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Evidence from binding studies for β1-adrenoceptors associated with glomeruli isolated from rat kidney
Life Sciences, Vol. 33, pp. 87-94 Printed in the U.S.A.
Pergamon Press
EVIDENCE FROM BINDING S~JDIES FOR BI-ADRENOCEPTORS ASSOCIATED WITH GLOMERULI ISOLATED FROM RAT KIDNEY Grant A. McPherson and Roger J. Summers University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospial, Heidelberg, V i c t o r i a , 3084, Australia (Received in final form April 20, 1983) Summary The B adrenoceptor antagonist radioligand [3H] dihydroalprenolol (DHA) has been used to characterise B adrenoceptors in membranes prepared from rat renal glomeruli. Association of the ligand was rapid and had reached equilibrium within 10 rains at 37°C. Dissociation occurred in
two d i s t i n c t phases, a rapidly dissociating phase (low a f f i n i t y s i t e ) and a slowly dissociating phase (hiqh a f f i n i t y s i t e ) . The KD value for the hiqh a f f i n i t y site calculated from the kinetic experiments was 0,8 nM. Saturation analysis of binding gave comparable values for KD (1.77 riM) and demonstrated that membranes from qlon~ruli had four times the density of binding sites measured in renal cortex, In all saturation studies H i l l coefficients were not s i q n i f i c a n t l y different from unity. Bindinq was stereoselective with respect to the (-) isomers of isoprenaline and propranolol and the potency of the selective displacinq aqents betaxolol (B1 adrenoceptors) and ICI 118,551 (B 2 adrenoceptors) indicated that the receptors are of the BI subtype. Radioliqands have been useful for the characterization, l o c a l i z a t i o n , and quantitation of m-adrenoceptors in kidney ( I - 6 ) . While there are many reports which describe the binding of radioliqands to ~-adrenoceptors in a wide variety of other tissues there have been r e l a t i v e l y few in kidney and none where the binding has bee9 localised to p a r t i c u l a r s t r ~ u r e s . The Badrenoceptor antagonists I~ ~ [°H]dihydroalprenolol (DHA), [~t~l] cyanopindolol (CYP) and [ L I] hydroxybenzylpindolol (HYP) and have been used to label stereoselective binding sites in rat kidney membrane preparations which have the molecular characteristics of B-adrenoceptors (7,8). Although i t was suggested that the B adrenoceptors in rat kidney membrane preparations are of the BI suhtype (7,8) more recent evidence suggests that both B1 and B2 subtypes are present (9). B-Adrenoceptors have been shown to have a number of functions in the kidney. Renin secretion from the kidney is enhanced by B-adrenoceptor agonists and decreased by B-adrenoceptor antagonists (10-13) by actinq either on the sympathetic nerve terminals or directly on the juxta qlomerular cells (14). In many species includinq man the B-adrenoceptors controlling renin release appear to be of the Bl - subtype (for references see 12). Fluid and electrolyte balance is also-influenced by Badrenoceptors since aqonists are known to increase f l u i d reabsorption (15} and affect tubular sodium transport (16). B-Adrenoceptor stimulated 0024-3205/83 $3.00 + o00 Copyright (c) 1983 Pergamon Press Ltd.
