European Journal of Pharmacology, 175 (1990) 13-20 Elsevier
13
EJP 51094
Characterization of ct2-adrenoceptors in the rat: proximal tubule, renal membrane and whole kidney studies C a r m e n K. S t a n k o
2 Marilyne
I. V a n d e l 1, R a t n a Bose 1 a n d D o n a l d D. S m y t h 1,2
Departments of 1 Pharmacology and Therapeutics and 2 Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 Received 28 August 1989, accepted 10 October 1989
In the present study, a2-adrenoceptors have been characterized in rat renal proximal tubules which were isolated by a Percoll gradient technique. Competitive binding curves with [3H]rauwolscine (0.5 nM) and az-adrenoceptor agonists and antagonists were consistent with an az~-adrenoceptor subtype. However, the rank order of potency ( K i ) for clonidine and UK 14,304 was reversed from that reported for other tissues (clonidine, 48 nM > UK 14,304, 330 nM). This rank order was confirmed in a crude renal membrane preparation consisting of whole kidney as well as separated medullary and cortical segments. An intrarenal infusion of clonidine at 11 nmol/kg per rain resulted in a greater diuresis and natriuresis than an equimolar dose of UK 14,304 suggesting that clonidine also had a greater affinity in the collecting tubules. Further displacement studies in proximal tubules with [3H]rauwolscine and calcium channel blockers demonstrated that verapamil was the most potent (Ki, 2.3/tM), followed by diltiazem (48% displacement at 100 /zM) and then nifedipine (no displacement at 100 ~tM). These studies indicated that az-adrenoceptor in the rat proximal tubule may be of the a2B-adrenoceptor subtype. Further studies will be required to determine whether the reverse rank order of potency of clonidine and UK 14,304 is consistent with an az-adrenoceptor subtype which is different from that found in other tissue. az-Adrenoceptors; Ca 2+ channel blockers; Kidney; Proximal tubule; Clonidine; UK 14,304; Diuresis;
(Radioligand binding); (Rat)
1. Introduction a2-Adrenoceptors have been identified with radioligand binding studies in the rat kidney (Schmitz et al., 1981) and subsequently characterized in the renal cortex (Woodcock and Johnston, 1982; Snavely and Insel, 1982). In the rat and guinea pig kidney, c~2-adrenoceptors are heavily concentrated in the cortex, in association with proximal tubules (Young and Kuhar, 1980;
Correspondence to: Donald D. Smyth, Departments of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, 770 Bannatyne Ave, Winnipeg, Manitoba, Canada R3E 0W3.
Muntz et al., 1986). Activation of a2-adrenoce ptors in this tissue inhibited parathyroid hormonestimulated cAMP production (Woodcock et al., 1984; Umemura et al., 1985). a2-Adrenoceptors have been identified in a number of other nephron segments (Pettinger et al., 1987). Physiological studies suggest that a substantial number of a 2adrenoceptors exist in the area of the collecting tubules and inhibit the renal effects of vasopressin (Chabardes et al., 1984; Umemura et al., 1985). Since az-adrenoceptors have been identified in a number of nephron segments, it would be desirable to study the characteristics of this receptor in a pure tubular preparation. Recently, both a tand az-adrenoceptors have been described in a pure suspension of proximal tubules, which had
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
14
been isolated with a percoll gradient technique (Sundaresan et al., 1987). [3H]Rauwolscine bound with reversibility and saturability to c~2-adrenoceptors in this proximal tubule suspension. We have extended these findings to include a characterization of c~2-adrenoceptors in the proximal tubule, using [3 H]rauwolscine in competition with a-adrenoceptor agonists and antagonists. Since a2-adrenoceptors have been associated with calcium channels in other tissues and in a crude renal membrane preparation (Van Meel et al., 1981a; Motulsky et al., 1983; Van Zwieten and Timmermans, 1988) we have also investigated the competitive displacement of [3H]rauwolscine with calcium channel blockers in a pure tubular suspension. The reverse rank order of affinity for clonidine and U K 14,304 documented in proximal tubules and renal membranes in the present study was investigated in collecting tubules. This was evaluated by comparing the diuretic effects of clonidine and U K 14,304, an effect which has been demonstrated to be mediated in the collecting tubules by the inhibition of the tubular effects of vasopressin (Blandford and Smyth, 1988). The present findings suggest that the az-adrenoceptors found in the proximal tubule of the rat are of the azB subtype. As well, clonidine had an apparent greater affinity for the az-adrenoceptor than U K 14,304, a result which is in contrast to non-renal tissue (Grant and Scrutton, 1980; Van Meel et al., 1981b). This reverse rank order was also documented with a crude renal membrane preparation and in the collecting tubule with in vivo physiological studies of renal sodium and water excretion.
2. Materials and methods 2.1. Proximal tubule and renal membrane preparation
The experimental protocol for the isolation of renal proximal tubules has been previously documented (Vinay et al., 1981; Sundaresan et al., 1987). Briefly, three to four male Sprague-Dawley rats (250-300 g) were utilized in each experiment.
On the day of the study, the animals were anesthetized (Nembutal, BDH, 50 m g / k g i.p.). The kidneys were rapidly removed, decapsulated and placed in ice-cold Krebs-Henseleit solution (KHS) of p H 7.4. Cortical slices were chopped (McIlwain Tissue Chopper) and placed in KHSenzyme solution (15 ml KHS, 1 ml 10% bovine serum albumin (BSA), 30 mg collagenase). The mixture was gassed for 2 rain (95% 02-5% CO2), capped with a rubber stopper and shaken in a Dubnoff metabolic shaker at 100 r.p.m, for 45 min at 37 ° C. Digestion was stopped by the addition of 30 ml of ice-cold KHS. The suspension was filtered through a tea strainer and centrifuged at 60 × g for 30 s at 4°C. The pellet was resuspended in KHS, centrifuged and washed 3 additional times. The resulting pellet was then suspended in 6 times its volume in KHS (0.5% BSA) for 5 min at 4 ° C. The suspension was spun again and the resulting pellet suspended in a percoll gradient. The percoll solution (120 ml) was prepared by mixing equal parts of percoll and a solution of double strength Krebs, gassed with 95% 02-5% CO 2 for 45 min and adjusted to a pH of 7.4. The tissue-percoll mixture was spun at 27 000 × g for 30 rain at 4 ° C. Four distinct bands were seen. The fourth or bottom band was removed and resuspended in buffer K (25 mM NaHzPO4, 25 mM K2HPO4, 1 mM MgCI2). The proximal tubular fraction was centrifuged at 90 × g for 30 s and washed 3 times. The final pellet was diluted in the appropriate volume of buffer K and homogenized with a Teflon pestle. Renal membranes from whole kidney and cortical and medullary segments were prepared by the method of Schmitz et al. (1981). Kidneys from male Sprague-Dawley rats were removed, immediately frozen in a mixture of dry ice and alcohol, and stored at - 8 0 ° C. On the day of the experiment, the kidney was thawed, minced in sucrose buffer (0.25 M sucrose, 5 mM Tris-HC1, 1 mM MgC12, pH 7.40), and homogenized 4 times with a polytron for 10 s. In one series of experiments the cortex and medulla were separated prior to the mincing and homogenation. After filtering through gauze, the homogenate was centrifuged for 10 rain at 482 × g. The pellet was discarded and the supernatant centrifuged for 10 rain at
15 29 000 x g. The pellet was washed twice by resuspension in buffer K, homogenized with a Teflon pestle and recentrifuged at 29000 x g. The final pellet was resuspended and homogenized in the appropriate amount of buffer K. Protein was measured by the Biuret method (Gornall et al., 1949) with bovine serum albumin as the standard.
