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J Appl Physiol 97: 1237-1244, 2004. First published May 14, 2004; doi:10.1152/japplphysiol.00401.2004
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Differential modulation by estrogen of {alpha}2-adrenergic and I1-imidazoline receptor-mediated hypotension in female rats

Mahmoud M. El-Mas and Abdel A. Abdel-Rahman

Department of Pharmacology, School of Medicine, East Carolina University, Greenville, North Carolina 27858

Submitted 15 April 2004 ; accepted in final form 14 May 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We have recently shown that estrogen negatively modulates the hypotensive effect of clonidine (mixed {alpha}2-/I1-receptor agonist) in female rats and implicates the cardiovascular autonomic control in this interaction. The present study investigated whether this effect of estrogen involves interaction with {alpha}2- and/or I1-receptors. Changes evoked by a single intraperitoneal injection of rilmenidine (600 µg/kg) or {alpha}-methyldopa (100 mg/kg), selective I1- and {alpha}2-receptor agonists, respectively, in blood pressure, hemodynamic variability, and locomotor activity were assessed in radiotelemetered sham-operated and ovariectomized (Ovx) Sprague-Dawley female rats with or without 12-wk estrogen replacement. Three time domain indexes of hemodynamic variability were employed: the standard deviation of mean arterial pressure as a measure of blood pressure variability and the standard deviation of beat-to-beat intervals (SDRR) and the root mean square of successive differences in R-wave-to-R-wave intervals as measures of heart rate variability. In sham-operated rats, rilmenidine or {alpha}-methyldopa elicited similar hypotension that lasted at least 5 h and was associated with reductions in standard deviation of mean arterial pressure. SDRR was reduced only by {alpha}-methyldopa. Ovx significantly enhanced the hypotensive response to {alpha}-methyldopa, in contrast to no effect on rilmenidine hypotension. The enhanced {alpha}-methyldopa hypotension in Ovx rats was paralleled with further reduction in SDRR and a reduced locomotor activity. Estrogen replacement (17{beta}-estradiol subcutaneous pellet, 14.2 µg/day, 12 wk) of Ovx rats restored the hemodynamic and locomotor effects of {alpha}-methyldopa to sham-operated levels. These findings suggest that estrogen downregulates {alpha}2- but not I1-receptor-mediated hypotension and highlight a role for the cardiac autonomic control in {alpha}-methyldopa-estrogen interaction.

rilmenidine; {alpha}-methyldopa; blood pressure; hemodynamic variability

CENTRALLY ACTING ANTIHYPERTENSIVE agents, such as clonidine, act primarily within the rostral part of the ventrolateral medulla (RVLM) to reduce sympathetic outflow to peripheral organs. Initial studies suggested that the hypotensive effect of clonidine is due to activation of central {alpha}2-adrenoceptors (7, 33). Bousquet et al. (4, 5) have proposed that nonadrenergic receptors in the RVLM, specifically sensitive to imidazolines, mediate the hypotensive and sympathoinhibitory effects of centrally acting antihypertensive agents of the clonidine type. Ernsberger et al. (18) proposed two subtypes of imidazoline receptors, I1 and I2 sites, that are predominantly labeled by [3H]clonidine and [3H]idazoxan, respectively. I1-imidazoline binding sites appear to mediate the decrease in arterial pressure elicited by imidazolines in the RVLM (5, 17). Rilmenidine and moxonidine are second-generation, centrally acting antihypertensive drugs that are structurally related to clonidine (7). Compared with clonidine, rilmenidine and moxonidine are more selective for the I1 site than for {alpha}2-adrenoceptors (16). The I1-to-{alpha}2-receptor affinity ratio of rilmenidine and moxonidine are 30 and 33, respectively, vs. only 3.8 in the case of clonidine (16). The relatively lower affinity of rilmenidine and moxonidine, compared with clonidine, to {alpha}2-adrenoceptors has been suggested as the reason for their lesser sedative side effect (36).