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adenylate cyclase a c t i v i t y has been measured in seqments of isolated renal tubules and was observed in d i s t a l tubules, c o l l e c t i n g duct and segments of the loop of Henle (17). The present study~has been carried out with the followinq objectives in mind. To use ( - ) [aH]DHA as a molecular probe to examine whether Radrenoceptors are associated with qlomeruli isolated from rat kidney, to ascertain what proportion of bindinq t h i s tissue contributes to that observed in the whole renal cortex and to characterize the 6-adrenoceptor subtype(s) involved. Methods Preparation of Glomeruli: In each experiment ten Spraque-Oawley rats (200250 g) of e i t h e r sex were k i l l e d by a blow to the head. The kidneys were removed through a midline i n c i s i o n , cleared of f a t and connective t i s s u e , and placed on ice. The renal cortex was dissected from the rest of the kidney at the cortico-medullary border using a scalpel blade. The pooled c o r t i c a l t i s s u e was forced through nylon mesh (212 um diameter) which disrupted most of the tubular structures but l e f t qlomeruli i n t a c t . The minced t i s s u e was suspended in 50 ml ice-cold normal saline (NaCI 0.9% w/v) and 5 ml of t h i s suspension taken as the control ( F I ) . The rest was centrifuged at 100 q for 1 min in a MSE Mistral 4 l_ centrifuqe. A 5 ml aliquot of the supernatant was collected to represent the 'qlomerulus free' f r a c t i o n (F2). The p e l l e t was resuspended in 50 ml of normal saline (4°C) and gently passed through 149 um nylon mesh. This retained undisrupted clumps of cortex but allowed qlomeruli to pass through (rat glomeruli diameter approximately 120 um). Material l e f t on top of the mesh was discarded. The suspension was then recentrifuged and washed in normal saline a f u r t h e r three times before pouring through a ~5 um nylon mesh which retained the glomeruli. Microscopic examination of the preparation (F3) showed glomeruli with r e l a t i v e l y l i t t l e tubular contamination. Each f r a c t i o n (FI, F2, F3) was made up to 20 ml with normal saline and homogenised in an U l t r a t u r r a x TR50 tissue homogeniser (30 sec. f u l l speed). The suspension was then centrifuged at 45,000 g for i0 min in an IEC B20A centrifuge. The p e l l e t mass was determined and the p e l l e t resuspended in 20 volumes of ice-cold Krebs phosphate b u f f e r (containing in mM NaCI, 119; KCI, 4.8; MgS04, 1.2; NaH2P04, i0; CaCI2, 1.27; pH 7.4) and recentrifuged. A f i n a l membrane suspension was made by resuspending the p e l l e t in 50 volumes of Krebs phosphate b u f f e r . The protein concentration of each f r a c t i o n was determined (18) using BSA as the standard. Mean protein concentrations were 0.64, 0.82 and 0.91 mg/ml f o r F3, F? and F1 respect i vel y. Saturation Studies: One ml aliquots of membrane suspension were added to an equal volume of Kreb§ phosphate buffer containing EDTA ( 0 . I mM) and increasing amounts of [°H]dihydroalprenolol ([aH]DHA - f i n a l concentration 0.05 - 5 nM). At each concentration of radioligand, non-specific bindinq was determined using ( - ) i s o p r e n a l i n e (~OI) uM). Membranes were incubated f o r 30 rain at 37% and all determinations were performed in duplicate. At liqand concentrations between 0.1 and 5 nM the percentage of specific to t o t a l binding decreased from 63 to 36 in FI, 54.9 to 23 in F2 and 76.2 to 49.7 in F3 membrane f r a c t i o n s . Competition Studies: Competition studies were performed to assess the a ~ i l i t y of s e l e c t i v e 6-adrenoceptor agonists and antagonists to displace [~H]DHA binding from membranes prepared from a p u r i f i e d glomerulus preparation (F3). 0.5 ml aliquots of membrane suspension were combined with an equal volume of Krebs phosphate b u f f e r containing I nM of [aH]DHA
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81-adrenoceptors in Rat Renal Glomeruli
with or without 6-8 concentrations of displacing drug. Me~ranes were incubated for 30 rain at 37% before f i l t r a t i o n and washing Association and Dissociation of ~3H]DHA: The association rate of [3H]DHA to membranes prepared from the F3 fraction was determined at 0.5, 1, 2, 4, 6, 8, 10 and 20 rain in duplicate samples. Non-specific binding was determined in duplicate at each time point using 200 ~M (-) isoprenaline. Dissociation rates were determined by adding 200 uM (-) isoprenaline to samples after incubation to equilibrium (30 rain) and continuinq the incubation for 0.5, I , 2, 4, 6, 8, 10 and 20 min before rapid f i l t r a t i o n and washing. The components of the dissociation curve were separated using the polyexponential curve f i t t i n g programme, ESTRIP (19). Sample Counting and Calculation of Results: After incubation, bound and free ligand were separated by f i l t r a t i o n through Whatman GF/B f i l t e r s and washed with 3 x 5 ml aliquots of the ice cold buffer. Filters were l e f t under partial vacuum for five minutes, transferred to scintillation vials and the radioactivity eluted by 3 ml of 2-methoxyethanol followed by 10 ml of the s c i n t i l l a t i o n cocktail (PPO, 0.4%; POPOP, 0.01% w/v in toluene). Samples were counted at ~45% efficiency in a Searle Delta 300 liquid s c i n t i l l a t i o n counter. P r e l i m i n a r y estimates of binding constants were obtained f o r s a t u r a t i o n data by Scatchard and H i l l a n a l y s i s and f o r drug displacement experiments using an i t e r a t i v e curve f i t t i n g programme and the Cheng and Prusoff equation (20) as p r e v i o u s l y described (6, 21). The n o n - l i n e a r curve f i t t i n g programme 'LIGAND' (22) was used to obtain f i n a l parameter est i mat es.
