Evidence for the Involvement of Central I₁ Imidazoline Receptor in Ethanol Counteraction of Clonidine Hypotension in Spontaneously Hypertensive Rats

Abstract

 

Our previous studies have shown that ethanol counteracts centrally mediated hypotensive responses to clonidine. In this study, we investigated the relative roles of central α2-adrenergic and I₁ imidazoline receptors in the antagonistic ethanol-clonidine hemodynamic interaction. The effects of selective blockade of α₂- or I₁ receptor by 2-methoxyidazoxan and efaroxan, respectively, on the blood pressure and heart rate responses to clonidine and subsequent ethanol administration were evaluated in conscious spontaneously hypertensive rats. Intracisternal administration of clonidine (1.5 μg/kg) produced significant (30 mm Hg; p < 0.05) and sustained (at least 60 min) decreases in blood pressure and heart rate. Systemic ethanol (1 g/kg), administered 10 min after clonidine, counteracted the hypotensive response and restored blood pressure to the preclonidine levels. Treatment with 2-methoxyidazoxan (0.16 μg/kg, intracisternal) or efaroxan (0.45 μg/kg, intracisternal) produced similar attenuation of the hypotensive and bradycardic responses to clonidine. The ability of ethanol to counteract the hypotensive action of clonidine was significantly (p < 0.05) attenuated in rats pretreated with efaroxan. The pressor response to ethanol lasted only 10 min compared with at least 60 min in the absence of efaroxan. In contrast, ethanol counteraction of clonidine-evoked hypotension was not altered when α₂-adrenoceptors were blocked by 2-methoxyidazoxan. These findings suggest that centrally mediated hypotensive and bradycardic effects of clonidine in conscious spontaneously hypertensive rats involve activation of both α₂-adrenergic and I₁ imidazoline receptors. Furthermore, the findings suggest the dependence of a fully expressed ethanol counteraction of the hypotensive action of clonidine on functional I₁ receptor within the central nervous system.

Reported findings have shown that regular use of ethanol is associated with inadequate control of blood pressure in patients treated for hypertension (^1,2). These clinical observations have been supported by experimental findings from our laboratory that highlighted the ability of ethanol to counteract the hypotensive effect of centrally acting antihypertensive agents such as clonidine and guanabenz (^3–6). This adverse effect of ethanol on centrally mediated hypotensive responses is demonstrated in conscious aortic barodenervated (^5,6) and spontaneously hypertensive rats (SHRs) (^3,4). In contrast, peripherally mediated hypotensive responses (e.g., hydralazine, nitroprusside, or hexamethonium) were not affected by ethanol (^3,4,7). These findings suggest that the ability of ethanol to adversely affect centrally mediated hypotensive responses involves, at least in part, the central nervous system. This view is further supported by ethanol counteraction of the centrally mediated sympathoinhibition elicited by clonidine. Ethanol counteracted clonidine-evoked reductions in plasma norepinephrine levels (^4,5) and in norepinephrine electrochemical signal in the rostral ventrolateral medulla (⁸). It is notable, however, that the possible involvement of the peripheral hemodynamic effects of ethanol, e.g. vasodilatation of cutaneous blood vessels (⁹), in their interaction with antihypertensive drugs cannot be overlooked. Acute ethanol administration may cause decreases (¹⁰), increases (¹¹), or no change (¹²) in blood pressure.

It is generally believed that clonidine lowers blood pressure via activation of α₂-adrenoceptors and I₁ imidazoline receptors in the central nervous system (^13–16). Given the lack of selectivity of clonidine to either receptor (¹⁷), whether one receptor site (I₁ or α₂-) plays a greater role in ethanol-clonidine hemodynamic interaction is not possible to ascertain from previous findings (^3,5–7). This issue was addressed in more recent studies from our laboratory (^18,19), which investigated the effect of ethanol on the hemodynamic and sympathoinhibitory responses to rilmenidine and α-methylnorepinephrine (α-MNE), selective I₁ and α₂-adrenergic receptor agonists, respectively. Interestingly, the results of these studies showed that ethanol counteracted decreases in blood pressure and peripheral vascular resistance elicited by central administration of rilmenidine whereas it had little or no effect on α-MNE-mediated responses (^18,19). Ethanol given after α-MNE produced a pressor effect that lasted 10 min only versus at least 60 min in case of rilmenidine (^18,19). Furthermore, the counteraction by ethanol of the rilmenidine-mediated sympathoinhibition was significantly greater (threefold) than its effect on α-MNE-mediated sympathoinhibition (¹⁹). These findings suggested a selective interaction of ethanol with central pathways that are essential for the elicitation of I₁ receptor-mediated hypotension and sympathoinhibition (^18,19).

