Evidence of histamine receptor function in isolated horse penile dorsal arteries

The effect of histamine (10(-9)-10(-3) M) on horse penile dorsal artery was evaluated. Precontracted vessels showed a biphasic response (relaxation-contraction) to histamine, while at basal tone, histamine only induced a contractile effect. The H1 receptor agonist, 2-pyridylethylamine (PEA) (10(-9)-10(-3) M), induced concentration-dependent relaxation in precontracted rings and provoked vasoconstriction at basal tone. Mepyramine (10(-9)-10(8) M), an H1 receptor antagonist, competitively antagonized the relaxant response to histamine (pA2 = 9.7) and PEA (pA2 = 9.2). At basal tone, mepyramine (10(-10)-10(-8) M) also caused a rightward shift in the histamine contraction curve (pA2 = 10.1). Mepyramine (10(-9)-10(-8) M)/PEA Schild plots for resting vessels yielded a pA2 value of 9.4. A regulatory role for H2 and H3 receptors was precluded since there was no response to their agonists (dimaprit (10(-9)-10(-3) M), (R)-alpha-methylhistamine (10(-10)- 3 x 10(-4) M)), and antagonists (cimetidine (10(-5) M), thioperamide (10(-6) M)) did not affect control curves. Removal of the endothelium abolished the relaxant component causing a leftward shift in the contractile component in precontracted rings, with no effect on maximum contraction. Inhibitors of nitric oxide (NO) synthesis, L-NAME (3 x 10(-4) M) and L-NOARG (3 x 10(-4) M), modified the relaxant response while contraction was unaffected. L-Arginine (3 x 10(-4) M) potentiated maximum relaxation but did not affect contraction in precontracted rings. Effects of a prostanoid and K+ channels were ruled out. The biphasic response of precontracted vessels persisted in the presence of indomethacin (3 x 10(-6) M), tetraethylammonium (10(-3) M) and gliblenclamide (10(-5) M). L-NAME plus indomethacin, or this combination plus TEA or glibenclamide produced similar effects as isolated treatments. In resting vessels, histamine contraction was also unaffected by the lack of endothelium, or L-NAME, L-arginine or indomethacin pretreatment. The biphasic response to histamine is probably mediated by H1 receptors with a partial role for NO in the relaxant response in precontracted vessels. In the absence of tone, the contractile effect may be mediated by direct action on smooth muscle.     

Mechanisms of vascular wall calcium relaxation.

Using the method of isometric tension measurement in isolated blood vessels, we investigated some mechanisms of action of high calcium concentrations (>3 mM) on the mechanical activity of small branches of the rat mesenteric artery. Calcium in concentrations up to 30 mM caused relaxation of the arteries (calcium relaxation). The amplitude of the effect decreased in the presence of ouabain (10(-4) M), tetraethylammonium (10(-3) M), charibdotoxin (10(-7) M) and in the potassium-free external solution in intact and denuded rings. Glibenclamide (10(-6) M), 4-aminopyridine (10(-3) M), barium (10(-3) M) and cesium (2.10(-2) M) were inefficient. Calcium relaxation of intact vessels was impaired in the presence of N(omega)-nitro-L-arginine (10(-4) M) or methylene blue (10(-4) M) but not in the presence of indomethacin (10(-5) M). The attenuation of calcium relaxation to the same extent was observed in denuded mesenteric arteries. We conclude that calcium can cause relaxation of vascular smooth muscle cells by two mechanisms. The first is mediated via the cell membrane hyperpolarization due to the activation of Na+/K(+)-ATPase and Ca(2+)-activated potassium channels. The second mechanism is endothelium-mediated and depends on the nitrogen monoxide-guanylate cyclase pathway.     

