17-Beta estradiol regulation of myocardial glutathione and its role in protection against myocardial stunning in dogs.

We studied the effect of 2-week treatment with estradiol 17beta on myocardial glutathione concentration in dogs and isolated perfused rat heart subjected to brief coronary ischemia and reperfusion. Estradiol protected against ischemia/reperfusion-induced myocardial systolic shortening and malonylaldehyde production and increased myocardial glutathione concentration and glucose-6-phosphate dehydrogenase enzyme activity. Reduction of myocardial glutathione with buthionine sulfoximine to levels seen in the absence of estrogen reversed the protective effect of estradiol against myocardial dysfunction and lipid peroxidation associated with ischemia/reperfusion. These results suggest that the antioxidant effect of estradiol in ischemia/reperfusion may be mediated by regulation of myocardial glutathione metabolism.

17 beta-Estradiol prevents dysfunction of canine coronary endothelium and myocardium and reperfusion arrhythmias after brief ischemia/reperfusion.

BACKGROUND: Brief myocardial ischemia is associated with myocardial and coronary endothelial dysfunction caused by oxygen free radicals released during reperfusion. Estrogen, known to have antioxidant activity, may prevent these complications. METHODS AND RESULTS: We assessed the effect of 2 weeks of treatment with 17 beta-estradiol (E, 100 micrograms.kg-1.d-1, n = 12) or placebo (P, n = 15) on myocardial and coronary endothelial function during the first 2 hours of reperfusion in dogs subjected to 15 minutes of ischemia induced by occlusion of the left anterior descending coronary artery (LAD). Our results show that the incidence of ventricular arrhythmias significantly decreased in E (3 of 12) compared with P (11 of 15). Systolic shortening, significantly depressed in P during early reperfusion, was maintained at preischemic levels in E. During reperfusion, the increase in LAD flow to acetylcholine, attenuated in P (60 +/- 6%), was preserved in E endothelium. (151 +/- 28%) and was associated with increased serum nitrite/nitrate concentration. n-Pentane in exhaled gas in vivo, an index of lipid peroxidation, increased significantly during early reperfusion in P (from 9.1 +/- 1.9 to 41.6 +/- 13.0 ppb, P < .05) but not in E (23.0 +/- 6.9 ppb). In vitro, arterial segments from E generated significantly less superoxide anion after hypoxia/reoxygenation than those from P. Ischemic/reperfused LAD segments from E also revealed a better preservation of endothelium-dependent relaxation in vitro (maximum relaxation, 42 +/- 4% versus 24 +/- 4% in P; P < .05). CONCLUSIONS: Estrogen protects against endothelial and myocardial dysfunction resulting from brief ischemia/reperfusion. This protection may relate to an antioxidant effect of estrogen.

L-arginine reverses the adverse pregnancy changes induced by nitric oxide synthase inhibition in the rat.

OBJECTIVE: Inhibition of nitric oxide synthase with N omega-nitro-L-arginine methyl ester (L-NAME) induces a preeclampsia-like syndrome of hypertension, proteinuria, intrauterine growth restriction, and renal glomerular capillary endothelial lesions in pregnant rats. We attempted to reverse these changes with late-pregnancy administration of L-arginine. STUDY DESIGN: Sprague Dawley rats with timed pregnancies received infusions of either saline solution (n = 12) (group SC) or L-NAME (n = 12) (group LC) (160 mg/kg per day) on gestational day 10 through term. On gestational day 16 half of the saline solution group (group SA) and half of the L-NAME group (group LA) received L-arginine (21 mg/kg per day) through delivery. Systolic blood pressures were determined via tail cuff on days 10, 16, and 21. Pup weights were assessed at delivery, serum and urine were collected and analyzed for nitrites and nitrates, and renal tissue was processed for histologic examination. Data were analyzed with the one-way analysis of variance and the Newman-Keuls test for multiple comparisons. RESULTS: In the L-NAME-treated animals L-arginine significantly lowered systolic blood pressure at late pregnancy (125 +/- 2.42 vs 153 +/- 3.0 mm Hg) (p < 0.01), increased mean pup weight (5.6 +/- 0.11 gm in group LA vs. 5.0 +/- 0.02 gm in group LC) (p < 0.001), decreased the degree of proteinuria (2+ vs trace), and decreased the proportion of injured glomeruli (7% vs 64%) (p < 0.001). CONCLUSIONS: Lesions induced by chronic inhibition of endothelium-derived nitric oxide synthesis (hypertension, intrauterine growth restriction, proteinuria, renal glomerulus injury) are reversed by treatment with L-arginine. These findings lend support to the potential for use of nitric oxide donors in the treatment and prevention of preeclampsia.