The main objective of the present study was to extend these previous studies to gain further support for the hypothesis that ethanol selectively interacts with central pathways involved in I₁ receptor-mediated hypotension. To achieve this goal, in the present study we used one agonist, clonidine, which exerts similar affinities at α₂-and I₁ receptor (¹⁷). The effect of the selective α₂-receptor antagonist, 2-methoxyidazoxan, and the I₁/α₂-antagonist, efaroxan, on the counteraction by ethanol of clonidine hypotension was evaluated. We hypothesize that a preferential attenuation by efaroxan of ethanol-clonidine hemodynamic interaction would suggest the involvement of I₁ imidazoline receptor in ethanol-clonidine hemodynamic interaction. Alternatively, a similar attenuation of the pressor effect of ethanol by efaroxan or 2-methoxyidazoxan would favor a major role for α₂-adrenoceptors in the interaction between ethanol and centrally acting hypotensive agents.

METHODS

Intracisternal cannulation

Four to 5 days before starting the experiment, a stainless steel guide cannula was implanted into the cisterna magna under methohexital anesthesia (50 mg/kg i.p.). The steel cannula (23G; Small Parts, Miami, FL, U.S.A.) was passed between the occipital bone and the cerebellum so that its tip protruded into the cisterna magna. The cannula was secured in place with small metal screws and dental acrylic cement (Durelon; Thompson Dental Supply, Raleigh, NC, U.S.A.) as described in our previous studies (^18,19). The guide cannula was considered patent when spontaneous outflow of cerebrospinal fluid was observed and by gross postmortem histologic verification following injection of 5 μl of fast green dye (EM Science, Cherry Hill, NJ, U.S.A.). After intracisternal (i.c.) cannulation, the rats were housed individually. Intravascular cannulation was performed 2 to 3 days later as described in the next section.

Intravascular cannulation

For measurement of blood pressure, the method described in our previous studies (^18,19) was adopted. Briefly, the rats were anesthetized by methohexital (50 mg/kg, i.p.). Catheters (polyethylene 50) were placed in the abdominal aorta and vena cava via the femoral artery and vein for measurement of blood pressure and i.v. administration of drugs, respectively. The catheters were inserted about 5 cm into the femoral vessels and secured in place with sutures. The arterial catheter was connected to a Gould-Statham pressure transducer (Oxnard, CA, U.S.A.) and blood pressure was displayed on a Grass polygraph (model 7D, Grass Instruments, Quincy, MA, U.S.A). Heart rate was computed from blood pressure waveforms by a Grass tachograph and was displayed on another channel of the polygraph. The catheters were tunneled subcutaneously and exteriorized at the back of the neck between the scapulae. The catheters were flushed with heparin (200 U/ml) and plugged by stainless steel pins. Incisions were closed with surgical clips and swabbed with povidone-iodine solution. Each rat received a subcutaneous injection of the analgesic buprenorphine hydrochloride (Buprenex, Reckitt & Coleman Pharmaceuticals, Inc., Richmond, VA, U.S.A.; 0.3 μg/rat) and an i.m. injection of 60,000 U of penicillin G benzathine and penicillin G procaine in an aqueous suspension (Durapen, Vedco, Overland Park, KS, U.S.A.) and was housed in a separate cage. The experiment started 48 h later. Experiments were performed in strict accordance with institutional animal care and use guidelines.

Protocol and experimental groups

A total of 37 male SHRs (300–350 g; Charles River, Raleigh, NC, U.S.A.) were used in the present study. On the day of the experiment, the arterial catheter was connected to a pressure transducer for measurement of blood pressure and heart rate as mentioned previously. A period of 30 min was allowed at the beginning of the experiment for stabilization of blood pressure and heart rate.

