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Am J Physiol Regul Integr Comp Physiol 275: R1867-R1874, 1998;
0363-6119/98 $5.00
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Vol. 275, Issue 6, R1867-R1874, December 1998

Central alpha 2-receptor mechanisms contribute to enhanced renal responses during ketamine-xylazine anesthesia

Antonio De Melo Cabral1, Daniel R. Kapusta2, Velga A. Kenigs2, and Kurt J. Varner2

1 Department of Physiological Sciences, Federal University of Espirito Santo, Brazil 29040-090; and 2 Department of Pharmacology and Experimental Therapeutics and the Neuroscience Center of Excellence, Louisiana State University Medical Center, New Orleans, Louisiana 70112

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

We have recently developed an experimental approach to study central opioid control of renal function in anesthetized rats. This model system uses the intravenous infusion of the alpha 2-agonist xylazine to enhance basal levels of urine flow rate and urinary sodium excretion in ketamine-anesthetized rats. This study examined the contribution of central and peripheral alpha 2-adrenergic receptor mechanisms in mediating the enhanced renal excretory responses produced by xylazine. In ketamine-anesthetized rats, the enhanced levels of urine flow rate and urinary sodium excretion produced by the intravenous infusion of xylazine were reversed by the intravenous bolus injection of the alpha 2-adrenoceptor antagonist yohimbine but not by the alpha 1-adrenoceptor antagonist terazosin. In separate animals the intracerebroventricular administration of yohimbine only reduced urine flow rate by ~50% but did not alter urinary sodium excretion. The decrease in urine flow rate produced by intracerebroventricular yohimbine was reversed by the intravenous injection of a selective V2-vasopressin receptor antagonist. In a separate group of ketamine- and xylazine-anesthetized rats, the bilateral microinjection of yohimbine into the hypothalamic paraventricular nucleus (PVN) also significantly decreased urine flow rate by 54% without altering urinary sodium excretion. The microinjection of the beta -adrenoceptor antagonist propranolol into the PVN did not alter either renal excretory parameter. These results suggest that during intravenous infusion, xylazine increases urine flow rate by activating alpha 2-adrenergic receptors in the PVN, which in turn decrease vasopressin release. The ability of alpha -adrenergic mechanisms in the PVN to selectively influence the renal handling of water, but not sodium, may contribute to the reported dissociation of the natriuretic and diuretic responses of alpha 2-adrenoceptor agonists.

hypothalamic paraventricular nucleus; yohimbine; urine flow rate; urinary sodium excretion; renal excretory function

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

WE HAVE RECENTLY demonstrated that the intravenous infusion of the alpha 2-adrenergic agonist xylazine significantly enhances basal levels of urine flow rate and urinary sodium excretion in ketamine-anesthetized rats (7). The enhanced and sustained renal responses attained in ketamine-anesthetized rats receiving xylazine infusion were in marked contrast to the low renal excretory levels of water and sodium observed in rats anesthetized with ketamine or pentobarbital alone (7). Similar to these findings, a number of studies have shown that other alpha 2-agonists (e.g., clonidine, guanabenz, BHT-933, rilmenidine, etc.) also produce a diuretic and natriuretic response in anesthetized (and conscious) animals and humans (13, 17, 18, 31). Despite these findings, the mechanisms by which these compounds change the renal excretion of water and sodium have not been completely elucidated.

Reduced renal excretory responses occur in surgically operated animals and humans anesthetized with different anesthetic agents (4, 10, 32, 45). Because surgery and anesthesia are potent stimuli for vasopressin release, alpha 2-agonists may produce diuretic and natriuretic responses during anesthesia and surgery by inhibiting the central nervous system (CNS) secretion and/or renal tubular action(s) of vasopressin. In regard to this latter possibility, considerable evidence indicates that the activation of renal alpha 2-adrenoceptors is a predominant mechanism by which selective alpha 2-agonists produce diuretic and natriuretic responses in several mammalian species (14, 15, 40, 41, 43). Stimulation of renal alpha 2-adrenoceptors inhibits vasopressin-mediated cAMP formation and the subsequent effects on water and sodium excretion (27, 28, 40). Based on these findings, the intravenous infusion of xylazine may enhance renal excretory function in ketamine-anesthetized rats by stimulating alpha 2-adrenoceptors in the collecting duct and thus modulating the antidiuretic effect of vasopressin in this nephron segment.

In addition to a direct renal action, it is possible that a portion of the diuretic and/or natriuretic response elicited by the intravenous infusion of xylazine in ketamine-anesthetized rats is mediated by a pathway involving alpha 2-adrenoceptors located in the CNS. More specifically, the increase in urine flow rate produced by intravenous xylazine may result, at least in part, from a central action of the drug to inhibit the secretion of vasopressin. Such a mechanism would be consistent with the results of a number of studies showing that the activation of central adrenergic receptors, in particular the alpha 2-receptor subtype, inhibits the release of vasopressin in conscious and anesthetized animals (6, 20, 21, 38, 39). Although changes in circulating vasopressin levels may also contribute, it appears that the natriuretic response produced by alpha 2-adrenoceptor agonists is mediated by an alternative mechanism(s) independent of this hormone (3, 9, 22, 24, 39, 43).