In a recent study from our laboratory (15), we provided the first experimental evidence that highlights a role for the female hormone estrogen in the modulation of clonidine hypotension. In that study, we investigated the influence of long-term (12-wk) ovariectomy and estrogen replacement on the acute hypotensive effect of clonidine in conscious female rats. The results showed that the hypotensive effect of clonidine was augmented in ovariectomized (Ovx) rats and restored to sham-operated levels after estrogen replacement (15). Because the blood pressure effects of clonidine were paralleled with similar changes in the time domain indexes of hemodynamic variability, it is concluded that alterations in the cardiovascular autonomic control might contribute to the clonidine-estrogen hemodynamic interaction (15). Notably, in addition to its important role in clonidine hypotension, sympathoinhibition has also been implicated in the clonidine-induced reductions in blood pressure and heart rate (HR) variability, as determined by frequency and time domain analyses in previous studies, including our own (8, 14, 20, 21, 34). It is believed that the variability of blood pressure and HR is a measure of cardiovascular autonomic balance, and oscillation abnormalities in these hemodynamic variables are known to associate life-threatening cardiovascular events such as sudden stroke, ventricular arrhythmias, and myocardial infarction (29). The reduction in hemodynamic variability by clonidine is important at least in two clinical settings. First, the long-term reduction in blood pressure variability by clonidine may contribute to the regression of ventricular and vascular hypertrophy in hypertensive patients (30, 32). Second, by reducing blood pressure and HR variability, clonidine promotes hemodynamic stability in patients undergoing surgery and reduces anesthetic requirements (25).

Given that both I1-imidazoline receptors and {alpha}2-adrenoceptors have been implicated in the hypotensive effect of clonidine (4, 5, 7, 13, 33), it is not clear whether the modulation by estrogen of clonidine hypotension (15) involves interaction with either or both types of receptors. The main objective of this study was to investigate the relative contribution of I1-imidazoline receptors and {alpha}2-adrenoceptors to the hemodynamic interaction of estrogen with centrally acting antihypertensive agents in female rats. This was accomplished by investigating the effect of long-term estrogen depletion and repletion on the hypotensive and hemodynamic variability responses to {alpha}-methyldopa, a pure {alpha}2-adrenoceptor agonist (33), and rilmenidine, an oxazoline derivative with a higher I1-receptor-to-{alpha}2-receptor affinity ratio compared with clonidine (16). Blood pressure was measured by the radiotelemetry technique, which allows continuous monitoring of hemodynamics in freely moving, untethered animals.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Female Sprague-Dawley rats (9–10 wk; 190–225 g; Harlan, Indianapolis, IN) were used in the present study. On arrival, the rats were housed individually in standard plastic cages and allowed free access to water and Purina chow and were maintained on a 12:12-h light-dark cycle with light off at 7:00 PM. The room temperature was maintained at 22 ± 1°C. After 1-wk acclimatization, rats were fed a standard Lieber-DeCarli high-protein liquid diet (Dyets, Bethlehem, PA) (24) for another week before Ovx or sham operation. Rats were pair-fed to allow similar nutrient and fluid intakes (11, 14). Rats received the diet daily at 8:30 AM. Fresh diets were prepared every other day and stored in the refrigerator until dispensed. Experiments were carried out in accordance with the Declaration of Helsinki and with the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the US National Institutes of Health.

Ovariectomy and estrogen replacement.   Three groups of rats [sham-operated, Ovx, and Ovx with estrogen replacement (OvxE2); n = 6–7 each], matched for body weight, were used in the present study. For bilateral ovariectomy, the ovaries were isolated, tied off with sterile suture, and removed (10, 12). The sham operation was performed by exposing the ovaries without isolation. Pellets of 17{beta}-estradiol (1.7 mg, 14.2 µg/day, 120-day release, Innovative Research of America, Sarasota, FL) were implanted subcutaneously at the back of the neck in one Ovx group. Previous studies from our laboratory showed that this estrogen regimen produces physiological levels of the hormone (10, 12). The other two groups (sham-operated and Ovx) received placebo pellets. Telemetry transmitter implantation was performed 9 wk after Ovx or estrogen replacement. Rats were left for 3 additional wk before starting the experiment (i.e., saline, {alpha}-methyldopa, or rilmenidine administration).

