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Am J Physiol Regul Integr Comp Physiol 281: R98-R107, 2001;
0363-6119/01 $5.00
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Vol. 281, Issue 1, R98-R107, July 2001

Activation of alpha 2-receptors in the rostral ventrolateral medulla evokes natriuresis by a renal nerve mechanism

Rubia Grecco Menegaz1, Daniel R. Kapusta2, Helder Mauad1, and Antonio de Melo Cabral1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The contribution of alpha 2-receptor mechanisms in the rostral ventrolateral medulla (RVLM) in mediating the enhanced renal excretory responses evoked by the intravenous infusion of the alpha 2-receptor agonist xylazine was examined in ketamine-anesthetized rats. In ketamine-anesthetized rats, the bilateral microinjection of the alpha 2-receptor antagonist yohimbine into the RVLM significantly reduced the enhanced levels of urine flow rate (V) and urinary sodium excretion (UNaV) produced by xylazine. In contrast, microinjection of yohimbine into the RVLM of chronically bilaterally renal-denervated rats significantly reduced the xylazine-evoked diuretic, but not natriuretic, response. In separate ketamine-anesthetized rats, intravenous xylazine infusion produced a near complete inhibition of renal sympathetic nerve activity (RSNA). The subsequent microinjection of yohimbine into the RVLM reversed this neural response and concurrently decreased V and UNaV. Together, these results indicate that during intravenous infusion, xylazine activates alpha 2-receptor mechanisms in the RVLM to selectively promote urinary sodium excretion by a renal nerve-dependent pathway. In contrast, activation of alpha 2-receptor in the RVLM affects the renal handling of water by a pathway independent of the renal nerves. This latter pathway may involve an interaction with other brain regions involved in antidiuretic hormone release (e.g., paraventricular nucleus of the hypothalamus).

renal sympathetic nerves; renal excretory function; urine flow rate; urinary sodium excretion; central nervous system; microinjection


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE STIMULATION OF alpha 2-receptors increases urine flow rate and urinary sodium excretion in animals and humans (2, 3, 16, 18, 19, 42, 45). The diuretic and natriuretic responses produced by alpha 2-receptor agonists (e.g., clonidine, guanabenz, xylazine, etc.) involves the integration of complex peripheral, direct renal and central nervous system (CNS) mechanisms (5, 6, 12, 13, 44). In regards to a direct renal action, alpha 2-agonists inhibit vasopressin (antidiuretic hormone)-stimulated cAMP formation in the distal tubule (11, 15, 16, 37) and consequently aquaporin-mediated water reabsorption (31). In addition to an intrarenal pathway, alpha 2-agonists can affect the renal handling of water and sodium from a locus within the CNS. In this manner, alpha 2-agonists produce diuretic and natriuretic responses by a central action to inhibit vasopressin release (4, 16, 23, 27, 28, 41) and to inhibit central sympathetic outflow to the kidneys (14, 29, 30, 32, 33), respectively.

The mechanisms and central sites of action by which alpha 2-receptor agonists produce diuresis and natriuresis are not completely known. As an approach to study the role of central alpha 2-receptors in the control of renal function, we have developed an experimental model in which ketamine-anesthetized rats are continuously infused intravenously with the alpha 2-receptor agonist xylazine. Using this method, we can use stereotaxic microinjection techniques (e.g., glass multibarrel pipettes) to explore the specific brain sites and mechanisms by which xylazine produces an enhanced and sustained increase in urine output and urinary sodium excretion (7). In previous studies, we showed that the intracerebroventricular or paraventricular nucleus (PVN) microinjection of the alpha 2-receptor antagonist yohimbine significantly reduced the enhanced level of urine flow rate, but not urinary sodium excretion, to xylazine infusion (5). The decrease in urine flow rate produced by intracerebroventricular yohimbine was reversed by the intravenous injection of a selective V2-vasopressin receptor antagonist (5). In contrast, the intravenous bolus injection of yohimbine (but not terazosin, a selective alpha 1-adrenoceptor antagonist) completely reversed both renal responses (5). These findings suggest that during intravenous infusion, xylazine increases urine flow rate, at least in part, by activating alpha 2-receptors in the PVN, which in turn decreases the secretion of vasopressin. In addition, these findings indicate that xylazine utilizes an alternative pathway(s) and/or CNS site of action(s) other than the PVN to produce natriuresis (2, 29, 30).