The purpose of the present study was to examine the contribution of central alpha 2-adrenergic receptor mechanisms in mediating the enhanced renal excretory responses produced by the intravenous infusion of xylazine in ketamine-anesthetized rats. For this purpose, we compared the changes in renal function produced by the intravenous and intracerebroventricular injection of the alpha 2-adrenergic receptor antagonist yohimbine in ketamine- and xylazine-anesthetized rats. Microinjection techniques were then used to determine whether alpha 2-adrenoceptors in the hypothalamic paraventricular nucleus (PVN) play a role in mediating the enhanced renal responses to xylazine. The PVN was chosen as the focus of these studies because the PVN contains neurons that synthesize and release vasopressin in the posterior pituitary (44). Vasopressin secretion is known to be markedly enhanced under conditions of anesthesia and surgery (8, 11, 30, 35). In addition, the vasopressinergic neurons in the PVN receive dense catecholaminergic projections from the A1 and other nuclei (44) and contain large numbers of alpha 2-adrenoceptors (1, 34, 37).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Subjects

Experiments were performed using male Sprague-Dawley rats (275-325 g, Harlan). All procedures were done in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Care and Use Committee at Louisiana State University Medical Center. The rats were housed in groups in a temperature- and humidity-controlled room with a 12:12-h light-dark cycle. Standard rat chow (Na+ content 163 meq/kg) and tap water were available ad libitum.

Surgical Procedures

Intracerebroventricular cannula implantation. Five to seven days before the experiment certain rats were anesthetized (ketamine 30 mg/kg im in combination with xylazine 3 mg/kg im) and a stainless steel cannula was implanted into the right lateral cerebral ventricle. The cannula was implanted 0.3 mm posterior to the bregma, 1.3 mm lateral to the midline, and 4.5 mm below the skull surface (36). The cannula was fixed into position by using jewelers' screws and cranioplastic cement. Verification of the cannula position in the lateral ventricle was made by observing the leakage of cerebrospinal fluid from the cannula after removal of the obturator (7).

Catheter implantation. On the day of the experiment rats were anesthetized with sodium methohexital (Brevital, 35 mg/kg ip, supplemented with 10 mg/kg iv as needed; Lilly, Indianapolis, IN). Catheters (PE-50 fused to PE-10) were placed in the femoral artery and vein for the recording of arterial pressure and administration of drugs, respectively. The catheters were tunneled subcutaneously to the back of the neck, flushed, and plugged. A suprapubic incision was then made, and a bladder catheter (flanged PE-240) was inserted and sutured into the urinary bladder. The bladder catheter was then exteriorized and secured by suturing it to adjacent muscle and skin. After surgical preparation, the rat was placed in a rat holder, which permitted the collection of urine. Rats then received ketamine (40 mg/kg iv) over a 5-min period. A supplemental intravenous infusion (55 µl/min) of isotonic saline containing ketamine (1.0 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1) was then started and continued throughout the experiment. The arterial and venous catheters were connected to a pressure transducer (model P23 Db, Statham, Oxnard, CA) and an infusion pump (model 944, Harvard Apparatus, South Natick, MA), respectively. Mean and pulsatile arterial pressures were recorded on a Grass 7D polygraph (Grass Instruments, Quincy, MA). Heart rate was determined from the arterial pressure signal by a Grass model 7P4 tachograph. During surgery and experimental procedures body temperature was maintained at 37 ± 1°C using a water-filled heating pad and/or a heat lamp.

Microinjection procedures. Certain studies were performed in ketamine- and xylazine-anesthetized rats in which drugs were microinjected into the PVN of the hypothalamus. For these studies rats were anesthetized (Brevital, 35 mg/kg ip, supplemented with 10 mg/kg iv as needed) and chronically implanted with catheters in the bladder and the femoral artery and vein as described previously. After the catheters were implanted, the rats were administered ketamine (40 mg/kg iv) over a 5-min period. An intravenous infusion (55 µl/min) of isotonic saline containing ketamine (1.0 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1) was then started and continued throughout the experiment. Ketamine- and xylazine-anesthetized rats were then placed prone in a stereotaxic apparatus with the bite bar 3.5 mm below the interaural line. Drugs were microinjected bilaterally into PVN using three-barreled glass micropipettes (0.4 mm ID, 0.75 mm OD) having a composite tip diameter of <40 µm. The pipettes were lowered stereotaxically into PVN through two burr holes drilled through the skull 2.0 mm posterior to bregma and 0.4 mm lateral to either side of the midline. The pipettes were lowered 7.9 mm below the surface of the skull. The barrels of the pipettes were filled by vacuum with saline (barrel 1), 1% solution of Pontamine sky-blue dye (barrel 2), and one of the test antagonists (barrel 3). All drugs were injected in a volume of 60 nl over a period of 0.5-1 min by using a pneumatic pressure injection system (General Valve). The speed and volume of the injection were controlled by watching the movement of the fluid meniscus in the pipette with a stereomicroscope and a gradicule affixed to the pipette.

Experimental Protocols

In previous studies we have shown that in ketamine-anesthetized rats the intravenous infusion of xylazine significantly increases urine flow rate and urinary sodium excretion (7). The enhanced levels of these renal excretory parameters tended to stabilize ~120 min after the start of drug infusion and remained relatively constant for an additional 90 min (longer time control periods not studied) (7). Therefore, in the experimental protocols described, rats were allowed to stabilize for at least 2 h after starting the intravenous ketamine and xylazine infusion before starting the experiment.

Effects of intravenous yohimbine or terazosin administration on renal excretory function. Experiments were performed to determine the role of alpha 2- and/or alpha 1-adrenergic receptor mechanisms in the renal responses produced by intravenous xylazine infusion in ketamine-anesthetized rats. After equilibration and stabilization of renal excretory responses, two consecutive control urine samples were collected (10 min each). The selective alpha 2-adrenoceptor antagonist yohimbine (0.5 mg/kg, n = 6) or the alpha 1-adrenoceptor antagonist terazosin (0.5 mg/kg, n = 5) was then administered as an intravenous bolus. After waiting 15 min for drug distribution, five consecutive experimental urine samples (10 min each) were collected.

