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Vol. 281, Issue 1, 48-53, 1997
Departments of Pharmacology and Therapeutics (H.D.I., D.D.S.) and Internal Medicine (D.D.S.), University of Manitoba, Winnipeg, Manitoba, Canada R3E 0W3
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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We have previously demonstrated that the osmolar and free water responses to an intrarenal infusion of clonidine could be dissociated pharmacologically into naltrexone-sensitive and prazosin-sensitive responses, respectively. These results supported the notion that two distinct alpha-2 adrenoceptor sites were mediating the effects of clonidine. The ability of prazosin to selectively block the increase in free water clearance suggested the involvement of the alpha-2b subtype. Based on the identification by others of only the alpha-2a/d and alpha-2b subtypes in the rat kidney, the osmolar response was, by deduction only, speculated but not proven to involve the alpha-2a/d subtype. To provide evidence that the alpha-2a/d subtype mediated osmolar clearance, we investigated the effects of intrarenal infusion of the selective alpha-2a/d adrenoceptor agonist guanfacine. Studies were conducted in anesthetized Sprague-Dawley rats that were unilaterally nephrectomized 7 to 10 days before the experiment. The infusion of guanfacine (3.0 nmol/kg/min) into the remaining renal artery increased urine flow without altering blood pressure or creatinine clearance. The increase in urine flow was associated with an increase in osmolar clearance but no increase in free water clearance. The effects of the alpha-2a/d adrenoceptor selective antagonist, RX-821002, on the renal actions of guanfacine were determined. RX-821002 (3.0 mg/kg) attenuated the ability of guanfacine to increase urine flow rate and osmolar clearance. Similarly to the increase in osmolar clearance observed with clonidine, the guanfacine-induced increase in osmolar clearance was attenuated by naltrexone (3.0 mg/kg) and unaltered by prazosin (0.15 mg/kg) pretreatment (i.e., naltrexone-sensitive and prazosin-insensitive). These results were consistent with the alpha-2a/d adrenoceptor subtype in the rat kidney which mediated an increase in osmolar clearance. A physiological function of this alpha-2a/d adrenoceptor subtype may therefore involve regulation of solute/sodium excretion.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Alpha-2 adrenoceptor
agonists have been reported to increase urine flow rate (Strandhoy
et al., 1982
; Gellai and Ruffolo, 1987; Stanton
et al., 1987; Blandford and Smyth, 1988, 1991) reflecting the sum of increases in osmolar and free water clearance. These renal
effects were postulated to involve two distinct sites and/or mechanisms. Clonidine, an alpha-2 adrenoceptor agonist, accordingly increased urine flow rate secondary to increases in free water and
osmolar clearance. Previous studies from our laboratory supported the
contention that two sites were involved in the renal response to
clonidine (Blandford and Smyth, 1988; 1990; 1991). Whether these
effects were caused by two sites and/or two unique alpha-2 adrenoceptors has not been delineated. We recently demonstrated that
these two sites mediating the osmolar and free water effects of
clonidine could be dissociated pharmacologically. The increase in
osmolar clearance was naltrexone sensitive (opioid receptor antagonist), whereas the increase in free water clearance was prazosin
sensitive (Intengan and Smyth, 1996). The observation that these
disparate effects could be selectively antagonized further supported
the possibility of two alpha-2 adrenoceptor subtypes being
involved. The specific subtypes remained unknown.
Based on the ability of prazosin to attenuate the free water effect of
clonidine, this response was tentatively attributed to the
alpha-2b adrenoceptor subtype (Intengan and Smyth, 1996). Prazosin, an alpha-1 adrenoceptor antagonist, has
selectivity for the alpha-2b subtype over other
alpha-2 subtypes (Bylund, 1985). Alpha-1
adrenoceptor agonists have been shown to have no effect on free water
clearance (Gellai and Ruffolo, 1987), which leaves the
alpha-2b subtype as a next logical possibility. The site
mediating the osmolar response to clonidine also remained unclear. The
inability of prazosin to block this response was consistent with the
alpha-2b subtype not being involved. In the rat kidney, it
was reported that only the alpha-2a/d and
alpha-2b adrenoceptor subtypes exist (Uhlén and
Wikberg, 1991a, b). This report is supported by in situ
hybridization studies which indicated a wide distribution of the mRNAs
of the alpha-2a/d and alpha-2b subtypes with very
low levels of the alpha-2c subtype (Meister et
al., 1994). We therefore hypothesized that the remaining
alpha-2 subtype in the kidney, the alpha-2a/d
subtype, mediated the osmolar response to clonidine. In support of
this, a low dose of UK-14,304, an agonist with debatable selectivity
for the alpha-2a/d subtype (Bylund and Ray-Prenger, 1989;
MacKinnon et al., 1994), selectively increased osmolar
clearance without altering base-line levels of free water clearance.
