Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO.

The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat. Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide [LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung] and killed 4 h later. SPA (300- to 600-micrometer outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2alpha, and exposed to either severe (bath PO2 8 +/- 3 mmHg) or milder (21 +/- 3 mmHg) hypoxia. In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME), 10(-3) M]. In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by L-NAME. The milder hypoxia elicited no increase in vascular tone. These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA. The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS. The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease

Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels.

Chronic nitric oxide synthase inhibition and carotid artery distensibility in renal hypertensive rats.

The goal of the present study was to examine the viscoelastic properties of the carotid artery in genetically identical rats exposed to similar levels of blood pressure sustained by different mechanisms. Eight-week old male Wistar rats were examined 2 weeks after renal artery clipping (two-kidney, one clip [2K1C] Goldblatt rats, n = 53) or sham operation (n = 49). One half of the 2K1C and sham rats received the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1.48 mmol/L) in their drinking water for 2 weeks after the surgical procedure. Mean blood pressure increased significantly in the 2K1C-water (182 mm Hg), 2K1C-L-NAME (197 mm Hg), and sham-L-NAME (170 mm Hg) rats compared with the sham-water rats (127 mm Hg). Plasma renin activity was not altered by L-NAME but significantly enhanced after renal artery clipping. A significant and similar increase in the cross-sectional area of the carotid artery was observed in L-NAME and vehicle-treated 2K1C rats. L-NAME per se did not modify cross-sectional area in the sham rats. There was a significant upward shift of the distensibility-pressure curve in the L-NAME- and vehicle-treated 2K1C rats compared with the sham-L-NAME rats. L-NAME treatment did not alter the distensibility-pressure curve in the 2K1C rats. These results demonstrate that the mechanisms responsible for artery wall hypertrophy in renovascular hypertension are accompanied by an increase in arterial distensibility that is not dependent on the synthesis of nitric oxide.

Nitric oxide synthase (NOS) inhibition for one week improves renal sodium and water excretion in cirrhotic rats with ascites

Normalization of the increased vascular nitric oxide (NO) generation with low doses of NG-nitro-L-arginine methyl ester (L-NAME) corrects the hemodynamic abnormalities of cirrhotic rats with ascites. We have undertaken this study to investigate the effect of the normalization of vascular NO production, as estimated by aortic cyclic guanosine monophosphate (cGMP) concentration and endothelial nitric oxide synthase (eNOS) protein expression in the aorta and mesenteric artery, on sodium and water excretion. Rats with carbon tetrachloride-induced cirrhosis and ascites were investigated using balance studies. The cirrhotic rats were separated into two groups, one receiving 0.5 mg/kg per day of L-NAME (CIR-NAME) during 7 d, whereas the other group (CIR) was administrated the same volume of vehicle. Two other groups of rats were used as controls, one group treated with L-NAME and another group receiving the same volume of vehicle. Sodium and water excretion was measured on days 0 and 7. On day 8, blood samples were collected for electrolyte and hormone measurements, and aorta and mesenteric arteries were harvested for cGMP determination and nitric oxide synthase (NOS) immunoblotting. Aortic cGMP and eNOS protein expression in the aorta and mesenteric artery were increased in CIR as compared with CIR-NAME. Both cirrhotic groups had a similar decrease in sodium excretion on day 0 (0.7 versus 0.6 mmol per day, NS) and a positive sodium balance (+0.9 versus +1.2 mmol per day, NS). On day 7, CIR-NAME rats had an increase in sodium excretion as compared with the CIR rats (sodium excretion: 2.4 versus 0.7 mmol per day, P < 0.001) and a negative sodium balance (-0.5 versus +0.8 mmol per day, P < 0.001). The excretion of a water load was also increased after L-NAME administration (from 28+/-5% to 65+/-7, P < 0.05). Plasma renin activity, aldosterone and arginine vasopressin were also significantly decreased in the CIR-NAME, as compared with the CIR rats. The results thus indicate that normalization of aortic cGMP and eNOS protein expression in vascular tissue is associated with increased sodium and water excretion in cirrhotic rats with ascites.

Effect of selective phosphodiesterase inhibitors on response of ovine pulmonary arteries to prostaglandin E2.