Effects of intravenous V2-vasopressin receptor antagonist administration on renal excretory function. Studies were performed to examine the role of vasopressin in mediating the changes in renal excretory function produced by the intravenous infusion of xylazine in ketamine-anesthetized rats. After stabilization of urine flow rate and urinary sodium excretion, two consecutive 10-min control urine samples were collected. Yohimbine (0.5 mg/kg iv) was then administered and allowed to distribute for 15 min. Urine was then collected during four consecutive experimental yohimbine periods. After the fourth experimental yohimbine urine sample was collected, the V2-vasopressin receptor antagonist [d(CH2)5,D-Ile2,Ile4,Arg8]-vasopressin (1 nmol/kg) was injected intravenously. Immediately after injection of the vasopressin antagonist, three consecutive 10-min urine samples were collected.

Effects of intracerebroventricular yohimbine administration on renal excretory function. Experiments were performed to examine the contribution of central alpha 2-adrenoceptor mechanisms to the diuretic and/or natriuretic response elicited by the intravenous infusion of xylazine in ketamine-anesthetized rats. After stabilization of urine flow rate and urinary sodium excretion, two consecutive 10-min control urine samples were collected. After these control periods the alpha 2-adrenoceptor antagonist yohimbine was injected (20 µg/kg icv, n = 6) and allowed to distribute for 15 min. Five consecutive experimental urine samples (10 min each) were then collected. In control experiments the study was repeated with the exception that the same dose of yohimbine (20 µg/kg, n = 4) was administered as an intravenous bolus.

Renal excretory responses elicited by the microinjection of yohimbine into PVN. Studies were performed to determine whether activation of alpha 2-adrenoceptor mechanisms in the PVN contribute to the enhanced diuretic and natriuretic responses produced by xylazine infusion in ketamine-anesthetized rats. After the ketamine and xylazine infusion was started and steady-state renal excretory responses were obtained, urine samples were collected during two consecutive 10-min control periods. After these control collections, the alpha 2-adrenergic receptor antagonist yohimbine (60 ng in 60 nl) was bilaterally microinjected into the PVN. The drug was then allowed 10 min for distribution. The experimental protocol was then completed by collecting five consecutive 10-min experimental urine samples. Preliminary experiments using 30, 60, and 90 ng of yohimbine showed that the 60-ng dose of yohimbine produced maximal changes in renal excretory function in ketamine- and xylazine-anesthetized rats. At the end of the experiment, injection sites in PVN were marked bilaterally by microinjecting Pontamine sky-blue dye through the third barrel of the pipette.

Two types of control groups were used in the microinjection studies. In the first, yohimbine (60 ng, n = 6) was bilaterally microinjected into sites superior, dorsal, or caudal to PVN. In a separate group of rats the mixed beta -adrenoceptor antagonist propranolol (60 ng, n = 7) was bilaterally microinjected into the PVN.

Histological Processing

At the end of the microinjection experiments the rats were deeply anesthetized and perfused transcardially with normal saline followed by 4% phosphate-buffered Formalin. The brains were removed and stored at 4°C for at least 2 days in the Formalin solution and an additional 2 days in a 4% sucrose solution. The brains were then frozen and sectioned (40 µm) using a cryostat microtome. The sections were then placed on glass slides and stained with neutral red. Microinjection sites were identified microscopically from the stained sections using the atlas of Paxinos and Watson (36) as a reference.

Data Analysis

Changes in mean arterial pressure and heart rate, before and after drug administration, were calculated directly from the polygraph records. The kidneys were removed, decapsulated, and weighed for normalization of renal excretory data. In microinjection studies the kidneys were removed before the rats were perfused. Urine volume was determined gravimetrically. Urine sodium concentration was measured by flame photometry (Instrumentation Laboratories, model 943).

All data are expressed as means ± SE. The data were statistically analyzed using repeated measures analysis of variance for the main effects and interactions and Scheffé's test for pairwise comparisons among the means (46). Statistical significance was defined as P < 0.05.

Drugs Used

The drugs used in this study were yohimbine hydrochloride (Sigma Chemical, St. Louis, MO), terazosin (generous gift from Abbott Laboratories, Abbott Park, IL), propranolol hydrochloride (Sigma), sodium methohexital (Brevital, Lilly, Indianapolis, IN), ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA), and xylazine (Butler, Columbus, OH). Yohimbine, terazosin, and propranolol were dissolved in normal saline (0.9%).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

The cardiovascular and renal responses produced by the intravenous bolus administration of the alpha 2-receptor antagonist yohimbine (0.5 mg/kg, n = 6) or the alpha 1-receptor antagonist terazosin (0.5 mg/kg, n = 5) in Sprague-Dawley rats receiving continuous infusion (55 µl/min iv) of ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1) are shown in Fig. 1. Mean data ± SE are shown for each cardiovascular and renal excretory parameter during two consecutive 10-min control periods (C1 and C2) and during five consecutive experimental periods (10 min each) beginning 15 min after intravenous bolus drug administration (25-65 min). The injection of yohimbine produced an immediate and profound decrease in urine flow rate and urinary sodium excretion. Intravenous yohimbine significantly reversed the enhanced renal excretory levels of water for ~55 min (time points 25-55 min). Concurrent with the changes in urine flow rate, intravenous yohimbine significantly decreased urinary sodium excretion at the 25- and 35-min time points before returning to control levels. In addition, intravenous yohimbine tended to reduce mean arterial pressure and increase heart rate, although these changes were not statistically significant. In contrast the intravenous injection of the alpha 1-receptor antagonist terazosin failed to alter either renal excretory parameter at any time. Terazosin did, however, produce a slight, but insignificant, decrease in mean arterial pressure and increase in heart rate.


