Mahmoud M. El-Mas

Estrogen enhancement of baroreflex sensitivity is centrally mediated.

We have recently shown that estrogen enhances baroreceptor control of reflex bradycardia in conscious rats. The present study replicated this finding in pentobarbital sodium-anesthetized rats, and the study was extended to investigate whether this effect of estrogen is centrally or peripherally mediated. Hemodynamic responses to electrical stimulation of the central end of the aortic depressor or the vagal efferent nerve were evaluated in pentobarbital sodium-anesthetized sham-operated (SO), ovariectomized (OVX), and OVX estradiol-treated Sprague-Dawley rats. Phenylephrine (1-16 microgram/kg iv) elicited dose-dependent pressor and bradycardic responses. Regression analysis of the baroreflex curves, relating changes in mean arterial pressure and heart rate, revealed a significantly smaller baroreflex sensitivity in OVX compared with SO anesthetized rats (-0.54 +/- 0.05 and -0.91 +/- 0.12 beats. min-1. mmHg-1, respectively; P < 0.05). Treatment of OVX rats with 17beta-estradiol (E2, 50 microgram. kg-1. day-1 for 2 days subcutaneously) significantly enhanced baroreflex sensitivity to a level similar to that of SO rats (P < 0.05). The enhancing effect of E2 on the baroreflex-mediated bradycardia, observed in conscious and anesthetized rats, seems to be selective because the baroreflex-mediated tachycardic responses measured in a separate group of conscious rats were not altered by ovariectomy or E2 administration. Electrical stimulation of the aortic nerve elicited frequency-dependent depressor and bradycardic responses that were significantly smaller in OVX compared with SO values (P < 0.05). Treatment of OVX rats with E2 restored the hemodynamic responses to aortic stimulation to near SO levels. On the other hand, hemodynamic responses to vagal stimulation were not affected by OVX or treatment with E2. These findings suggest that enhancement of reflex bradycardia by estrogen is centrally mediated and involves interaction with central projections of the aortic nerve.We have recently shown that estrogen enhances baroreceptor control of reflex bradycardia in conscious rats. The present study replicated this finding in pentobarbital sodium-anesthetized rats, and the study was extended to investigate whether this effect of estrogen is centrally or peripherally mediated. Hemodynamic responses to electrical stimulation of the central end of the aortic depressor or the vagal efferent nerve were evaluated in pentobarbital sodium-anesthetized sham-operated (SO), ovariectomized (OVX), and OVX estradiol-treated Sprague-Dawley rats. Phenylephrine (1-16 μg/kg iv) elicited dose-dependent pressor and bradycardic responses. Regression analysis of the baroreflex curves, relating changes in mean arterial pressure and heart rate, revealed a significantly smaller baroreflex sensitivity in OVX compared with SO anesthetized rats (-0.54 ± 0.05 and -0.91 ± 0.12 beats ⋅ min-1 ⋅ mmHg-1, respectively; P < 0.05). Treatment of OVX rats with 17β-estradiol (E2, 50 μg ⋅ kg-1 ⋅ day-1for 2 days subcutaneously) significantly enhanced baroreflex sensitivity to a level similar to that of SO rats ( P < 0.05). The enhancing effect of E2 on the baroreflex-mediated bradycardia, observed in conscious and anesthetized rats, seems to be selective because the baroreflex-mediated tachycardic responses measured in a separate group of conscious rats were not altered by ovariectomy or E2 administration. Electrical stimulation of the aortic nerve elicited frequency-dependent depressor and bradycardic responses that were significantly smaller in OVX compared with SO values ( P < 0.05). Treatment of OVX rats with E2 restored the hemodynamic responses to aortic stimulation to near SO levels. On the other hand, hemodynamic responses to vagal stimulation were not affected by OVX or treatment with E2. These findings suggest that enhancement of reflex bradycardia by estrogen is centrally mediated and involves interaction with central projections of the aortic nerve.

Testosterone facilitates the baroreceptor control of reflex bradycardia: role of cardiac sympathetic and parasympathetic components.