Telemetry system.   The telemetry system (Data Sciences International, St. Paul, MN) used in this study has been described in our previous studies (10, 12, 14). The system consists of five major components: 1) implantable transmitter unit for measurement of blood pressure, 2) radio receiver to receive telemetered signals, 3) ambient pressure monitor to measure absolute atmospheric pressure, 4) a consolidation matrix to multiplex multiple cage signals to the computer, and 5) a personal computer-based data-acquisition system to process signals. The implanted sensor consisted of a fluid-filled catheter (0.7 mm diameter, 15 cm long; model TA11PA-C40) connected to a highly stable, low-conductance, strain gauge pressure transducer, which measured the absolute arterial pressure relative to a vacuum, and a radio-frequency transmitter. The tip of the catheter was filled with a viscous gel that prevented blood reflux and was coated with an antithrombogenic film to inhibit thrombus formation and maintain patency. The distal 1 cm of the catheter consisted of a thin-walled thermoplastic membrane, whereas the remainder of the catheter was composed of a thick-walled, low-compliance urethane. The implants (2.5-cm length and 1.2-cm diameter) weighed 9 g and had a typical battery life of 6 mo. Implants were gas sterilized and provided precalibrated (relative to vacuum) by the manufacturer, and calibrations were verified to be accurate within 3 mmHg (6). A radio receiver platform (RLA1010, Data Sciences International) connected the radio signal to digitized input that was sent to a dedicated personal computer (Compaq, Pressario 9548). Arterial pressures were calibrated by using an input from an ambient-pressure monitor (C11PR, Data Sciences International).

Transmitter implantation.   The method described in our previous studies (10, 12, 14) was adopted. The rats were anesthetized with intraperitoneal injection of a mixture of ketamine (90 mg/kg; Ketaject) and xylazine (10 mg/kg; Xyla-ject). The abdomen was opened with a midline incision (4 cm). Another incision (1.5 cm) was made along the inner thigh to expose the femoral artery. The abdominal wall was pierced with a large-bore syringe needle (15 gauge) from the femoral side into the peritoneal cavity. The implant body was placed in the peritoneal cavity, and the catheter (15 cm) was passed caudally through the syringe needle into the thigh area. A 5-cm portion of the catheter was inserted into the femoral artery and secured in place with sutures. The abdominal muscle was closed with nonabsorbable suture incorporating the implant suture rib with alternating stitches. The skin (abdomen and thigh) was closed with surgical clips. Each rat received a subcutaneous injection of the analgesic ketorolac tromethamine (2 mg/kg; Toradol) and an intramuscular injection of 60,000 units of penicillin G benzathine and penicillin G procaine in an aqueous suspension (Durapen). Individual rat cages were placed on the top of the radio receivers, and all data were collected by using a computerized data-acquisition system (Dataquest ART, Data Sciences International). The system is designed to cycle from animal to animal.

Measurement of plasma norepinephrine.   Blood (0.4 ml) was withdrawn from the tail vein 1 day before drug administration. The blood was collected into heparinized tubes containing 10 µl of glutathione (60 mg/ml) and 10 µl of perchloric acid (0.1 M). The collected blood was centrifuged at 5,000 rpm for 5 min, and the plasma was aspirated and stored at –80°C until analysis. Norepinephrine (pg/ml) was measured by ultrafiltration of the plasma followed by high-performance liquid chromatography with electrochemical detection, as described in our previous studies (10, 11).

Hemodynamic effects of {alpha}-methyldopa or rilmenidine.   This experiment investigated the influence of long-term Ovx and estrogen replacement on the acute hemodynamic effects of {alpha}-methyldopa or rilmenidine on locomotor activity, blood pressure, HR, and their variability in conscious, telemetered female rats. After 12 wk of sham operation, Ovx, or estrogen replacement, each rat received a single intraperitoneal injection of saline (1 ml/kg), rilmenidine (600 µg/kg), or {alpha}-methyldopa (100 mg/kg) at 3-day intervals. Blood pressure, HR, and locomotor activity were followed for 7 h after drug or saline administration. Waveforms of blood pressure for each rat were sampled at a rate of 500 Hz for 10 s every 10 min. Changes in mean arterial pressure (MAP) and HR from baseline values were averaged in 20-min blocks (i.e., the average of two successive measurements) for analysis, as in previous studies (14, 15). Baseline values of different hemodynamic variables were taken as the average of the 3-h period (9:00 AM to 12:00 PM) that preceded the administration of drugs.