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  		Fig. 1.   Cardiovascular and renal excretory responses produced by intravenous administration of yohimbine or terazosin in ketamine- and xylazine-anesthetized Sprague-Dawley rats. Values are means ± SE, illustrating systemic cardiovascular and renal responses produced by intravenous bolus injection of selective alpha 2-receptor antagonist yohimbine (0.5 mg/kg, n = 6) or the selective alpha 1-receptor antagonist terazosin (0.5 mg/kg, n = 5), in rats anesthetized (40 mg/kg iv bolus over 5 min) and infused (55 µl/min iv) with isotonic saline containing ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1). Urine samples were collected during control (C1-C2, 10 min each) and 15 min after intravenous injections of drugs (consecutive 10-min urine samples for 65 min). HR, heart rate; MAP, mean arterial pressure; V, urine flow rate; UNaV, urinary sodium excretion. * P < 0.05, significantly different from corresponding control.

The changes in cardiovascular and renal excretory function produced by the sequential intravenous administration of yohimbine and the V2-vasopressin receptor antagonist [d(CH2)5,D-Ile2,Ile4,Arg8]-vasopressin in ketamine- and xylazine-anesthetized rats are shown in Fig. 2. Mean data are shown for each parameter during consecutive 10-min urine collection periods during control (C1 and C2), 15 min after intravenous administration of yohimbine (time points 25-55), and the subsequent intravenous bolus administration of the V2-vasopressin receptor antagonist (time points 65-85). Similar to that shown in Fig. 1, the intravenous bolus administration of yohimbine produced a significant reduction in urine flow rate (25-55 min) and urinary sodium excretion (Fig. 2, 25-35 min). Subsequent administration of the V2-vasopressin receptor antagonist immediately reversed the yohimbine-induced antidiuresis to levels significantly above those observed during control (time points 65 and 75 min). Despite the effects on urine flow rate, the V2-vasopressin receptor antagonist did not alter urinary sodium excretion (65-85 min). Neither yohimbine nor the V2-vasopressin receptor antagonist significantly altered heart rate or mean arterial pressure.


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  		Fig. 2.   Effects of intravenous administration of a V2-vasopressin receptor antagonist on yohimbine-induced cardiovascular and renal responses in ketamine- and xylazine-anesthetized Sprague-Dawley rats. Values are means ± SE, illustrating changes in systemic cardiovascular and renal excretory function produced by intravenous administration yohimbine (0.5 mg/kg) and subsequent administration of selective V2-vasopressin receptor antagonist, [d(CH2)5,D-Ile2,Ile4,Arg8]-vasopressin (1 nmol/kg, n = 6), in ketamine- and xylazine-anesthetized rats. Experiments were performed during continuous intravenous infusion of isotonic saline (55 µl/min) containing ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1). Consecutive 10-min urine samples were collected during control (C1-C2), 15 min after intravenous yohimbine injection (time points 25-55 min), and immediately after intravenous injection of the V2-vasopressin receptor antagonist (time points 65-85 min). Abbreviations as in Fig. 1. * P < 0.05, significantly different from corresponding control.

The cardiovascular and renal responses produced by the intracerebroventricular injection of yohimbine (20 µg/kg), in 6 ketamine- and xylazine-anesthetized rats are shown in Fig. 3. Mean data are depicted for each parameter during two consecutive control periods (C1 and C2, 10 min each) and during five consecutive experimental periods (10 min each) beginning 15 min after the intracerebroventricular injection of yohimbine (25-65 min). The intracerebroventricular administration of yohimbine produced a significant decrease in urine flow rate that was rapid in onset and persisted for 45 min (experimental periods 25-45) before returning to control levels. Although intracerebroventricular yohimbine tended to decrease urinary sodium excretion, these changes were not statistically significant. Concurrent with the renal excretory changes, yohimbine increased mean arterial pressure 25 and 35 min after intracerebroventricular injection, but did not significantly change heart rate. The intravenous bolus administration of the same dose of yohimbine (20 µg/kg) failed to alter any cardiovascular or renal excretory parameter (Fig. 3).


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  		Fig. 3.   Cardiovascular and renal excretory responses produced by intracerebroventricular or intravenous administration of yohimbine in ketamine- and xylazine-anesthetized Sprague-Dawley rats. Values are means ± SE, illustrating systemic cardiovascular and renal responses produced by intracerebroventricular (20 µg/kg, n = 6) or intravenous (20 µg/kg, n = 4) injection of selective alpha 2-receptor antagonist yohimbine in ketamine- and xylazine-anesthetized rats. Experiments were performed during continuous intravenous infusion of isotonic saline (55 µl/min) containing ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1). Consecutive 10-min urine samples were collected during control (C1-C2) and 15 min after intracerebroventricular or intravenous injection of drug (time points 25-65). Abbreviations as in Fig. 1. * P < 0.05, significantly different from corresponding control.

The cardiovascular and renal responses produced by the bilateral microinjection of the alpha 2-receptor antagonist yohimbine (60 ng, n = 8) into PVN are shown in Fig. 4. After stabilization of renal excretory function, two consecutive 10-min urine samples (C1 and C2) were collected. Compared with control levels the microinjection of yohimbine into PVN produced an immediate and significant decrease (54%) in urine flow rate by the first experimental collection (compare C2 vs. the 20-min time point). The decrease in urine flow rate was maximal within 20 min after injection and returned to control levels by the fourth experimental period (time point 50 min). In contrast, microinjection of yohimbine into PVN did not alter urinary sodium excretion at any time period. Yohimbine failed to alter any cardiovascular parameter. The microinjection of yohimbine (60 ng, n = 5) into sites dorsal or caudal to PVN did not significantly change heart rate, mean arterial pressure, or renal excretory function (Fig. 4). The histologically identified sites into which yohimbine was microinjected are shown in Fig. 6.