2.2. Radioligand binding studies [3H]Rauwolscine (74.05 Ci/mmol, NEN Research Products, DuPont, Boston MA) was used to label az-adrenoceptors. Binding assays were performed by incubating 200 btl of proximal tubule suspension or renal membrane (protein concentration, 0.5-3.0 mg/ml), 50 /~1 of [3H]rauwolscine and 50/~1 of either buffer K or drug to yield a final volume of 300/~1. The mixture was incubated at 25 ° C for 30 min. The reaction was terminated by the addition of 5 ml of ice-cold buffer and instantaneous filtration through Whatman G F / C glass fiber filters. The filters were washed with two additional 5 ml aliquots of cold buffer, placed in scintillation vials and counted in 4 ml of tritontoluene aqueous scintillation cocktail, with a counting efficiency of 40%. The radioligand and drugs were diluted to the appropriate concentration in the assay buffer. Specific binding was determined as the binding which was in excess of nonspecific binding in the presence of 10 /zM phentolamine. Competition experiments were performed with varying concentrations of cold a 1- and az-adrenoceptor agonists and antagonists in the presence of 0.5 nM [3H]rauwolscine. At least seven concentrations of cold displacing drug were tested in triplicate. Competition experiments with calcium channel blockers were performed in triplicate with at least six concentrations of cold drug and 0.5 nM [3H]rauwolscine. The stock solution of calcium channel blocker was prepared in ethyl alcohol and diluted in buffer to the appropriate assay concentrations. Corresponding total counts and nonspecific binding were determined in the same buffer solution.
2.3. In vivo renal function Sprague-Dawley rats (250 g) were unilaterally nephrectomized (right kidney) under ether anesthesia 7-10 days prior to the experiment. On the day of the experiment, rats were anesthetized (Nembutal, BDH; 50 m g / k g i.p.), tracheotomized and placed on a respirator, if necessary. Body temperature was maintained (38°C) by placing the rat on a small animal heating blanket with a rectal thermometer connected to a Harvard Animal Blanket Control Unit. A polyethylene catheter (PE60) was placed in the left carotid artery and blood pressure recorded with a Statham pressure transducer (Model P23Dc) connected to a Grass polygraph Model V. A catheter was placed in the left jugular vein for the infusion of saline. The remaining left kidney was exposed by a flank incision and the left ureter cannulated (PE50). A 30 gauge needle was inserted into the abdominal aorta and advanced into the renal artery. The needle was secured with glue. This line was used for the infusion of vehicle or study drugs (clonidine or U K 14,304). A baseline level of sodium and water excretion was established by the infusion of saline at 0.097 m l / m i n immediately following the completion of surgery and continued for the duration of the experiment. Immediately following a 45 min stabilization period, five consecutive 15 rain urine collections were obtained. Following the first urine collection, the intrarenal infusion of vehicle or study drug was started and continued for the duration of the experiment at 0.0034 m l / m i n . The clonidine and U K 14,304 were infused at 11 n m o l / k g per min. Sodium and potassium concentrations in the plasma and urine were determined with a Beckman Klina Flame Photometer. Creatinine concentrations were determined by a modified Jaff6 method with a Beckman Creatinine Analyzer Model 2. Urine osmolality was measured with a microOsmette (Precision Systems). The drugs and their sources were as follows: rauwolscine ( A t o m e r g i c C h e m i c a l s Corp., Plainview, NY); prazosin (Pfizer Canada Inc., Kirkland, Quebec); U K 14,304 (Pfizer Central Research, Sandwich, England); verapamil (Knoll AG, Eudwigshafen, FRG); diltiazem (Nordic Lab.
16
Inc., Kirkland Quebec); nifedipine, clonidine, yohimbine and phenylephrine (Sigma Chemical Company, St. Louis, MO). Analysis of competition experiments was performed with LIGAND, a computer-assisted iterative curve fitting program (Munson and Rodbard, 1980; McPherson, 1983). The in vivo renal function experiments were analyzed with a repeated measures analysis of variance. Significant interactions were analyzed further with Tukey's HSD test. All data are presented as the means + S.E.M., unless otherwise stated.
3. Results
3.1. Radioligand binding studies Competition binding studies were performed with [3H]rauwolscine (0.5 nM) and various aadrenoceptor agonists and antagonists. [3H]Rauwolscine bound with specificity to ch-adrenoceptors in the rat renal proximal tubule. The relative order of potency (Ki) for antagonists was: rauwolscine (2.1 + 0.6 nM) > yohimbine (5.6 + 1.2 nM) > prazosin (26 + 5 nM); and for agonists was: clonidine (48 + 6 n M ) > U K 14,304 (330 + 4 0 nM) > > phenylephrine (28 000 + 8 000 nM) (fig. 1). The rank order of potency of clonidine and U K 14,304 was reversed as compared to that reported for other tissues. Clonidine was 7 times more potent than U K 14,304 in the competition for
[3H]rauwolscine-labelled a2-adrenoceptor binding sites in the proximal tubule. This finding was confirmed in crude renal membranes from the rat. Clonidine was again more potent than U K 14,304 in this whole kidney tissue preparation (Ki, 49 + 11 and 278 _+ 29 nM respectively). Similar differences were observed in the cortex (K~, 66 _+ 18 and 240 + 18 nM) and medulla (K i 61 + 26 and 250_+ 28 nM) for clonidine and U K 14,304 respectively. Competition binding studies with [3H]rauwolscine and calcium channel blockers indicated that calcium channel blockers interact with ch-adrenoceptors in varying degrees (fig. 2). Verapamil was the most potent, as demonstrated by a complete displacement of [3H]rauwolscine binding, with a K i of 2.3/~M. Diltiazem at the highest concentration tested (100 /~M) displaced [3H]rauwolscine binding by only 48%. Nifedipine had no interaction with a2-adrenoceptor sites at the highest concentration tested (100/zM).