Similar to clonidine, this osmolar effect was naltrexone sensitive and
prazosin insensitive (Intengan and Smyth, 1996). However, these
findings were not considered definitive for the attribution of the
alpha-2a/d adrenoceptor subtype to this response, because
other studies have failed to detect the selectivity of UK-14,304 for
the alpha-2a/d subtype over the alpha-2b subtype
(Uhlén and Wikberg, 1991a).
In this study, we used guanfacine and RX-821002 to address the role of the alpha-2a/d adrenoceptor in the regulation of osmolar clearance. Guanfacine and RX-821002 were selected because of approximately 60- and 7-fold selectivities, respectively, for the alpha-2a/d over the alpha-2b subtype. Guanfacine was predicted to increase osmolar clearance without affecting free water clearance. Such a response was expected to be antagonized by RX-821002. Furthermore, based on previous studies with clonidine and UK-14,304, we postulated that an osmolar response to guanfacine would be attenuated by pretreatment with naltrexone but insensitive to prazosin pretreatment. The present results indicated that the alpha-2a/d adrenoceptor in the rat kidney is involved in the regulation of solute excretion. These findings may have implications in the significance of altered alpha-2a/d adrenoceptors in human hypertension and animal models of hypertension.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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Experimental preparation.
The general procedures have been
described previously (Intengan and Smyth, 1996). Male Sprague-Dawley
rats (200-225 g) were obtained from the University of Manitoba
(Charles River Breeding Stock) and cared for according to regional
animal care standards protocol. The animals were fed a standard Purina
rat chow diet with free access to tap water in cages at 22°C with a
12-hr light/dark cycle. Seven to ten days before the experiment, the
right kidney was removed under ether anesthesia via a flank
incision.
1 i.p.). Additional anesthetic was administered as
required in an i.v. bolus dose of 3.0 mg kg1. The rats
were placed on a Harvard Animal Blanket Control Unit with a rectal
thermometer probe, and the temperature was set for 37.5°C. A
tracheotomy was performed, after which the animals were allowed to
breathe spontaneously. The left carotid artery was cannulated with
PE-50 tubing and connected to a Statham pressure transducer (model
P23Dc) and a Grass model 5 polygraph for the monitoring of blood
pressure. The left jugular vein was cannulated with PE-160 tubing for
the infusion of normal saline at 97 µl min1 and
additional anesthetic as required. A left-flank incision was performed
and the remaining kidney exposed. The ureter was catheterized to
facilitate the collection of urine into preweighed vials. Urine volume
was determined gravimetrically. A 31-gauge stainless steel needle was
advanced into the renal artery for the infusion of the agonist of
interest or vehicle with a Harvard sage pump.
The preparation was allowed to stabilize for 45 min. Antagonists were
administered immediately after the beginning of (prazosin), or 15 min
into (RX-821002 and naltrexone), the stabilization period as a slow
intravenous bolus (0.2 ml) over 1 min. Immediately after the
stabilization period, a 30-min control urine collection was obtained.
After this first collection period, the intrarenal infusion (3.4 µl
min1) of agonist (guanfacine) or vehicle was initiated
and maintained for the remainder of the experiment. During this time,
two consecutive 30-min urine collections were obtained.
Drug dosage.
Preliminary experiments were conducted with
four doses of guanfacine (0.3, 1.0, 3.0 and 10.0 nmol kg1
min1, data not shown). The dose subsequently chosen (3.0 nmol kg1 min1) was a moderate dose which
elicited renal effects without blood pressure effects. The dose of
RX-821002 (3.0 mg kg1) was chosen as the lowest effective
dose. Naltrexone (3.0 mg kg1) and prazosin (0.15 mg
kg1) were administered in doses previously shown to block
the renal effects of clonidine (Intengan and Smyth, 1996).
Renal response to guanfacine; effects of RX-821002.
Animals
were randomly assigned to one of four study groups, each consisting of
six rats. Group 1, the vehicle control group, received an intrarenal
infusion of normal saline at 3.4 µl min1. Group 2 received RX-821002 (3.0 mg kg1 i.v.) alone. Group 3 received an intrarenal infusion of guanfacine (3.0 nmol
kg1 min1). Group 4 received pretreatment
with RX-821002, followed by guanfacine.
Effects of prazosin or naltrexone on the renal effects of
guanfacine.
Animals were randomly assigned to one of six study
groups, each consisting of six rats. Group 1, the vehicle control
group, received an intrarenal infusion of normal saline at 3.4 µl
min1. Groups 2 and 3 received naltrexone (3.0 mg
kg1) or prazosin (0.15 mg kg1) alone,
respectively. Group 4 received an intrarenal infusion of guanfacine
(3.0 nmol kg1 min1). Groups 5 and 6 received pretreatment with prazosin or naltrexone, respectively,
followed by an infusion of guanfacine.