Drugs and Chemicals: (-) [3H]Dihydroalprenolol (Spec. Act. 41.8 Ci/mmol New England Nuclear); (-) and (+) isoprenaline bitartrate (Sigma and Sterling Winthrop); (-)propranolol hydrochloride, (+)propranolol hydrochloride, atenolol, practolol, ICI 118,551 hydrochloride (Imperial Chemical Industries); betaxolol (SL 75212 Synthelabo). All other chemicals were of analytical grade. Results Kinetics of [3H]DHA BindiQ9 in Membranes from Rat Isolated Glomeruli The association of [oI4]DHA to sites in membranes prepared from rat qlomeruli was rapid and reversible. Binding had reached 50% of its final BOUND(dpm/1000) B
FIG. 1 Association of [3H]DHA to membranes prepared from rat renal glomeruli in a reDresentati ve experiment. M~mbranes were incubated with
i
15
TIMEIMin)
i
30
[~H] DHA (~ 1 nM) f o r 1-30 min at 37°C. The points shown are the means of d u p l i c a t e dete rmi nat i o ns.
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value within I min of the start of incubation and reached plateau levels within 4 tR 10. mins as shown in Figure 1. The association rate constant K1 (2.48 x 10° M-I min-I) was derived from the equation KI = (Kobs - ._ I)/([3H]DHA conc.) where Kobs is the slope of the plot In (Beq/(Beq-Bt)) vs. time, where B~n is the specific bindinq at equilibrium and Bt binding at time t , taken fPom the computed line of best f i t . [3H]DHA dissociated from its binding site at two different rates as shown in Fiqure 2. The rate constant (K_ I) determined by the polyexponential curve f i t t i n g programme (19) fo~ the high a f f i n i t y s i t e (slowly dissociating phase) was 0.2 ± 0.03 mi~ - I and for the low a f f i n i t y s i t e ( f a s t dissociating phase) 7.7 ± 2.2 rain- . The K0 (dissociation rate constant) calculated from the equation KD = K 1/KI values of 0.80 nM for the high a f f i n i t y s i t e and 31 nM f o r the low a f f i n i t y s i t e . % BOUNO 100
FIG. 2
30
i
5 TIME(Min)
i
10
Dissociation of [3H]DHA from membranes prepared from rat renal glomeruli in a representati ve experiment. M~mbranes were incubated with [~H]DHA (~ 1 riM) for 30 rain at 37°C. Dissociation of the liqand is shown at various times a f t e r addition of 200 uM (-) isoprenaline. The points shown are the means of duplicate determination and the line is the computed line of best f i t (19).
Binding of [3H]DHA to Tissue Fractions From Rat Kidney Cortex Three fractions were prepared from rat renal cortex, F1 (whole cortex), F2 (~raction with qlomeruli removed) and F3 (qlomerulus enriched fraction). [ H]DHA bound with hiqh a f f i n i t y to sites present in each of the three regions. Analys~s of data by 'LIGAND' indicated that over the concentration range used [~H]DHA was binding to a single population of sites in each f r a c t i o n , and t h i s is also shown by the l i n e a r i t y of the Scatchard plots (Figure 3). The apparent dissociation constants (KD) obtained in each region 1.58 ± 0.19, 1.28 ± 0.07 and 1.77 ± 0.04 nM for FI, F2 and F3 were not s i g n i f i c a n t l y d i f f e r e n t from each other (n=3). They also showed good agreement with the value obtained f o r the high a f f i n i t y s i t e (0.8 nM) in the kinetic experiments. There were however marked differences in the density of binding sites obtained i~ each f r a c t i o n which were 74.6 ± 3.9, 43.2 ± 10.3 and 280 _+ 24 fmoles mq-~ protein f o r FI, F2 and F3 respectively. The decrease in bindinq sites in F2 (most qlomeruli removed) and the increase in F3 (qlomeruli enriched) were s i g n i f i c a n t l y (P < 0.02 and P < 0.005 respectively~ d i f f e r e n t from control (n=3, paired t - t e s t ) . The H i l l c o e f f i c i e n t s in all three fractions were not s i g n i f i c a n t l y d i f f e r e n t from
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Bl-adrenoceptors in Rat Renal Glomeruli
unity, indicating a lack of cooperati~ity in binding. These studies indicate a marked enrichment of (-) [OH]DHA binding sites in membranes prepared from the glomerulus enriched f r a c t i o n . BIF
0:12
\\
FIG. 3 Scatchard plots obtained by addition of increasing amounts of [~H]DHA (0.05-5 nM) to membranes prepared from renal cortex (o), cortex with qlomeruli removed (A) or glomeruli (e). Incubation was for 30 rain at 37°C and non specific binding was defined by (-) isoprenaline (200 uM).