3.2 In vivo renal function The effects of an intrarenal infusion of clonidine or U K 14,304 on renal sodium and water excretion has been summarized in fig. 3. The data obtained during the third collection period (75-90 rain) is representative of the difference observed between the groups following the intrarenal drug infusions and is discussed in detail. During this collection period, blood pressure was not increased by U K 14,304 (124 + 4 mm Hg) as corn-
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Fig. 1. Displacement of [3 H]rauwolscine (0.5 nM) by a-adrenoceptor agonists and antagonists. (A) Relative potencies of antagonists, rauwolscine (O), yohimbine (o) and prazosin (A). (B) Relative potencies of agonists, clonidine (e), UK 14,304 (o) and phenylephrine (A). Each data point represents the mean of at least four experiments, each done in triplicate. 100% binding is defined as [ 3H]rauwolscine binding in the absence of displacer.
17
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Fig. 2. Displacement of [3H]rauwolscine (0.50 nM) by calcium channel blockers, verapamil (O), diltiazem (0) and nifedipine (,). Competition experiments with calcium channel blockers were performed in triplicate with at least six concentrations of cold drug. 100% binding is defined as [3 H]rauwolscine binding in the absence of displacer. The maximum dose of displacer was 100 /iM. At this concentration, diltiazem displaced [3H]rauwolscine by only 48%• Nifedipine at the highest dose tested had no interaction with [3 H]rauwolscine.
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Experimental Per~o~ {~in) Fig. 3. Effect of intrarenal infusions of saline vehicle (I) and equimolar infusions (11 nmol/kg per min) of clonidine (zx) and UK 14,304 (O) on sodium and water excretion in the anesthetized rat. Values represent the means_+S.E.M, of five to six experiments.
pared to the group receiving vehicle (117 +_ 3 mm Hg). Clonidine, however, at an equimolar infusion rate increased blood pressure (147 + 4 mm Hg) from control. Creatinine clearance was not altered by any of these experimental interventions. Urine flow, as compared to the control group (34 _+ 6 ~tl/min), was increased to a greater extent following the clonidine (210 + 15 btl/min) than the U K 14,304 (150 +_ 18 ~tl/min) infusion (fig. 3). Similarly, clonidine increased sodium excretion (18 _+ 2 # E q / m i n ) more than U K 14,304 (9 + 1/~Eq/min). Clonidine also resulted in a greater increase in free water clearance than U K 14,304 (56 +_ 24 vs. 0 _+ 8 /tl/min) as compared to the control group ( - 6 7 _+ 7/~l/min).
4. Discussion
Autoradiographic studies have localized a 2adrenoceptors in the renal cortex, especially the proximal tubule (Young and Kuhar, 1980; Stephenson and Summers, 1985; Muntz et al., 1986). Isolation of proximal tubules by a Percoll gradient technique produces a pure suspension of proximal tubules, confirmed by microscopic examination, enzyme studies and the ability of parathyroid hormone to stimulate cAMP (Vinay et al., 1981; Gesek et al., 1987; Sundaresan et al., 1987). Displacement of [3H]rauwolscine binding was used in the present study to characterize a2-adrenoceptors in this proximal tubular suspension. Clonidine had a greater affinity than U K 14,304 for [3H]rauwolscine-labelled a2-adrenoceptors in the proximal tubule. This was not secondary to the isolation procedure for proximal tubules since this reversed rank order was confirmed in a crude renal membrane preparation. The antagonists demonstrated a relative order of potency wtfich was consistent with that reported by others (Lefkowitz, 1979; Motulsky and Insel, 1982; Timmermans and Van Zwieten, 1982; Pettinger et al., 1987). Moreover, these az-adrenoce ptors displayed characteristics consistent with that of an a2B subtype, a2-Adrenoceptor subtypes have recently been described (Bylund, 1985; Bylund, 1988; Bylund et al., 1988). The a2B subtype, in the neonatal rat lung, was characterized by rauwol-
18
scine being 3 times more potent than yohimbine and prazosin having a relatively high affinity. In our tubular supension, rauwolscine had a 2.7 times greater affinity than yohimbine and prazosin had a relatively high affinity for this site (Ki, 26 nM). U K 14,304, a full ~2-adrenoceptor agonist, has been demonstrated to be more potent than the partial agonist clonidine in eliciting physiologic responses in tissues such as rabbit pulmonary artery, guinea pig ileum, rat heart and human platelet (Grant and Scrutton, 1980; Cambridge, 1981; Van Meel et al., 1981a). Radioligand binding studies in a cultured opossum cell line, which is similar to proximal tubular epithelia, demonstrated that clonidine had a slightly higher affinity than UK 14,304 for c~2-adrenoceptors with K i values of 14 and 22 nM respectively (Murphy and Bylund, 1986). In this tissue, U K 14,304 was able to inhibit parathyroid hormone stimulated cAMP production by 70%, whereas clonidine had no effect. These findings were consistent with clonidine binding to a population of c~2-adrenoceptors in the proximal tubule which were not associated with the antagonism of the parathyroid hormoneactivated adenylate cyclase. Our finding of a reverse rank order of potency for clonidine and U K 14,304 in the proximal tubule of the rat was confirmed by experiments in rat renal membranes, where the K~ values for clonidine and U K 14,304 were approximately 49 and 278 nM respectively. The order of potency for clonidine and U K 14,304 in the medullary segments, however, was unknown. In this regard, the diuretic and natriuretic effect of c~2-adrenoceptor agonists has been attributed to an inhibition of vasopressin-mediated increases in cAMP in the collecting ducts (Blandford and Smyth, 1988). Although clonidine produced a greater diuresis and natriuresis than U K 14,304, this may have been secondary to the increase in blood pressure observed with the clonidine. The ability of these agonists to decrease urine osmolality to less than 200 m O s m / k g suggested that the site of vasopressin antagonism was the medullary collecting ducts. This would indicate that free water clearance rather than urine volume and sodium excretion may give a better indication of the degree of inhibition of the renal effects of vasopressin in the collecting
ducts. Clonidine produced a significantly greater increase in free water clearance than UK 14,304 in these experiments. Thus, the renal function data were consistent with the reverse rank order of potency being maintained throughout both the medullary and cortical segments. Previous radioligand binding studies with a crude renal membrane preparation demonstrated an interaction between calcium channel blockers and renal ~2-adrenoceptors (Motulsky et al., 1983). In the present study, this interaction between calcium channel blockers and c~2-adrenoceptors was confirmed in a pure proximal tubular suspension. As documented in a crude renal membrane preparation (Motulsky et al., 1983), we found verapamil was the most potent, displacing [3H]rauwolscine with K i value of 2.3 /~M. Diltiazem, on the other hand, at the highest concentration tested (100 #M) displaced only 48% of the bound [3H]rauwolscine. Nifedipine had no effect on [3H] rauwolsince binding at concentrations as high as 100/~M. In vivo and in vitro experiments reveal that calcium channel blockers can inhibit a2-adrenoceptor-mediated functions such as vasoconstriction (Van Meel et al., 1981a; Cavero et al., 1983; Scarborough et al., 1984; Van Zwieten et al., 1985; 1986; Van Zwieten and Timmermans, 1988), clonidine induced sodium chloride absorption in the rabbit ileum (Homaidan et al., 1988) and epinephrine induced platelet aggregation (Barnathan et al., 1982). Our findings suggest that physiological studies involving the investigation of calcium channels associated with c~2-adrenoceptors in proximal tubules should not utilize calcium channel blockers of the verapamil class. Antagonsim of the physiological effects of c~2 agonists may be attributed to blockade of the calcium channel when in fact it is secondary to displacement of binding of the a 2 agonist. In summary, az-adrenoceptors in the proximal tubule of the rat have been characterized. This adrenoceptor has characteristics resembling that of an a2B-adrenoceptor subtype. Whether the relatively higher affinity of clonidine over U K 14,304 suggests that renal a2-adrenoceptors in the proximal tubule may represent another subtype, which may be differentiated by this reversed rank order of potency, remains to be determined.