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  		Fig. 4.   Effects of microinjection of yohimbine into paraventricular nucleus (PVN) on cardiovascular and renal excretory function in ketamine- and xylazine-anesthetized Sprague-Dawley rats. Values are means ± SE, illustrating systemic cardiovascular and renal excretory responses produced by bilateral microinjection of yohimbine (60 ng/60 nl) into PVN (n = 8) or into sites superior, dorsal, or caudal to PVN (missed injections, n = 5) in ketamine- and xylazine-anesthetized rats. Experiments were performed during continuous intravenous infusion of isotonic saline (55 µl/min) containing ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1). Consecutive 10-min urine samples were collected during control (C1-C2) and 10 min after microinjection of yohimbine (time points 20-60). Abbreviations as in Fig. 1. * P < 0.05, significantly different from corresponding control.


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  		Fig. 6.   Histologically verified sites into which yohimbine and propranolol were microinjected into PVN and into adjacent control sites. bullet , Sites of yohimbine microinjection; open circle , control injections of yohimbine; , site of propranolol microinjection. Horizontal calibration is 1 mm.

Figure 5 demonstrates the cardiovascular and renal excretory responses produced by the bilateral microinjection of propranolol (60 ng, n = 7) into PVN. Compared with control periods (C1 and C2, 10 min each), the microinjection of propranolol into PVN did not significantly alter any cardiovascular or renal excretory parameter.


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  		Fig. 5.   Effects of microinjection of propranolol into PVN on cardiovascular and renal excretory function in ketamine- and xylazine-anesthetized Sprague-Dawley rats. Values are means ± SE, illustrating systemic cardiovascular and renal excretory responses produced by bilateral microinjection of nonselective beta -adrenoceptor antagonist propranolol (60 ng/60 nl) into PVN (n = 7) in ketamine- and xylazine-anesthetized rats. Experiments were performed during continuous intravenous infusion of isotonic saline (55 µl/min) containing ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1). Consecutive 10-min urine samples were collected during control (C1-C2) and 10 min after microinjection of propranolol (time points 20-60). Abbreviations as in Fig. 1.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

We have previously shown that the intravenous infusion of the alpha 2-adrenergic agonist xylazine increases urine flow rate and urinary sodium excretion in ketamine-anesthetized rats (7). The results of the present studies demonstrate that these renal excretory responses are mediated, at least in part, by the activation of alpha 2-receptors in the CNS. Moreover, it appears that the diuretic, but not the natriuretic, response produced by the intravenous infusion of xylazine is mediated by a pathway that involves alpha 2-receptors located in the PVN. This premise is based on the findings that the microinjection of the alpha 2-receptor antagonist yohimbine into PVN reduced urine flow rate in these rats by ~50% but had no effect on urinary sodium excretion. The magnitude of the decrease in urine flow rate produced by bilateral microinjection of yohimbine (60 ng) into PVN was comparable to that observed when this antagonist was administered intracerebroventricularly (20 µg/kg; compare Figs. 4 and 3, respectively). The diuretic and natriuretic responses produced by xylazine infusion were not affected by microinjection of propranolol into PVN, thus excluding the possibility that beta -adrenoceptor mechanisms in this nucleus contributed to the xylazine-induced renal excretory responses.

The results of the present studies suggest that the diuretic response elicited by xylazine in ketamine-anesthetized rats involves a central pathway in which xylazine inhibits vasopressin secretion. Although not tested directly, our findings are consistent with such a mechanism since the enhanced basal level of urine flow rate produced by intravenous infusion of xylazine was significantly decreased by the microinjection of yohimbine into the PVN. The PVN is a brain nucleus that is dense in alpha 2-receptors and that is known to participate in the control of vasopressin secretion (1, 37, 44). These findings suggest that xylazine activates alpha 2-adrenergic receptors in the PVN and thereby inhibit vasopressin secretion. The subsequent microinjection of yohimbine into the PVN reduced urine flow rate by ~50% by preventing the inhibitory action of xylazine on vasopressin secretion and thus increasing circulating levels of this hormone. This later premise is supported by our observation that the reduction in urine flow rate produced by yohimbine administration was reversed by the subsequent intravenous administration of a V2-vasopressin receptor antagonist (see Fig. 2).

As suggested previously, the PVN is at least one brain site that is involved in mediating the enhanced urine flow rate response produced by xylazine infusion. In light of previously published findings regarding the role of the PVN in vasopressin synthesis, storage, and release, these results were not entirely unforeseen. Whether other CNS sites are also involved has not been determined. It is apparent that there is also a peripheral component by which xylazine infusion augments urine flow rate. This was made apparent by the observation that both the intracerebroventricular and PVN injection of yohimbine only reduced urine flow rate by ~50%. This was in contrast to the profound reduction of urine flow rate (and urinary sodium excretion) that was produced when yohimbine was injected intravenously (Fig. 1). Thus both central (i.e., PVN) and peripheral alpha 2-adrenergic mechanisms appear to contribute to the enhanced diuretic response produced by the intravenous infusion of xylazine in ketamine-anesthetized rats. Note, however that because these studies did not establish whether all CNS alpha 2-receptors were blocked by intracerebroventricular yohimbine, these results only estimate the extent to which central vs. peripheral alpha 2-receptor mechanisms contribute to the diuretic response produced by xylazine infusion. Therefore, it is possible that this treatment only reduced but did not abolish the CNS component of the diuresis. In this manner, intracerebroventricular yohimbine may not have affected alpha 2-receptor mechanisms in other brain regions (i.e., the supraoptic nucleus) to the same extent as in the PVN. Despite these possibilities, it is important to consider that while the peripheral (19, 38, 39, 42, 43) and central (5, 6, 20, 21) administration of alpha 2-agonists reduce vasopressin secretion, the majority of studies in rats indicate that alpha 2-agonists have a direct renal action to produce a diuresis by modulating the hydrosmotic effect of vasopressin (3, 5, 12, 14-16, 33, 40). Thus it appears that the diuretic response produced by intravenous xylazine infusion may involve integration of complex peripheral, direct renal, and CNS mechanisms of action. This premise is in accord with proposed mechanisms by which other alpha 2-receptor agonists (e.g., clonidine, BHT-933, guanabenz, rilmenidine) affect the renal handling of water after their peripheral administration (3, 39, 43).