\ 00~
i
i
IO0
200
3OO
Bound { fmol rncj-4prot )
Bi ndi nq Recovery The total binding possible in sieved whole cortex {FI) could be calculated from the density of binding sites and the total protein Dresent before i s o l a t i o n of the glomeruli. The amount of protein recovered in the F2 f r a c t i o n (glomeruli free) was 65.0 +_ 6.5% and that in the F3 f r a c t i o n (glomeruli) 8.0 _+ 1.7% of that in the s t a r t i n g material. The total binding in F2 membranes was 36.7 ± 8.8% and in F3 membranes 30.7 ± 8.7% of that in the s t a r t i n g material. No correction has been made f o r the 27% of protein discarded during the i n i t i a l sieving procedures or with the subsequent washes. Thus in the F3 f r a c t i o n some 30.7% of the binding was to 8.0% of the total protein. Charac~erisation of [3H]DHA Bindin$ in Membranes from Isolated Glomeruli [ HI DHA binding was displaced by drugs known to act at B adrenoceptors. All compgunds tested were capable of completely displacing all s p e c i f i c a l l y bound [JH]DHA and the rank order of potency f o r a series of antagonists was (-) propranolol > betaxolol > ICI 118,551 > (+) propranolol > atenolol > practolol (Table I ) . Kn values f o r each antaqonist were obtained using computer a s s i s t e d - i t e r a t i v e curve f i t t i n g (22). In all cases the minimum residual variance was obtained by assuming a single s i t e model although some deviation from ideal behaviour was noted at high concentrations of competitor probably related to the influence of the low a f f i n i t y s i t e observed in the dissociation studies. (-) Isoprenaline also displaced with a Ki of 84 nM a l b e i t with a low pseudo H i l l c o e f f i c i e n t (0.63). S t e r e o - s e l e c t i v i t y was shown to both aoonist and antagonist competitors with the (+) isomers of isoprenaline and propranolol being 14 and 90 times less effective than t h e i r corresponding (-) isomers. _Drugs acting on other receptor systems were without significant effect on [3H] DHA binding. These included phentolamine and rauwolscine (~adrenoceptors) ; atropi ne and pempidi ne (acetylcholi ne receptors) ; parqyli ne (MAO i nhibitor) ; dopamine and d-butaclamol (dopamine receptors) ; serotonin, histamine and the opiate antagonist naloxone. All these agents had Ki values in excess of 10 uM.
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Table I Displacement of [3H] (-) DHA Binding from Membranes Prepared from Rat Isolated Renal Glomeruli by F~ Adrenoceptor Antagonists.