Time domain analyses.   Three time domain parameters were employed to measure hemodynamic variability, as described in previous studies, including ours (14, 27, 29, 37). The standard deviation of the MAP (SDMAP) was taken as a measure of blood pressure variability. HR variability was determined by computing the standard deviation of beat-to-beat intervals (SDRR) and the root mean square of successive beat-to-beat differences in R-wave-to-R-wave (R-R) interval durations (rMSSD). The R-R intervals were computed from the HR values (i.e., the reciprocal of HR in ms) as in our previous studies (14, 15). Our previous studies and others have shown that the time domain indexes of blood pressure and HR variability correlate well with the frequency-domain measurements (11, 27, 29). The SDRR is comparable to the total power of the spectrum of R-R variability, which measures the overall autonomic (sympathetic and parasympathetic) balance of the heart. The rMSSD correlates with the high-frequency power of the spectrum and, therefore, more specifically quantifies the vagal influence on HR variability (27, 29). Changes from baseline values evoked by each treatment (saline, {alpha}-methyldopa, or rilmenidine) in the short-term variability of MAP and HR were calculated by averaging each 1-h values (i.e., 6 successive measurements at 10-min intervals) of SDMAP, SDRR, and rMSSD for a total of 7 h, as described in our previous studies (14, 15).

Drugs.   {alpha}-Methyldopa (Sigma Chemical, St. Louis, MO), Ketaject (ketamine), Xyla-ject (xylazine; Phoenix Pharmaceuticals, St. Joseph, MI), Toradol (ketorolac tromethamine; Abbott Laboratories, Chicago, IL), and Durapen (penicillin G benzathine and penicillin G procaine; Vedco, Overland Park, KS) were purchased from commercial vendors. Rilmenidine dihydrogen phosphate was a gift from Servier Pharmaceutical.

Statistical analysis.   All values are expressed as means ± SE. The repeated-measures two-way ANOVA followed by a Newman-Keuls post hoc test was used to analyze the effects of Ovx and estrogen replacement on hemodynamic responses to {alpha}-methyldopa or rilmenidine. These analyses were performed by SAS software release 6.04 (SAS Institute, Cary, NC), as in our previous studies (14, 15). Probability levels <0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline data.   Baseline values of locomotor activity, blood pressure, HR, and their variability indexes (SDMAP, SDRR, and rMSSD), measured in conscious female rats 12 wk after sham operation, Ovx, or estrogen replacement are shown in Table 1. The values of these variables in the three groups of rats were not statistically different except for significantly higher SDRR values in OvxE2 compared with sham-operated rats (Table 1). The body weights of rats before sham operation or Ovx were similar (sham, 196.6 ± 2.7 g; Ovx, 199.2 ± 3.4 g; OvxE2, 194.9 ± 3.9 g). By the end of the study, the body weight of Ovx rats (338.7 ± 12.6 g) was slightly but significantly higher than that of sham-operated (299.5 ± 7.2 g) or OvxE2 (283.6 ± 5.6 g) rats. Baseline plasma norepinephrine levels measured 12 wk after sham, Ovx, or OvxE2 were not statistically different (435.9 ± 27.8, 544.6 ± 56.2, and 516.9 ± 62.3 pg/ml, respectively).