The vascular protective effects of estrogen.

There is now strong epidemiological evidence that estrogen replacement therapy has a protective effect in postmenopausal women. The cardiovascular protective action of estrogen is reported to be mediated indirectly by an effect on lipoprotein metabolism and by a direct effect on the vessel wall itself. Estrogen is active both in vascular smooth muscle and endothelium. Functionally competent estrogen receptors have been identified in vascular smooth muscle cells, and specific binding sites have been demonstrated in endothelium. Estrogen administration promotes vasodilation both in human and experimental animals, in part by stimulating] prostacyclin and nitric oxide synthesis. Both the prostaglandin synthase and the constitutive nitric oxide synthase were recently reported to be induced by estrogen treatment. In vitro, estrogen exerts a direct inhibitory effect on the smooth muscle by inhibiting calcium influx. In addition, estrogen inhibits vascular smooth muscle cell proliferation. In vivo, estradiol-17 beta prevents neointimal thickening after balloon injury and in rabbit cardiac transplant allografts. These data are consistent with in vitro studies wherein estrogen inhibits [3H]thymidine uptake by arterial segments from porcine coronary artery as well as proliferation of rabbit aortic vascular smooth muscle cells induced by hyperlipedemic serum. Recent studies have also reported an effect of estrogen on directed vascular smooth muscle cell migration. Furthermore, like other steroids, the effect of estrogen on the vessel wall has a rapid nongenomic component involving membrane phenomena, such as alteration of membrane ionic permeability and activation of membrane-bound enzymes, as well as the classical genomic effect involving estrogen receptor activation and gene expression. The nature of these estrogen response genes in the vessel wall and their relation to vasodilation and antiproliferation remain to be determined.

Estradiol effect on rate of proliferation of rat carotid segments: effect of gender and tamoxifen.

Using organ culture of carotid artery segments from sexually mature male and female rats, we examined the effect of estradiol 17 beta on proliferation. The index of cell proliferation was [3H]thymidine uptake. Estradiol 17 beta (0.18-0.36 microM) inhibited the uptake of thymidine in a concentration-dependent manner (p < 0.05). Estradiol 17 beta inhibited [3H]thymidine uptake only in the absence of the weak estrogen receptor agonist phenol red and in carotid artery segments from sexually mature female (p < 0.01) but not male rats. Tamoxifen (0.1 and 1 microM), a partial agonist of estrogen receptors, significantly inhibited thymidine uptake (p < 0.01). However, preincubation of the segments with tamoxifen (0.1 and 1.0 microM) for 4 h before the exposure to estradiol, blocked estradiol 17 beta-induced inhibition of thymidine uptake (p < 0.05 and p < 0.01 for 0.1 and 1.0 microM, respectively). The cyclooxygenase inhibitor indomethacin (5 microM) did not affect either the basal [3H]thymidine uptake or the estradiol 17 beta-induced inhibition of that uptake. This latter finding suggests that prostacyclin or prostaglandin E2 does not mediate the inhibitory response to estradiol 17 beta. The results of these experiments suggest that estradiol 17 beta-induced inhibition of proliferation of rat carotid artery segments is mediated through activation of estrogen receptors.

Non-genomic effects of estrogen and the vessel wall.

Estrogen, like other steroids, is now believed to possess rapid membrane effects independent of the classical gene activation pathway of steroid action. The presence of membrane estrogen receptors has been demonstrated in different cell types, but not yet in vascular tissue. In vivo, estrogen administration rapidly promotes acetylcholine-induced vasodilation of the coronary and peripheral vascular beds of postmenopausal women. Estrogen also causes relaxation of precontracted isolated arterial segments and perfused organ preparations, within minutes of administration of the hormone. These rapid vasomotor effects of estrogen may be related to blockade of the cell membrane voltage-dependent calcium channels, resulting in inhibition of extracellular Ca2+ mobilization and flux. Recently, estradiol has been shown to rapidly affect cyclic nucleotide turnover in vascular segments, smooth muscle, and epithelial cell cultures, suggesting the possibility of a "cross-talk" between membrane-mediated events and nuclear receptor activation.

Estradiol increases cyclic adenosine monophosphate in rat pulmonary vascular smooth muscle cells by a nongenomic mechanism.