The activation of alpha 2-receptors in the CNS inhibits sympathetic outflow to the kidneys and thereby reduces renin release, the renal tubular reabsorption of sodium and water, and renal vascular resistance (9). Several regions in the CNS, including the rostral ventrolateral medulla (RVLM), have been suggested as sites where alpha 2-agonists act to reduce peripheral sympathetic drive (1, 17, 20-22, 39). Microinjection of alpha 2-agonists (e.g., clonidine) into the RVLM of anesthetized animals evokes concurrent decreases in renal sympathetic nerve activity, heart rate, and arterial blood pressure (20-22). These observations and others support the generally held view that the RVLM is the major site of sympathoinhibitory action of centrally acting antihypertensive agents (e.g., clonidine). However, despite these findings it remains unknown as to whether activation of alpha 2-adrenoceptor pathways in the RVLM increases the renal excretion of sodium and/or water by inhibiting sympathetic outflow to the kidneys. This possibility is of importance because alterations in renal sympathetic nerve activity can have marked affects on urine flow rate and urinary sodium excretion (9).

The purpose of the present investigations was to determine whether alpha 2-receptor mechanisms in the RVLM contribute to the enhanced levels of urine flow rate and/or urinary sodium excretion produced by the intravenous infusion of xylazine in ketamine-anesthetized rats and to establish the role of the renal nerves in mediating these responses. For this purpose we first examined the changes in renal excretory function evoked by the microinjection (glass multibarrel pipettes) of the alpha 2-receptor antagonist yohimbine into the RVLM of rats infused intravenously with ketamine and xylazine. To critically investigate whether the renal excretory responses produced by activation of alpha 2-receptors in the RVLM are coupled to a neural pathway that requires an intact renal innervation, we repeated microinjection studies with yohimbine in chronically bilaterally renal-denervated rats (RDNX). Finally, in pilot studies we showed that the intravenous infusion of xylazine, like other alpha 2-adrenoceptor agonists, produces a profound reduction in renal sympathetic nerve activity. To test whether xylazine acts within the RVLM to affect the renal handling of water and sodium by changing central sympathetic outflow to the kidneys, we measured changes in renal sympathetic nerve activity (direct recording) and renal excretory function in ketamine- and xylazine-anesthetized rats before and after the microinjection of yohimbine into the RVLM.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects

Experiments were performed on male Wistar rats (240-260 g) (Federal University of Espirito Santo and Harlan, Indianapolis, IN). All procedures were conducted in accordance with the biomedical research guidelines for the care and use of laboratory animals as stated by the Federation of Brazilian Societies of Experimental Biology, the Louisiana State University Health Sciences Center, and the National Institutes of Health. The rats were housed in groups in a temperature- and humidity-controlled room with a 12-h light-dark cycle. Standard rat chow (Na+ content 163 meq/kg) and tap water were available ad libitum.

Surgical Procedures

Catheter implantation. On the day of the experiment, rats were anesthetized with sodium thiopental (Tiopental 50 mg/kg, supplemented intravenously as needed; Cristalia, São Paulo, Brazil). Catheters (PE-50 fused to PE-10) were placed in the femoral artery and vein for the recording of arterial blood pressure and the administration of drugs and isotonic saline infusion, respectively. As is standard procedure in our laboratory, 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.

Microinjection procedures for studies in ketamine- and xylazine-anesthetized rats. After implantation of catheters, rats were administered ketamine (40 mg/kg iv) over a 5-min period. An infusion (55 µl/min iv) 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. Each ketamine- and xylazine-anesthetized rat was then placed prone in a stereotaxic apparatus with the bite bar 11.0 mm below the interaural line. An occipital craniotomy was performed to expose the dorsal surface of the brain stem and cerebellum. The dura was opened and retracted, exposing the calamus scriptorius, whose vertex was taken as a landmark for stereotaxic coordinates.

Drugs were microinjected bilaterally into the RVLM using a triple-barrelled glass micropipette (0.4 mm ID, 0.75 mm OD) having a composite tip diameter of <40 µm. The pipette was directed to the desired stereotaxic position (anteroposterior, 3.0 mm rostral and 1.8 mm lateral to the obex, 3.4 mm dorsal). The three barrels of the pipette were filled by vacuum with saline (barrel 1), yohimbine (1 mg/ml, barrel 2), and Evans blue dye (barrel 3). All drugs were injected in a volume of 60 nl over a period of 0.5-1.0 min by a pneumatic pressure injection system (Pneumatic Picopump, World Precision Instruments, Sarasota, FL). We controlled the speed and volume of the injection by watching the movement of the fluid meniscus in the pipette with a stereomicroscope and a gradicule affixed to the pipette.