19
Acknowledgements CKS is the recipient of a Sandoz Clinical Research Fellowship. DDS is the recipient of a Canadian Heart Foundation Scholarship. Supported by MRC of Canada and the Manitoba Heart Foundation.
References Barnathan, E.S., V.P. Addonizio and S.J. Shattil, 1982, Interaction of verapamil with human platelet alpha-adrenergic receptors, Am. J. Physiol. 242, H19. Blandford, D.E. and D.D. Smyth, 1988, Dose selective dissociation of water and solute excretion after alpha-2 adrenoceptor stimulation, J. Pharmacol. Exp. Ther. 247, 1181. Bylund, D.B., 1985, Heterogeneity of alpha-2 adrenergic receptors, Pharmacol. Biochem. Behav. 22, 835. Bylund, D.B., 1988, Subtypes of alpha2-adrenoceptors: pharmacological and molecular biological evidence converge, Trends Pharmacol. Sciences. 9, 356. Bylund, D.B., C. Ray-Prenger and T.J. Murphy, 1988, Alpha-2A and alpha-2B adrenergic receptor subtypes: Antagonist binding in tissues and cell lines containing only one subtype, J. Pharmacol. Exp. Ther. 245, 600. Cambridge, D., 1981, UK-14,304, a potent and selective alpha2-agonist for the characterization of alpha-adrenoceptor subtypes, European J. Pharmacol. 72, 413. Cavero, I., N. Shepperson, F. Lefevre-Borg, and S.Z. Langer, 1983, Differential inhibition of vascular smooth muscle responses to alphal- and alpha2-adrenoceptor agonists by diltiazem and verapamil, Circ. Res. 52, 69. Chabardes, D., M. Montegut, M. Imbert-Teboul and F. Morel, 1984, Inhibition of alpha2-adrenergic agonists on AVP-induced cAMP accumulation in isolated collecting tubule of the rat kidney, Mol. Cell. Endocrinol. 37, 263. Gesek, F.A., D.W. Wolff and J.W. Strandhoy, 1987, Improved separation method for rat proximal and distal renal tubules, Am. J. Physiol. 253, F358. Gornall, A.G., C. Bardawill and M. David, 1949, Determination of serum proteins by means of the biuret reaction, J. Biol. Chem. 177, 751. Grant, J.A. and M.C. Scrutton, 1980, Interaction of selective alpha-adrenoceptor agonists and antagonists with human and rabbit blood platelets, Br. J. Pharmacol. 71, 121. Homaidan, F.R., M. Donowitz, J. Wicks, S. Cusolito, M. E1 Sabban, G.A. Weiland and G.W. Sharp, 1988, Ca 2+ channel blockers interact with alpha2-adrenergic receptors in rabbit ileum, Am. J. Physiol. 254, G586. Lefkowitz, R.J., 1979, Direct binding studies of adrenergic receptors: Biochemical, physiologic, and clinical implications, Ann. Intern. Med. 91,450. McPherson, G.A., 1983, A practical computer-based approach to the analysis of radioligand binding experiments, Comput. Programs Biomed. 17, 107.