Experiment 1: effect of efaroxan or 2-methoxyidazoxan on the hypotensive effect of clonidine

Three groups of rats (n = 5–9, Table 1) were used in this experiment to investigate the effect of selective blockade of α₂- or I₁ receptor (by 2-methoxyidazoxan and efaroxan, respectively) on the hemodynamic responses to i.c. administration of clonidine (1.5 μg/kg) in conscious freely moving SHRs. The 1.5-μg/kg dose of clonidine was shown in our previous studies to elicit approximately 80% of the maximal depressor response after i.c. administration (^20,21). Ten minutes after clonidine administration, efaroxan (0.45 μg/kg), 2-methoxyidazoxan (0.16 μg/kg), or the vehicle (saline) was injected i.c. These doses of efaroxan and 2-methoxyidazoxan were found in preliminary studies to elicit similar attenuation of the clonidine-evoked hypotension. The injection volume for administered drugs i.c. was 5 μl. Changes evoked in mean arterial pressure (MAP) and heart rate by clonidine and subsequent efaroxan or 2-methoxyidazoxan administration were followed for 80 min.

TABLE 1: Baseline values of mean arterial pressure (MAP, mm Hg) and heart rate (HR, beats/min)

Experiment 2: effect of efaroxan or 2-methoxyidazoxan on ethanol-clonidine hemodynamic interaction

Three groups of SHRs (n = 5–8, Table 1) were used in this experiment to investigate the role of α₂-adrenoceptors and I₁ imidazoline receptors in ethanol counteraction of centrally mediated hypotension. The protocol described in the previous experiment was followed in this experiment, with the exception that ethanol (1 g/kg, i.v. over 1 min) was injected 10 min after the administration of efaroxan, 2-methoxyidazoxan, or the vehicle. Changes in blood pressure and heart rate evoked by ethanol were followed for another 60 min. Ethanol (1 g/kg) was administered as 95% in a volume of 1.3 ml/kg body weight as in our previous studies (^18,19).

Drugs

Clonidine hydrochloride, 2-methoxyidazoxan hydrochloride (Sigma Chemical, St. Louis, MO, U.S.A.), efaroxan hydrochloride (Research Biochemical Int., Natick, MA, U.S.A.), methohexital sodium (Brevital, Eli Lilly & Co., Indianapolis, IN, U.S.A.), povidone-iodine solution (Norton Co., Rockford, IL, U.S.A.), Durapen (Vedco, Overland Park, KS, U.S.A.), and ethanol (Midwest Grain Products Co., Weston, MO, U.S.A.) were purchased from commercial vendors.

Statistical analysis

Values are presented as mean ± SEM. MAP was calculated as diastolic pressure + one third pulse pressure (systolic-diastolic pressures). Repeated-measures analysis of variance followed by a Newman-Keuls post hoc analysis was used to analyze the effects of subsequent ethanol or saline administration on hemodynamic responses (blood pressure and heart rate) evoked by clonidine in the absence and presence of efaroxan or 2-methoxyidazoxan. Probability levels < 0.05 were considered significant.

RESULTS

Baseline values of MAP and heart rate were similar in all groups of rats used in this study (Table 1).

Experiment 1: effect of efaroxan or 2-methoxyidazoxan on the hypotensive effect of clonidine

Changes in MAP and heart rate evoked by clonidine and the subsequent administration of efaroxan or 2-methoxyidazoxan in conscious freely moving SHRs are shown in Figure 1. Intracisternal administration of clonidine (1.5 μ/kg) elicited decreases in MAP and heart rate (Fig. 1). The decrease in MAP started 2–3 min after clonidine administration, reached its maximal (29 ± 4 mm Hg) after 10 min, and remained at this level for the following 70 min (Fig. 1). The hypotensive effect of clonidine was associated with a reduction in heart rate that amounted to 35 ± 13 beats/min at 10 min and showed more reductions thereafter (Fig. 1). Subsequent blockade of α₂-adrenergic or I₁ imidazoline receptor (10 min later) by 2-methoxyidazoxan (0.16 μg/kg, i.c.) or efaroxan (0.45 μg/kg, i.c.), respectively, attenuated the hypotensive and bradycardic responses to clonidine (Fig. 1). Conversely, i.c. administration of equal volume of the vehicle (saline) had no effect on the hemodynamic responses to clonidine (Fig. 1). Preliminary studies were performed using a series of doses of efaroxan (0.24–1.36 μg/kg) and 2-methoxyidazoxan (0.08–0.24 μg/kg) so as to select doses of the two antagonists that would elicit similar attenuation of clonidine hypotension (data not shown). As shown in Figure 1, the chosen doses of efaroxan (0.45 μg/kg) or 2-methoxyidazoxan (0.16 μg/kg) administered following clonidine produced similar increases in MAP and heart rate, in terms of both duration and magnitude (Fig. 1). At these dose levels, efaroxan or 2-methoxyidazoxan caused approximately 60% attenuation of the initial hypotensive response to clonidine.