Sample analysis. At the end of each experiment, a blood sample was collected through the carotid artery catheter. Dye was injected through the renal artery line to confirm proper positioning of the intrarenal needle. Creatinine levels in the urine and plasma were measured with a Beckman Creatinine 2 Analyzer. Urine and plasma osmolalities were determined with a Precision Systems Micro Osmometer. The sodium concentrations in urine and plasma were measured with a Nova Electrolyte Analyzer (13+).
Statistical analysis. Data are presented as the mean ± S.E. Data were analyzed by repeated measures of analysis of variance (drug treatment × time) with the Systat software, version 5.0. Significant interactions were further analyzed by a Tukey's Multiple Comparison Test. Significance is denoted with "*" representing P < .05 and "**" representing P < .01.
Drugs. Guanfacine (Wyeth-Ayerst), RX-821002 (Research Biochemicals International, Natick, MA), prazosin (Sigma Chemical Co., St. Louis, MO) and naltrexone (Sigma) were used in the present studies.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Preparation controls. Data from the first collection period were analyzed to determine the similarities between groups after the surgery. Base-line values of blood pressure, creatinine clearance and other parameters of interest are shown in tables 1 and 2. Base-line levels for all parameters were similar except for the lower base-line blood pressure observed for the prazosin pretreatment alone group and a slightly greater urine flow rate in the group that later received guanfacine alone. The data have therefore been presented as the difference between base-line and final collection values (that is, deltas) to highlight the magnitude of responses between groups.
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Renal response to guanfacine; effects of RX-821002.
Blood
pressure and creatinine clearance were unaltered by the intrarenal
infusion of guanfacine (selective alpha-2a/d subtype agonist) in the presence or absence of RX-821002 (selective
alpha-2a/d subtype antagonist). Similarly, RX-821002
pretreatment alone failed to alter these parameters (fig.
1).
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Effects of prazosin or naltrexone on the renal effects of
guanfacine.
Blood pressure and creatinine clearance were unaltered
by the experimental interventions (fig. 4). Intrarenal
infusion of guanfacine increased urine flow rate and sodium excretion
(fig. 5). This response was caused solely by an increase
in osmolar clearance, whereas free water clearance remained unaltered
(fig. 6). Prazosin pretreatment failed to significantly
alter the renal response to guanfacine. Naltrexone pretreatment
completely abolished the increases in urine flow rate, sodium excretion
and osmolar clearance produced by guanfacine (figs. 5 and 6). Neither
prazosin nor naltrexone pretreatment alone affected these parameters
(data not shown).
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In anesthetized rats, the increase in urine flow rate observed
after an intrarenal infusion of clonidine has been reported to be
secondary to an increase in both free water clearance and osmolar
clearance. Previous studies from our laboratory suggested that these
effects involved at least two distinct sites and/or receptors
(Blandford and Smyth, 1988, 1990,1991). Recently, we demonstrated that
the free water and osmolar effects of clonidine could be
pharmacologically dissociated into prazosin- and naltrexone-sensitive responses, respectively (Intengan and Smyth, 1996). These renal effects
of clonidine appeared to be mediated by two alpha-2
adrenoceptor sites.
In the rat kidney, radioligand binding studies have suggested the
existence of only the alpha-2a/d and alpha-2b
adrenoceptor subtypes (Uhlén and Wikberg, 1991a,b). Selective
blockade by prazosin of only the clonidine-induced increase in free
water clearance indicated that the alpha-2b subtype was
involved in the free water response (Intengan and Smyth, 1996). By
deduction, it was postulated that the increase in osmolar clearance
after clonidine was mediated by the alpha-2a/d adrenoceptor.
Based on the ability of UK-14,304 to selectively increase osmolar
clearance (Intengan and Smyth, 1996) and the suggestion that UK-14,304
was slightly selective for the alpha-2a/d adrenoceptor
subtype (Bylund and Ray-Prenger, 1989; MacKinnon et al.,
1994), we hypothesized that the naltrexone-sensitive increase in
osmolar clearance was mediated by the alpha-2a/d
adrenoceptor subtype. However, although UK-14,304 has been speculated
to be alpha-2a/d selective, it has also been reported that
this drug is nonselective between the alpha-2a/d and
alpha-2b adrenoceptor subtypes (Uhlén and Wikberg, 1991a).