Bilateral renal denervation. Certain studies were performed in ketamine- and xylazine-anesthetized rats in which the influence of the renal nerves on renal excretory function was removed. For this purpose, rats underwent chronic bilateral renal denervation 5-7 days before the experiment. Under ketamine (30 mg/kg im) and xylazine (3 mg/kg im) anesthesia, each rat had its left kidney exposed via a flank incision. The adventitia surrounding the renal artery and vein were stripped, and all visible renal nerves were cut under a microscope (World Precision Instruments 13301). The vessels were then treated with alcohol solution containing phenol (10%). After completion of renal denervation, the flank incision was sutured closed, and the procedure was repeated on the opposite side to denervate the right kidney. This renal denervation procedure prevents the renal vasoconstrictor response to suprarenal lumbar sympathetic nerve stimulation, prevents the antinatriuretic response to environmental stress, and reduces renal tissue norepinephrine concentration to <5% of control for up to 15 days postdenervation (10). Our laboratories previously verified that this renal denervation procedure completely removes the influence of the renal nerves on kidney function (24, 25).

Method for implanting the renal nerve recording electrode in ketamine- and xylazine-anesthetized rats. To verify the role of the renal nerves in mediating the renal excretory responses produced by bilateral microinjection of yohimbine into the RVLM, we implanted a recording electrode on a renal nerve bundle for measurement of changes in multifiber renal sympathetic nerve activity. On the morning of the experiment, rats were anesthetized with sodium thiopental (50 mg/kg, supplemented intravenously as needed) and implanted with arterial, venous, and bladder catheters as previously stated. After catheter implantation, the left kidney was exposed through a left incision via a retroperitoneal approach. With the use of a dissecting microscope (×25), a renal nerve branch from the aorticorenal ganglion was isolated and carefully dissected. The renal nerve branch was then placed on a bipolar platinum wire (Cooner Wire, Chatsworth, CA) electrode and fixed with a dentistry impression material (Coltene President). The electrode cable was then secured in position by suturing it to the abdominal trunk muscles. Finally the electrode cable was exteriorized, and the flank incision was closed in layers.

Extracellular action potentials from renal sympathetic nerves were amplified (10,000-50,000×), filtered (low, 30 Hz; high, 3,000 Hz) with a Grass P511 band-pass amplifier (Grass Instruments, Quincy, MA). The amplified and filtered signal was channeled to a Tektronix 5113 oscilloscope (Tektronix, Beaverton, OR) and Grass model 7DA polygraph for visual evaluation, to an audio amplifier-loudspeaker (Grass model AM 8 Audio Monitor) for auditory evaluation, and to a rectifying voltage integrator (Grass model 7P10). The integrated voltage signals were displayed on the Grass polygraph, and data acquisition for renal sympathetic nerve activity measurements were performed with a commercially available software package (Acknowledge for Windows, Biopac, Santa Barbara, CA). Integrated renal sympathetic nerve activity was expressed as microvolt seconds per 1-s intervals. For each 10-min experimental period, the values for integrated renal sympathetic nerve activity were sampled over the entire collection period, and the numbers were averaged. The data for renal sympathetic nerve activity are expressed as the percentage of the baseline value obtained during ketamine-alone anesthesia (denoted K in Fig. 2), with this being expressed as 100% for each animal. We determined the background noise level of renal sympathetic nerve activity by observing the neural signal that remained 10 min after the start of the intravenous infusion of ketamine and xylazine.

Experimental Protocols

Renal excretory responses elicited by the microinjection of yohimbine into the RVLM of ketamine- and xylazine-anesthetized rats. Experiments were performed in ketamine-anesthetized rats to determine whether alpha 2-receptor mechanisms are activated in the RVLM and contribute to the enhanced natriuretic and/or diuretic responses to xylazine infusion. The intravenous infusion of xylazine enhances the renal excretion of water and sodium, and these levels tend to stabilize and remain constant ~120 min from the beginning of infusion (7). Therefore, after equilibration and stabilization of renal excretory responses, two consecutive control urine samples were collected (10 min each). The alpha 2-receptor antagonist yohimbine (60 ng in 60 nl, n = 6) was then microinjected bilaterally into the RVLM. The drug was allowed 5 min for distribution. The experimental period then entailed collection of urine during six consecutive 10-min experimental periods.

We repeated the same protocol in RDNX to examine the role of an intact renal innervation in mediating the renal excretory responses elicited by the microinjection of yohimbine into the RVLM of ketamine- and xylazine-anesthetized rats.