DRUG
KD nM
(-) propranolol betaxolol ICI 118,551 (+) propranolol atenolol practolol
2.65 7.81 llO 240 310 622
_+ 0.27 _+ 0.12 _+ 36 _+ 72 _+ I I 0 ± 84
Displacement curves were obtained using #-8 concentrations of displacing druq and approximately I nM [°H] (-) DHA. Values qiven are the mean ~ s.e.m, f o r 3-4 experiments, conducted in duplicate. K0 values were obtained usinq 'LIGAND' (22). Best f i t s (residual variance range 3-F~9) were obtained by adopting a single s i t e model. Discussion The results from t h i s study suqqest that [3H]DHA bindinq is markedly enriched in membranes prepared from rat isolated qlomeruli compared to cortex. A preparation similar to that used here has been previously characterized both morphologically and in terms of renin content (23). Morphologically three types of glomeruli are seen and most lack Bowmans capsule. Glomeruli with no apparent attachments were most common, then qlomeruli with a single a r t e r i o l a r attachment and f i n a l l y and least commonly, qlomeruli with both a r t e r i o l e s attached. Microscopical examination of the qlomeruli obtained in the present study confirmed these observations. Only 14% of the renin present in renal cortex is recovered in the qlomeruli (23) i n d i c a t i n g e i t h e r that only 14% of the qlomeruli retain renin containing cells or that all renin containing cells are present but r e t a i n only 14% of t h e i r o r i g i n a l content. Most probably the proportion of qlomeruli with renin containing c e l l s s t i l l attached would f a l l between these two extremes. There are severa~ cell types w i t h i n the qlomerulus which could be responsible for the [°H]DHA binding including the c a p i l l a r i e s and the JGA. The B-adrenoceptors associated with the JGA a l t e r renin release. Stimulation of the sympathetic nerve supply to the kidney increases renin release (I0, 12) and at least part of t h i s response is due to stimulation of intrarenal 8-adrenoceptors by noradrenaline released from the nerves. In isolated perfused rat kidney (24), renal slices (25) or isolated glomeruli (23), noradrenaline, adrenaline and isoprenaline increase the rate of renin release and t h i s e f f e c t is blocked s t e r e o s e l e c t i v e l y hy propranolol (24, 25). The evidence suggests that these adrenoceptors are of the (31-subtype in rat (12, 13, 26, 27), dog (28, 29) cat (30), and human ( f o r ~eferences see 12). Radioliqands have been used in only ~ few studies to characterise 8adrenoceptors in rat kidney (7, 8, 9). [JH]DHA binding was s t e r e o s e l e c t i v e l y displaced by propranolol, isoprenaline, adrenaline and
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B1-adrenoceptors in Rat Renal Glomeruli
noradrenaline (7, 8) and i t was claimed that these adrenoceptors are of the BI type and localized to kidney tubular cell membranes (7). In membranes prepared from whole rat kidney the use of selective antagonists allows both B and B~ adrenoceptors to be characterised and in competition studies blst f i t ~ are obtained usinq a two site model (9). The present studies confirm the presence of B adrenoceptors in the kidney and indicate that in the rat a significant proportion of these are associated with g~omeruli. The kinetic studies indicated that both high and low a f f i n i t y [aH]DHA binding sites are present but these were not seen in the saturation studies where relativeIv low (< 5 nM) concentrations of liqand were used. The low a f f i n i t y sites probably represent binding associated with the lipophilic character of the ligand (34). The use of selective antagonists to characterise the B adrenoceptor subtypes present indicated that the receptor in glomerular membranes is of the B1 subtype. This conclusion is based f i r s t l y on the observation that the best computer curve f i t s were obtained assuming a single site model even for highly selective displacers and secondly~on the relative potency of a series of antagonists in displacing [JH]DHA binding. The f i r s t point can be challenged on the basis that owing to the small amounts of membrane material recovered only 6-8 points were used to construct displacement curves but the low a f f i n i t y of IC1118,551 (KD 110 nM) and the high a f f i n i t y of betaxolol (KF) 7.8 nM) strongly suggest that the receptor is of the BI subtype. I t is likely that at least some of these receptors control renin release. The KD values for the antagonists (-) propranolol, betaxolol, atenolol and practolol were similar to those found at el-receptors in guinea pig lunq and l e f t ventricle (31). Betaxolol ~as been shown to be some 100 times less potent at ~p-adrenoceptors (32). Althouqh thg Bp selective antagonist IC1118,551 was ~ relatively potent displacer of [~H]t)HA binding in qlomerular membranes the KD value of 110 nM is considerably less than aqainst B2adrenoceptors in cerebral microvessels (Culvenor and Jarrott, personal communication) or rat erythrocytes (32). This potency ratio is also observed in pharmacoloqical studies (33~. I t is also clear that the B]adrenoceptors in rat kidney are not confined to tubular membranes since a~ much as 30% of total hindinq in kidney cortex was to the glomerular fraction. The tubular membranes as prepared in previous studies (7) may well have contained significant glomerular contamination since no attempt was made to remove these organelles durinq preparation. In conclusion, therefore, [3H]DHA binding is markedly enriched in membranes from rat isolated glomeruli. Competition studies with selective agents suggest that the binding site has recognition characteristics of a Bl-adrenoceptor. At least a proportion of these receptors are probably B1adrenoceptors known from pharmacological studies to be directly associate~ with the JGA and controlling renin release. Ac k now I ed 9ement S
The authors wish to thank the National Health and Medical Research Council of Australia of which R.J.S. is a Senior Research Fellow for a grant-in-aid, ICI Ltd for gifts of propranolol isomers and IC1118,551, and Synthelabo for a q i f t of betaxolol. Refere nces I. 2. 3.