In contrast to activation of alpha 2- or beta -adrenergic receptor mechanisms in the PVN, the present studies suggest that xylazine utilizes an alternative pathway(s) and/or CNS site of action(s) to produce natriuresis. It has been proposed that the natriuretic action of peripherally administered alpha 2-agonists may involve both central inhibition of vasopressin release in combination with a second action of the compound that leads to inhibition of the renal tubular reabsorption of sodium (2, 9, 39, 43). More specifically, it has been proposed that the natriuretic action of alpha 2-agonists is independent of an action of vasopressin (9, 29). For example, alpha 2-agonists inhibit efferent renal sympathetic nerve activity (22-25), with sympathetic withdrawal resulting in natriuresis due to attenuation of the neural release and postsynaptic tubular action of norepinephrine on sodium transport in the kidneys (26). Although we have shown that intravenous infusion of xylazine produces a decrease in directly recorded renal sympathetic nerve activity (unpublished observation), it remains to be established whether renal sympathoinhibition contributes to the natriuresis observed in ketamine- and xylazine-anesthetized rats. It is apparent, however, that during intravenous infusion xylazine does not act within the PVN to affect sodium excretion because the microinjection of yohimbine into this nucleus failed to alter urinary sodium excretion (see Fig. 4). It should be noted that in addition to a CNS action, it is clear that alpha 2-agonists can inhibit renal tubular sodium reabsorption by modulating sodium (and water) transport in the renal nephron (2, 5, 14, 16, 39, 40).

Perspectives

The hypotension and reduction in glomerular perfusion pressure during anesthesia and surgery (a major stimulus to vasopressin secretion) substantially reduce urine flow rate and urinary sodium excretion. The impaired renal function during anesthesia and surgery may result, at least in part, from the ability of these stressors to attenuate tonic alpha 2-adrenergic receptor-mediated inhibition of vasopressin secretion in PVN or other areas such as supraoptic nucleus. The administration of xylazine appears to counteract the disinhibitory action of these stressors. Whether xylazine attenuates vasopressin release by presynaptically inhibiting excitatory inputs to the PVN or by activating inhibitory postsynaptic alpha 2-receptor on vasopressinergic neurons remains to be determined. At the level of the kidneys, it is likely that xylazine physiologically antagonizes the hydrosmotic effect of vasopressin by stimulating alpha 2-receptors in the collecting duct. Together, the maintenance of a continuous alpha 2-agonist influence on renal function produced by the intravenous infusion of xylazine may restore the ability of the kidneys to excrete water and sodium by reinstating these inhibitory actions on vasopressin mechanisms.

In summary, we have previously demonstrated that intravenous infusion of the alpha 2-agonist xylazine produces a marked increase in urine flow rate and urinary sodium excretion in ketamine-anesthetized rats. The present study extends these findings and indicates that the enhanced renal excretory responses produced by xylazine are mediated via activation of complex peripheral and CNS alpha 2-adrenergic receptor systems. In regard to central mechanisms, the findings of these studies also demonstrate that xylazine activates alpha 2-adrenergic receptors in the PVN of the hypothalamus to contribute to the increase in urine flow rate, but not urinary sodium excretion. The diuretic response produced by xylazine is presumably caused by a decrease in vasopressin release subsequent to PVN alpha 2-receptor stimulation. The inability of the microinjection of propranolol into PVN to alter either renal excretory response indicates that beta -adrenergic receptors in this brain nucleus are not involved in mediating the renal responses produced by intravenous xylazine. The action of alpha 2-adrenergic mechanisms in the PVN to selectively influence the renal handling of water, but not sodium, may contribute to the reported dissociation of the natriuretic and diuretic responses of alpha 2-adrenoceptor agonists.

    ACKNOWLEDGEMENTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-43337 to D. R. Kapusta, the National Institute on Drug Abuse Grant DA-08255 to K. J. Varner, and the American Heart Association-Louisiana Affiliate (LA-06-GS-14) to D. R. Kapusta and K. J. Varner.

    FOOTNOTES

This work was performed while A. de Melo Cabral was a visiting professor in the Department of Pharmacology and Experimental Therapeutics at the Louisiana State University Medical Center. Doctor Cabral was supported by a fellowship from CNPq, Brazil.

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. §1734 solely to indicate this fact.

Address for reprint requests: K. J. Varner, Dept. of Pharmacology and Experimental Therapeutics, Louisiana State Univ. Medical Center, 1901 Perdido St., New Orleans, LA 70112.