Motulsky, H.J. and P.A. Insel, 1982, Adrenergic receptors in man, N. Engl. J. Med. 307, 18. Motulsky, H.J., M.D. Snavely, R.J. Hughes and P.A. Insel, 1983, Interaction of verapamil and other calcium channel blockers with alphal- and alpha2-adrenergic receptors, Circ. Res. 52, 226. Munson, P.J. and D. Rodbard, 1980, LIGAND: A versatile computerized approach for characterization of ligand-binding systems, Anal. Biochem. 107, 220. Muntz, K.H., L. Meyer, S. Gadol and T.A. Calianos, 1986, Alpha-2 adrenergic receptor localization in the rat heart and kidney using autoradiography and tritiated rauwolscine, J. Pharmacol. Exp. Ther. 236, 542. Murphy, T.J. and D.B. Bylund, 1988, Characterization of alpha-2 adrenergic receptors in the OK cell, and opossum kidney cell line, J. Pharmacol. Exp. Ther. 244, 571. Pettinger, W.A., S. Umemura, D.D. Smyth and W.B. Jeffries, 1987, Renal alpha2-adrenoceptors and the adenylate cyclase-cAMP system: biochemical and physiological interactions, Am. J. Physiol. 252, F199. Scarborough, N.L. and G.O. Carrier, 1984, Nifedipine and alpha adrenoceptors in rat aorta. 1. Role of extracellular calcium in alpha-1 and alpha-2 adrenoceptor-mediated contraction, J. Pharmacol. Exp. Ther. 231, 597. Schmitz, J.M., R. Graham, A. Sagalowsky and W.A. Pettinger, 1981, Renal alpha-1 and alpha-2 adrenergic receptors: Biochemical and pharmacological correlations, J. Pharmacol. Exp. Ther. 219, 400. Snavely, M.D. and P.A. Insel, 1982, Characterization of alpha-adrenergic receptor subtypes in the rat renal cortex, Mol. Pharmacol. 22, 532. Stephenson, J.A. and R.J. Summers, 1985, Light microscopic autoradiography of the distribution of 3H-rauwolscine binding to alpha2-adrenoceptors in rat kidney, European J. Pharmacol. 116, 271. Sundaresan, P.R., T. Fortin and S.L. Kelvie, 1987, Alpha- and beta-adrenergic receptors in proximal tubules of rat kidney, Am. J. Physiol. 253, F848. Timmermans, P.B. and P.A. Van Zwieten, 1982, Alpha2 adrenoceptors: Classification, localization, mechanisms, and targets for drugs, J. Med. Chem. 25, 1389. Umemura, S., D. Marver, D.D. Smyth and W.A. Pettinger, 1985, Alpha2-adrenoceptors and cellular cAMP levels in single nephron segments from the rat, Am. J. Physiol. 249, F28. Van Meel, J.C., A. De Jonge, H. Kalkman, B. Wilffert, P. Timmermans and P. Van Zwieten, 1981a, Organic and inorganic calcium antagonists reduce vasoconstriction in vivo mediated by postsynaptic alpha2-adrenoceptors, Naunyn-Schmiedeb. Arch. Pharmacol. 316, 288. Van Meel, J.C., A. De Jonge, P.B. Timmermans and P.A. Van Zwieten, 1981b, Selectivity of some alpha adrenoceptor agonists for peripheral alpha-1 and alpha-2 adrenoceptors in the normotensive rat, J. Pharmcol. Exp. Ther. 219, 760. Van Zwieten, P.A., P. Timmermans, M. Thoolen, B. Wilffert and A. De Jonge, 1985, Calcium dependency of vasoconstriction mediated by alpha1- and alpha2-adrenoceptors, J. Cardiovasc. Pharmacol. 7, Sl13.
20 Van Zwieten, P.A., P. Timmermans, M. Thoolen, B. Wilffert and A. De Jonge, 1986, Inhibitory effect of calcium antagonist drugs on vasoconstriction induced by vascular alpha2-adrenoceptor stimulation, Am. J. Cardiol. 57, 11D. Van Zwieten, P.A. and P. Timmermans, 1988, Receptor subtypes involved in the action of calcium entry blockers, Ann. N.Y. Acad. Sci., 522, 351. Vinay, P., A. Gougoux and G. Lemieux, 1981, Isolation of a pure suspension of rat proximal tubules, Am. J. Physiol. 241, F403.
Woodcock, E.A. and C.I. Johnston, 1982, Characterization of adenylate cyclase-coupled alpha2-adrenergic receptors in rat renal cortex using 3H-yohimbine, Mol. Pharmacol. 22, 589. Woodcock, E.A., J.K. McLeod and C.I. Johnston, 1984, Demonstration of proximal tubular alpha-adrenoceptors in vivo, Clin. Exp. Pharmacol. Physiol. 11, 399. Young, W.S. and M.J. Kuhar, 1980, Alpha-2 adrenergic receptors are associated with renal proximal tubules, European J. Pharmacol. 67, 493.
13
EJP 51094
Characterization of ct2-adrenoceptors in the rat: proximal tubule, renal membrane and whole kidney studies C a r m e n K. S t a n k o
2 Marilyne
I. V a n d e l 1, R a t n a Bose 1 a n d D o n a l d D. S m y t h 1,2
Departments of 1 Pharmacology and Therapeutics and 2 Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E OW3 Received 28 August 1989, accepted 10 October 1989
In the present study, a2-adrenoceptors have been characterized in rat renal proximal tubules which were isolated by a Percoll gradient technique. Competitive binding curves with [3H]rauwolscine (0.5 nM) and az-adrenoceptor agonists and antagonists were consistent with an az~-adrenoceptor subtype. However, the rank order of potency ( K i ) for clonidine and UK 14,304 was reversed from that reported for other tissues (clonidine, 48 nM > UK 14,304, 330 nM). This rank order was confirmed in a crude renal membrane preparation consisting of whole kidney as well as separated medullary and cortical segments. An intrarenal infusion of clonidine at 11 nmol/kg per rain resulted in a greater diuresis and natriuresis than an equimolar dose of UK 14,304 suggesting that clonidine also had a greater affinity in the collecting tubules. Further displacement studies in proximal tubules with [3H]rauwolscine and calcium channel blockers demonstrated that verapamil was the most potent (Ki, 2.3/tM), followed by diltiazem (48% displacement at 100 /zM) and then nifedipine (no displacement at 100 ~tM). These studies indicated that az-adrenoceptor in the rat proximal tubule may be of the a2B-adrenoceptor subtype. Further studies will be required to determine whether the reverse rank order of potency of clonidine and UK 14,304 is consistent with an az-adrenoceptor subtype which is different from that found in other tissue. az-Adrenoceptors; Ca 2+ channel blockers; Kidney; Proximal tubule; Clonidine; UK 14,304; Diuresis;
(Radioligand binding); (Rat)
1. Introduction a2-Adrenoceptors have been identified with radioligand binding studies in the rat kidney (Schmitz et al., 1981) and subsequently characterized in the renal cortex (Woodcock and Johnston, 1982; Snavely and Insel, 1982). In the rat and guinea pig kidney, c~2-adrenoceptors are heavily concentrated in the cortex, in association with proximal tubules (Young and Kuhar, 1980;
Correspondence to: Donald D. Smyth, Departments of Pharmacology and Therapeutics, Faculty of Medicine, University of Manitoba, 770 Bannatyne Ave, Winnipeg, Manitoba, Canada R3E 0W3.