B. JARROTT and R.J. SUMMERS,Br. J. Pharmacol., 64, 418-419P (1978). B. JARROTT, W.J. L(IUIS and R.J. SUMMERS,B r i t . J. Pharmacol. 65, 663670 (1979). D.C. U'PRICHARD and S.H. SNYDER, Life Sci. 2_99, 79-88 (1979).
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4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
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W. SCOTT YOUNG 111 and M.J. KUHAR, Eur. J. Phamac., 6_7_7,493-495 (1980). G.A. McPHERSON and R.J. SUMMERS, Biochem. Pharmac., 3]_1, 583-587 (1982). G.A. McPHERSON and R.J. SUMMERS, Br. J. Pharmacol., 7_]_7, 177-184 (1982). S. GAVENDO, S. KAPULER, I . SERBAN, A. IAINA, E. BEN-DAVID and H. ELIAHOU, Kidney I n t . , 1_7_7,764-770 (1980). E.A. WOODCOCKand C.I. JOHNSTON, Biochim. Biophys. Acta., 631, 317-326 (1980). M.D. SNAVELY, H.J. MOTULSKY, E. MOUSTAFFA, L.C. MAHAN and P.A. INSEL., Circ. Res., 5__I_I,504-513 (1982). J.A. JOHNSON, J.O., DAVIS, R.W. GOTSWALL, T.E. LOHMEIER, J.L. DAVIS, B. BRAVERMANand G.E. TEMPLE, Am. J. Physiol., 230, 410-418 (1976). M.S. TAHER, L.G. MclAIN, K.M. McDONALD and R.Wo SCHRIER, J. Clin. Invest., 57, 459-465 (1976). T.K. KEET~ and W.B. CAMPBELL, Pharmacol. Rev., 32, 81-227 (1980). H.F. OATES, L.M. STOKER, J.C. MONAGHANand G.S. STOKES, Archs.lnt. Pharmacodyn., 234, 205-213 (1978). I.A. REID, B.J. MORRIS and W.F. GANONG, Ann. Rev. Physiol., 4(], 337410 (1978). E. BELLO-REUSS, Am. J. Physiol., 23__88,F347-F352 (1980). G.F. DI BONA, In New Trends in Arterial Hypertension. Inserm Symposium No. 17, Ed. M. Worcel. (1981). F. MOREL, Am. J. Physiol., 240, F159-164 (1981). O.H. LOWRY, N.J. ROSEBROUGFF;-'D-.L. FARR and R.J. RANDALL, J. Biol. Chem., 193 , 265-275 (1951). R.D. BROWN and J.E. MANNO, J. Pharmaceutical Sci., 6_7_7,1687-1691 (1978). Y. CHENG and W.H. PRUSOFF, Biochem. Pharmacol., 22, 3099-3108 (1973). G.A. McPHERSON, Comput. Prog. Biomed. in press. P.J. MUNSONand D. RODBARD, Anal. Biochem., 197, 220-239, (1980). B.J. MORRIS, R.L. NIXON and C.I. JOHNSTON, Clin. Exp. Pharmacol. Physiol., 3, 37-47 (1976). R. VANDONGEN,W.S. PEART and G.W. BOYD, Circ. Res., 3_22, 290-296 (1973). M. WEINBERGER, W. AOI and C. GRIM, Circ. Res., 37, 318-324 (1975). E. DESAULLES, F. MIESCH and J. SCHWARTZ, Dr. J.~harmacol., 6__3, 421425 (1978). G. HAEUSLER, Naunyn-Schmiedeberq's Arc. Pharmacol., 302, R52 (1978). N. HIMORI, A. IZUMI and T. ISHIMORI, Am. J. Physiol., 238, F387-F393 (1980). U. KOPP, M. AURELL, I.M. NILSSON and I3. ABLAD, Pfluqers Arch., 4387, 107-113 (1980). E.J. JOHNS, Dr. J. Pharmacol., 73, 749-754 (1981). G. ENGEL, D. HOYER, R. BERTHOLD and H. WAGNER, Naunyn-Schmiedberq's Arch. Pharmacol., 317, 277-285 (1981). K. DICKINSON, A. RICHARDSON and S.R. NAHORSKI, MoI. Pharmacol., 19, 194-204 (1981). S.R. O'DONNELL and J.C. WANSTALL, Life Sci., 27, 671-677 (1980). E.M. DAX and J.S. PARTILLA, MoI. Pharmacol., 2_~, 5-7 (1982).