Received 13 May 1998; accepted in final form 4 August 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Aoki, C., C. G. Go, C. Venkatesan, and H. Kurose. Perikaryal and synaptic localization of alpha2A-adrenergic receptor-like immunoreactivity. Brain Res. 650: 181-204, 1994[Medline].

2.   Barr, J. G., and M. L. Kauker. Renal tubular site and mechanism of clonidine-induced diuresis in rats: clearance and micropuncture studies. J. Pharmacol. Exp. Ther. 209: 389-395, 1979[Free Full Text].

3.   Blandford, D. E., and D. D. Smyth. Dose selective dissociation of water and solute excretion after renal alpha-2 adrenoceptor stimulation. J. Pharmacol. Exp. Ther. 247: 1181-1186, 1988[Abstract/Free Full Text].

4.   Brewster, W. R., Jr., J. P. Isaacs, and T. Waino-Andersen. The present effect of ether on myocardium of the dog and its modification by reflex release of epinephrine and nor-epinephrine. Am. J. Physiol. 175: 399-414, 1953.

5.   Brooks, D. P., R. M. Edwards, P. D. Depalma, T. A. Fredrickson, J. P. Hieble, and M. Gellai. The water diuretic effect of the alpha-2 adrenoceptor agonist, AGN 190851, is species-dependent. J. Pharmacol. Exp. Ther. 259: 1277-1282, 1991[Abstract/Free Full Text].

6.   Brooks, D. P., L. Share, and J. T. Crofton. Central adrenergic control of vasopressin release. Neuroendocrinology 42: 416-420, 1986[Medline].

7.   Cabral, A. M., K. J. Varner, and D. R. Kapusta. Renal excretory responses produced by central administration of opioid agonists in ketamine and xylazine-anesthetized rats. J. Pharmacol. Exp. Ther. 282: 609-616, 1997[Abstract/Free Full Text].

8.   Calogero, A. E., J. A. Norton, B. C. Sheppard, S. J. Listwak, D. T. Cromack, R. Wall, R. T. Jensen, and G. P. Chrousos. Pulsatile activation of the hypothalamic-pituitary-adrenal axis during major surgery. Metabolism 41: 839-845, 1992[Medline].

9.   Dawson, R., Jr., and D. Wallace. Central and peripheral actions of alpha-2 adrenergic agonists on renal function in Long-Evans and Brattleboro rats. Pharmacology 39: 240-252, 1989[Medline].

10.   DeBodo, R. C., and K. F. Prescott. The antidiuretic action of barbiturates (phenobarbital, amytal, pentobarbital) and the mechanism involved in the action. J. Pharmacol. Exp. Ther. 85: 222-233, 1945[Abstract/Free Full Text].

11.   Deutsch, S., R. D. Bastron, E. C. Pierce, Jr., and L. D. Vandam. The effects of anaesthesia with thiopentone, nitrous oxide, narcotics and neuromuscular blocking drugs on renal function in normal man. Br. J. Anaesth. 41: 807-815, 1969[Abstract/Free Full Text].

12.   Edwards, R. M., E. J. Stack, M. Gellai, and D. P. Brooks. Inhibition of vasopressin-sensitive cAMP accumulation by a2-adrenoceptor agonists in collecting tubules is species dependent. Pharmacology 44: 26-32, 1992[Medline].

13.   Evans, R. G., A. Shweta, S. C. Malpas, S. M. Fitzgerald, and W. P. Anderson. Renal effects of rilmenidine in volume-loaded anaesthetized dogs. Clin. Exp. Pharmacol. Physiol. 24: 64-67, 1997[Medline].

14.   Gellai, M. Modulation of vasopressin antidiuretic action by renal a2-adrenoceptors. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F1-F8, 1990[Abstract/Free Full Text].

15.   Gellai, M., and R. M. Edwards. Mechanism of a2-adrenoceptor agonist-induced diuresis. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F317-F323, 1988[Abstract/Free Full Text].

16.   Gellai, M., and R. R. Ruffolo. Renal effects of selective alpha-1 and alpha-2 adrenoceptor agonists in normotensive, conscious rats. J. Pharmacol. Exp. Ther. 240: 723-728, 1987[Abstract/Free Full Text].

17.   Hamaya, Y., T. Nishikawa, and S. Dohi. Diuretic effect of clonidine during isoflurane, nitrous oxide, and oxygen anesthesia. Anesthesiology 81: 811-819, 1994[Medline].

18.   Hayashi, Y., and M. Maze. Alpha2 adrenoceptor agonists and anaesthesia. Br. J. Anaesth. 71: 108-118, 1993[Free Full Text].

19.   Humphreys, M. H., and I. A. Reid. Suppression of antidiuretic hormone secretion by clonidine in the anesthetized dog. Kidney Int. 7: 405-412, 1975[Medline].

20.   Kimura, T., L. Share, B. C. Wang, and J. T. Crofton. The role of central adrenoceptors in the control of vasopressin release and blood pressure. Endocrinology 108: 1829-1836, 1981[Abstract/Free Full Text].

21.   Kimura, T., M. Shoji, K. Iitake, K. Ota, K. Matsui, and K. Yoshinaga. The role of central a1- and a2-adrenoceptors in the regulation of vasopressin release and the cardiovascular system. Endocrinology 114: 1426-1432, 1984[Abstract/Free Full Text].

22.   Kline, R. L., and D. F. Cechetto. Renal effects of rilmenidine in anesthetized rats: importance of renal nerves. J. Pharmacol. Exp. Ther. 266: 1556-1562, 1993[Abstract/Free Full Text].

23.   Kline, R. L., J. van der Mark, and D. F. Cechetto. Natriuretic effect of rilmenidine in anesthetized rats. Am. J. Cardiol. 74: 20A-24A, 1994[Medline].