Effects of alpha calcitonin gene-related peptide in human bronchial smooth muscle and pulmonary artery

Although airway and pulmonary vessel tone are regulated predominantly by cholinergic and adrenergic impulses, biologically active peptides such as calcitonin gene-related peptide (CGRP) may significantly influence human smooth muscle tone in normal and pathophysiological states. In the present study, the expression of CGRP and its receptor CGRPR-1 and the biological effect of the peptide were investigated in human airways and pulmonary arteries. Immunohistochemistry revealed the presence of CGRP in human airway nerves and neuro-epithelial cells, whereas the receptor was found in epithelial cells and smooth muscle myocytes of the bronchi and in pulmonary artery endothelium. On precontracted bronchi (3-4 mm in diameter) alpha-CGRP (0.01-10 nM) caused a concentration-dependent contraction on epithelium-denuded bronchi, whereas no significant effect was recorded in bronchi with intact epithelium. In pulmonary arteries (2-6 mm in diameter), alpha-CGRP caused a concentration-dependent relaxation of endothelium intact and denuded vessels. Pre-treatment with indomethacin, but not with l-NAME, prevented the relaxation induced by alpha-CGRP in pulmonary arteries suggesting that prostaglandins but not nitric oxide (NO) are involved in the intracellular signal transduction pathway. The effects induced by alpha-CGRP in bronchi and vessels were prevented by application of the antagonist CGRP((8-37)). In summary, the present studies examined the biological function of CGRP in human airways and demonstrated a constrictory effect of CGRP only in epithelium-denuded airway smooth muscle indicating an alteration of CGRP airway effects in respiratory tract pathological states with damaged epithelium such as chronic obstructive pulmonary disease or bronchial asthma.     

Sphingomyelinase and ceramide analogs induce contraction and rises in [Ca(2+)](i) in canine cerebral vascular muscle

Studies were designed to investigate effects of neutral sphingomyelinase (N-SMase) and ceramide analogs as well as phosphorylcholine on vascular tone and Ca(2+) mobilization in isolated canine cerebral arterial smooth muscle. N-SMase (0.001-0.1 U/ml) provoked a gradual but sustained vasoconstriction of arterial rings in a concentration-related manner that was endothelium independent. Incubation of denuded arterial rings in Ca(2+)-free medium or pretreatment with verapamil in extracellular Ca(2+) resulted in a reduction of the N-SMase-evoked constriction. Exposure of arterial rings to 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid (BAPTA)-AM did not, however, result in a reduction of N-SMase-induced constriction. Both staurosporine and bisindolymaleimide I attenuated N-SMase-induced contractions to 66% and 72% of control, respectively. N-SMase caused gradual and sustained rises in intracellular Ca(2+) concentration ([Ca(2+)](i)) in primary cultured cerebral vascular smooth muscle cells. Pretreatment of these cultured cells with nimodipine and verapamil caused a steady decline in N-SMase-induced rises in [Ca(2+)](i). Exposure of the cells to Ca(2+)-free solution reversed the [Ca(2+)](i)-induced rise triggered by N-SMase to the resting baseline. Both C(8) and C(16) ceramide (10(-9)-10(-6) M), but not phosphorylcholine, constricted denuded canine arterial rings in a concentration-related manner and elevated [Ca(2+)](i). Our results suggest that the sphingomyelin-signaling pathway, via a probable release of ceramide molecules, may play an important role in regulation of cerebral arterial wall tone.     

Guanylyl cyclase mediates ANP-induced vasoconstriction of murine splenic vessels

We have previously shown that ANP causes differential constriction of the splenic vasculature of the rat (veins greater than arteries), which may be inhibited by blocking the production of cGMP with A7195. In this paper, we report experiments done on vessels derived from guanylyl cyclase (GC)-A knockout mice. Small splenic arteries ( approximately 150-microm diameter) and veins ( approximately 250-microm diameter) were dissected from male GC-A-deficient 129sv mice or age-matched wild-type controls and mounted in a wire myograph. In the wild-type mice, ANP exhibited higher potency in the veins than in the arteries (EC(50) values wild-type mice: artery, 8 +/- 3 x 10(-9) M, n = 5 vs. vein, 6 +/- 4 x 10(-10) M, n = 5; P < 0.05). The concentration-response curve for ANP-induced vasoconstriction was also shifted leftward in denuded compared with intact arteries (EC(50) values: denuded artery: 5 +/- 3 x 10(-10) M, n = 5 vs. intact artery, 8 +/- 3 x 10(-9) M, n = 5; P < 0.05), i.e., the denuded vessels were more reactive. By contrast, ANP caused no significant change in tension from baseline in intact splenic arteries, intact splenic veins, or denuded splenic arteries derived from the GC-A-deficient mice, although these vessels did show normal concentration-dependent increases in tension to phenylephrine. We conclude that ANP causes vasoconstriction in the splenic vasculature by an endothelium-independent mechanism, mediated via guanylyl cyclase.     