Reported clinical and experimental findings have shown that baroreflex control of heart rate is attenuated in women compared with men. This study investigated whether the sexual dimorphism in baroreflex function relates to the ability of the male hormone testosterone to facilitate baroreflex responsiveness. Relative contributions of the vagal and sympathetic autonomic components to testosterone modulation of baroreflex function were also investigated. Baroreflex curves relating changes in heart rate to increases or decreases in blood pressure evoked by phenylephrine and sodium nitroprusside, respectively, were constructed in sham-operated rats and castrated rats with and without testosterone replacement. Slope of the curves was taken as an index of baroreflex sensitivity (BRS PE and BRS NP ). Castration (for 10 days) significantly reduced plasma testosterone levels and attenuated reflex bradycardia, as indicated by significantly smaller BRS PE in castrated rats compared with values in sham-operated rats (−0.85 ± 0.07 vs. −1.51 ± 0.10 beats/min per mm Hg). Testosterone replacement in castrated rats restored plasma testosterone and BRS PE to levels similar to those of sham-operated rats. Muscarinic blockade by atropine caused 55% reduction in BRS PE in sham-operated rats, an effect that was significantly (p < 0.05) attenuated in castrated rats and restored to intact levels after testosterone supplementation. &bgr;-Adrenergic blockade by propranolol caused slight and insignificant decreases in BRS PE . Castration and testosterone supplementation had no effect on BRS NP , ruling out a modulatory effect of testosterone on reflex tachycardia. These data provide the first experimental evidence of a favorable role for testosterone in baroreceptor control of reflex bradycardia. Further, baroreflex modulation by testosterone appears to be autonomically mediated and involves an enhancement of cardiomotor vagal activity.

Estrogen enhances baroreflex control of heart rate in conscious ovariectomized rats.

In previous studies, we have shown that the baroreflex control of heart rate is significantly attenuated in females compared with age-matched males. This study investigated the role of estrogen in the modulation of baroreflex function in conscious unrestrained rats. Baroreflex-mediated decreases in heart rate in response to increments in blood pressure evoked by phenylephrine were evaluated in conscious freely moving male and female Sprague-Dawley rats as well as in ovariectomized rats. The effect of a 2-day 17 beta-estradiol (50 micrograms.kg-1.day-1, s.c.) or vehicle treatment on baroreflex sensitivity was investigated in ovariectomized rats. Intravenous bolus doses of phenylephrine (1-16 micrograms/kg) elicited dose-dependent pressor and bradycardic responses in all groups of rats. Regression analysis of the baroreflex curves relating increments in blood pressure to the associated heart rate responses revealed a significantly (p < 0.05) smaller baroreflex sensitivity in female compared with male rats (-1.22 +/- 0.07 and -1.85 +/- 0.15 beats.min-1.mmHg-1, respectively), suggesting an attenuated baroreflex function in females. In age-matched ovariectomized rats, baroreflex sensitivity showed further reduction (-0.93 +/- 0.02 beats.min-1.mmHg-1). Treatment of ovariectomized rats with 17 beta-estradiol significantly (p < 0.05) enhanced the baroreflex sensitivity (-1.41 +/- 0.16 beats.min-1.mmHg-1) to a level that was slightly higher than that of sham-operated female rats. Furthermore, baroreflex sensitivity of ovariectomized estradiol-treated rats was not significantly different from that of age-matched male rats. The vehicle, on the other hand, had no effect on baroreflex sensitivity of ovariectomized rats. These data support our earlier findings that sexual dimorphism exists in baroreflex control of heart rate. More importantly, the present study provides experimental evidence that suggests a facilitatory role for estrogen in the modulation of baroreflex function.

Testosterone depletion contributes to cyclosporine-induced chronic impairment of acetylcholine renovascular relaxations

The immunosuppressant drug cyclosporine causes nephrotoxicity mainly via alterations of renovascular reactivity. This study investigated whether this effect of cyclosporine is modulated by the male gonadal hormone testosterone. The endothelium-dependent and -independent relaxations evoked by acetylcholine and sodium nitroprusside, respectively, were evaluated in phenylephrine-preconstricted isolated perfused kidneys obtained from sham-operated, castrated, and testosterone-replaced castrated (CAS+T) male rats in the absence and presence of cyclosporine. Compared with sham-operated values, short-term (10 days) castration or cyclosporine treatment caused significant and equivalent reductions in plasma testosterone levels and vasorelaxant responses to acetylcholine. Treatment of castrated rats with cyclosporine caused no further attenuation of acetylcholine relaxations. Testosterone replacement of castrated (CAS+T) or cyclosporine-treated castrated (CAS+CyA+T) rats restored plasma testosterone and acetylcholine relaxations to near-sham-operated levels. On the other hand, castration caused significant increases in nitroprusside relaxations versus no effect for cyclosporine. The relaxant responses to nitroprusside in castrated rats were restored to sham-operated levels after testosterone replacement. Plasma urea and creatinine were not affected by castration but were significantly increased by cyclosporine. These findings suggest that testosterone exerts directionally opposite modulatory effects on endothelium-dependent and -independent renal relaxations. Further, the results demonstrate that testosterone depletion may contribute, at least partly, to the inhibitory effect of cyclosporine on renovascular endothelial function. These data are clinically important because endothelial dysfunction contributes to vascular abnormalities associating cyclosporine therapy.