At the end of experiments in intact and RDNX, we marked sites of drug or vehicle injection bilaterally by microinjecting Evans blue dye (60 nl) through the third barrel of the pipette. Only the rats whose microinjection site was located bilaterally in the RVLM were used for data analysis. In rats with the microinjection misplaced into areas adjacent to the RVLM or in rats that received bilateral microinjection of isotonic saline (60 nl) into the RVLM, no changes in renal or cardiovascular parameters were observed.

For each study, ketamine and xylazine were infused intravenously for the duration of the experiment via an infusion pump (model 600-900V, Harvard Apparatus, Dover, MA). The arterial catheter was connected to a pressure transducer (model P23Db, Statham, Oxnard, CA). Throughout the experiment, mean arterial pressure and heart rate were continuously recorded on a polygraph (Sensormedics Dynograf Recorder R 711). During surgery and the experimental protocol, the body temperature of rats was maintained at 37 ± 1°C by use of a heat lamp.

Renal excretory and renal nerve responses elicited by the microinjection of yohimbine into the RVLM of ketamine- and xylazine-anesthetized rats. Additional studies were performed to investigate whether changes in renal sympathetic nerve activity are involved in producing the renal excretory responses elicited by the microinjection of yohimbine into the RVLM of ketamine- and xylazine-anesthetized rats.

After completion of surgical implantation procedures (i.e., catheters, renal nerve recording electrode) performed under thiopental, rats were administered ketamine (40 mg/kg iv) over 5 min. Rats were then placed in a stereotaxic apparatus, and baseline cardiovascular and renal sympathetic nerve activity parameters were continuously measured. An infusion (55 µl/min iv) 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. After equilibration and stabilization of renal excretory responses (~2 h), two consecutive control urine samples were collected (10 min each). The alpha 2-receptor antagonist yohimbine (60 ng in 60 nl, n = 5) was then microinjected bilaterally into the RVLM. The drug was allowed 5 min for distribution. The experimental phase then entailed collection of urine during six consecutive 10-min experimental periods. Heart rate, mean arterial pressure, and renal sympathetic nerve activity were measured throughout the experiment and displayed on a Grass polygraph. At the end of experiments, sites of drug or vehicle injection were marked bilaterally by microinjecting Evans blue dye (60 nl) through the third barrel of the pipette.

Histological Processing

At the end of the microinjection studies, anesthetized rats were perfused transcardially with normal saline followed by 10% formaldehyde. The brains were removed and stored for at least 2 days in the formaldehyde solution. The brains were then frozen and sectioned (40 µm) with a cryostat microtome. The sections were then placed on glass slides and stained with neutral red 1%. We used the atlas of Paxinos and Watson (38) as a reference to identify microinjection sites microscopically from the stained sections.

Data Analysis

Changes in mean arterial pressure and heart rate produced by drug administration were calculated directly from the polygraph records. Before the rats were perfused, the kidneys were removed, decapsulated, and weighed for normalization of renal excretory data. Urine volume was determined gravimetrically. Urine sodium concentration was measured by flame photometry (Micronal, model B, São Paulo, Brazil, or model 943, Instrumentation Laboratories, Lexington, MA).

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

Drugs Used

The drugs used in this study were yohimbine hydrochloride (Sigma, St. Louis, MO), ketamine hydrochloride (Ketaset, Fort Dodge Laboratories, Fort Dodge, IA), sodium thiopental (Tiopental, Cristalia, São Paulo, Brazil), and xylazine (Butler, Columbus, OH). Yohimbine and xylazine were dissolved in normal saline (0.9%).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Figure 1 shows the systemic cardiovascular and renal excretory responses produced by the bilateral microinjection of the alpha 2-receptor antagonist yohimbine (60 ng, n = 6) into the RVLM of intact and RDNX. Mean data ± SE are shown for each cardiovascular and renal excretory parameter during two consecutive 10-min control periods (C1 and C2) and six consecutive experimental periods (10 min each) beginning 5 min after microinjection (time points 15-65 min). Compared with control levels (Fig. 1, C2), the bilateral microinjection of yohimbine into the RVLM of intact rats (n = 6, ) produced an immediate and profound decrease in urine flow rate [-60%: C2, 60 ± 9 µl · min-1 · g kidney wt-1 (Kw); yohimbine 15 min, 24 ± 5 µl · min-1 · gKw-1] and urinary sodium excretion (-56%: C2, 5.7 ± 0.7 µeq · min-1 · gKw-1; yohimbine 15 min, 2.5 ± 0.5 µeq · min-1 · gKw-1) that occurred by the first experimental period. In a similar manner, the microinjection of yohimbine into the RVLM of RDNX (Fig. 1, odot , n = 6), significantly (P < 0.01) reduced urine flow compared with the predrug control level (-38%: C2, 61 ± 5 µl · min-1 · gKw-1; yohimbine 15 min, 38 ± 6 µeq · min-1 · gKw-1). In contrast, in RDNX (Fig. 1), the microinjection of yohimbine into the RVLM did not alter urinary sodium excretion at any time period (C2, 4.0 ± 0.4 µeq · min-1 · gKw-1; yohimbine 15 min, 3.9 ± 0.5 µeq · min-1 · gKw-1). The microinjection of yohimbine into the RVLM failed to alter any cardiovascular parameter in either intact or renal-denervated groups (Fig. 1).