Acetylcholine but not adenosine triggers preconditioning through PI3-kinase and a tyrosine kinase

Acrolein is a highly reactive aldehyde pollutant and an endogenous product of lipid peroxidation. Increased generation of, or exposures to, acrolein incites pulmonary and vascular injury. The effects of acrolein on the vasomotor responses of rat aortic rings were studied to understand its mechanism of action. Incubation with acrolein (10-100 microM) alone did not affect the resting tone of aortic vessels; however, a dose-dependent relaxation of phenylephrine-precontracted aortic rings was observed. Acrolein-induced relaxation was slow and time dependent and the extent of relaxation after 100 min of application was 44.7 +/- 4.1% (10 microM), 56.0 +/- 5.6% (20 microM), 61.0 +/- 7.9% (40 microM), and 96.1 +/- 2.1 (80 microM), respectively, versus 14.2 +/- 3.3% relaxation in the absence of acrolein. Acrolein-induced vasorelaxation was prevented by endothelial denudation and was abolished on pretreatment with the nitric oxide synthase inhibitor Nomega-nitro-L-arginine methyl ester, the guanylyl cyclase inhibitor 1H-[1,2,4]oxidazolo[4,3-a]quinoxaline-1-one, or the cyclooxygenase inhibitor indomethacin. Inhibition of K+ channels (by tetrabutylammonium) or Na+-K+-ATPase (by ouabain) did not significantly prevent acrolein-mediated vasorelaxation. Exposure to acrolein in the presence or absence of other compounds elicited slow wave vasomotor effect in 77% of aortic vessels versus 1.4% in control. Vasomotor responses were also studied on aortic rings prepared from rats fed 2 mg. kg-1. day-1 acrolein for 3 alternate days by oral gavage. These vessels developed a significantly lower contractile response to phenylephrine compared with controls. Together, these results indicate that acute acrolein exposure evokes delayed vasorelaxation due to a nitric oxide- and prostacyclin-dependent mechanism, whereas in vivo acrolein exposure compromises vessel contractility.     

Relaxing effect of insulin in renal arteries from diabetic rats

Abnormal renal vasomotor tone exists in the early stages of diabetes mellitus. Insulin has been proposed to modulate renal function and to possess vasodilatory effects. The present study was initiated in order to evaluate the direct effect of insulin on isolated renal arteries. Twelve insulin-treated streptozotocine diabetic rats with diabetes for 50 days were compared with 15 weight-matched control rats. The contractile responses to 60 mM K+ and 10(-4) M noradrenaline, and the insulin- (0.8-6.4 I.U./ml) induced relaxation of vessels precontracted with noradrenaline, were similar in diabetic and control rats. There was a tendency towards greater relaxation in diabetic (71%) than in control rats (54%). Nw-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) given before noradrenaline tended to attenuate the insulin-induced relaxation, while addition of L-arginine (10(-6) M) to L-NAME attenuated the relaxation in diabetic but increased it in control rats (P < 0.05). The effect of insulin was tested further in control rats and was not influenced by administration of a single dose (10(-6) M) of indomethacin or propranolol given instead of L-NAME. The effect of a single dose of methylene-blue, given before noradrenaline, was tested in control rats in varying doses between 2 x 10(-6) and 2 x 10(-4) M. In the highest concentration it made no difference whether insulin was given or not and there was a similar relaxing effect in diabetic and control arteries. In conclusion, the present study showed that insulin per se has a relaxing effect on renal arteries. There was a tendency to greater relaxation in diabetic than in control rats, an effect which was attenuated by in-vitro-pretreatment with L-NAME as well as with L-NAME and L-arginine in diabetic vessels, while relaxation was increased in control vessels. This may indicate that the effect of insulin may be mediated through nitric oxide in diabetic but not in control rats. The effects of insulin in control vessels were not modified in vitro by indomethacin, propranolol or methylene-blue.     