Several adenosine 3',5'-cyclic monophosphate (cAMP)-hydrolyzing phosphodiesterase isozymes are present in the pulmonary vasculature. The present study was designed to determine the effect of selective inhibitors of phosphodiesterase subtypes on prostaglandin E2 (PGE2)-induced relaxation of isolated fourth-generation pulmonary arteries of newborn lambs. PGE2 and forskolin caused pulmonary arteries to relax and induced an increase in the intracellular cAMP content in the vessels. The relaxation and change in cAMP content were augmented by milrinone and rolipram, inhibitors of phosphodiesterase type 3 (PDE3) and type 4 (PDE4), respectively. The augmentation in relaxation and the increase in cAMP content caused by milrinone plus rolipram was greater than the sum of the responses caused by either of the inhibitors alone. 8-Methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine, an inhibitor of phosphodiesterase type 1, had no effect on relaxation and change in cAMP induced by PGE2 and forskolin. Acetylcholine alone had no effect on cAMP content in the vessels but augmented the relaxation and the increase in cAMP induced by PGE2 and forskolin in arteries with endothelium. This effect was not observed in arteries without endothelium or in arteries with endothelium treated with NG-nitro-L-arginine. These results suggest that PDE3 and PDE4 are the primary enzymes hydrolyzing cAMP of pulmonary arteries of newborn lambs and that an inhibition of both PDE3 and PDE4 would result in a greater effect than that caused by inhibition of either one of the subtype isozymes alone. Furthermore, endothelium-derived nitric oxide may enhance cAMP-mediated relaxation by inhibition of PDE3.

Coronary vasomotor responses to isometric handgrip exercise are primarily mediated by nitric oxide: a noninvasive MRI test of coronary endothelial function.

Endothelial cell release of nitric oxide (NO) is a defining characteristic of nondiseased arteries, and abnormal endothelial NO release is both a marker of early atherosclerosis and a predictor of its progression and future events. Healthy coronaries respond to endothelial-dependent stressors with vasodilatation and increased coronary blood flow (CBF), but those with endothelial dysfunction respond with paradoxical vasoconstriction and reduced CBF. Recently, coronary MRI and isometric handgrip exercise (IHE) were reported to noninvasively quantify coronary endothelial function (CEF). However, it is not known whether the coronary response to IHE is actually mediated by NO and/or whether it is reproducible over weeks. To determine the contribution of NO, we studied the coronary response to IHE before and during infusion of N(G)-monomethyl-l-arginine (l-NMMA, 0.3 mg·kg(-1)·min(-1)), a NO-synthase inhibitor, in healthy volunteers. For reproducibility, we performed two MRI-IHE studies ∼8 wk apart in healthy subjects and patients with coronary artery disease (CAD). Changes from rest to IHE in coronary cross-sectional area (%CSA) and diastolic CBF (%CBF) were quantified. l-NMMA completely blocked normal coronary vasodilation during IHE [%CSA, 12.9 ± 2.5 (mean ± SE, placebo) vs. -0.3 ± 1.6% (l-NMMA); P < 0.001] and significantly blunted the increase in flow [%CBF, 47.7 ± 6.4 (placebo) vs. 10.6 ± 4.6% (l-NMMA); P < 0.001]. MRI-IHE measures obtained weeks apart strongly correlated for CSA (P < 0.0001) and CBF (P < 0.01). In conclusion, the normal human coronary vasoactive response to IHE is primarily mediated by NO. This noninvasive, reproducible MRI-IHE exam of NO-mediated CEF promises to be useful for studying CAD pathogenesis in low-risk populations and for evaluating translational strategies designed to alter CAD in patients.

Anticonvulsant and proconvulsant roles of nitric oxide in experimental epilepsy models

The effect of acute (120 mg/kg) and chronic (25 mg/kg, twice a day, for 4 days) intraperitonial injection of the nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-L-arginine (L-NOARG) was evaluated on seizure induction by drugs such as pilocarpine and pentylenetetrazole (PTZ) and by sound stimulation of audiogenic seizure-resistant (R) and audiogenic seizure-susceptible (S) rats. Seizures were elicited by a subconvulsant dose of pilocarpine (100 mg/kg) only after NOS inhibition. NOS inhibition also simultaneously potentiated the severity of PTZ-induced limbic seizures (60 mg/kg) and protected against PTZ-induced tonic seizures (80 mg/kg). The audiogenic seizure susceptibility of S or R rats did not change after similar treatments. In conclusion, proconvulsant effects of NOS inhibition are suggested to occur in the pilocarpine model and in the limbic components of PTZ-induced seizures, while an anticonvulsant role is suggested for the tonic seizures induced by higher doses of PTZ, revealing inhibitor-specific interactions with convulsant dose and also confirming the hypothesis that the effects of NOS inhibitors vary with the model of seizure

