha, E2, E2, F1 beta and an analog of PGH2 upon calcium release from mitochondria isolated from bovine intrapulmonary vein and contraction of helical strips of the same tissue were determined. The order of activity of the prostaglandins for calcium release was similar to that for contraction with the exception of the PGH2 analog. It is suggested that prostaglandin A2, F2 alpha, E2 and A1 induced release of mitochondrial calcium may influence the contractile state of bovine intrapulmonary vein. However, the PGH2 analog has a subcellular mechanism other than or in addition to mitochondrial calcium release and is different from the other prostaglandins.

The influence of endothelium on angiotensin II-induced contraction was investigated in rings of rat aorta, bovine coronary artery, bovine intrapulmonary artery and bovine intrapulmonary vein. Destruction of endothelium significantly enhanced angiotensin II-induced contraction in rat aorta and bovine coronary artery, but not in bovine intrapulmonary artery and bovine intrapulmonary vein. Indomethacin (10(-5) M) did not alter angiotensin II-induced contraction in rat aorta or bovine coronary artery. However, hemoglobin (10(-5) M) or methylene blue (10(-5) M) significantly enhanced angiotensin II-induced contraction in rat aorta and bovine coronary artery with, but not without, endothelium. Intimal rubbing did not affect stimulation of phosphoinositide hydrolysis by angiotensin II in rat aorta. The findings demonstrate that angiotensin II-induced contraction in vascular rings can be modulated by endothelium. However, the effect of endothelium apparently depends upon the species and vascular bed from which the vessel is isolated. Results obtained using inhibitors suggest that in rat aorta and bovine coronary artery release of endothelium-derived relaxant factor (EDRF), rather than cyclooxygenase products, is involved in mediating the inhibitory influence of endothelium. Further, similar stimulation of phosphoinositide hydrolysis in intimally rubbed and unrubbed rat aorta suggests that EDRF does not modulate angiotensin II-induced contraction in this vessel by inhibiting angiotensin II stimulation of phosphoinositide hydrolysis.

Recent studies have shown that N-nitroso compounds can activate arterial guanylate cyclase and relax isolated arterial smooth muscle; however, the effects of these substances on the cardiovascular system in the anesthetized cat are unknown. The present study was undertaken to compare the effects of several nitrosoguanidines and a nitrosamine, N-nitrosodimethylamine, on arterial guanylate cyclase activity, isolated arterial smooth muscle tone, and systemic vascular resistance in the anesthetized cat. Intravenous injections and infusions of the nitrosoguanidines glyceryl trinitrate (GTN) and sodium nitroprusside (SNP) decreased systemic arterial pressure. During intravenous infusion of the nitrosoguanidines GTN and SNP, cardiac output was unchanged at the peak of the decrease in aortic pressure, indicating that the nitrosoguanidines GTN and SNP both reduced systemic vascular resistance. In addition, intraarterial injections of the nitrosoguanidines produced dose-dependent decreases in perfusion pressure in the feline mesenteric vascular bed perfused at constant flow. These substances were potent relaxants of isolated arterial smooth muscle and markedly activated arterial guanylate cyclase. In contrast, N-nitrosodimethylamine was devoid of vasodilator activity in vivo and exerted only minimal effects on isolated arterial smooth muscle tone or on arterial guanylate cyclase activity. The present data demonstrate a relationship between guanylate cyclase activation and arterial smooth muscle relaxation and suggest that the vasodilator effects on resistance vessels in vivo in response to selected N-nitroso compounds may involve such a mechanism. Although the significance of the presently reported cardiovascular responses to N-nitroso compounds is uncertain, N-nitroso compounds may represent a previously unrecognized class of substances which can be formed in the body and which possess marked vasodilator activity. It is possible that this vasodilator activity may involve the relaxation of vascular smooth muscle through activation of guanylate cyclase.