Control of cardiovascular function in the icefish Chionodraco hamatus: involvement of serotonin and nitric oxide

The involvement of nitric oxide (NO) in the branchial circulation and cardiac performance of the Antarctic hemoglobinless icefish Chionodraco hamatus was investigated using isolated and perfused head and working heart preparations. In the branchial vasculature under basal (i.e. unstimulated conditions), the nitric oxide synthase (NOS) inhibitor L-NIO (L-N(5)-(1-iminoethyl) ornithine, 10(-5) and 10(-4) M), caused a marked vasoconstriction (20%), indicating a basal nitrergic vasodilator tone, while the dose-response curve of the NO donor SIN-1 (3-morpholinosydnonimine) showed a dose-dependent vasodilator effect. Acetylcholine induced a dose-dependent branchial vasoconstriction mediated by muscarinic receptors, since the effects were abolished by pre-treatment with atropine (10(-4) M). Serotonin (5-HT) induced a dose-dependent branchial methysergide-sensitive vasoconstriction which was abolished by pre-treatment with L-NIO, indicating a NO-dependent mechanism. On the isolated heart, the NOS inhibitor L-NMMA (N(G)-monomethyl-L-arginine) 10(-4) M produced a small, but significant decrease of heart rate and, as a consequence, a decrease of power output, while the NO donor sodium nitroprusside (SNP) 10(-4) M elicited increases of stroke volume, stroke work and power output, suggesting an exogenous NO-dependent positive inotropism. Exposure of the bulbus arteriosus to L-NMMA was without any appreciable effect. In contrast, SNP produced a notable relaxation of the bulbus wall with a marked increase of its stiffness, as indicated by the increase of systolic and diastolic dP/dt max (23 and 104%, respectively).     

BK(Ca) channels compensate for loss of NOS-dependent coronary artery relaxation in cardiomyopathy

Previously, we showed that development of myocardial necrotic lesions is associated with impaired endothelium-dependent coronary artery relaxation in young cardiomyopathic hamsters. Since active necrosis declines with aging, this study was designed to determine whether coronary artery endothelium-dependent relaxation to ACh is restored and to identify the mechanisms mediating this effect. Intraluminal diameter was recorded in coronary arteries (150-250 micrometer) from control (C, 297 +/- 5 days old) and cardiomyopathic (M, 296 +/- 4 days old) hamsters. Relaxation to ACh (10(-9)-3 x 10(-5) M) was similar in vessels from C and M hamsters. However, mechanisms mediating relaxation to ACh were altered. Inhibition of nitric oxide synthase (NOS) activity with N-nitro-L-arginine (1 mM) had a greater inhibitory effect in vessels from C hamsters, indicating a reduction in NOS-dependent relaxation in vessels from M hamsters. Conversely, inhibition of large Ca(2+)-dependent K(+) (BK(Ca)) channels with charybdotoxin (CTX, 0.1 microM) had a greater inhibitory effect in vessels from M hamsters. In the presence of both N-nitro-L-arginine and CTX, relaxation to ACh was abolished in both groups. CTX (0.1 micrometer) produced a 50 +/- 4 and 30 +/- 3% contraction of vessels from M and C hamsters, respectively, indicating an enhanced role for BK(Ca) channels in regulation of coronary artery tone in M hamsters. Finally, vasodilatory cyclooxygenase products contributed to ACh-induced relaxation in vessels from M, but not C, hamsters. In conclusion, NOS-dependent relaxation of coronary small arteries is reduced in the late stage of cardiomyopathy. An increase in relaxation mediated by BK(Ca) channels and vasodilatory cyclooxygenase products compensates for this effect.     