Estrogen, like other steroids, may induce rapid nongenomic cellular effects. We studied the effect on intracellular cAMP of short-term exposure (5 min) of cultured rat pulmonary vascular smooth muscle cells (VSMC) to estradiol 17 beta. At confluence, VSMC were incubated in phosphate buffer saline for 1 hr before exposure to different hormones. The reaction was stopped with 0.1 N HCl and cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The 5-min incubation with estradiol 17 beta (0.3-30 microM) significantly increased basal intracellular cAMP in a concentration-dependent manner. The stimulatory effect of estradiol on cAMP was time-dependent, increasing with prolonged exposure to the hormone, and was not affected by the protein synthesis inhibitor, actinomycin D (5 micrograms/ml), at 5 and 30 min. Comparable concentrations of testosterone or estradiol 17 alpha had no significant effect on cAMP. The estrogen receptor partial agonist, tamoxifen also significantly increased basal cAMP in a concentration-dependent manner, but inhibited the effect of estradiol. Furthermore, forskolin elicited a concentration-dependent increase in cAMP (396.6 +/- 53% at 10 microM concentration), which was significantly potentiated in presence of estradiol. The effect of estradiol is unlikely to be mediated by G-protein activation, because the G protein inhibitor, pertussis toxin (100 ng/ml), did not significantly affect estradiol-induced increase in cAMP. Removal of Ca++ from the incubation medium inhibited the stimulatory effect of estradiol 17 beta suggesting that estradiol may increase pulmonary VSMC cAMP via a Ca(++)-dependent pathway. We suggest that the effect of estradiol 17 beta in these experiments is nongenomic in nature, and is possibly mediated by direct interaction of the hormone with specific membrane binding sites.

Effect of testosterone treatment on vasoconstrictor response of left anterior descending coronary artery in male and female pigs.

Androgens may be risk factors in the pathogenesis of coronary artery disease by promoting coronary vasoconstriction. We studied the effect of testosterone treatment on coronary vascular reactivity of male and female domestic pigs treated for 2 weeks with either placebo or testosterone 10 mg/kg subcutaneous (s.c.) pellets. Vascular reactivity was studied in ring segments (4-5 mm) isolated from the left anterior descending coronary artery (LAD). No significant sex difference was noted in the response of LAD segments from placebo-treated male and female animals to KCl and prostaglandin F2 alpha (PGF2 alpha). Androgen treatment increased the maximum response (Tmax) of intact vessels to KCl from 3,647 +/- 689 mg in controls to 8,939 +/- 1,284 mg in testosterone-treated males (p < 0.01) and from 3,405 +/- 669 to 10,524 +/- 1,663 mg in testosterone-treated female pigs (p < 0.01). Testosterone similarly increased the response to PGF2 alpha 10(-6) M from a mean of 2,149 +/- 1,036 to 3,163 +/- 867 mg in males (p < 0.05) and from 2,076 +/- 810 to 3,565 +/- 578 mg in female segments (p < 0.05). Endothelial denudation significantly decreased the potentiating effect of testosterone treatment in males to both KCl and PGF2 alpha (p < 0.05), but not in segments from females. Our data show that testosterone treatment potentiates contractility of porcine LAD segments to both receptor- and nonreceptor-mediated agonists. In male pigs, this effect may be mediated by an effect on endothelium.

Effect of estradiol 17 beta on pressor responses of rat mesenteric bed to norepinephrine, K+, and U-46619.

We reinvestigated the effect of estradiol 17 beta on the responses of adrenergic and nonadrenergic vasoconstrictors characterized it in terms of steroid specificity, time course, and the role of classic estrogen receptors. We evaluated the effect of estradiol 17 beta on the pressor responses of isolated perfused rat mesenteric vascular bed (McGregor's preparation). Estradiol 17 beta (7-700 nM) significantly increased the pressor response to bolus applications of norepinephrine (NE) (p < 0.05). However, estradiol 17 beta did not significantly increase the responses to endogenous NE release induced by electrical field stimulation. Other steroids, testosterone, and the 17 alpha isomer of estradiol (7 and 700 nM) were ineffective. Estradiol 17 beta (700 nM) also significantly increased the maximum pressor response of rat mesenteric preparation to both the prostaglandin endoperoxide analogue U-46619 and to K+. The potentiation by estradiol 17 beta of mesenteric vasoconstriction elicited by NE, K+, and U-46619 was rapid (2-8 min), suggesting that a nuclear receptor may not be involved. This notion received further support in that significant potentiation of the NE-induced pressor response was also observed with estradiol 17 beta conjugated to albumin (700 nM), but not when electrical field stimulation was used. The conjugate increased the effect of all NE concentrations. Its effect was also more consistent (p < 0.01) than that elicited by free estradiol 17 beta. The dose-response curve was shifted to the left, and the maximum effect was increased. These data suggest that estradiol 17 beta may possess rapid nongenomic actions unrelated to nuclear receptor binding and gene transcription.