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  		Table 1. Baseline values of MAP, HR, SDMAP, SDRR, rMSSD, and locomotor activity

 
Effects of Ovx and estrogen replacement on the hemodynamic effects of {alpha}-methyldopa.   The changes elicited by {alpha}-methyldopa in blood pressure, HR, hemodynamic variability, and locomotor activity in telemetered sham-operated or Ovx female rats with or without estrogen replacement are shown in Figs. 1–3. Compared with the corresponding time course effects of saline, {alpha}-methyldopa (100 mg/kg ip) produced significant reductions in MAP in sham-operated rats that started at 40 min and continued for the remaining duration of the experiment (Fig. 1A). The hypotensive effect of {alpha}-methyldopa was significantly enhanced in rats subjected to long-term Ovx and restored to intact (sham-operated) levels after estrogen replacement (Fig. 1A). The maximum hypotensive response elicited by {alpha}-methyldopa was increased twofold in Ovx rats compared with sham-operated and OvxE2 rats (–30 ± 5, –16 ± 2, and –15 ± 2 mmHg, respectively). On the other hand, the HR showed significant increases by {alpha}-methyldopa in sham-operated rats during the first 2 h (Fig. 1B), an effect that was not altered by Ovx or estrogen replacement (Fig. 1B).



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  		Fig. 1. Changes ({Delta}) in mean arterial pressure (MAP; A) and heart rate (HR; B) evoked by {alpha}-methyldopa (100 mg/kg ip) in conscious, telemetered sham-operated, ovariectomized (Ovx), and estrogen-replaced ovariectomized (OvxE2) rats. Values are means ± SE of 6–7 observations. P < 0.05 vs. * saline, # sham-operated, and + OvxE2 values.

 


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  		Fig. 3. Changes in the locomotor activity evoked by intraperitoneal {alpha}-methyldopa (100 mg/kg; A) or rilmenidine (600 µg/kg; B) in conscious, telemetered sham-operated, Ovx, and OvxE2 rats. Values are means ± SE of 6–7 observations. P < 0.05 vs. * saline, # sham-operated, and + OvxE2 values.

 
The MAP variability index (SDMAP, Fig. 2A) and the overall HR variability index (SDRR, Fig. 2B) were significantly reduced by {alpha}-methyldopa at 4 h in sham-operated rats. The reduction in SDMAP by {alpha}-methyldopa was not altered by Ovx or estrogen replacement (Fig. 2A). In contrast, the {alpha}-methyldopa-evoked reduction in SDRR was enhanced in Ovx rats, in terms of both magnitude and duration (Fig. 2B). Compared with control (saline) values, the reduction in SDRR appeared at 2 h after {alpha}-methyldopa administration to Ovx rats and continued for the following 4 h (Fig. 2B). Also, the reductions in SDRR by {alpha}-methyldopa in Ovx rats were statistically significant compared with sham-operated values at 5 and 6 h and compared with OvxE2 values at 5 h (Fig. 2B). No differences in the SDRR responses to {alpha}-methyldopa were demonstrated between sham-operated and OvxE2 rats (Fig. 2B). {alpha}-Methyldopa had no effect on the variability of the adjacent interbeat intervals (rMSSD) in any of the three rat groups (Fig. 2C). The locomotor activity was significantly reduced by {alpha}-methyldopa only in Ovx rats in contrast to no effect in sham-operated or OvxE2 rats (Fig. 3A).



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  		Fig. 2. Changes in the time domain variability indexes of MAP [standard deviation of MAP (SDMAP); A] and HR [standard deviation of beat-to-beat intervals (SDRR; B), and the root mean square of successive beat-to-beat differences (rMSSD; C)] evoked by {alpha}-methyldopa (100 mg/kg ip) in conscious, telemetered sham-operated, Ovx, and OvxE2 rats. Values are means ± SE of 6–7 observations. P < 0.05 vs. * saline, # sham-operated, and + OvxE2 values.

 
Effects of Ovx and estrogen replacement on the hemodynamic effects of rilmenidine.   The intraperitoneal injection of rilmenidine (600 µg/kg) to sham-operated rats elicited significant reductions in MAP (Fig. 4A) and HR (Fig. 4B) with maximum responses of –17 ± 2 mmHg and –42 ± 14 beats/min, respectively, observed at 60 min. The hypotensive and bradycardic effects of rilmenidine were not influenced by Ovx or estrogen replacement (Fig. 4). The SDMAP (Fig. 5A), but not SDRR (Fig. 5B) or rMSSD (Fig. 5C), was significantly and similarly reduced by rilmenidine in sham-operated, Ovx, and OvxE2 rats. Also, the locomotor activity was not altered by rilmenidine in the three groups of rats (Fig. 3B).