Nitric oxide-mediated vasorelaxation by Rhizoma Ligustici wallichii in isolated rat thoracic aorta.

The vasorelaxant effect of Rhizoma Ligustici wallichii and its possible mechanism of action on the vasomotor tone of the rat thoracic aortic rings were examined in an organ bath. Chloroform extracts of Rhizoma Ligustici wallichii (Ch1LW) elicited a dose-dependent, transient, relaxing response in endothelium-intact rat aorta contracted with norepinephrine (NE). This relaxant effect was abolished by removal of the endothelium and also by pretreatment with nitric oxide synthase inhibitors. Neither a muscarinic receptor antagonist nor a cyclooxygenase inhibitor altered the Ch1LW-induced relaxation. Tetramethylpyrazine, derived from Rhizoma Ligustici wallichii as a potent vasodilating component, induced a complete relaxation in both endothelium-intact and denuded rat aortas contracted by NE, but nitric oxide synthase inhibitors did not affect the relaxation. Ch1LW-induced endothelium-dependent relaxation was mediated by nitric oxide released from the endothelium, and could be caused by component(s) other than tetramethylpyrazine.     

Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several mediators of vasodilation, which include prostacyclin (PGI(2)), nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently defined the role of nitric oxide and PGI(2) in the dilation of submucosal intestinal arterioles from patients with normal bowel function. However, significant endothelium-dependent dilator capacity to ACh remained after inhibiting both these mediators. The current study was designed to examine the potential role of EDHF in human intestinal submucosal arterioles. ACh elicited endothelium-dependent relaxation in the presence of inhibitors of nitric oxide synthase and cyclooxygenase (23 +/- 10%, n = 6). This ACh-induced relaxation was inhibited and converted to constriction by catalase (-53 +/- 10%, n = 6) or KCl (-30 +/- 3%, n = 7), whereas 17-octadecynoic acid and 6-(2-propargylloxyphenyl) hexanoic acid, two inhibitors of cytochrome P450 monooxygenase, had no significant effect (3 +/- 1% and 20 +/- 8%, n = 5, respectively). Exogenous H(2)O(2) elicited dose-dependent relaxation of intact microvessels (52 +/- 10%, n = 7) but caused frank vasoconstriction in arterioles denuded of endothelium (-73 +/- 8%, n = 7). ACh markedly increased the dichlorofluorescein fluorescence in intact arterioles in the presence of nitric oxide synthase and cyclooxygenase inhibitors compared with control and compared with catalase-treated microvessels (363.6 +/- 49, 218.8 +/- 10.6, 221.9 +/- 27.9, respectively, P < 0.05 ANOVA, n = 5 arbitrary units). No changes in the dichlorofluorescein fluorescence were recorded in vessels treated with ACh alone. These results indicate that endothelial production of H(2)O(2) occurs in response to ACh in human gut mucosal arterioles but that H(2)O(2) is not an EDHF in this tissue. Rather, we speculate that it stimulates the release of a chemically distinct EDHF.     

Folic acid improves acetylcholine-induced vasoconstriction of coronary vessels isolated from hyperhomocysteinemic mice: an implication to coronary vasospasm