FIG. 1.: Effect of subsequent intracisternal (i.c.) administration of efaroxan (0.45 μg/kg), 2-methoxyidazoxan (MI; 0.16 μg/kg), or an equal volume of saline on the hypotensive (top panel) and bradycardic (bottom panel) responses to clonidine (1.5 μg/kg, i.c.) in conscious spontaneously hypertensive rats. Clonidine was administered at time 0 followed by efaroxan, MI, or saline 10 min later. Values are mean ± SEM and number of rats in each group is shown in parentheses. ^*p < 0.05 versus corresponding saline values.

Experiment 2: effect of efaroxan or 2-methoxyidazoxan on ethanol-clonidine interaction

Figures 2, 3, and 4 depict the effect of systemic administration of ethanol, in the absence and presence of efaroxan or 2-methoxyidazoxan, on hemodynamic responses to clonidine in conscious SHRs. Ethanol (1 g/kg, i.v.) counteracted the hypotensive effect of clonidine and caused significant (p < 0.05) increases in MAP that were sustained over the remaining period (60 min) of the study (Fig. 2). The bradycardic response to clonidine was significantly (p < 0.05) increased, compared with corresponding saline values, for 20 min by subsequent ethanol administration (Fig. 2).

FIG. 2.: Effect of subsequent administration of ethanol (1 g/kg, i.v.) or equal volume of saline on the hypotensive (top panel) and bradycardic (bottom panel) responses to clonidine (1.5 μg/kg, i.c.) in conscious spontaneously hypertensive rats. Clonidine was administered at time 0 followed by ethanol 10 min later. Values are mean ± SEM and number of rats in each group is shown in parentheses.^*p < 0.05 versus corresponding saline values.

FIG. 3.: Effect of efaroxan on ethanol-clonidine hemodynamic interaction in conscious spontaneously hypertensive rats. Intracisternal (i.c.) clonidine (1.5 μg/kg) was administered at time 0 followed by i.c. efaroxan (0.45 μg/kg, single arrow) plus ethanol (1 g/kg, i.v.) or equal volume of saline (double arrows). Values are mean ± SEM and number of rats in each group is shown in parentheses. ^*p < 0.05 versus corresponding saline values.

FIG. 4.: Effect of 2-methoxyidazoxan (MI) on ethanol-clonidine hemodynamic interaction in conscious spontaneously hypertensive rats. Intracisternal (i.c.) clonidine (1.5 μg/kg) was administered at time 0 followed by i.c. 2-methoxyidazoxan (0.16 μg/kg, single arrow), plus ethanol (1 g/kg, i.v.) or equal volume of saline (double arrows). Values are mean ± SEM and number of rats in each group is shown in parentheses. ^*p < 0.05 versus corresponding saline values.

Blockade of I₁ imidazoline receptor by efaroxan (0.45 μg/kg, i.c.), administered 10 min after clonidine, resulted in a significant (p < 0.05) attenuation of the pressor response to subsequently administered ethanol (Fig. 3). In the presence of efaroxan, ethanol elicited a pressor effect that lasted 10 min, after which the MAP returned to levels that were similar or even lower (at 70 and 80 min) than preethanol and postsaline values (Fig. 3). In contrast, the ability of ethanol to counteract clonidine-evoked hypotension was not altered when α₂-adrenoceptors were blocked by 2-methoxyidazoxan. As shown in Figure 4, systemic administration of ethanol (1 g/kg), 10 min after 2-methoxyidazoxan (0.16 μg/kg, i.c.), elicited significant increases in MAP that continued for the remaining period of the study. Conversely, the bradycardic effect of ethanol was not affected by prior administration of efaroxan (Fig. 3) or 2-methoxyidazoxan (Fig. 4). The elevation in MAP evoked by ethanol was similar, in magnitude and duration, in rats pretreated with 2-methoxyidazoxan or saline. In contrast, pretreatment with efaroxan caused drastic attenuation of the pressor response to ethanol compared with 2-methoxyidazoxan or saline-treated values. The pressor response to ethanol was demonstrated at only 10 min in efaroxan-treated rats and disappeared thereafter.