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  		Fig. 1.   Effects of bilateral microinjection of yohimbine into the rostral ventrolateral medulla (RVLM) of ketamine- and xylazine-anesthetized rats with intact and bilaterally denervated (DNX) kidneys. Values are means ± SE, illustrating the systemic cardiovascular and renal responses produced by the bilateral microinjection of the alpha 2-receptor antagonist, yohimbine (60 ng in 60 nl), in ketamine- and xylazine-anesthetized rats with intact (, n = 6) and bilaterally renal denervated (odot , n = 6) kidneys. Experiments were performed in chronically instrumented ketamine-anesthetized rats (ketamine 40 mg/kg iv over 5 min) that were continuously infused with isotonic saline (55 µl/min iv) containing ketamine (1 mg · kg-1 · min-1) and xylazine (50 µg · kg-1 · min-1). Two hours after the start of the infusion, consecutive 10-min urine samples were collected from a urinary bladder catheter during control (C1, C2) and 5 min after the microinjection of yohimbine into the RVLM (time points 15-65 min). HR, heart rate; MAP, mean arterial pressure; V, urine flow rate; UNaV, urinary sodium excretion. *P < 0.05, tP < 0.05, significantly different from corresponding control (C2) period for each group.

The bilateral microinjection of yohimbine into sites rostral, dorsal, or caudal to RVLM did not significantly change any cardiovascular or renal excretory parameter compared with predrug control periods (data not shown). Moreover, in additional studies, the bilateral microinjection of isotonic saline vehicle (60 nl) into the RVLM of intact or RDNX did not change any cardiovascular or renal excretory parameter over the course of the experiment (data not shown).

Figure 2 illustrates the cardiovascular, renal excretory, and renal sympathetic nerve responses produced by the microinjection of yohimbine in ketamine- and xylazine-anesthetized rats. Shown are the measurements for each parameter observed during ketamine-alone anesthesia (K). A continuous intravenous infusion of ketamine and xylazine was then started. After ~2 h of equilibration, consecutive urine samples (10 min each) were then collected during control (C1, C2) and 5-min after the microinjection of yohimbine into the RVLM (time points 15-65). Compared with levels observed during ketamine-alone anesthesia (K), the intravenous infusion of xylazine produced a marked decrease in heart rate, mean arterial pressure, and renal sympathetic nerve activity (expressed as %K), and an increase in urine flow rate and urinary sodium excretion. Compared with respective control values, the bilateral microinjection of yohimbine into the RVLM (60 ng in 60 nl; n = 5; ) produced a significant decrease in urine flow rate and urinary sodium excretion. The magnitude and pattern of these renal responses were similar to those observed in intact rats depicted in Fig. 1. In addition, the microinjection of yohimbine into the RVLM of ketamine- and xylazine-anesthetized rats produced a significant increase in renal sympathetic nerve activity that peaked by 25 min after the microinjection of yohimbine. The action of yohimbine to reverse the sympathoinhibitory effect of xylazine tended to correspond to the yohimbine-induced antinatriuresis and antidiuresis. In contrast to these responses, the microinjection of yohimbine into brain sites outside of the RVLM (Fig. 2; missed injection; n = 5; odot ) did not significantly alter renal excretory function or renal sympathetic nerve activity. Finally, the microinjection of yohimbine into the RVLM or into sites rostral, dorsal, or caudal to RVLM did not change any cardiovascular parameter.


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  		Fig. 2.   Effects of bilateral microinjection of yohimbine into the RVLM of ketamine- and xylazine-anesthetized rats. Values are means ± SE, illustrating the systemic cardiovascular and renal responses produced by the bilateral microinjection of the alpha 2-receptor antagonist yohimbine (60 ng in 60 nl) into the RVLM (, n = 5) or brain sites outside of the RVLM (odot , missed injections, n = 5) in ketamine-anesthetized rats infused intravenously with ketamine and xylazine (see Fig. 1 legend). K, levels for each parameter measured during ketamine-alone anesthesia. Hash marks indicate a lapse of 2 h during which rats were continuously infused intravenously with ketamine and xylazine. Consecutive 10-min urine samples were collected from a urinary bladder catheter during control (C1, C2) and 5 min after the microinjection of yohimbine into the RVLM (time points 15-65 min). RSNA, renal sympathetic nerve activity; other abbreviations as in Fig. 1. *P < 0.05, significantly different from corresponding group control (C2) period.