Imidazoline I1 receptor-induced activation of phosphatidylcholine-specific phospholipase C elicits mitogen-activated protein kinase phosphorylation in PC12 cells

In the present study, we tested the hypothesis that the activation of imidazoline I(1)-receptor, which is coupled to phosphatidylcholine-specific phospholipase C, results in downstream activation of mitogen-activated protein kinase (p42(mapk) and p44(mapk) isoforms) in PC12 cells. PC12 cells pretreated with nerve growth factor (50 ng/ml, 48 h) to initiate neuronal differentiation were incubated with [methyl-3H]choline and [3H]myristate. Activation of imidazoline I(1) receptor by rilmenidine (10 microM) caused time-dependent increases in diacylglycerol accumulation and phosphocholine release. The Western blotting analysis showed that rilmenidine (10 microM) produced a time-dependent activation of p42(mapk) and p44(mapk) that reached its maximum at 15 min and returned to control levels after 30 min. This finding was confirmed by immunofluorescence labeling of activated mitogen-activated protein kinase in the same model system. Efaroxan (imidazoline I(1)-receptor antagonist) or tricyclodecan-9-yl-xanthogenate (D609, phosphatidylcholine-specific phospholipase C inhibitor) attenuated the phosphorylation of p42(mapk) and p44(mapk) induced by rilmenidine. Nerve growth factor-induced phosphorylation of both mitogen-activated protein kinase isoforms was not affected by D609. These results support the hypothesis that the activation of the imidazoline I(1) receptor coupled phosphatidylcholine-specific phospholipase C results in the downstream activation of mitogen-activated protein kinase.

Facilitation of myocardial PI3K/Akt/nNOS signaling contributes to ethanol-evoked hypotension in female rats.

BACKGROUND The mechanism by which ethanol reduces cardiac output (CO) and blood pressure (BP) in female rats remains unclear. We tested the hypothesis that enhancement of myocardial phosphatidylinositol 3-kinase (PI3K)/Akt signaling and related neuronal nitric oxide synthase (nNOS) and/or endothelial nitric oxide synthase (eNOS) activity constitutes a cellular mechanism for the hemodynamic effects of ethanol. METHODS We measured the level of phosphorylated eNOS (p-eNOS) and p-nNOS in the myocardium of ethanol (1 g/kg intragastric, i.g.) treated female rats along with hemodynamic responses [BP, CO, stroke volume, (SV), total peripheral resistance, (TPR)], and myocardial nitrate/nitrite levels (NOx) levels. Further, we investigated the effect of selective pharmacological inhibition of nNOS with N(omega)-propyl-l-arginine (NPLA) or eNOS with N(5)-(1-iminoethyl)-l-ornithine (l-NIO) on cellular, hemodynamic, and biochemical effects of ethanol. The effects of PI3K inhibition by wortmannin on the cardiovascular actions of ethanol and myocardial Akt phosphorylation were also investigated. RESULTS The hemodynamic effects of ethanol (reductions in BP, CO, and SV) were associated with significant increases in myocardial NOx and myocardial p-nNOS and p-Akt expressions while myocardial p-eNOS remained unchanged. Prior nNOS inhibition by NPLA (2.5 or 12.5 microg/kg) attenuated hemodynamic effects of ethanol and abrogated associated increases in myocardial NOx and cardiac p-nNOS contents. The hemodynamic effects of ethanol and increases in myocardial p-Akt phosphorylation were reduced by wortmannin (15 microg/kg). On the other hand, although eNOS inhibition by l-NIO (4 or 20 mg/kg) in a dose-dependent manner attenuated ethanol-evoked hypotension, the concomitant reductions in CO and SV remained unaltered. Also, selective eNOS inhibition uncovered dramatic increases in TPR in response to ethanol, which appeared to have offset the reduction in CO. Neither NPLA nor l-NIO altered plasma ethanol levels. CONCLUSIONS These findings implicate the myocardial PI3K/Akt/nNOS signaling in the reductions in BP and CO produced by ethanol in female rats.