24.   Kline, R. L., and P. F. Mercer. Contribution of renal nerves to the natriuretic and diuretic effect of alpha-2 adrenergic receptor activation. J. Pharmacol. Exp. Ther. 253: 266-271, 1990[Abstract/Free Full Text].

25.   Koepke, J. P., and G. F. DiBona. Central adrenergic receptor control of renal function in conscious hypertensive rats. Hypertension 8: 133-141, 1986[Abstract/Free Full Text].

26.   Kopp, U. C., and G. F. DiBona. The neural control of renal function. In: The Kidney: Physiology and Pathophysiology (2nd Ed.), edited by D. W. Seldin, and G. Giebisch. New York: Raven, 1992, p. 1157-1204.

27.   Krothapalli, R. K., W. B. Duffy, H. O. Senekjian, and W. N. Suki. Modulation of the hydro-osmotic effect of vasopressin on the rabbit cortical collecting tubule by adrenergic agents. J. Clin. Invest. 72: 287-294, 1983.

28.   Krothapalli, R. K., and W. N. Suki. Functional characterization of the alpha adrenergic receptor modulating the hydroosmotic effect of vasopressin on the rabbit cortical collecting tubule. J. Clin. Invest. 73: 740-749, 1984.

29.   Leander, J. D., R. L. Zerbe, and J. C. Hart. Diuresis and suppression of vasopressin by kappa opioids: comparison with mu and delta opioids and clonidine. J. Pharmacol. Exp. Ther. 234: 463-469, 1985[Abstract/Free Full Text].

30.   Maktabi, M. A., F. F. Elboki, F. M. Faraci, and M. M. Todd. Halothane decreases the rate production of cerebrospinal fluid. Anesthesiology 78: 72-82, 1993[Medline].

31.   Maze, M., and W. Tranquilli. Alpha-2 adrenoceptor agonists: defining the role in clinical anesthesia. Anesthesiology 74: 581-605, 1991[Medline].

32.   Mazze, R. I., F. D. Schwartz, H. C. Slocum, and K. G. Barry. Renal function during anesthesia and surgery. The effects of halothane anesthesia. Anesthesiology 24: 279-284, 1963.

33.   Miller, M. Clonidine-induced diuresis in the rat: evidence for a renal site of action. J. Pharmacol. Exp. Ther. 214: 608-613, 1980[Abstract/Free Full Text].

34.   Nicholas, A. P., V. Peribone, and T. Holfelt. Distributions of mRNAs for alpha-2 adrenergic receptor subtypes in rat brain: an in situ hybridization study. J. Comp. Neurol. 328: 575-594, 1993[Medline].

35.   Papper, S., and E. M. Papper. The effects of preanesthetic, anesthetic, and postoperative drugs on renal function. Clin. Pharmacol. Ther. 5: 205-215, 1964.

36.   Paxinos, G., and C. Watson. The Rat Brain in Stereotaxic Coordinates (2nd Ed.). Perth, Western Australia: Academic, 1986.

37.   Pieribone, V. A., A. P. Nicholas, A. Dagerlind, and T. Hokfelt. Distribution of alpha-1 adrenoceptors in rat brain revealed by in situ hybridization experiments utilizing subtype-specific probes. J. Neurosci. 14: 4252-4268, 1994[Abstract].

38.   Reid, I. A., P. L. Nolan, J. A. Wolf, and L. C. Keil. Suppression of vasopressin secretion by clonidine: effects of alpha-adrenoceptor antagonists. Endocrinology 104: 1403-1406, 1979[Abstract/Free Full Text].

39.   Roman, R. J., A. W. Cowley, Jr., and C. Lechene. Water diuretic and natriuretic effect of clonidine in the rat. J. Pharmacol. Exp. Ther. 211: 385-393, 1979[Free Full Text].

40.   Smyth, D. D., S. Umemura, and W. A. Pettinger. Alpha2-adrenoceptor antagonism of vasopressin-induced changes in sodium excretion. Am. J. Physiol. 248 (Renal Fluid Electrolyte Physiol. 17): F767-F772, 1985.

41.   Stanton, B., E. Puglisi, and M. Gellai. Localization and mechanism of alpha 2-adrenoceptor-mediated increase in renal Na+, K+, and water excretion. Am. J. Physiol. 252 (Renal Fluid Electrolyte Physiol. 21): F1016-F1021, 1987[Abstract/Free Full Text].

42.   Strandhoy, J. W. Role of alpha-2 receptors in the regulation of renal function. J. Cardiovasc. Pharmacol. 7: S28-S33, 1985.

43.   Strandhoy, J. W., M. Morris, and V. M. Buckalew, Jr. Renal effects of the antihypertensive, guanabenz, in the dog. J. Pharmacol. Exp. Ther. 221: 347-352, 1982[Free Full Text].

44.   Swanson, K. L. W., and P. E. Sawchenko. Hypothalamic integration: Organization of the paraventricular and supraoptic nuclei. Annu. Rev. Neurosci. 6: 269-324, 1983[Medline].

45.   Udelsman, R., J. A. Norton, S. E. Jelenich, D. S. Goldstein, W. M. Linehan, D. L. Loriaux, and G. P. Chrousos. Responses of the hypothalamic-pituitary-adrenal and renin-angiotensin axes and the sympathetic system during controlled surgical and anesthetic stress. J. Clin. Endocrinol. Metab. 64: 986-994, 1987[Abstract/Free Full Text].

46.   Wallenstein, S., C. L. Zucker, and J. L. Fleiss. Some statistical methods useful in circulation research. Circ. Res. 47: 1-9, 1980[Abstract/Free Full Text].

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