Protection by oestradiol against the development of cardiovascular changes associated with monocrotaline pulmonary hypertension in rats.

1. We studied the effects of oestradiol 17 beta on the development of pulmonary vascular changes and right ventricular (RV) hypertrophy in response to monocrotaline in male Sprague-Dawley rats. 2. Rats were treated with either placebo or oestradiol 17 beta (10 mg) in the form of slow release pellets implanted subcutaneously 48 h before monocrotaline administration. Rats were injected with either saline or a single dose of monocrotaline (60 mg kg-1, i.m.). Pulmonary vascular changes and RV hypertrophy were studied at 4 weeks following monocrotaline administration. 3. Monocrotaline induced a significant increase in the ratio of right ventricle (RV) to left ventricle-plus-septum (LV + S) weights. Monocrotaline-treated rats also showed significant myointimal proliferation in small pulmonary arteries, decrease of arterial numbers and increase in the number of abnormal alveolar macrophages. 4. Oestradiol 17 beta attenuated myointimal hyperplasia in pulmonary vessels, decreased the RV/(LV + S) ratio in monocrotaline-treated rats. Oestradiol 17 beta had no significant effect on control animals. 5. Oestradiol treatment prevented the increase in lung wet to dry weight ratio, observed 7 days post monocrotaline administration. 6. These results suggest that oestradiol 17 beta protects against the pulmonary vascular remodelling and RV hypertrophy associated with monocrotaline-induced pulmonary hypertension in the rat. Oestradiol also protects against microvascular leak observed in the early days of lesion.

In vitro effect of oestradiol on thymidine uptake in pulmonary vascular smooth muscle cell: role of the endothelium.

1. The effect of different concentrations of oestradiol-17 beta (3-300 nM) on [3H]-thymidine uptake was studied in segments from canine pulmonary artery, and cultures of rat pulmonary vascular smooth muscle cells (VSMC). 2. Incubation with oestradiol-17 beta for 24 h, potentiated in a concentration-dependent manner [3H]-thymidine uptake in VSMC cultures. 3. Oestradiol-17 beta potentiated thymidine uptake by pulmonary arterial segments but only when the endothelium had been removed. Autoradiography showed dense incorporation of radioactive thymidine in the vascular smooth muscle cells of the media. 4. The non-steroidal oestrogen, stilboestrol (300 nM), also significantly potentiated [3H]-thymidine uptake, in both VSMC cultures and pulmonary artery segments. Testosterone was ineffective at a similar concentration. 5. Pre-incubation of the pulmonary VSMC with the anti-oestrogen tamoxifen (1 microM) antagonized the potentiating effect of oestradiol-17 beta on [3H]-thymidine incorporation. The effect of tamoxifen was less pronounced in pulmonary arterial segments. 6. These data suggest that oestrogen may promote proliferation of pulmonary VSMC. Endothelial injury or dysfunction may be an important factor in the expression of the oestrogenic effect. 7. We speculate that plasma oestrogen may be a contributing factor to the proliferative lesion observed in certain forms of pulmonary vascular injury in women.

Estradiol potentiates the vasopressor response of the isolated perfused rat lung to the thromboxane mimic U-46619.

The high incidence of primary pulmonary hypertension in young women suggests a role of female sex hormones in the pathogenesis of this disease. Thromboxane A2 is a potent pulmonary vasoconstrictor, and a possible mediator of pulmonary hypertension. We studied the pulmonary vascular expression of thromboxane in isolated perfused lungs from sexually mature male and female rats, as well as the modulation of this expression by estrogen. Our data show that lungs from female rats exhibited a significantly (P less than .05) greater pressor response to the thromboxane mimic U46619, but not to angiotensin II, than those from their male cohorts. The difference in the response to U46619, however, was abolished by indomethacin (10 microM). Addition of 10 nM concentrations of estradiol 17-beta or diethylstilbestrol to the perfusate significantly potentiated (55 and 63%, respectively) the pressor response to U46619. Similarly, perfusion with diethylstilbestrol enhanced the pressor response to angiotensin II (70%). Estradiol 17-alpha or testosterone, however, were ineffective at similar concentrations. Ovariectomy, on the other hand, depressed, whereas chronic administration of estradiol 17-beta enhanced the pressor response to the thromboxane mimic, but had no effect on the response to angiotensin II. These data show a gender difference in pulmonary vascular expression of thromboxane in isolated perfused rat lungs, possibly modulated by estradiol. Thus, changes in estradiol levels may play a role in the development of primary pulmonary hypertension in young women.