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  		Fig. 4. {Delta}MAP (A) and {Delta}HR (B) evoked by rilmenidine (600 µg/kg ip) in conscious, telemetered sham-operated, Ovx, and OvxE2 rats. Values are means ± SE of 6–7 observations. * P < 0.05 vs. corresponding saline values.

 


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  		Fig. 5. {Delta}SDMAP (A), {Delta}SDRR (B), and {Delta}rMSSD (C) evoked by rilmenidine (600 µg/kg ip) in conscious, telemetered sham-operated, Ovx, and OvxE2 rats. Values are means ± SE of 6–7 observations. * P < 0.05 vs. corresponding saline values.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Evidence of a negative modulatory effect of estrogen on the hypotensive action of the centrally acting antihypertensive agent clonidine in female rats has been established in a recent study from our laboratory (15). Because clonidine lowers blood pressure via activation of {alpha}2-adrenoceptors and I1-imidazoline receptors (4, 5, 7, 13, 33) and exhibits a relatively low I1-receptor-to-{alpha}2-receptor affinity ratio (16), whether I1- and {alpha}2-receptors play different roles in the hemodynamic interaction between estrogen and centrally evoked hypotension cannot be ascertained from our previous report (15). {alpha}-Methyldopa and rilmenidine, by virtue of their higher affinity at {alpha}2-adrenoceptors and I1-imidazoline receptors (16, 33), respectively, were used to address this issue. Therefore, experiments were undertaken to investigate the effect of 12-wk Ovx and estrogen replacement on the hypotensive and hemodynamic variability responses to {alpha}-methyldopa or rilmenidine in telemetered female rats. The most important conclusion of the present study is that estrogen differentially influences hypotensive and hemodynamic variability responses mediated via activation of {alpha}2-adrenergic and I1-imidazoline receptors. This conclusion is based on the following findings: 1) the hypotensive effect of {alpha}-methyldopa, but not rilmenidine, was enhanced in Ovx rats and resorted to sham-operated levels after estrogen supplementation; 2) the enhancement of {alpha}-methyldopa hypotension in Ovx rats was paralleled with significant increases in the associated reductions in the overall HR variability (SDRR), whereas the adjacent interbeat intervals (rMSSD) were not altered; and 3) locomotor activity was significantly reduced by {alpha}-methyldopa only in Ovx rats. These findings suggest a selective restraining influence of estrogen on {alpha}2-adrenergic-receptor-mediated hypotension, possibly via alteration of the cardiac autonomic control.

The time domain measures of hemodynamic variability were employed in the present study to reflect changes in the cardiovascular autonomic control and its possible contribution to the hemodynamic effects of centrally acting antihypertensive agents and their interaction with estrogen. The results in sham-operated rats showed that {alpha}-methyldopa and rilmenidine elicited similar decreases in blood pressure, but their effect on hemodynamic variability was variable. The hypotensive effect of {alpha}-methyldopa was accompanied by reductions in the variability of both blood pressure (SDMAP) and HR (SDRR), in contrast to a reduction only in the blood pressure variability-associating rilmenidine hypotension. Because the time domain index of parasympathetic activity rMSSD was not affected by either drug, the findings are consistent with an inhibitory effect of the two drugs on sympathetic activity. The reduction by {alpha}-methyldopa in both SDMAP and SDRR may reflect inhibition of vascular and cardiac sympathetic activity by the drug. On the other hand, SDMAP, but not SDRR, was reduced by rilmenidine, suggesting a selective inhibitory effect on vascular sympathetic activity. These results demonstrate differential modulation of cardiovascular autonomic control by I1- and {alpha}2-receptors in female rats. Notably, earlier reports established a link between the reductions in blood pressure (9, 33) and hemodynamic variability (8, 19, 21, 34) caused by clonidine and related drugs on the one hand, and the inhibition of central sympathetic tone on the other. The reduced blood pressure variability produced by rilmenidine in hypertensive humans coincides with a predominant reduction in blood pressure fluctuations in the midfrequency range (0.1 Hz), which reflects sympathoinhibition (19). Furthermore, time or frequency domain measurements highlight a role for sympathoinhibition in the reduction in hemodynamic variability that parallels centrally evoked hypotension (8, 21, 34).