The principal objective of this study was to test the hypothesis that nitroprusside relaxes vascular smooth muscle via the reactive intermediate, nitric oxide (NO), and that the biologic action of NO is associated with the activation of guanylate cyclase. Nitroprusside, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and NO elicit concentration-dependent relaxation of precontraced helical strips of bovine coronary artery. Nitroprusside, MNNG and NO also markedly activate soluble guanylate cyclase from bovine coronary arterial smooth muscle and, thereby, stimulate the formation of cyclic GMP. Three heme proteins, hemoglobin, methemoglobin and myoglobin, and the oxidant, methylene blue, abolish the coronary arterial relaxation elicited by NO. Similarly, these heme proteins, methylene blue and another oxidant, ferricyanide, markedly inhibit the activation of coronary arterial guanylate cyclase by NO, nitroprusside and MNNG. The following findings support the view that certain nitroso-containing compounds liberate NO in tissue:heme proteins, which cannot permeate cells, inhibit coronary arterial relaxation elicited by NO, but not by nitroprusside or MNNG; the vital stain, methylene blue, inhibits relaxation by NO, nitroprusside and MNNG; heme proteins and oxidants inhibit guanylate cyclase activation by NO, nitroprusside and MNNG in cell-free mixtures. The findings that inhibitors of NO-induced relaxation of coronary artery also inhibit coronary arterial guanylate cyclase activation suggest that cyclic GMP formation may be associated with coronary arterial smooth muscle relaxation.

Strips of bovine intrapulmonary artery (IPA), intrapulmonary vein (IPV), trachealis muscle and parenchyma were mounted for recording of isometric tension and responses to several vascular smooth muscle relaxants were measured and compared. Although nitroglycerin (GTN) and sodium nitroprusside (SNP) induced concentration-related (10(-11) to 10(-5)) relaxation (up to 80-100%) in IPA, IPV and trachealis muscle, 10(-5) M GTN or SNP induced only 4-5% relaxation in parenchyma. In contrast, all four tissues relaxed well in response to isoproterenol (10(-11) to 10(-5) M), forskolin (10(-5) M), 8-bromo-cyclic GMP (10(-4) M) and 1-methyl-3-isobutylxanthine (10(-5) M). When compared to IPA, IPV and trachealis muscle, the marked hyporesponsiveness of parenchyma to GTN and SNP suggests that the contractile cells which are primarily responsible for potassium-induced tone in bovine parenchymal strips differ markedly from smooth muscle cells in trachealis muscle and large, central IPA and IPV in their responsiveness to the vasodilators, GTN and SNP.

The contractile effects of serotonin (5-hydroxytryptamine, 5-HT) were compared in helical strips of intrapulmonary artery (IPA) and vein (IPV) isolated from lungs of dog, rabbit, cow, and man. 5-HT (10(-8) - 10(-4) M) increased force generation by IPA and IPV from the four species in a concentration-related manner. Sensitivities to 5-HT of IPA and IPV from the four species were similar, with median effective concentrations ranging from 3 X 10(-8) to 3 X 10(-7) M. However, maximal and contractile responses to 5-HT relative to those elicited by potassium were greater in canine and human IPA and bovine IPA and IPV than in rabbit IPA and IPV and canine and human IPV. Results obtained using specific pharmacology blocking agents suggest that 5-HT-induced contraction of canine and rabbit intrapulmonary vessels does not involve alpha-adrenergic mechanisms except, possibly, in rabbit IPV. The contractile effects of 5-HT observed in the present study suggest that 5-HT released from platelets could contribute to increased vascular resistance during pulmonary thromboembolism by actively constricting arterial and venous segments of the pulmonary vascular bed in a variety of species, including man.

Glyceryl trinitrate specifically required cysteine, whereas NaNO2 at concentrations less than 10 mM required one of several thiols or ascorbate, to activate soluble guanylate cyclase from bovine coronary artery. However, guanylate cyclase activation by nitroprusside or nitric oxide did not require the addition of thiols or ascorbate. Whereas various thiols enhanced activation by nitroprusside, none of the thiols tested enhanced activation by nitric oxide. S-Nitrosocysteine, which is formed when cysteine reacts with either NO-2 or nitric oxide, was a potent activator of guanylate cyclase. Similarly, micromolar concentrations of the S-nitroso derivatives of penicillamine, GSH and dithiothreitol, prepared by reacting the thiol with nitric oxide, activated guanylate cyclase. Guanylate cyclase activation by S-nitrosothiols resembled that by nitric oxide and nitroprusside in that activation was inhibited by methemoglobin, ferricyanide and methylene blue. Similarly, guanylate cyclase activation by glyceryl trinitrae plus cysteine, and by NaNO2 plus either a thiol or ascorbate, was inhibited by methemoglobin, ferricyanide and methylene blue. These data suggest that the activation of guanylate cyclase by each of the compounds tested may occur through a common mechanism, perhaps involving nitric oxide. Moreover, these findings suggest that S-nitrosothiols could act as intermediates in the activation of guanylate cyclase by glyceryl trinitrate, NaNO2 and possibly nitroprusside.