DISCUSSION

The current study presents three main findings that pertain to the role of central α₂-adrenoceptors and I₁ imidazoline receptors in the mediation of the hypotensive response to clonidine and its counteraction by ethanol in conscious SHRs. First, blockade of α₂- or I₁ receptors produced similar counteraction of the hypotensive and bradycardic responses to clonidine. Second, blockade of central α₂-receptor did not influence ethanol counteraction of clonidine-evoked hypotension whereas blockade of I₁ receptor virtually abolished the interaction. Third, ethanol potentiated the bradycardic response to clonidine and this effect was not influenced by prior α₂- or I₁ receptor blockade. These findings suggest that clonidine hypotension involves activation of α₂- and I₁ receptors and that the function of central I₁ receptor is altered by acute ethanol administration.

Several reports from our laboratory established that short- or long-term ethanol administration counteracts centrally but not peripherally mediated hypotension (^3–6,22). Recent studies showed that the ability of ethanol to adversely affect hemodynamic responses to centrally acting drugs is not generalizable and depends on the type of receptor, I₁ or α₂, involved in the response (^18,19). This view gains support from the findings that ethanol counteracts hypotensive and sympathoinhibitory responses to rilmenidine but not α-MNE, selective I₁- and α₂-adrenergic receptor agonists, respectively (^18,19). Based on these latter findings, we hypothesized that ethanol interacts selectively with neural pathways involved in I₁-mediated hypotension (^18,19). As shown in the present study, which is in full agreement with reported findings (^13–16), both central I₁ and α₂-adrenergic receptors contribute to the hypotensive action of clonidine. The present study sought further evidence to support this conclusion by evaluating the effect of selective blockade of I₁ or α₂-adrenergic receptors (by efaroxan and 2-methoxyidazoxan, respectively) on the capacity of ethanol to counteract clonidine hypotension. The dose of (1 g/kg) ethanol employed in the present study has been shown in our previous studies (^11,19) to produce blood ethanol concentration similar to that achieved in humans after moderate alcohol consumption (²³).

The finding that ethanol counteracted the hypotensive action of clonidine in conscious freely moving SHRs agrees with our previous findings in the same rat strain and supports the hypothesis that ethanol compromises centrally mediated hypotensive responses (^3,18,22). The current study also presents two observations that lend further support to and extend our recent findings (^18,19) concerning the selective interaction of ethanol with central I₁ receptor neural pathways. First, the use of the I₁/α₂-antagonist efaroxan virtually abolished the ability of ethanol to counteract clonidine-evoked hypotension. The pressor effect of ethanol lasted only 10 min in the presence of efaroxan compared with at least 60 min in its absence. Second, the ability of ethanol to counteract clonidine hypotension was preserved after selective blockade of α₂-adrenoceptors by 2-methoxyidazoxan. The differential effect of efaroxan (inhibition) and 2-methoxyidazoxan (no effect) on the antagonistic ethanol-clonidine hemodynamic interaction cannot be attributed to a difference in blood pressure prior to ethanol administration. In effect, clonidine produced similar falls in blood pressure (about 30 mm Hg) in all groups of rats. More importantly, we chose doses of efaroxan and 2-methoxyidazoxan that elicited similar attenuation of the hypotensive response to clonidine to facilitate data interpretation. These present findings that the I₁/α₂-antagonist efaroxan, but not the selective α₂-antagonist 2-methoxyidazoxan, drastically shortened the pressor effect of ethanol in clonidine-treated SHRs argue against a major contribution of α₂-adrenoceptors in ethanol-clonidine hemodynamic interaction. Similarly, these findings strongly support the hypothesis that ethanol influences the neuronal signaling triggered by the I₁ imidazoline receptor.