Figure 3 depicts an original tracing in which the cardiovascular (heart rate and pulsatile and mean arterial pressure) and renal nerve (integrated renal sympathetic nerve activity) responses to the bilateral microinjection of yohimbine into the RVLM were examined in a single rat infused intravenously with ketamine and xylazine. Figure 3A shows that in the rat anesthetized with ketamine alone, the start of the intravenous infusion of ketamine and xylazine produced a marked decrease in heart rate and arterial blood pressure and a near complete inhibition of renal sympathetic nerve activity. Figure 3B shows that after 2 h of infusion of ketamine and xylazine, the levels for these parameters still remained low before the administration of yohimbine. However, the bilateral microinjection of yohimbine into the RVLM produced a profound increase in renal sympathetic nerve activity that tended to approach preinfusion levels observed during ketamine-alone anesthesia (Fig. 3A). In contrast to renal sympathetic nerve activity, the microinjection of yohimbine into the RVLM did not antagonize heart rate or arterial pressure responses to intravenous ketamine and xylazine infusion.


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  		Fig. 3.   Polygraph tracing for a single ketamine-anesthetized rat showing HR, arterial pressure (AP), MAP, and integrated renal sympathetic nerve activity (RSNA) responses produced during the start of intravenous infusion of ketamine and xylazine (A) and after bilateral microinjection of yohimbine (60 ng) into the RVLM (B). Time lapse between A and B: 2 h. PE, intravenous bolus injection of phenylephrine (2 µg).

The photomicrograph in Fig. 4 shows the dye-marked injection site into which 60 ng yohimbine was microinjected into the RVLM of the same rat for which the tracing is presented in Fig. 3. The histologically identified sites in which the drugs were microinjected in ketamine- and xylazine-anesthetized rats with intact and bilaterally denervated kidneys (from Fig. 1) are shown in Fig. 5.


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  		Fig. 4.   Photomicrograph of a coronal section though the rat medulla showing a microinjection site in the RVLM (arrow) of the same rat in which the tracing is shown in Fig. 3. Horizontal calibration is 1 mm. Amb, nucleus ambiguus; Rpa, nucleus raphe pallidus; py, pyramidal tract.



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  		Fig. 5.   Histologically verified sites into which the alpha 2-adrenoceptor antagonist yohimbine (60 ng in 60 nl) was microinjected into the RVLM of rats with intact and bilaterally denervated kidneys for which data are presented in Fig. 1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The purpose of the present study was to examine the role of alpha 2-receptor mechanisms in the RVLM in producing changes in the renal excretion of water and sodium. To investigate this question, we used an experimental protocol in which the continuous intravenous infusion of the alpha 2-agonist xylazine produces an enhanced and sustained increase in urine flow rate and urinary sodium excretion in ketamine-anesthetized rats (7). In previous studies, it was shown that intravenous xylazine increases urine flow rate in part by activating alpha 2-receptors in the PVN of the hypothalamus, this response acting to decrease the secretion/release of vasopressin (5). In these studies, the natriuretic response was shown to be due to an alternative pathway(s) and/or CNS site of action of xylazine (8, 29, 30). The results of the present study extend these findings and demonstrate that alpha 2-receptor mechanisms in the RVLM play an important role in contributing to the natriuretic and diuretic effects produced by intravenous xylazine infusion in ketamine-anesthetized rats. In this regard, these studies demonstrate that xylazine activates alpha 2-receptors within the RVLM to enhance urinary sodium excretion and that this response is mediated by a renal nerve-dependent pathway that involves suppression of sympathetic outflow to the kidneys. In contrast to the effects on sodium, it appears that alpha 2-receptor mechanisms in the RVLM can influence the renal handling of water by a pathway that is independent of the renal nerves. When previous findings are considered (5), this suggests that in ketamine-anesthetized rats the enhanced renal excretory responses produced by xylazine infusion involve both central alpha 2-receptor pathways in the RVLM and the PVN and a potential interaction between these brain regions.