Role of nitric oxide in hypoxia-induced hyperventilation and hypothermia: participation of the locus coeruleus

Hypoxia elicits hyperventilation and hypothermia, but the mechanisms involved are not well understood. The nitric oxide (NO) pathway is involved in hypoxia-induced hypothermia and hyperventilation, and works as a neuromodulator in the central nervous system, including the locus coeruleus (LC), which is a noradrenergic nucleus in the pons. The LC plays a role in a number of stress-induced responses, but its participation in the control of breathing and thermoregulation is unclear. Thus, in the present study, we tested the hypothesis that LC plays a role in the hypoxia-induced hypothermia and hyperventilation, and that NO is involved in these responses. Electrolytic lesions were performed bilaterally within the LC in awake unrestrained adult male Wistar rats weighing 250-350 g. Body temperature and pulmonary ventilation (VE) were measured. The rats were divided into 3 groups: control (N = 16), sham operated (N = 7) and LC lesioned (N = 19), and each group received a saline or an NG-nitro-L-arginine methyl ester (L-NAME, 250 µg/µl) intracerebroventricular (icv) injection. No significant difference was observed between control and sham-operated rats. Hypoxia (7% inspired O2) caused hyperventilation and hypothermia in both control (from 541.62 ± 35.02 to 1816.18 ± 170.7 and 36.3 ± 0.12 to 34.4 ± 0.09, respectively) and LC-lesioned rats (LCLR) (from 694.65 ± 63.17 to 2670.29 ± 471.33 and 36 ± 0.12 to 35.3 ± 0.12, respectively), but the increase in VE was higher (P<0.05) and hypothermia was reduced (P<0.05) in LCLR. L-NAME caused no significant change in VE or in body temperature under normoxia, but abolished both the hypoxia-induced hyperventilation and hypothermia. Hypoxia-induced hyperventilation was reduced in LCLR treated with L-NAME. L-NAME also abolished the hypoxia-induced hypothermia in LCLR. The present data indicate that hypoxia-induced hyperventilation and hypothermia may be related to the LC, and that NO is involved in these responses.

Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

Chronic hypertension alters the expression of Cx43 in cardiovascular muscle cells

Connexin43 (Cx43), the predominant gap junction protein of muscle cells in vessels and heart, is involved in the control of cell-to-cell communication and is thought to modulate the contractility of the vascular wall and the electrical coupling of cardiac myocytes. We have investigated the effects of arterial hypertension on the expression of Cx43 in aorta and heart in three different models of experimental hypertension. Rats were made hypertensive either by clipping one renal artery (two kidney, one-clip renal (2K,1C) model) by administration of deoxycorticosterone and salt (DOCA-salt model) or by inhibiting nitric oxide synthase with NG-nitro-L-arginine methyl ester (L-NAME model). After 4 weeks, rats of the three models showed a similar increase in intra-arterial mean blood pressure and in the thickness of the walls of both aorta and heart. Analysis of heart mRNA demonstrated no change in Cx43 expression in the three models compared to their respective controls. The same 2K,1C and DOCA-salt hypertensive animals expressed twice more Cx43 in aorta, and the 2K,1C rats showed an increase in arterial distensibility. In contrast, the aortae of L-NAME hypertensive rats were characterized by a 50% decrease in Cx43 and the carotid arteries did not show increased distensibility. Western blot analysis indicated that Cx43 was more phosphorylated in the aortae of 2K,1C rats than in those of L-NAME or control rats, indicating a differential regulation of aortic Cx43 in different models of hypertension. The data suggest that localized mechanical forces induced by hypertension affect Cx43 expression and that the cell-to-cell communication mediated by Cx43 channels may contribute to regulating the elasticity of the vascular wall.