The effects of the prostaglandin endoperoxide, PGH2, and a stable endoperoxide analog were compared on the canine pulmonary vascular bed under conditions of controlled blood flow. The analog produced dose-related increases in lobar arterial and small vein pressure when injected into the perfused lobar artery. The increase in resistance in response to the analog was greater during retrograde perfusion than during forward perfusion. These data suggest that the analog increases resistance to flow by constricting pulmonary veins and upstream vessels. The increase in resistance was similar when the lung was perfused with dextran or blood and the response was not different when the lung was ventilated or collapsed. The response was notattenuated by indomethacin, an inhibitor of prostaglandin synthesis. These data suggest that the response to the analog is not related to changes in bronchomotor tone, platelet aggregation, or enhanced prostaglandin synthesis. Although the analog was a potent pressor substance, PGH2 itself produced only small increases in pulmonary vascular resistance. However, PGH2 had greater contractile activity than the analog on isolated helical segments of canine intrapulmonary vein. The present study suggests that endoperoxides may represent an active form of the prostaglandins in the lung but that their effects would be localized in the tissue in which they are formed because of their extremely short biologic half-life.

Relaxation by nitroglycerin, sodium nitrite, and amyl nitrite of bovine coronary arterial smooth muscle was inhibited by the oxidant methylene blue. Methylene blue also inhibited activation of bovine coronary arterial soluble guanylate cyclase by nitroglycerin, which required addition of cysteine. At concentrations less than 10 mM, sodium nitrite required the addition of one of several thiols or ascorbate to activate guanylate cyclase from bovine coronary artery. Guanylate cyclase activation by large amounts (50 microL) of saturated amyl nitrite gas did not require, but was enhanced by, the addition of thiols or ascorbate. However, similar to sodium nitrite, guanylate cyclase activation by smaller amounts (5 microL) of saturated amyl nitrite gas did require the addition of one of various thiols or ascorbate. Methylene blue markedly inhibited guanylate cyclase activation by sodium nitrite in the presence of cysteine or ascorbate and similarly inhibited enzyme activation by amyl nitrite either in the absence or presence of cysteine or ascorbate. These data support the hypothesis that nitrates and nitrites relax vascular smooth muscle by stimulating cyclic GMP formation. The results further suggest that, similar to relaxation and guanylate cyclase activation by nitroso-containing compounds, relaxation and enzyme activation by nitrates and and nitrites may involve the formation of nitric oxide or complexes of nitric oxide as active intermediates.

In anesthetized, mechanically ventilated, and vagotomized cats, we studied the distribution of the bronchodilatory effects of prostaglandin I2 (PGI2) in central and peripheral airways. In the intact-chest cat, bolus intravenous injection of PGI2 in a range of doses from 3 to 100 micrograms produced dose-dependent reversal of 5-hydroxytryptamine- (5HT) induced bronchoconstriction. Bronchodilatory effects were manifested by a pronounced decrease in central airways flow resistance, as estimated by measurement of lung resistance and by an increase in dynamic lung compliance (Cdyn). A similar distribution and magnitude of airway effects were produced by intravenous administration of PGE1 in a range of doses from 0.3 to 10 micrograms. PGI2 significantly reversed the frequency dependence of compliance induced by infusion of 5HT. The PGI2 metabolite, 6-keto-PGF1 alpha, was without effect on pulmonary mechanics. Comparison of right- versus left-heart administration of PGI2 did not point to a significant role of the bronchial circulation in the airway effects of PGI2. Comparison of the effects of PGI2 and PGE1 on cat bronchial and parenchymal strips contracted by 5HT showed both prostaglandins to have predominant action on bronchial smooth muscle with PGI2 being 3-10 times less active than PGE1. These data suggest that PGI2 has central airway bronchodilator activity, similar to, but less potent than PGE1. In addition, they suggest that PGI2 has significant dilator activity in certain peripheral areas of the lung, as well.