The RVLM is a region in the brain stem in which cell bodies of a group of sympathoexcitatory neurons involved in the central regulation of cardiovascular function are located. The neurons that originate from this nucleus are essential for the generation and modulation of sympathetic tone. From the RVLM, regulation of autonomic function is achieved by a subset of neurons that project directly to the intermediolateral nucleus of the thoracic spinal cord. In addition, neuronal cell bodies in the RVLM receive afferent input from other brain regions that are also involved in the regulation of cardiovascular function (17, 26, 35, 40). The microinjection of alpha 2/imidazoline receptor agonists into the RVLM produces bradycardia, hypotension, and a reduction in renal sympathetic nerve activity. Based on these latter observations, the RVLM has been suggested to be a predominant brain site involved in mediating the antihypertensive effect of alpha 2/imidazoline receptor agonists (20-22, 39).

The renal sympathoinhibitory response produced by the microinjection of alpha 2-receptor agonists into the RVLM is of interest in that changes in renal sympathetic nerve activity can evoke significant alterations in the renal handling of water and sodium (9). In this manner, alterations (e.g., a decrease) in renal sympathetic nerve activity can produce reciprocal changes in the renal excretion of urine flow rate and urinary sodium excretion (e.g., diuresis and natriuresis) (9). Despite evidence demonstrating that the stimulation of alpha 2-receptors in the RVLM suppresses sympathetic outflow to the kidneys (20-22), previous studies have not specifically examined whether this response evokes an alteration in renal excretory function. Instead, in these previous investigations, renal sympathetic nerve activity was measured as a means to establish the relationship between the changes in central sympathetic outflow produced by activation of alpha 2-receptors in the RVLM and changes in cardiovascular function (i.e., heart rate and arterial blood pressure).

Because the stimulation of alpha 2-receptors in the RVLM decreases renal sympathetic nerve activity, it is possible that during intravenous infusion xylazine activates a similar inhibitory neural mechanism in the RVLM to enhance renal excretory function. In this case, it would be anticipated that the microinjection of the alpha 2-receptor antagonist yohimbine into the RVLM would reverse xylazine's effects on renal excretory function. The results of the present study support this hypothesis by demonstrating that the microinjection of yohimbine into the RVLM of intact rats markedly reduced the enhanced basal levels of urine flow rate and urinary sodium excretion produced by intravenous xylazine. The reduction in renal excretory function evoked by yohimbine was rapid in onset and slow to recover, despite the continued infusion of xylazine. The microinjection of isotonic saline vehicle into the RVLM, or the injection of yohimbine into sites outside this brain nucleus, failed to alter the xylazine-induced renal responses. Together, these findings indicate that during intravenous infusion of xylazine, alpha 2-receptors located in the RVLM are activated and have an important role in contributing to the enhanced level of sodium and water excretion.

To determine whether the renal excretory responses produced by microinjection of yohimbine into the RVLM require an intact renal innervation, we performed additional microinjection studies in RDNX. In these studies (Fig. 1), the microinjection of yohimbine into the RVLM of renal-denervated rats had no effect on the enhanced levels of urinary sodium excretion. This is in contrast to the sharp reduction in urinary sodium excretion produced by the microinjection of yohimbine in intact rats. These findings indicate that yohimbine acts within the RVLM to reverse the xylazine-induced natriuresis by a renal nerve-dependent pathway.

As noted above, an intact renal innervation is required to mediate the antinatriuresis produced by the microinjection of yohimbine into the RVLM. These findings suggest that in ketamine-anesthetized rats, xylazine activates alpha 2-adrenoceptors in the RVLM to suppress renal sympathetic nerve activity and consequently produce natriuresis. To further evaluate this premise, we performed microinjection studies with yohimbine in which renal sympathetic nerve activity was directly recorded. In these studies, it was demonstrated that the sympatholytic effect of xylazine infusion was reversed by the microinjection of yohimbine into the RVLM (Figs. 2 and 3) and that this response occurred over a time frame in which renal excretory responses were reduced. These findings demonstrate that yohimbine antagonized the action of xylazine to inhibit renal sympathetic nerve activity and thus restored the influence of the renal nerves on the renal tubular handling of sodium (and water). It is interesting to note that in these studies, the microinjection of yohimbine into the RVLM did not restore heart rate and arterial blood pressure to levels observed before the start of the xylazine infusion. These observations indicate that during intravenous infusion of xylazine, there are additional sites (central and/or peripheral) in which xylazine activates alpha 2-adrenoceptor pathways (neural and or humoral) that affect heart rate and blood pressure.