Pancreatic nitric oxide and oxygen free radicals in the early stages of streptozotocin-induced diabetes mellitus in the rat

The objective of the present study was to explore the regulatory mechanisms of free radicals during streptozotocin (STZ)-induced pancreatic damage, which may involve nitric oxide (NO) production as a modulator of cellular oxidative stress. Removal of oxygen species by incubating pancreatic tissues in the presence of polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) (1 U/ml) produced a decrease in nitrite levels (42%) and NO synthase (NOS) activity (50%) in diabetic but not in control samples. When NO production was blocked by N G-monomethyl-L-arginine (L-NMMA) (600 µM), SOD activity increased (15.21 ± 1.23 vs 24.40 ± 2.01 U/mg dry weight). The increase was abolished when the NO donor, spermine nonoate, was added to the incubating medium (13.2 ± 1.32). Lipid peroxidation was lower in diabetic tissues when PEG-SOD was added (0.40 ± 0.02 vs 0.20 ± 0.03 nmol/mg protein), and when L-NMMA blocked NOS activity in the incubating medium (0.28 ± 0.05); spermine nonoate (100 µM) abolished the decrease in lipoperoxide level (0.70 ± 0.02). We conclude that removal of oxygen species produces a decrease in pancreatic NO and NOS levels in STZ-treated rats. Moreover, inhibition of NOS activity produces an increase in SOD activity and a decrease in lipoperoxidation in diabetic pancreatic tissues. Oxidative stress and NO pathway are related and seem to modulate each other in acute STZ-induced diabetic pancreas in the rat.

Role of nitric oxide and prostaglandin in the maintenance of cortical and renal medullary blood flow

This study was undertaken in anesthetized dogs to evaluate the relative participation of prostaglandins (PGs) and nitric oxide (NO) in the maintenance of total renal blood flow (TRBF), and renal medullary blood flow (RMBF). It was hypothesized that the inhibition of NO should impair cortical and medullary circulation because of the synthesis of this compound in the endothelial cells of these two territories. In contrast, under normal conditions of perfusion pressure PG synthesis is confined to the renal medulla. Hence PG inhibition should predominantly impair the medullary circulation. The initial administration of 25 µM kg-1 min-1 NG-nitro-L-arginine methyl ester produced a significant 26% decrease in TRBF and a concomitant 34% fall in RMBF, while the subsequent inhibition of PGs with 5 mg/kg meclofenamate further reduced TRBF by 33% and RMBF by 89%. In contrast, the initial administration of meclofenamate failed to change TRBF, while decreasing RMBF by 49%. The subsequent blockade of NO decreased TRBF by 35% without further altering RMBF. These results indicate that initial PG synthesis inhibition predominantly alters the medullary circulation, whereas NO inhibition decreases both cortical and medullary flow. This latter change induced by NO renders cortical and RMBF susceptible to a further decrease by PG inhibition. However, the decrease in medullary circulation produced by NO inhibition is not further enhanced by subsequent PG inhibition.

Consequences of cerebroventricular insulin injection on renal sodium handling in rats: effect of inhibition of central nitric oxide synthase

In the present study, we investigated the effects of acute intracerebroventricular (icv) insulin administration on central mechanisms regulating urinary sodium excretion in simultaneously centrally NG-nitro-L-arginine methylester (L-NAME)-injected unanesthetized rats. Male Wistar-Hannover rats were randomly assigned to one of five groups: a) icv 0.15 M NaCl-injected rats (control, N = 10), b) icv dose-response (1.26, 12.6 and 126 ng/3 µL) insulin-injected rats (N = 10), c) rats icv injected with 60 µg L-NAME in combination with NaCl (N = 10) or d) with insulin (N = 10), and e) subcutaneously insulin-injected rats (N = 5). Centrally administered insulin produced an increase in urinary output of sodium (NaCl: 855.6 ± 85.1 Δ%/min; 126 ng insulin: 2055 ± 310.6 Δ%/min; P = 0.005) and potassium (NaCl: 460.4 ± 100 Δ%/min; 126 ng insulin: 669.2 ± 60.8 Δ%/min; P = 0.025). The urinary sodium excretion response to icv 126 ng insulin microinjection was significantly attenuated by combined administration of L-NAME (126 ng insulin: 1935 ± 258.3 Δ%/min; L-NAME + 126 ng insulin: 582.3 ± 69.6 Δ%/min; P = 0.01). Insulin-induced natriuresis occurred by increasing post-proximal sodium excretion, despite an unchanged glomerular filtration rate. Although the rationale for decreased urinary sodium excretion induced by combined icv L-NAME and insulin administration is unknown, it is tempting to suggest that perhaps one of the efferent signals triggered by insulin in the CNS may be nitrergic in nature.