Human atherosclerotic coronary vessels elicited vasoconstriction to acetylcholine (Ach) and revealed a phenomenon of vasospasm. Homocysteine (Hcy) levels are elevated in the atherosclerotic plaque tissue, suggesting its pathological role in endothelial damage in atherosclerotic diseases. Accordingly, we examined the role hyperhomocysteinemia in coronary endothelial dysfunction, vessel wall thickness, lumen narrowing, leading to acute/chronic coronary vasospasm. The therapeutic potential and mechanisms of folic acid (FA) using hyperhomocysteinemic cystathionine beta synthase heterozygote (CBS-/+) and wild type (CBS+/+) mice were addressed. The CBS-/+ and CBS+/+ mice were treated with or without a Hcy lowering agent FA in drinking water (0.03 g/L) for 4 weeks. The isolated mouse septum coronary artery was cannulated and pressurized at 60 mmHg. The wall thickness and lumen diameters were measured by Ion-Optic. The vessels were treated with Ach (10(-8) -10(-5) M) and, for comparison, with non-endothelial vasodilator sodium nitroprusside (10(-5) M). The endothelium-impaired arteries from CBC-/+ mice constricted in response to Ach and this vasoconstriction was mitigated with FA supplementation. The level of endothelial nitric oxide synthase (eNOS) was lower in coronary artery in CBS-/+ than of CBS+/+ mice. Treatment with FA increased the levels of Ach-induced NO generation in the coronary artery of CBS-/+ mice. The results suggest that Ach induced coronary vasoconstriction in CBS-/+ mice and this vasoconstriction was ameliorated by FA treatment. The mechanisms for the impairment of vascular function and therapeutic effects of FA may be related to the regulation of eNOS expression, NO availability and tissue homocysteine.     

Differential effects of adenosine and verapamil on histamine vascular contractions

The present study was undertaken to compare the effects of adenosine and verapamil on histamine-induced contractions in rabbit vascular smooth muscle. Ring segments of rabbit femoral artery were isometrically mounted and contractile responses to histamine (10(-7) to 10(-4) M) were recorded. Verapamil (10(-5) to 10(-4) M) and adenosine (10(-5) to 10(-4) M) produced significant (P less than 0.05) shifts to the right of the histamine dose-response curve in normal physiological salt solution (PSS). Adenosine (10(-4) M) had no effect on the contractile responses to histamine in calcium-deplete PSS but significantly (P less than 0.01) increased the rate of relaxation (-dT/dt, 16.1 +/- 2.3 mg/s before adenosine, 53.7 +/- 7.0 mg/s during adenosine). In calcium-free PSS, verapamil (10(-4) M) had no effects on histamine-induced contractions, nor did it affect the spontaneous rate of relaxation. These findings suggest that the relaxant responses to adenosine, like verapamil, are partially mediated through blockade of external calcium influx, while adenosine, unlike verapamil, appears to have an additional intracellular mode of action.     

Endothelial modulation and tolerance development in the vasorelaxant responses to nitrate of rabbit aorta

We investigated the endothelial modulations in nitrate tolerance in isolated rabbit aorta. Nitrate tolerance was induced by a 72-h treatment with transdermal nitroglycerin (NTG, 0.4 mg/h) in conscious rabbits, which was verified by a 20-fold increase in the EC50 values [NTG tolerance (6.1 +/- 0.8) x 10(-7) M vs control (3.0 +/- 0.6) x 10(-8) M]. The relaxations to NTG in tolerant and nontolerant aortic strips were enhanced when their endothelia were denuded [E(-)]. In the presence of endothelium [E(+)], NTG-tolerant vessels were not tolerant to acetylcholine (ACh), which can release endothelial nitric oxide (NO), exogenous NO or 8-bromo (Br)-cGMP. In NTG-tolerant and nontolerant vessels with endothelium, concentration-response curves for NO were the same as those in endothelium-absent tolerant vessels. In both NTG-tolerant and nontolerant vessels, treatment with superoxide dismutase (SOD, 20 units/ml), an O2-. scavenger, unaffected the responses to NTG reduced in the presence of endothelium, but treatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), an NO synthase (NOS) inhibitor, reversed these reductions. Thus, our data did not indicate that an increased endothelial superoxide O2-. production contributes to nitrate tolerance. Our study suggested that (i) an impaired biotransformation process from NTG to NO is responsible for the occurrence of nitrate tolerance and (ii) vascular response to NTG enhanced by endothelial removal is related to blocked endothelial NO release.     

ET-1-induced pulmonary vasoconstriction shifts from ET(A)- to ET(B)-receptor-mediated reaction after preconstriction.