Findings of the present study showed that the ability of estrogen to modulate hemodynamic responses to centrally acting drugs depended on the type of receptor, I1 or {alpha}2, involved. Estrogen attenuated the hypotensive response elicited by activation of {alpha}2-receptors ({alpha}-methyldopa) in contrast to no effect on I1 (rilmenidine)-mediated hypotension. This view is supported by the observations that 1) long-term ovariectomy, which causes a considerable reduction in plasma estrogen levels (10, 12), remarkably potentiated the hypotensive effect of {alpha}-methyldopa; 2) in Ovx rats, estrogen replacement restored the hypotensive effect of {alpha}-methyldopa to sham-operated levels; and 3) the hypotensive effect of rilmenidine was not dependent on hormonal status of the female rat; i.e., it was not altered by ovariectomy or estrogen replacement. These findings, together with our previous observation that estrogen downregulates the hypotensive response to the mixed {alpha}2-/I1-receptor agonist clonidine (15), may suggest a selective interaction of estrogen with central neuronal pathways involved in {alpha}2-receptor-mediated hypotension. It is noteworthy that the estrogen regimen employed in the present study produced plasma estrogen concentrations in our previous studies (10, 12) similar to those demonstrated during the proestrus phase in rats (1).

The selective modulatory effect of estrogen on the hypotensive response to {alpha}2-receptor activation might be accounted for, at least in part, by the associated changes in hemodynamic variability and locomotor activity. Similar to its effects on {alpha}-methyldopa hypotension, Ovx increased the magnitude and duration of the {alpha}-methyldopa-evoked reduction in SDRR, whereas the reduction in SDMAP remained unaffected. These findings may implicate the cardiac sympathetic control in {alpha}-methyldopa-estrogen hemodynamic interaction. Unlike {alpha}-methyldopa, rilmenidine had no effect on cardiac autonomic activity, as indicated by the lack of any changes in the HR variability indexes. A reduction in the variability of blood pressure (SDMAP) was demonstrated after rilmenidine administration, but this effect was independent of ovarian hormones, i.e., remained unaffected by Ovx or estrogen replacement. Also, the telemetric measurement of the locomotor activity provided information that may explain the facilitated hemodynamic effects of {alpha}-methyldopa in Ovx rats. The locomotor activity (whole body movement) was obtained by telemetric monitoring of changes in the received signal strength, which occurred on the animal's movement. Changes in signal strength beyond a predetermined level generate a digital pulse, which is counted by the Dataquest system. The present study demonstrated that the locomotor activity was reduced by {alpha}-methyldopa in Ovx rats but not in sham-operated or estrogen-replaced Ovx rats. It is believed that {alpha}-methyldopa and clonidine, owing to their {alpha}2-agonistic activity, produce sedation, which favors hemodynamic stability (15, 21). The relatively lower affinity of rilmenidine, compared with {alpha}-methyldopa or clonidine, to {alpha}2-adrenoceptors may account for its lesser sedative side effect (36), which is supported by the lack of rilmenidine effect on locomotor activity in the present study.

Given the critical role of sympathoinhibition in {alpha}-methyldopa-evoked hypotension (33), the possibility must be considered that a higher preexisting sympathetic activity may have accounted for the enhanced hypotensive action of {alpha}-methyldopa in Ovx rats. This assumption appeared unlikely because plasma norepinephrine (index of sympathetic activity) and blood pressure values measured before drug administration were not significantly altered by Ovx or estrogen replacement. Notably, the present study and others (10) showed that Ovx rats gained more weight over the course of the study compared with sham or OvxE2 rats. It is worth mentioning that the pair-feeding paradigm was utilized in the present study to allow similar fluid and nutrient intakes (11, 14) and, therefore, circumvent the impact of differences in the magnitude of fluid intake on hemodynamics (3). The question of whether differences in the body weight gain played a part in the estrogen modulation of {alpha}-methyldopa hypotension is not clear. It remains also to be investigated whether changes in body fat, glucose, or insulin profiles contribute to the hormonal modulation of {alpha}-methyldopa-induced hypotension.