Pharmacological characterization of the relaxant effect induced by adrenomedullin in rat cavernosal smooth muscle

The aim of the present study was to determine the mechanisms underlying the relaxant effect of adrenomedullin (AM) in rat cavernosal smooth muscle (CSM) and the expression of AM system components in this tissue. Functional assays using standard muscle bath procedures were performed in CSM isolated from male Wistar rats. Protein and mRNA levels of pre-pro-AM, calcitonin receptor-like receptor (CRLR), and Subtypes 1, 2 and 3 of the receptor activity-modifying protein (RAMP) family were assessed by Western immunoblotting and quantitative real-time polymerase chain reaction, respectively. Nitrate and 6-keto-prostaglandin F1α (6-keto-PGF1α; a stable product of prostacyclin) levels were determined using commercially available kits. Protein and mRNA of AM, CRLR, and RAMP 1, -2, and -3 were detected in rat CSM. Immunohistochemical assays demonstrated that AM and CRLR were expressed in rat CSM. AM relaxed CSM strips in a concentration-dependent manner. AM22-52, a selective antagonist for AM receptors, reduced the relaxation induced by AM. Conversely, CGRP8-37, a selective antagonist for calcitonin gene-related peptide receptors, did not affect AM-induced relaxation. Preincubation of CSM strips with NG-nitro-L-arginine-methyl-ester (L-NAME, nitric oxide synthase inhibitor), 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, quanylyl cyclase inhibitor), Rp-8-Br-PET-cGMPS (cGMP-dependent protein kinase inhibitor), SC560 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazole, selective cyclooxygenase-1 inhibitor], and 4-aminopyridine (voltage-dependent K+ channel blocker) reduced AM-induced relaxation. On the other hand, 7-nitroindazole (selective neuronal nitric oxide synthase inhibitor), wortmannin (phosphatidylinositol 3-kinase inhibitor), H89 (protein kinase A inhibitor), SQ22536 [9-(tetrahydro-2-furanyl)-9H-purin-6-amine, adenylate cyclase inhibitor], glibenclamide (selective blocker of ATP-sensitive K+ channels), and apamin (Ca2+-activated channel blocker) did not affect AM-induced relaxation. AM increased nitrate levels and 6-keto-PGF1α in rat CSM. The major new contribution of this research is that it demonstrated expression of AM and its receptor in rat CSM. Moreover, we provided evidence that AM-induced relaxation in this tissue is mediated by AM receptors by a mechanism that involves the nitric oxide-cGMP pathway, a vasodilator prostanoid, and the opening of voltage-dependent K+ channels.

Deficiency of sex hormones does not affect 17-ß-estradiol-induced coronary vasodilation in the isolated rat heart

The relaxation of coronary arteries by estrogens in the coronary vascular beds of naive and hypertensive rats has been well described. However, little is known about this action in gonadectomized rats. We investigated the effect of 17-ß-estradiol (E2) in coronary arteries from gonadectomized rats, as well as the contributions of endothelium-derived factors and potassium channels. Eight-week-old female and male Wistar rats weighing 220-300 g were divided into sham-operated and gonadectomized groups (n=9−12 animals per group). The baseline coronary perfusion pressure (CPP) was determined, and the vasoactive effects of 10 μM E2 were assessed by bolus administration before and after endothelium denudation or by perfusion with NG-nitro-L-arginine methyl ester (L-NAME), indomethacin, clotrimazole, L-NAME plus indomethacin, L-NAME plus clotrimazole or tetraethylammonium (TEA). The CPP differed significantly between the female and sham-operated male animals. Gonadectomy reduced the CPP only in female rats. Differences in E2-induced relaxation were observed between the female and male animals, but male castration did not alter this response. For both sexes, the relaxation response to E2 was, at least partly, endothelium-dependent. The response to E2 was reduced only in the sham-operated female rats treated with L-NAME. However, in the presence of indomethacin, clotrimazole, L-NAME plus indomethacin or L-NAME plus clotrimazole, or TEA, the E2 response was significantly reduced in all groups. These results highlight the importance of prostacyclin, endothelium-derived hyperpolarizing factor, and potassium channels in the relaxation response of coronary arteries to E2 in all groups, whereas nitric oxide may have had an important role only in the sham-operated female group.