Muntz et al., 1986). Activation of a2-adrenoce ptors in this tissue inhibited parathyroid hormonestimulated cAMP production (Woodcock et al., 1984; Umemura et al., 1985). a2-Adrenoceptors have been identified in a number of other nephron segments (Pettinger et al., 1987). Physiological studies suggest that a substantial number of a 2adrenoceptors exist in the area of the collecting tubules and inhibit the renal effects of vasopressin (Chabardes et al., 1984; Umemura et al., 1985). Since az-adrenoceptors have been identified in a number of nephron segments, it would be desirable to study the characteristics of this receptor in a pure tubular preparation. Recently, both a tand az-adrenoceptors have been described in a pure suspension of proximal tubules, which had
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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been isolated with a percoll gradient technique (Sundaresan et al., 1987). [3H]Rauwolscine bound with reversibility and saturability to c~2-adrenoceptors in this proximal tubule suspension. We have extended these findings to include a characterization of c~2-adrenoceptors in the proximal tubule, using [3 H]rauwolscine in competition with a-adrenoceptor agonists and antagonists. Since a2-adrenoceptors have been associated with calcium channels in other tissues and in a crude renal membrane preparation (Van Meel et al., 1981a; Motulsky et al., 1983; Van Zwieten and Timmermans, 1988) we have also investigated the competitive displacement of [3H]rauwolscine with calcium channel blockers in a pure tubular suspension. The reverse rank order of affinity for clonidine and U K 14,304 documented in proximal tubules and renal membranes in the present study was investigated in collecting tubules. This was evaluated by comparing the diuretic effects of clonidine and U K 14,304, an effect which has been demonstrated to be mediated in the collecting tubules by the inhibition of the tubular effects of vasopressin (Blandford and Smyth, 1988). The present findings suggest that the az-adrenoceptors found in the proximal tubule of the rat are of the azB subtype. As well, clonidine had an apparent greater affinity for the az-adrenoceptor than U K 14,304, a result which is in contrast to non-renal tissue (Grant and Scrutton, 1980; Van Meel et al., 1981b). This reverse rank order was also documented with a crude renal membrane preparation and in the collecting tubule with in vivo physiological studies of renal sodium and water excretion.
2. Materials and methods 2.1. Proximal tubule and renal membrane preparation
The experimental protocol for the isolation of renal proximal tubules has been previously documented (Vinay et al., 1981; Sundaresan et al., 1987). Briefly, three to four male Sprague-Dawley rats (250-300 g) were utilized in each experiment.
On the day of the study, the animals were anesthetized (Nembutal, BDH, 50 m g / k g i.p.). The kidneys were rapidly removed, decapsulated and placed in ice-cold Krebs-Henseleit solution (KHS) of p H 7.4. Cortical slices were chopped (McIlwain Tissue Chopper) and placed in KHSenzyme solution (15 ml KHS, 1 ml 10% bovine serum albumin (BSA), 30 mg collagenase). The mixture was gassed for 2 rain (95% 02-5% CO2), capped with a rubber stopper and shaken in a Dubnoff metabolic shaker at 100 r.p.m, for 45 min at 37 ° C. Digestion was stopped by the addition of 30 ml of ice-cold KHS. The suspension was filtered through a tea strainer and centrifuged at 60 × g for 30 s at 4°C. The pellet was resuspended in KHS, centrifuged and washed 3 additional times. The resulting pellet was then suspended in 6 times its volume in KHS (0.5% BSA) for 5 min at 4 ° C. The suspension was spun again and the resulting pellet suspended in a percoll gradient. The percoll solution (120 ml) was prepared by mixing equal parts of percoll and a solution of double strength Krebs, gassed with 95% 02-5% CO 2 for 45 min and adjusted to a pH of 7.4. The tissue-percoll mixture was spun at 27 000 × g for 30 rain at 4 ° C. Four distinct bands were seen. The fourth or bottom band was removed and resuspended in buffer K (25 mM NaHzPO4, 25 mM K2HPO4, 1 mM MgCI2). The proximal tubular fraction was centrifuged at 90 × g for 30 s and washed 3 times. The final pellet was diluted in the appropriate volume of buffer K and homogenized with a Teflon pestle. Renal membranes from whole kidney and cortical and medullary segments were prepared by the method of Schmitz et al. (1981). Kidneys from male Sprague-Dawley rats were removed, immediately frozen in a mixture of dry ice and alcohol, and stored at - 8 0 ° C. On the day of the experiment, the kidney was thawed, minced in sucrose buffer (0.25 M sucrose, 5 mM Tris-HC1, 1 mM MgC12, pH 7.40), and homogenized 4 times with a polytron for 10 s. In one series of experiments the cortex and medulla were separated prior to the mincing and homogenation. After filtering through gauze, the homogenate was centrifuged for 10 rain at 482 × g. The pellet was discarded and the supernatant centrifuged for 10 rain at
15 29 000 x g. The pellet was washed twice by resuspension in buffer K, homogenized with a Teflon pestle and recentrifuged at 29000 x g. The final pellet was resuspended and homogenized in the appropriate amount of buffer K. Protein was measured by the Biuret method (Gornall et al., 1949) with bovine serum albumin as the standard.
2.2. Radioligand binding studies [3H]Rauwolscine (74.05 Ci/mmol, NEN Research Products, DuPont, Boston MA) was used to label az-adrenoceptors. Binding assays were performed by incubating 200 btl of proximal tubule suspension or renal membrane (protein concentration, 0.5-3.0 mg/ml), 50 /~1 of [3H]rauwolscine and 50/~1 of either buffer K or drug to yield a final volume of 300/~1. The mixture was incubated at 25 ° C for 30 min. The reaction was terminated by the addition of 5 ml of ice-cold buffer and instantaneous filtration through Whatman G F / C glass fiber filters. The filters were washed with two additional 5 ml aliquots of cold buffer, placed in scintillation vials and counted in 4 ml of tritontoluene aqueous scintillation cocktail, with a counting efficiency of 40%. The radioligand and drugs were diluted to the appropriate concentration in the assay buffer. Specific binding was determined as the binding which was in excess of nonspecific binding in the presence of 10 /zM phentolamine. Competition experiments were performed with varying concentrations of cold a 1- and az-adrenoceptor agonists and antagonists in the presence of 0.5 nM [3H]rauwolscine. At least seven concentrations of cold displacing drug were tested in triplicate. Competition experiments with calcium channel blockers were performed in triplicate with at least six concentrations of cold drug and 0.5 nM [3H]rauwolscine. The stock solution of calcium channel blocker was prepared in ethyl alcohol and diluted in buffer to the appropriate assay concentrations. Corresponding total counts and nonspecific binding were determined in the same buffer solution.