The present data support a role for the RVLM and the renal sympathetic nerves in mediating the enhanced renal excretory responses produced by intravenous xylazine infusion. It appears, however, that other brain sites and/or CNS/peripheral mechanisms also participate in mediating the diuretic and natriuretic responses to this alpha 2-agonist. This is apparent from the observation that intravenous xylazine infusion augmented the basal level of urine flow rate and urinary sodium excretion in ketamine-anesthetized rats with bilaterally denervated kidneys (Fig. 1). Thus, under conditions in which the influence of the renal sympathetic nerves on renal excretory function is entirely removed (e.g., renal denervation), xylazine utilizes alternative renal nerve-independent pathways to affect the renal handling of water and sodium. This finding is in agreement with those from other studies in which alpha 2-agonists have been shown to elicit diuretic and natriuretic responses in bilaterally renal denervated animals (36, 45, 46). Alternative to these nonneural pathways, it should be noted that in intact animals, alpha 2-agonists can also affect the renal handling of water and sodium by affecting renal nerve activity from a site(s) other than the RVLM. In this manner, alpha 2-agonists (e.g., guanabenz) lower the control level of renal sympathetic nerve activity and raise baseline urinary sodium excretion when microinjected into the central amygdaloid nucleus (34). Whether the diuretic/natriuretic responses produced by intravenous xylazine involve the stimulation of alpha 2-receptors in this or other brain regions has yet to be determined.

Perspectives

The microinjection of yohimbine into the RVLM of renal-denervated rats did not alter the natriuretic response to xylazine infusion, thus demonstrating that alpha 2-receptor mechanisms in this brain region require intact renal nerves to influence urinary sodium excretion. In contrast, however, in these same renal-denervated animals, yohimbine produced a significant decrease in urine flow rate (Fig. 1). This observation suggests that, during xylazine infusion, alpha 2-receptor pathways in the RVLM may participate in mediating an enhanced level of urine flow rate by activating a neural pathway that influences the activity of another brain site, potentially the PVN (and/or supraoptic nucleus). In accord with a hypothesis for an interaction between the RVLM and PVN, neuroanatomical studies using tract-tracing techniques have shown that the PVN sends neural projections to both the intermediolateral column of the spinal cord and RVLM (43). It also has been proposed that the RVLM not only receives afferent inputs from other brain sites but also sends projections to several other nuclei in the medulla and forebrain, including hypothalamic regions (17). Thus, although xylazine has been shown to stimulate alpha 2-receptor mechanisms in the PVN (5), the concurrent activation of alpha 2-receptor pathways in the RVLM may act synergistically to inhibit vasopressin release from the PVN of the hypothalamus and consequently evoke diuresis.

In summary, we have previously demonstrated that the intravenous infusion of the alpha 2-agonist xylazine produces a marked increase in renal excretory function in ketamine-anesthetized rats (7). The results of the present study demonstrate that the enhanced renal excretory responses produced by xylazine are markedly reduced by the bilateral microinjection of yohimbine into the RVLM. Thus stimulation of alpha 2-receptor mechanisms in the RVLM contributes to the diuresis and natriuresis produced by xylazine. In the RVLM, xylazine affects the renal excretion of sodium, but not water, exclusively by a renal nerve-dependent pathway that involves suppression of renal sympathetic nerve activity. This premise is supported by the observation that the microinjection of yohimbine into the RVLM reversed the renal sympathoinhibitory response to intravenous xylazine infusion in intact rats, and the natriuretic, but not diuretic, response to yohimbine was abolished in RDNX. These findings suggest that alpha 2-receptors in the RVLM are contained in a neural circuit (inhibitory) that involves the renal sympathetic nerves and that this pathway is particularly involved in the renal handling of sodium. On the other hand, activation of alpha 2-receptors in the RVLM can increase urine flow rate by an alternative CNS pathway, potentially by influencing the secretion of vasopressin from the PVN of the hypothalamus (5).


    ACKNOWLEDGEMENTS

The authors thank L. A. Dayan (Louisiana State University Health Sciences Center-New Orleans) for valuable technical assistance in studies that involved nerve recording.


    FOOTNOTES

This work was supported by grants from the Council for Science and Technology (CNPq) to A. M. Cabral and R. G. Menegaz and by the National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-43337 and DK-02605 to D. R. Kapusta.

Address for reprint requests and other correspondence: A. M. Cabral, Universidade Federal do Espirito Santo, Departamento de Ciencias Fisiologicas, Centro Biomedico, Av. Marechal Campos 1468, 29040-090 Vitoria, Espirito Santo, 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. Section 1734 solely to indicate this fact.

Received 30 May 2000; accepted in final form 8 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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