The attenuation by estrogen of {alpha}2-receptor-mediated hypotension may also be accounted for by the ability of estrogen to alter the binding activity of {alpha}2-adrenergic receptors. In effect, estrogen has been shown to reduce the binding activity (22, 28) and mRNA expression (23) of {alpha}2-adrenoceptors in the brain. Also, estrogen administration to Ovx rats decreases {alpha}2-receptor expression in brain tissues via increasing the activity of the G-protein-coupled receptor kinase (2), which is involved in the downregulation of G-protein-coupled receptors, including {alpha}2-adrenoceptors (26). On the other hand, whether estrogen alters the binding activity of central I1-receptors has not been investigated. Notably, the present study provides evidence that supports earlier findings, including ours (4, 5, 9, 13), concerning the role of I1-imidazoline receptors in the central regulation of blood pressure and as the main site of the hypotensive action of rilmenidine and related imidazolines. The ability of estrogen to attenuate hypotensive and hemodynamic variability responses to {alpha}-methyldopa but not rilmenidine suggests the involvement of different neuronal pathways and receptor sites, {alpha}2-adrenoceptors and I1-imidazoline receptors, respectively, in the hypotensive effects of these drugs.

The present finding that the hypotensive effect of {alpha}-methyldopa was associated with significant increases in HR deserves a comment. The tachycardic response to {alpha}-methyldopa does not appear to be a compensatory baroreflex response to the evoked hypotension because the peak increase in HR appeared 20 min after {alpha}-methyldopa administration and preceded the fall in blood pressure, as showed in this study (Fig. 1) and by others (35). Evidence is available that {alpha}-methyldopa-evoked tachycardia is a peripherally mediated effect and involves direct activation of cardiac {beta}-adrenoceptors following its conversion to {alpha}-methyldopamine in cardiac sympathetic neurons (35). In support of this view, {alpha}-methyldopa elicits no tachycardia when administered centrally (9, 35). Because the activation of cardiac {beta}-adrenoceptors elicits chronotropic and inotropic effects, it is possible that these effects might act to diminish the blood pressure-lowering action of {alpha}-methyldopa. In effect, the initial tachycardic response to {alpha}-methyldopa may help explain, at least partly, the delayed hypotensive and hemodynamic variability responses to the drug in the present study. The delayed hypotensive effect of {alpha}-methyldopa has traditionally been accounted for by the dependence of the drug effect on its central degradation into {alpha}-methylnorepinphrine, another metabolite of {alpha}-methyldopa (31).

In summary, findings of the present study undertaken in telemetered female rats suggest a differential effect of estrogen on hemodynamic responses elicited via activation of {alpha}2-adrenoceptors and I1-imidazoline receptors by {alpha}-methyldopa and rilmenidine, respectively. Long-term (12-wk) ovariectomy significantly potentiated the reductions in blood pressure and hemodynamic variability evoked by {alpha}-methyldopa in contrast to no effect on rilmenidine responses, suggesting a selective downregulatory effect of ovarian hormones on {alpha}2-receptor-mediated responses. The finding that estrogen replacement of Ovx rats restored the facilitated hemodynamic effects of {alpha}-methyldopa to sham operation levels provides direct evidence that implicates estrogen in the regulation of {alpha}-methyldopa responses. Alterations in the cardiac autonomic control and locomotor activity appear to contribute, at least partly, to the interaction. The alteration by estrogen of {alpha}-methyldopa-mediated effects should be considered when the drug is used to control hypertension in clinical conditions characterized by altered hormonal balance, such as menopause and pregnancy. Under these conditions, the use of the selective I1-receptor agonist rilmenidine may be advantageous because its effects on blood pressure and its variability index are not affected by estrogen and, unlike {alpha}-methyldopa, rilmenidine does not inhibit locomotor activity.


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 ABSTRACT
 METHODS
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This study was supported by National Institute on Alcohol Abuse and Alcoholism Grant AA-07839.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. A. Abdel-Rahman, Dept. of Pharmacology, School of Medicine, East Carolina Univ., Greenville, NC 27858 (E-mail: abdelrahmana{at}mail.ecu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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