OVARIECTOMY ALTERS THE CHRONIC HEMODYNAMIC AND SYMPATHETIC EFFECTS OF ETHANOL IN RADIOTELEMETERED FEMALE RATS

This study determined the chronic hemodynamic effects of ethanol in telemetered freely moving female Sprague-Dawley rats. The role of ovarian hormones and sympathetic activity in the modulation of ethanol responses was also investigated. Changes in blood pressure (BP), heart rate (HR), and plasma estrogen and norepinephrine (NE, as index of sympathetic activity) were evaluated in pair-fed sham-operated (SO) and ovariectomized (OVX) rats receiving liquid diet with or without ethanol (5% w/v) for 12 weeks. OVX caused a significant increase (about 40 g) in body weight, compared with the sham operation, which was apparent after two weeks and remained so for the duration of the study. The body weight showed gradual and similar increases in both ethanol and control groups. Ethanol feeding had no effect on the plasma estrogen level in SO or OVX rats. Daily ethanol intake was significantly (P < 0.05) less in OVX compared with SO rats whereas the blood ethanol concentration were similar in the two groups except for a significantly (P < 0.05) higher level in OVX rats at weeks 8, 10, and 11. Ethanol feeding caused significant (P < 0.05) decreases in BP in SO rats that started at week 1and reached maximal response (approximately 10 mmHg) at week 6 and remained at that level till the end of week 12. In OVX rats, ethanol had no effect on BPduring the first 5 weeks of the study. A slight but significant reduction in BP (about 5 mmHg) by ethanol in OVX rats started to appear at week 6 and remained for the following 5 weeks. The reduced hypotensive effect of ethanol in OVX rats was associated with an increase in the sympathetic activity as indicated by the significant (P < 0.05) increases in plasma NE levels. This sympathoexcitatory effect of ethanol was not demonstrated in SO rats. The HR was not affected by ethanol in the two groups of rats except for significant (P < 0.05) increases at weeks 1 through 3 in SO rats. The present findings suggest that the ovarian hormones modulate, at least partly, the hemodynamic and neurohumoral effects of chronic ethanol feeding in female rats. Ethanol lowers BP in female rats and this effect was delayed and diminished in OVX rats due possibly to the associated increase in sympathetic activity.

Ovariectomy abolishes ethanol-induced impairment of baroreflex control of heart rate in conscious rats

Our previous studies have shown that ethanol attenuates baroreflex control of heart rate in male rats. The present study investigated whether this effect of ethanol is gender-related, and whether it involves hormonal factors. The effect of intragastric administration of ethanol or equal volume of water on baroreflex-mediated decreases in heart rate in response to increments in blood pressure evoked by phenylephrine were evaluated in conscious age-matched male and female Sprague-Dawley rats as well as in ovariectomized rats. Baroreflex curves relating changes in blood pressure and associated heart rate responses were constructed, and the slopes of the regression lines were taken as a measure of baroreflex sensitivity. Phenylephrine (1-16 microg kg(-1), i.v.) elicited dose-dependent pressor responses that were similar in all groups of rats. However, the associated reflex bradycardic responses depended on the rat preparation and the dose of ethanol employed. In water-treated (control) animals, significantly (P < 0.05) lesser reflex bradycardic responses were observed in female compared with male rats (baroreflex sensitivity, -1.21 +/- 0.12 vs. -1.67 +/- 0.12 beats min(-1) mmHg(-1)). Ovariectomy resulted in a further reduction in baroreflex sensitivity (-0.82 +/- 0.06 beats min(-1) mmHg(-1)), suggesting a favorable role for ovarian hormones in baroreflex modulation. In male rats, ethanol (0.25, 0.5, or 1 g kg(-1), intragastric) elicited dose-related decreases in reflex bradycardic responses. The reduction in the regression coefficient obtained by the two higher doses (0.5 and 1 g kg(-1)) of ethanol was statistically significant compared with control values. The ability of ethanol to reduce baroreflex sensitivity appears to be gender-independent as it was similarly demonstrated in intact female rats. In contrast, ethanol had no effect on reflex bradycardic responses in ovariectomized rats at any of the doses tested. The data suggest that ethanol reduces baroreflex control of heart rate irrespective of the rat gender. Further, the lack of an effect of ethanol on baroreflex sensitivity in ovariectomized rats may suggest a role for ovarian hormones in ethanol-evoked baroreflex attenuation.