This study provides pharmacological evidence that stimulation of the
alpha-2a/d adrenoceptor subtype in the rat kidney will increase osmolar clearance. Guanfacine has been reported to be a
selective alpha-2a/d adrenoceptor agonist with 60-fold
greater affinity for the alpha-2a/d than the
alpha-2b subtype (Uhlén and Wikberg, 1991a). The
intrarenal administration of guanfacine in anesthetized rats
significantly increased urine flow by increasing osmolar clearance. No
effect on free water clearance was observed after guanfacine. Alone,
this observation suggested that the alpha-2a/d adrenoceptor
was involved in modulating osmolar clearance. In addition, the osmolar
response to guanfacine was attenuated by pretreatment with RX-821002,
an alpha-2 adrenoceptor agonist with approximately 7-fold
selectivity for the alpha-2a/d over the alpha-2b subtype (Uhlen and Wikberg, 1991a). The ability of RX-821002 to attenuate the osmolar response to guanfacine provided an additional line of evidence that the changes in osmolar clearance may be mediated
by the alpha-2a/d subtype.
We also investigated the similarity of the osmolar response to
guanfacine with that of clonidine and UK-14,304. The osmolar responses
to clonidine and UK-14,304 were attenuated by naltrexone and unaffected
by prazosin (Intengan and Smyth, 1996). In this study, we found that
the osmolar response to guanfacine was also naltrexone sensitive and
prazosin insensitive. This indicated that the sites mediating the
osmolar responses to the alpha-2 adrenoceptor agonists,
guanfacine, clonidine and UK-14,304, were conceivably the same site;
that is, the alpha-2a/d adrenoceptor subtype.
The observation that the increase in sodium excretion produced by guanfacine in the study with naltrexone/prazosin reached statistical significance, but not in the study with RX-821002, was confounding. These increases were in fact of similar magnitude (9.4 ± 1.1 and 10.0 ± 0.8 µEq/min, respectively). This effect may have been masked by the higher increase in sodium excretion in the control group of the guanfacine/RX-821002 study (6.4 ± 2.0 µEq/min) versus that of the guanfacine/naltrexone/prazosin study (2.5 ± 0.9 µEq/min). The elevated sodium excretion in the control group of the guanfacine/RX-821002 study can be attributed to two experiments which, in our experience, were much higher than normal.
The finding that the alpha-2a/d adrenoceptor subtype
mediates osmolar clearance has very strong implications with respect to
hypertension. Alterations in the alpha-2a/d adrenoceptor
gene have been linked to hypertension in various strains of genetically hypertensive rats, including the spontaneously hypertensive rat (Chun
et al., 1991; Pettinger et al., 1991) and the
Sabra salt-sensitive rat (Le Jossec et al., 1995). A second
allele for the alpha-2a/d adrenoceptor gene has been
identified as well in humans (Hoehe et al., 1988) with
changes of this subtype being correlated to hypertension in humans
(Lockette et al., 1995). In patients with essential
hypertension or children of hypertensive parents, platelet alpha-2a adrenoceptor densities were elevated as compared
with those with no history of hypertension (Fritschka et
al., 1987; Michel et al., 1989). This
"hypertensive" allele was also associated with diminished sodium
excretion induced by immersion in thermal neutral water (Freeman
et al., 1995). The present investigation established a
natriuretic function for the alpha-2a/d subtype. Although
diminished sodium excretion has been associated with an alteration
(second allele) of the alpha-2a/d subtype gene, such renal
and genetic alterations have also correlated with hypertension. Thus,
further investigation of the renal alpha-2a/d adrenoceptor is required in that any functional abnormalities may be involved in the
onset of human essential hypertension.
In conclusion, previous studies in our laboratory have led to the hypothesis that renal alpha-2a/d adrenoceptors mediate osmolar clearance. Convincing evidence to support this hypothesis was not available until the current data. The selective alpha-2a/d adrenoceptor agonist, guanfacine, increased the urine flow rate by increasing osmolar clearance without affecting free water clearance. This effect was blocked by the selective alpha-2a/d antagonist, RX-821002. The ability of naltrexone and the inability of prazosin to block the osmolar effect of guanfacine was identical with the blockade of the actions of clonidine and UK-14,304. These data implicated the alpha-2a/d adrenoceptor subtype in mediating osmolar clearance. The present findings describe a novel physiological function for the alpha-2a/d adrenoceptor subtype in the rat kidney which may involve regulation of solute/sodium excretion. The identification of this function in normal rats will allow the determination of the potential significance of altered regulation of this receptor in genetic models of hypertension.
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Footnotes |
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Accepted for publication December 13, 1996.
Received for publication May 29, 1996.
1 This work was supported by the Medical Research Council of Canada.
2 Recipient of a Canadian Hypertension Society/Pfizer/Medical Research Council of Canada Graduate Studentship.
3 Recipient of a Scientist Award from the Medical Research Council of Canada.
Send reprint requests to: Donald D. Smyth, Ph.D., Department of Pharmacology & Therapeutics, Faculty of Medicine, University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba, Canada. R3E 0W3.
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Abbreviations |
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RX, RX-821002; GF, guanfacine; PZ, prazosin; NX, naltrexone.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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2-adrenoceptor sites.
Br. J. Pharmacol.
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