2.3. In vivo renal function Sprague-Dawley rats (250 g) were unilaterally nephrectomized (right kidney) under ether anesthesia 7-10 days prior to the experiment. On the day of the experiment, rats were anesthetized (Nembutal, BDH; 50 m g / k g i.p.), tracheotomized and placed on a respirator, if necessary. Body temperature was maintained (38°C) by placing the rat on a small animal heating blanket with a rectal thermometer connected to a Harvard Animal Blanket Control Unit. A polyethylene catheter (PE60) was placed in the left carotid artery and blood pressure recorded with a Statham pressure transducer (Model P23Dc) connected to a Grass polygraph Model V. A catheter was placed in the left jugular vein for the infusion of saline. The remaining left kidney was exposed by a flank incision and the left ureter cannulated (PE50). A 30 gauge needle was inserted into the abdominal aorta and advanced into the renal artery. The needle was secured with glue. This line was used for the infusion of vehicle or study drugs (clonidine or U K 14,304). A baseline level of sodium and water excretion was established by the infusion of saline at 0.097 m l / m i n immediately following the completion of surgery and continued for the duration of the experiment. Immediately following a 45 min stabilization period, five consecutive 15 rain urine collections were obtained. Following the first urine collection, the intrarenal infusion of vehicle or study drug was started and continued for the duration of the experiment at 0.0034 m l / m i n . The clonidine and U K 14,304 were infused at 11 n m o l / k g per min. Sodium and potassium concentrations in the plasma and urine were determined with a Beckman Klina Flame Photometer. Creatinine concentrations were determined by a modified Jaff6 method with a Beckman Creatinine Analyzer Model 2. Urine osmolality was measured with a microOsmette (Precision Systems). The drugs and their sources were as follows: rauwolscine ( A t o m e r g i c C h e m i c a l s Corp., Plainview, NY); prazosin (Pfizer Canada Inc., Kirkland, Quebec); U K 14,304 (Pfizer Central Research, Sandwich, England); verapamil (Knoll AG, Eudwigshafen, FRG); diltiazem (Nordic Lab.
16
Inc., Kirkland Quebec); nifedipine, clonidine, yohimbine and phenylephrine (Sigma Chemical Company, St. Louis, MO). Analysis of competition experiments was performed with LIGAND, a computer-assisted iterative curve fitting program (Munson and Rodbard, 1980; McPherson, 1983). The in vivo renal function experiments were analyzed with a repeated measures analysis of variance. Significant interactions were analyzed further with Tukey's HSD test. All data are presented as the means + S.E.M., unless otherwise stated.
3. Results
3.1. Radioligand binding studies Competition binding studies were performed with [3H]rauwolscine (0.5 nM) and various aadrenoceptor agonists and antagonists. [3H]Rauwolscine bound with specificity to ch-adrenoceptors in the rat renal proximal tubule. The relative order of potency (Ki) for antagonists was: rauwolscine (2.1 + 0.6 nM) > yohimbine (5.6 + 1.2 nM) > prazosin (26 + 5 nM); and for agonists was: clonidine (48 + 6 n M ) > U K 14,304 (330 + 4 0 nM) > > phenylephrine (28 000 + 8 000 nM) (fig. 1). The rank order of potency of clonidine and U K 14,304 was reversed as compared to that reported for other tissues. Clonidine was 7 times more potent than U K 14,304 in the competition for
[3H]rauwolscine-labelled a2-adrenoceptor binding sites in the proximal tubule. This finding was confirmed in crude renal membranes from the rat. Clonidine was again more potent than U K 14,304 in this whole kidney tissue preparation (Ki, 49 + 11 and 278 _+ 29 nM respectively). Similar differences were observed in the cortex (K~, 66 _+ 18 and 240 + 18 nM) and medulla (K i 61 + 26 and 250_+ 28 nM) for clonidine and U K 14,304 respectively. Competition binding studies with [3H]rauwolscine and calcium channel blockers indicated that calcium channel blockers interact with ch-adrenoceptors in varying degrees (fig. 2). Verapamil was the most potent, as demonstrated by a complete displacement of [3H]rauwolscine binding, with a K i of 2.3/~M. Diltiazem at the highest concentration tested (100 /~M) displaced [3H]rauwolscine binding by only 48%. Nifedipine had no interaction with a2-adrenoceptor sites at the highest concentration tested (100/zM).
3.2 In vivo renal function The effects of an intrarenal infusion of clonidine or U K 14,304 on renal sodium and water excretion has been summarized in fig. 3. The data obtained during the third collection period (75-90 rain) is representative of the difference observed between the groups following the intrarenal drug infusions and is discussed in detail. During this collection period, blood pressure was not increased by U K 14,304 (124 + 4 mm Hg) as corn-
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Fig. 1. Displacement of [3 H]rauwolscine (0.5 nM) by a-adrenoceptor agonists and antagonists. (A) Relative potencies of antagonists, rauwolscine (O), yohimbine (o) and prazosin (A). (B) Relative potencies of agonists, clonidine (e), UK 14,304 (o) and phenylephrine (A). Each data point represents the mean of at least four experiments, each done in triplicate. 100% binding is defined as [ 3H]rauwolscine binding in the absence of displacer.
17
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Fig. 2. Displacement of [3H]rauwolscine (0.50 nM) by calcium channel blockers, verapamil (O), diltiazem (0) and nifedipine (,). Competition experiments with calcium channel blockers were performed in triplicate with at least six concentrations of cold drug. 100% binding is defined as [3 H]rauwolscine binding in the absence of displacer. The maximum dose of displacer was 100 /iM. At this concentration, diltiazem displaced [3H]rauwolscine by only 48%• Nifedipine at the highest dose tested had no interaction with [3 H]rauwolscine.
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Experimental Per~o~ {~in) Fig. 3. Effect of intrarenal infusions of saline vehicle (I) and equimolar infusions (11 nmol/kg per min) of clonidine (zx) and UK 14,304 (O) on sodium and water excretion in the anesthetized rat. Values represent the means_+S.E.M, of five to six experiments.
pared to the group receiving vehicle (117 +_ 3 mm Hg). Clonidine, however, at an equimolar infusion rate increased blood pressure (147 + 4 mm Hg) from control. Creatinine clearance was not altered by any of these experimental interventions. Urine flow, as compared to the control group (34 _+ 6 ~tl/min), was increased to a greater extent following the clonidine (210 + 15 btl/min) than the U K 14,304 (150 +_ 18 ~tl/min) infusion (fig. 3). Similarly, clonidine increased sodium excretion (18 _+ 2 # E q / m i n ) more than U K 14,304 (9 + 1/~Eq/min). Clonidine also resulted in a greater increase in free water clearance than U K 14,304 (56 +_ 24 vs. 0 _+ 8 /tl/min) as compared to the control group ( - 6 7 _+ 7/~l/min).
