Abdel A. Abdel-Rahman

Ethanol-induced hypertension involves impairment of baroreceptors.

We studied the effect of 12 weeks of ethanol feeding on arterial blood pressure and baroceptor reflex control of heart rate in Sprague-Dawley and Wistar rats. Baroceptor reflex sensitivity and pressor responsiveness were evaluated by evoking graded rises in mean arterial pressure with increasing doses of phenylephrine and angiotensin II. After 12 weeks of ethanol feeding there was a modest increase in mean arterial pressure with no change in heart rate in both strains. When angiotensin II or phenylephrine was used as the pressor agent, baroreceptor reflex curves (relationships between changes in mean arterial pressure and heart rate) of Wistar rats were shifted upward and had a markedly reduced slope compared with those of control rats, suggesting that impairment of baroreceptor reflex control of heart rate had occurred. This effect was less evident in the Sprague-Dawley rats. Ethanol-fed rats had a higher sympathetic activity, since beta-blockade with propranolol decreased heart rate to a greater degree than that seen in control rats. The pressor response curve of phenylephrine was shifted to the right in control rats challenged with ethanol (0.5 g/kg), implying the presence of alpha-blockade. This shift was not present in ethanol-fed rats, showing that tolerance had developed to this effect of ethanol. These findings show that attenuation of baroreceptor reflex function is associated with ethanol-induced hypertension but do not establish whether this is a cause or an effect of the developed hypertension.

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.

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.

Neuronal and cardiovascular responses to adenosine microinjection into the nucleus tractus solitarius

This study investigated neuronal, blood pressure, and heart rate responses to adenosine microinjection into caudal and rostral NTS of anesthetized rats. The site of recording and microinjection was verified chemically by observing the responses to a test dose of l-glutamate (5 nmol) and histologically at the conclusion of the experiment. Neuronal firing rate increased (+29.4 +/- 5.3%) and decreased (-48 +/- 9.4%) in response to l-glutamate microinjection into the rostral and caudal NTS, respectively. These opposite neuronal responses were followed by depressor (-32.4 +/- 8.3 vs. -36 +/- 5.5 mmHg) and bradycardic (-25.2 +/- 7.7 vs. -25.8 +/- 3.4 beats/min) responses to l-glutamate microinjection into the two subareas of the NTS. Microinjection of a submaximal dose (1 nmol) of adenosine into the NTS produced site-dependent cardiovascular responses which were preceded by similar inhibition of neuronal firing (-60 +/- 4 vs. -55.9 +/- 1.7%). Whereas adenosine microinjection into the rostral NTS elicited modest pressor (+10.1 +/- 2.1 mmHg) and tachycardic (+9 +/- 3.9 beats/min) responses, its microinjection into the caudal NTS produced depressor (-29.2 +/- 5.3 mmHg) and bradycardic (-14.6 +/- 1.7 beats/min) responses. These findings suggest that compared to l-glutamate, adenosine produces opposite (rostral) and similar (caudal) neuronal and cardiovascular effects in the two subareas of the NTS. In the caudal NTS, adenosine (0.1, 1, and 10 nmol) elicited dose-related inhibitory neuronal and cardiovascular responses that were attenuated by systemic theophylline but not 8-(p-sulfophenyl) theophylline (8-SPT) administration. The neuronal and cardiovascular responses to adenosine microinjection into the caudal NTS were also attenuated by microinjection of 8-SPT into the same area. Finally, single-unit activity inhibited by adenosine or l-glutamate microinjection into the caudal NTS was also inhibited by baroreceptor loading and excited by baroreceptor unloading. These findings suggest a) l-glutamate elicits opposite neuronal responses in the rostral and caudal NTS; b) the distinct hemodynamic responses elicited by adenosine in the two subareas may be related, at least in part, to their differing responses to l-glutamate; and c) the similarity between the neuronal responses to adenosine and l-glutamate microinjection into the caudal NTS and the response of the same neurons to baroreceptor activation support the hypothesis that adenosine plays a neuromodulatory role in the processing of baroreceptor information.

Impairment of baroreceptor reflex control of heart rate but not sympathetic efferent discharge by central neuroadministration of ethanol.