Endothelin-1 (ET-1) has been reported to induce pulmonary vasoconstriction via either ET(A) or ET(B) receptors, and vasorelaxation after ET-1 injection has been observed. Our study investigated the effects of ET-1 in isolated rabbit lungs, which were studied at basal tone (part I) and after preconstriction (U-46619; part II). Pulmonary arterial pressure (PAP) and lung weight gain were monitored continuously. In part I, ET-1 (10(-8) M; n = 6; control) was injected after pretreatment with the ET(A)-receptor antagonist BQ-123 (10(-6) M; n = 6) or the ET(B)-receptor antagonist BQ-788 (10(-6) M; n = 6). The same protocol was carried out in part II after elevation of pulmonary vascular tone. ET-1 induced an immediate PAP increase (DeltaPAP 4.3 +/- 0.4 mmHg at 10 min) that was attenuated by pretreatment with BQ-123 (P < 0.05 at 10 min and P < 0.01 thereafter) and that was more pronounced after BQ-788 (P < 0.01 at 10 min and P < 0.001 thereafter). In part II, ET-1 induced an immediate rise in PAP with a maximum after 5 min (DeltaPAP 6.3 +/- 1.4 mmHg), leveling off at DeltaPAP 3.2 +/- 0.2 mmHg after 15 min. Pretreatment with BQ-123 failed to attenuate the increase. BQ-788 significantly reduced the peak pressure at 5 min (0.75 +/- 0.4 mmHg; P < 0.001) as well as the plateau pressure thereafter (P < 0.01). We conclude that ET-1 administration causes pulmonary vasoconstriction independent of basal vascular tone, and, at normal vascular tone, the vasoconstriction seems to be mediated via ET(A) receptors. BQ-788 treatment resulted in even more pronounced vasoconstriction. After pulmonary preconstriction, ET(A) antagonism exerted no effects on PAP, whereas ET(B) antagonism blocked the PAP increase. Therefore, ET-1-induced pulmonary vasoconstriction is shifted from an ET(A)-related to an ET(B)-mediated mechanism after pulmonary vascular preconstriction.     

Possible role of nitric oxide and arachidonic acid pathways in hypoxia-induced contraction of rabbit coronary artery rings

In isolated coronary arteries, hypoxia induces an increase in tone by releasing an unidentified endothelium-derived contracting factor (EDCF). Isometric force was measured in an isolated rabbit coronary artery ring at 37 degrees C in control and high K+ (40 mM) pre-contracted conditions. Hypoxia (15 mmHg pO2) induced by equilibrating the perfusate with nitrogen. Hypoxia did not affect the resting tone but induced an endothelium-dependent contraction on pre-contracted rings. Inhibitors of nitric oxide (NO) were tested, L-NAME (10(-4) M) totally and L-NMMA (10(-4) M) partially convert the hypoxic contraction to an hypoxic relaxation. The addition of L-arginine (10(-4) or 10(-3) M) did not restore the response. Methylene blue (10( -5) M) and ODQ (1 H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one, 10(-5) M), both inhibitors of guanylate cyclase, also changed the hypoxic contraction into a hypoxic relaxation. Catalase (1200 U/ml), which decomposes hydrogen peroxide (H2O2), and superoxide dismutase (150 U/ml, SOD), a free radical scavenger, did not change the hypoxic response but quinacrine (50 microM), an inhibitor of phospholipase A2, significantly decreased it. Inhibitors of arachidonic acid metabolism (indomethacin, diethylcarbamazine, miconazole) however did not affect the hypoxic response. We conclude that in K+ pre-contracted rabbit coronary artery rings, hypoxia induces a contraction which is nitric oxide and arachidonic acid dependent.     