Endothelium-mediated effects of N-substituted arginines on the isolated perfused rat kidney.

Endothelium-derived relaxing factor (EDRF) has been suggested to be derived from the metabolism of arginine or an arginine-containing moiety. We have compared the vascular effects of arginine and some of its N-substituted derivatives on the perfusion pressure (Pp) of the isolated rat kidney preparation preconstricted with phenylephrine. Irrespective of the stereochemistry, high doses (10(-4) mol) of L- and D-arginine HCl produced a slight vasodilation. In contrast L- and D-arginine free base, at similar doses, further increased renal Pp. N-substituted L-arginine compounds, however, decreased Pp dose-dependently. Their order of potency (ED50) was as follows: N-alpha-benzoyl-L-arginine ethyl ester (BAEE, 3.8 x 10hm6 mol) greater than N-alpha-benzoyl-L-arginine methyl ester (2.5 x 10(-5) mol) greater than L-arginine ethyl ester (2.7 x 10(-5) mol) greater than L-arginine HCl (10(-4) mol). Methylene blue (10(-5) M), hemoglobin (10(-5) M) and NG-mono-methyl-L-arginine (5 mumol) antagonized the vasodilation elicited by infusion of BAEE. Similarly, injection of xanthine oxidase/xanthine (100 mU) reversed BAEE-induced renal vasodilation, but had no effect on dilation elicited by infusion of atrial natriuretic peptide. These data demonstrate that substituted arginine compounds are more potent renal vasodilators than L-arginine and their potency depends on the nature of the substitution. These compounds exert their effect, at least in part, via an endothelium-dependent mechanism. We conclude that exogenous L-arginine is a poor substrate for EDRF generation in the kidney, and that it may cause release of EDRF by another mechanism, possibly related to a change in the pH of the medium.

Vasodilatory property of N-alpha benzoyl-L-arginine ethyl ester in the rat isolated pulmonary artery and perfused lung.

L-arginine has been proposed to be the precursor of the endothelium-derived relaxing factor. In this study, we evaluated the pulmonary vascular effects of L-arginine-HCl and its benzoyl derivative N-alpha-benzoyl-L-arginine ethyl ester (BAEE) in the rat, in comparison with other vasodilators such as acetylcholine and sodium nitroprusside. In isolated pulmonary artery rings incubated with indomethacin (10 microM) and precontracted with phenylephrine (2 microM), BAEE (10(-6)-10(-5) M) significantly (P less than .05) relaxed the rings more than L-arginine. This effect was potentiated by the endothelium (P less than .05). The relaxing effect of BAEE (ED50 = 2.1 X 10(-6) M) and acetylcholine (ED50 = 2.4 X 10(-7) M) was significantly less potent than that of sodium nitroprusside (ED50 = 1.1 X 10(-8) M). Moreover, pretreatment with the soluble guanylate cyclase inhibitors methylene blue (10(-5) M) and hemoglobin (10(-5) M) antagonized BAEE-induced relaxation in intact pulmonary rings but had no effect on the relaxation elicited with atrial natriuretic peptide. In the isolated lung preparations perfused with the endoperoxide analog U46619 (5-10 nmol/min), sodium nitroprusside (10(-10)-10(-8) M) elicited potent vasodilation (ED50 = 2.8 X 10(-9) mol) whereas no vasodilation was observed with acetylcholine (10(-8)-10(-5) mol). BAEE (10(-6)-10(-5) M) decreased in a dose-dependent manner pulmonary perfusion pressure, and similar doses of L-arginine showed only a mild vasodilating effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of immunoreactive-neuropeptide by rat platelets.

Neuropeptide Y, a potent vasoconstrictor and cardiac depressant, is re-leased from sympathetic nerve endings. Its presence in megakaryocytes suggests this peptide might be stored in platelet granules and released during aggregation. Immunoreactive-neuropeptide Y was measured in platelet rich and platelet poor plasma, and was substantially greater in the former. Addition of collagen to platelets resulted in release of neuropeptide Y which paralleled, in a concentration-dependent manner, the degree of platelet aggregation. Adenosine diphosphate, at concentrations which induce only the first phase of aggregation and not the release reaction, caused only a minor release of neuropeptide Y. These results suggest that platelet release could be a major source of circulating neuropeptide Y and could contribute to hemodynamics of pathophysiological states involving platelet activation.