We investigated the acute hemodynamic effects of ethanol microinjection into brain areas known to influence cardiovascular function and reflexes. In chloralose-anesthetized rats, ethanol had no effect on baseline mean arterial pressure, heart rate, or sympathetic efferent discharge when microinjected into the nucleus tractus solitarius, the dorsal motor nucleus of the vagus, the rostral ventrolateral medulla, or the posterior hypothalamus. On the other hand, ethanol microinjection into the anterior hypothalamus caused a site-dependent pressor effect and an increase in sympathetic efferent discharge. Baroreceptor heart rate response but not sympathetic efferent discharge response was impaired by ethanol microinjection into the nucleus tractus solitarius, the dorsal motor nucleus of the vagus, and the rostral ventrolateral medulla, suggesting that ethanol involves one or more of these areas in its inhibitory effect on baroreceptor heart rate response and that ethanol has a selective action on baroreceptor reflex control of heart rate. The findings that 1) the effect was dose dependent and 2) injection of ethanol outside of, or an equal volume of cerebrospinal fluid into, the nucleus tractus solitarius had no effect on the response strongly suggest that the observed effect on baroreceptor heart rate response was ethanol mediated. Ethanol microinjection into the dorsal motor nucleus of the vagus impaired the heart rate response, thus raising the possibility that leakage of ethanol to that area from the nucleus tractus solitarius might have contributed to its effect. These findings show that ethanol has a pressor and sympathoexcitatory site of action within the anterior hypothalamus and that it selectively impairs baroreceptor heart rate response via a central site of action; the mechanisms by which ethanol produces these effects remain to be elucidated.

Role of the Sympathetic Nervous System in Ethanol-Induced Hypertension in Rats

The present study investigated the role of the sympathetic nervous system in the development of ethanol-induced hypertension (EIH) in the rat. Sympathetic nerve activity (SNA) as an index of central sympathetic tone was measured directly from the preganglionic fibers of the greater splanchnic nerve. Four weeks after starting ethanol feeding, and prior to the development of hypertension, SNA of the ethanol-fed rats was significantly greater than that of controls. The increase in SNA was also evident at the early stages of EIH, at 8 weeks, and in fully developed EIH, after 12 weeks of ethanol consumption. Baroreceptor reflex control of heart rate (HR) but not SNA was impaired prior to the development of EIH at 4 weeks. However, at 8 and 12 weeks, baroreflex control of HR and SNA was normal or slightly greater than that of control rats. Because arterial pressure of ethanol-fed rats was significantly higher than that of controls at 8 and 12 weeks, the data suggest that ethanol feeding caused baroreceptor resetting. Pressor responsiveness to phenylephrine was depressed before the development of EIH but was similar to that of control rats following the development of EIH. The data also shows that blood and plasma volumes of ethanol-fed rats at the times that coincided with the pre- and posthypertensive states were similar to those of control rats which suggests that the development of EIH does not involve an increase in plasma volume. It is concluded that an increase in SNA contributes to the development of EIH and that baroreceptor resetting evoked by ethanol feeding plays a permissive role in maintaining an elevated blood pressure in ethanol-fed rats.

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.

Environmental temperature variations cause degradations in epinephrine concentration and biological activity

This study determined the biological consequence of temperature induced epinephrine degradation. Two different epinephrine preparations (1:1,000 and 1:10,000) were exposed to either cold (5 degrees C) or hot (70 degrees C) temperature. The exposure occurred for 8-hour periods each day in 4-, 8-, and 12-week intervals. Samples and identical controls were then chemically evaluated using high-pressure liquid chromatography (HPLC), and biological activity of samples showing chemical degradation was assessed in conscious rats. Epinephrine (1:10,000) underwent a significant degradation and a loss of concentration of the parent compound after 8 weeks of heat treatment. By 12 weeks, 64% of the epinephrine was degraded. A smaller (30%) but significant loss of cardiovascular potency was determined by blood pressure and heart rate responses in conscious rats. The degradation of epinephrine (1:1,000) was not statistically significant even after 12 weeks of heat exposure. No change was noted from control in either epinephrine concentration when exposed to cold temperatures. In conclusion, epinephrine (1:10,000) deteriorates in the presence of elevated temperature and should be protected from high temperatures when carried by EMS providers. The degradation products may possess biological activity.

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.