Regulation of phospholipase D activity in synaptosomes permeabilized with Staphylococcus aureus α-toxin

In order to investigate the regulation of presynaptic phospholipase D (PLD) activity by calcium and G proteins, we established a permeabilization procedure for rat cortical synaptosomes using Staphylococcus aureus α-toxin (30–100 μg/ml). In permeabilized synaptosomes, PLD activity was significantly stimulated when the concentration of free calcium was increased from 0.1 μM to 1 μM. This activation was inhibited in the presence of KN-62 (1 μM), an inhibitor of calcium/calmodulin-dependent kinase II (CaMKII), but not by the protein kinase C inhibitor, Ro 31-8220 (1–10 μM). Synaptosomal PLD activity was also stimulated in the presence of 1 μM GTPγS. When Rho proteins were inhibited by pretreatment of the synaptosomes with Clostridium difficile toxin B (TcdB; 1–10 ng/ml), the effect of GTPγS was significantly reduced; in contrast, brefeldin A (10–100 μM), an inhibitor of ARF activation, was ineffective. Calcium stimulation of PLD was inhibited by TcdB, but GTPγS-dependent activation was insensitive to KN-62. We conclude that synaptosomal PLD is activated in a pathway which sequentially involves CaMKII and Rho proteins.     

Role of nitric oxide on the vasorelaxant effect of atrial natriuretic peptide on rabbit aorta basal tone

The role of nitric oxide (NO) on the vasorelaxant effect of atrial natriuretic peptide (ANP) on the basal tone of rabbit aortic rings conditioned to angiotensin II (Ang II) was studied. ANP aortic relaxation and nitrite release were measured in the presence and absence of endothelium and a NO-synthase inhibitor. Ang II at 10(-8) M triggered a contractile response, conditioning the vessel to a vasorelaxant effect of ANP (10(-8) M). This effect was significantly enhanced by endothelium removal, NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), and methylene blue (10(-5) M). ANP decrease of basal tone in Ang-II-sensitized aortic rings was improved when a higher concentration of Ang II was used (l0(-6) M). Basal and Ang-II-stimulated nitrite release were measured in stretched (S) and nonstretched (NS) aortic rings. Nitrite release was significantly increased in S rings (p < 0.001). L-NAME (10(-4) M) partially inhibited nitrite release in both basal and Ang-II-stimulated S aortic rings. In NS aortic rings, the NO inhibitor did not inhibit basal nitrite release but blunted the Ang-II-stimulated nitrite level. A significant negative correlation between nitrite release and the ANP vasorelaxant effect on basal tone was dependent on the Ang-II-sensitizing dose. The present results demonstrate that ANP relaxant effects on aortic basal tone are related to NO levels, which are regulated by S- and Ang-II-concentration-dependent NO generation and quenching.     

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.     

Vasoactive effects of substance P on isolated rabbit pulmonary artery

The vasoactive properties of substance P (SP) were studied in isolated rabbit pulmonary artery (PA) segments in vitro. In the absence of active base-line tone, noncumulative administration of SP (10(-11) to 10(-4) M) produced dose-dependent increases in PA tension. The peak isometric tension (Tmax) with SP was similar to the Tmax response to epinephrine; however, the doses of the agonist producing a threshold contraction and 25% of Tmax (ED25) were significantly lower for SP. In the presence of active base-line tone, induced by epinephrine or 5-hydroxytryptamine, SP produced transient PA relaxation which was directly related to the magnitude of the precontracted PA tension. Blockade of neurotransmission with tetrodotoxin (1 microgram/ml) and antagonists to alpha 1-adrenergic and histamine receptor binding had no effect on the contractile response to SP. On the other hand, PA contraction to an ED50 dose of SP was 1) inhibited by a mean of 33 +/- 10% (SE) following pretreatment with the cholinesterase inhibitor, neostigmine (10(-6) M) and 2) augmented by 52 +/- 21% with the cholinergic antagonist, atropine (10(-4) M). The latter also completely blocked the relaxation response to SP in precontracted PA. Similarly, removal of the PA endothelium also abolished the relaxation response to SP. In contrast, SP-induced contraction was markedly inhibited by the cyclooxygenase inhibitor, meclofenamate (1 microgram/ml), as well as the SP antagonist, D-Pro2, D-Trp7,9-SP.(ABSTRACT TRUNCATED AT 250 WORDS)