Nonetheless, the presence of a brief, but significant, pressor effect of ethanol in efaroxan-treated rats deserves a comment. It is possible that such a response represents a nonspecific pressor effect of ethanol or is a result of ethanol interaction with α₂-adrenergic receptor. A lack of a pressor effect of ethanol in rats whose blood pressure was lowered by the peripherally acting drugs in our previous studies (^3,4) argues against nonspecificity of ethanol action. Conversely, the α₂-adrenergic receptor may contribute to the brief pressor effect of ethanol. In a previous study from our laboratory, ethanol produced a similar brief pressor effect (approximately 10-min duration) when given following the selective α₂-receptor agonist α-MNE (¹⁷). Given the selectivity of efaroxan towards the I₁ imidazole receptor (^16,24), the dose chosen in the present study for this purpose may have had only a small effect on the α₂-adrenergic receptor, as we intended. The presence of the brief pressor response under these circumstances, along with our previous finding (¹⁷), may suggest a contributory role for the α₂-receptor in the brief pressor effect of ethanol, in efaroxan-treated rats, in the present study.

Conflicting reports are available regarding the central mechanisms involved in the hemodynamic responses to clonidine and whether they involve interaction with α₂-or I₁ receptors (^16,24–26). Currently, there is no selective I₁ receptor antagonist available. Therefore, as is the case in the present study, the relative role of the α₂- and I₁ receptor in centrally mediated hypotension has been based on findings with selective α₂-receptor antagonists and mixed I₁/α₂-receptor antagonists (^16,24). Binding (^27,28) and functional (²⁹) studies have confirmed the presence of imidazoline and α₂-adrenergic receptors in the rostral ventral lateral medulla, a brain stem area that plays a major role in the sympathoinhibitory and hypotensive actions of clonidine (^29,30). The present findings that efaroxan or 2-methoxyidazoxan attenuated the hypotensive effect of clonidine favor the assumptions that both receptors are involved in the blood pressure-lowering effect of clonidine. Furthermore, based on the same premise, the drastic reduction of clonidine hypotension by efaroxan suggests that the I₁ receptor containing neurons seem to be a neuroanatomic target for ethanol action.

The present study showed that, contrary to its hypotensive response, the bradycardic effect of clonidine was not attenuated but rather potentiated by subsequent administration of ethanol. It is notable that the bradycardic effect of clonidine involves the activation of central α₂-or I₁ receptor (^16,24–26). This view is supported by the present finding that blockade of central I₁ or α₂-receptor attenuated the bradycardic response. Furthermore, the effect of ethanol on the baroreflex may shed light on the opposite effects of ethanol on the blood pressure and heart rate responses to clonidine. Notably, the heart rate response to clonidine is complex and involves cardiac sympathetic inhibition, vagal facilitation, and the baroreflex response to the fall in blood pressure (^31,32). Our previous findings that ethanol compromised clonidine facilitation of the baroreflex (³) suggest a role for this action in ethanol counteraction of the hypotensive response to clonidine in the present and earlier (³) studies. It is also possible that ethanol counteraction of clonidine facilitation of the baroreflex (³) makes the heart more susceptible to the direct cardiodepressant action of ethanol (^33,34), which may explain the greater bradycardic response to clonidine in ethanol-treated rats. In support of this notion, α₂-receptor blockade, produced by 2-methoxyidazoxan, which is known to attenuate the baroreflex (^35,36), caused further enhancement of the bradycardic response to clonidine in ethanol-treated rats.

In conclusion, the present study was designed to investigate the relative contribution of central α₂-adrenoceptor and I₁ imidazoline receptor to the antagonistic hemodynamic interaction between ethanol and centrally mediated hypotension in conscious SHRs. By comparing the effects of the selective α₂-receptor antagonist, 2-methoxyidazoxan, and the mixed I₁/α₂-antagonist, efaroxan, the present findings suggest that ethanol compromises the hypotensive action of clonidine via a selective interaction with neuronal pools containing the I₁ receptor. Furthermore, ethanol seems to interact with the α₂-receptor, which may explain the initial brief pressor effect caused by ethanol when administered following clonidine or similar drugs. Conversely, clonidine-evoked bradycardia was potentiated by ethanol, an effect that may relate to the antagonistic effect of ethanol on the baroreflex enhancement caused by clonidine.

Acknowledgment:

Supported by Grant AA07839 from the National Institute on Alcohol Abuse and Alcoholism. The authors thank Ms. S. R. Vadlamudi for her technical assistance.