The present study investigated possible involvement of cysteine (CSH) and reduced glutathione (GSH) as critical cellular sulfhydryls which mediate nitroglycerin (GTN)-induced cyclic GMP accumulation and relaxation in bovine coronary artery (BCA). Tolerance to the relaxant effects of GTN was induced in BCA in vitro by preincubation with 1 mM GTN for 2 h. GTN-tolerant BCA were at least 100-fold less sensitive than non-tolerant BCA to the relaxant effects of GTN. Consistent with a relationship between tolerance to both GTN-induced cyclic GMP accumulation and relaxation, cyclic GMP accumulation induced by 1 microM GTN was markedly reduced in GTN-tolerant BCA when compared with non-tolerant BCA. Incubation with 1 mM CSH for 1 h did not significantly alter GTN-induced cyclic GMP accumulation or relaxation in either GTN-tolerant or non-tolerant BCA. Levels of CSH, GSH and glutathione-disulfide (GSSG) were measured in non-tolerant BCA, GTN-tolerant BCA and GTN-tolerant BCA incubated with 1 mM CSH for 1 h. Levels of CSH and GSH were lower in GTN-tolerant BCA than in non-tolerant BCA, whereas GSSG levels were similar in both. In GTN-tolerant BCA incubated with 1 mM CSH, CSH levels were more than 10-fold above, and GSH levels were similar to corresponding values obtained in non-tolerant BCA. These data indicate that although incubation with CSH did not significantly reverse tolerance to GTN-induced cyclic GMP accumulation and relaxation in BCA, it did effectively raise the level of CSH and GSH in GTN-tolerant BCA, at least to corresponding levels found in non-tolerant BCA. These results indicate that the relaxant effects of GTN in BCA do not correlate with tissue levels of CSH and GSH. The findings do not support the hypothesis that CSH and GSH are the cellular sulfhydryls involved in mediating GTN-induced guanylate cyclase activation, cyclic GMP accumulation and relaxation in intact BCA.

Bradykinin induced relaxation and cyclic GMP accumulation in both bovine intrapulmonary artery and vein. Both the relaxant responses and the accompanying cyclic GMP accumulations were abolished or markedly reduced by intimal rubbing or pretreatment with the guanylate cyclase inhibitor, methylene blue. These findings indicate that both bovine intrapulmonary artery and vein exhibit endothelium-dependent relaxation in response to bradykinin, and that the relaxant responses in both vessels are associated with cyclic GMP accumulation.

Nitrates are generally regarded as bronchial smooth muscle relaxants. However, reports about the clinical efficacy of the nitrates in the treatment of asthma are conflicting. In the present study, the relaxant effects of glyceryl trinitrate, isosorbide dinitrate, sodium nitroprusside, and isoproterenol were measured and compared in vitro in airway preparations isolated from variously sized airways in bovine lung. Strips of trachealis from cervical and thoracic ends of the trachea and rings of intrapulmonary bronchi of various diameters (14 to 1 mm outer diameters) were mounted for recording of isometric tension. Submaximal tone was induced using 10(-6) M carbachol, and responses to cumulatively increasing concentrations of glyceryl trinitrate, isosorbide dinitrate, sodium nitroprusside, or isoproterenol were measured. Isoproterenol (10(-8) to 10(-4) M) was equiactive as a relaxant in all preparations. Glyceryl trinitrate (10(-7) to 10(-5) M) relaxed trachealis, but was progressively and significantly less effective as a relaxant as the diameters of the bronchial rings decreased. Although less potent than isoproterenol, sodium nitroprusside (10(-6) to 10(-4) M) was effective in relaxing all the airway preparations. Similar to glyceryl trinitrate, sodium nitroprusside was less effective in small than in large airways. However, the relationship between relaxant responses to sodium nitroprusside and airway size was not as marked as for glyceryl trinitrate. Isosorbide dinitrate at concentrations as great as 10(-5) M did not relax carbachol-induced tone in any of the bronchial rings and only induced a small amount of relaxation in trachealis at 10(-5) M.(ABSTRACT TRUNCATED AT 250 WORDS)