Centrally mediated reduction in cardiac output elicits the enhanced hypotensive effect of clonidine in conscious aortic barodenervated rats

In a previous study, we showed that centrally mediated hypotensive responses are enhanced in aortic barodenervated (ABD) rats as compared with sham-operated (SO) rats. In the present study, we tested the hypothesis that the high basal total peripheral resistance (TPR) of ABD rats accounts for enhanced hypotensive responses to clonidine in this rat model. Aortic barodenervation resulted in acute increases in blood pressure (BP) and heart rate (HR) in anesthetized rats, associated with significant increases in plasma norepinephrine (NE) levels and TPR; cardiac index (CI) and stroke volume (SV) were not affected. After recovery from anesthesia, conscious ABD rats had significantly increased BP at 3 h after barodenervation; BP returned to SO levels by 48 h even though plasma NE levels and TPR remained significantly increased. On the other hand, CI and SV showed significant reductions, beginning at 3 h, and remained low throughout the postdenervation period (48 h); the reduction in CI offset the increase in TRP and may therefore account for the restoration of BP of ABD rats to normal levels. Beginning at similar baseline BP values, cumulative intracisternal (i.c.) doses of clonidine (0.02-2.5 micrograms) elicited greater decreases in BP and plasma NE levels in conscious ABD as compared with SO rats. These responses were centrally mediated because systemic administration of 0.12 micrograms clonidine, a dose that elicited near maximal hypotensive response after i.c. administration, affected neither BP nor plasma NE levels. Contrary to the hypothesis, the hypotensive effect of clonidine in ABD rats resulted exclusively from a reduction in CO (owing to reductions in both HR and SV) because TPR was not affected. These findings suggest that (a) in ABD rats, a reduction in CO offsets a sustained sympathetically mediated elevation in TPR and restores BP to normal levels; and (b) an enhanced hypotensive response to clonidine in ABD as compared with SO rats cannot be accounted for by a higher basal TRP but rather by elicitation of greater reductions in CO through a centrally mediated sympathoinhibitory action.

An association between the estrogen-dependent hypotensive effect of ethanol and an elevated brainstem c-jun mRNA in female rats

We have recently demonstrated that chronic ethanol administration lowers blood pressure (BP) in female rats and this effect is significantly attenuated by ovariectomy. The present study investigated whether ethanol hypotension is estrogen dependent. Further, since estrogen regulates AP-1 activity, the study was extended to determine whether estrogen/c-jun interaction is involved in the estrogen-dependent hypotensive effect of ethanol. Changes in BP and heart rate (HR) were evaluated in radiotelemetered pair-fed sham-operated (SO), ovariectomized (OVX), and OVX estradiol (E2)-treated rats receiving liquid diet with or without ethanol (5%, w/v) for 12 weeks. The in situ hybridization technique was used to measure the c-jun mRNA expression in two brainstem areas, the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla (RVLM). Ethanol feeding caused significant (P<0.05) decreases in BP in SO rats that started at week 1 and reached its maximum (approximately 10 mmHg) at week 6 and remained at that level till the end of week 12. In OVX rats, ethanol had no effect on BP during the first 5 weeks after which a decrease of 5 mmHg was demonstrated and remained thereafter. Estrogen replacement (17beta-estradiol subcutaneous pellet, 14.2 microg/day) restored the hypotensive effect of ethanol to a level similar to that of SO rats both in terms of magnitude and duration. Densitometric analysis of the in situ hybridization autoradiograms revealed that OVX and E2 replacement had no effect on c-jun mRNA expression in the NTS or RVLM. Ethanol feeding produced a significant (twofold) increase in c-jun mRNA expression in the RVLM of SO rats versus no effect in the NTS. The increased expression of c-jun mRNA observed following ethanol treatment in the RVLM of SO rats was abolished in OVX rats and restored to SO levels after E2 replacement. These findings suggest a link between the estrogen-dependent hypotensive effect of chronically administered ethanol and the increased expression of c-jun mRNA in the brainstem of female rats.