4. Discussion
Autoradiographic studies have localized a 2adrenoceptors in the renal cortex, especially the proximal tubule (Young and Kuhar, 1980; Stephenson and Summers, 1985; Muntz et al., 1986). Isolation of proximal tubules by a Percoll gradient technique produces a pure suspension of proximal tubules, confirmed by microscopic examination, enzyme studies and the ability of parathyroid hormone to stimulate cAMP (Vinay et al., 1981; Gesek et al., 1987; Sundaresan et al., 1987). Displacement of [3H]rauwolscine binding was used in the present study to characterize a2-adrenoceptors in this proximal tubular suspension. Clonidine had a greater affinity than U K 14,304 for [3H]rauwolscine-labelled a2-adrenoceptors in the proximal tubule. This was not secondary to the isolation procedure for proximal tubules since this reversed rank order was confirmed in a crude renal membrane preparation. The antagonists demonstrated a relative order of potency wtfich was consistent with that reported by others (Lefkowitz, 1979; Motulsky and Insel, 1982; Timmermans and Van Zwieten, 1982; Pettinger et al., 1987). Moreover, these az-adrenoce ptors displayed characteristics consistent with that of an a2B subtype, a2-Adrenoceptor subtypes have recently been described (Bylund, 1985; Bylund, 1988; Bylund et al., 1988). The a2B subtype, in the neonatal rat lung, was characterized by rauwol-
18
scine being 3 times more potent than yohimbine and prazosin having a relatively high affinity. In our tubular supension, rauwolscine had a 2.7 times greater affinity than yohimbine and prazosin had a relatively high affinity for this site (Ki, 26 nM). U K 14,304, a full ~2-adrenoceptor agonist, has been demonstrated to be more potent than the partial agonist clonidine in eliciting physiologic responses in tissues such as rabbit pulmonary artery, guinea pig ileum, rat heart and human platelet (Grant and Scrutton, 1980; Cambridge, 1981; Van Meel et al., 1981a). Radioligand binding studies in a cultured opossum cell line, which is similar to proximal tubular epithelia, demonstrated that clonidine had a slightly higher affinity than UK 14,304 for c~2-adrenoceptors with K i values of 14 and 22 nM respectively (Murphy and Bylund, 1986). In this tissue, U K 14,304 was able to inhibit parathyroid hormone stimulated cAMP production by 70%, whereas clonidine had no effect. These findings were consistent with clonidine binding to a population of c~2-adrenoceptors in the proximal tubule which were not associated with the antagonism of the parathyroid hormoneactivated adenylate cyclase. Our finding of a reverse rank order of potency for clonidine and U K 14,304 in the proximal tubule of the rat was confirmed by experiments in rat renal membranes, where the K~ values for clonidine and U K 14,304 were approximately 49 and 278 nM respectively. The order of potency for clonidine and U K 14,304 in the medullary segments, however, was unknown. In this regard, the diuretic and natriuretic effect of c~2-adrenoceptor agonists has been attributed to an inhibition of vasopressin-mediated increases in cAMP in the collecting ducts (Blandford and Smyth, 1988). Although clonidine produced a greater diuresis and natriuresis than U K 14,304, this may have been secondary to the increase in blood pressure observed with the clonidine. The ability of these agonists to decrease urine osmolality to less than 200 m O s m / k g suggested that the site of vasopressin antagonism was the medullary collecting ducts. This would indicate that free water clearance rather than urine volume and sodium excretion may give a better indication of the degree of inhibition of the renal effects of vasopressin in the collecting
ducts. Clonidine produced a significantly greater increase in free water clearance than UK 14,304 in these experiments. Thus, the renal function data were consistent with the reverse rank order of potency being maintained throughout both the medullary and cortical segments. Previous radioligand binding studies with a crude renal membrane preparation demonstrated an interaction between calcium channel blockers and renal ~2-adrenoceptors (Motulsky et al., 1983). In the present study, this interaction between calcium channel blockers and c~2-adrenoceptors was confirmed in a pure proximal tubular suspension. As documented in a crude renal membrane preparation (Motulsky et al., 1983), we found verapamil was the most potent, displacing [3H]rauwolscine with K i value of 2.3 /~M. Diltiazem, on the other hand, at the highest concentration tested (100 #M) displaced only 48% of the bound [3H]rauwolscine. Nifedipine had no effect on [3H] rauwolsince binding at concentrations as high as 100/~M. In vivo and in vitro experiments reveal that calcium channel blockers can inhibit a2-adrenoceptor-mediated functions such as vasoconstriction (Van Meel et al., 1981a; Cavero et al., 1983; Scarborough et al., 1984; Van Zwieten et al., 1985; 1986; Van Zwieten and Timmermans, 1988), clonidine induced sodium chloride absorption in the rabbit ileum (Homaidan et al., 1988) and epinephrine induced platelet aggregation (Barnathan et al., 1982). Our findings suggest that physiological studies involving the investigation of calcium channels associated with c~2-adrenoceptors in proximal tubules should not utilize calcium channel blockers of the verapamil class. Antagonsim of the physiological effects of c~2 agonists may be attributed to blockade of the calcium channel when in fact it is secondary to displacement of binding of the a 2 agonist. In summary, az-adrenoceptors in the proximal tubule of the rat have been characterized. This adrenoceptor has characteristics resembling that of an a2B-adrenoceptor subtype. Whether the relatively higher affinity of clonidine over U K 14,304 suggests that renal a2-adrenoceptors in the proximal tubule may represent another subtype, which may be differentiated by this reversed rank order of potency, remains to be determined.
19
Acknowledgements CKS is the recipient of a Sandoz Clinical Research Fellowship. DDS is the recipient of a Canadian Heart Foundation Scholarship. Supported by MRC of Canada and the Manitoba Heart Foundation.
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20 Van Zwieten, P.A., P. Timmermans, M. Thoolen, B. Wilffert and A. De Jonge, 1986, Inhibitory effect of calcium antagonist drugs on vasoconstriction induced by vascular alpha2-adrenoceptor stimulation, Am. J. Cardiol. 57, 11D. Van Zwieten, P.A. and P. Timmermans, 1988, Receptor subtypes involved in the action of calcium entry blockers, Ann. N.Y. Acad. Sci., 522, 351. Vinay, P., A. Gougoux and G. Lemieux, 1981, Isolation of a pure suspension of rat proximal tubules, Am. J. Physiol. 241, F403.
Woodcock, E.A. and C.I. Johnston, 1982, Characterization of adenylate cyclase-coupled alpha2-adrenergic receptors in rat renal cortex using 3H-yohimbine, Mol. Pharmacol. 22, 589. Woodcock, E.A., J.K. McLeod and C.I. Johnston, 1984, Demonstration of proximal tubular alpha-adrenoceptors in vivo, Clin. Exp. Pharmacol. Physiol. 11, 399. Young, W.S. and M.J. Kuhar, 1980, Alpha-2 adrenergic receptors are associated with renal proximal tubules, European J. Pharmacol. 67, 493.