Pergamon Press
EVIDENCE FROM BINDING S~JDIES FOR BI-ADRENOCEPTORS ASSOCIATED WITH GLOMERULI ISOLATED FROM RAT KIDNEY Grant A. McPherson and Roger J. Summers University of Melbourne, Clinical Pharmacology and Therapeutics Unit, Austin Hospial, Heidelberg, V i c t o r i a , 3084, Australia (Received in final form April 20, 1983) Summary The B adrenoceptor antagonist radioligand [3H] dihydroalprenolol (DHA) has been used to characterise B adrenoceptors in membranes prepared from rat renal glomeruli. Association of the ligand was rapid and had reached equilibrium within 10 rains at 37°C. Dissociation occurred in
two d i s t i n c t phases, a rapidly dissociating phase (low a f f i n i t y s i t e ) and a slowly dissociating phase (hiqh a f f i n i t y s i t e ) . The KD value for the hiqh a f f i n i t y site calculated from the kinetic experiments was 0,8 nM. Saturation analysis of binding gave comparable values for KD (1.77 riM) and demonstrated that membranes from qlon~ruli had four times the density of binding sites measured in renal cortex, In all saturation studies H i l l coefficients were not s i q n i f i c a n t l y different from unity. Bindinq was stereoselective with respect to the (-) isomers of isoprenaline and propranolol and the potency of the selective displacinq aqents betaxolol (B1 adrenoceptors) and ICI 118,551 (B 2 adrenoceptors) indicated that the receptors are of the BI subtype. Radioliqands have been useful for the characterization, l o c a l i z a t i o n , and quantitation of m-adrenoceptors in kidney ( I - 6 ) . While there are many reports which describe the binding of radioliqands to ~-adrenoceptors in a wide variety of other tissues there have been r e l a t i v e l y few in kidney and none where the binding has bee9 localised to p a r t i c u l a r s t r ~ u r e s . The Badrenoceptor antagonists I~ ~ [°H]dihydroalprenolol (DHA), [~t~l] cyanopindolol (CYP) and [ L I] hydroxybenzylpindolol (HYP) and have been used to label stereoselective binding sites in rat kidney membrane preparations which have the molecular characteristics of B-adrenoceptors (7,8). Although i t was suggested that the B adrenoceptors in rat kidney membrane preparations are of the BI suhtype (7,8) more recent evidence suggests that both B1 and B2 subtypes are present (9). B-Adrenoceptors have been shown to have a number of functions in the kidney. Renin secretion from the kidney is enhanced by B-adrenoceptor agonists and decreased by B-adrenoceptor antagonists (10-13) by actinq either on the sympathetic nerve terminals or directly on the juxta qlomerular cells (14). In many species includinq man the B-adrenoceptors controlling renin release appear to be of the Bl - subtype (for references see 12). Fluid and electrolyte balance is also-influenced by Badrenoceptors since aqonists are known to increase f l u i d reabsorption (15} and affect tubular sodium transport (16). B-Adrenoceptor stimulated 0024-3205/83 $3.00 + o00 Copyright (c) 1983 Pergamon Press Ltd.
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adenylate cyclase a c t i v i t y has been measured in seqments of isolated renal tubules and was observed in d i s t a l tubules, c o l l e c t i n g duct and segments of the loop of Henle (17). The present study~has been carried out with the followinq objectives in mind. To use ( - ) [aH]DHA as a molecular probe to examine whether Radrenoceptors are associated with qlomeruli isolated from rat kidney, to ascertain what proportion of bindinq t h i s tissue contributes to that observed in the whole renal cortex and to characterize the 6-adrenoceptor subtype(s) involved. Methods Preparation of Glomeruli: In each experiment ten Spraque-Oawley rats (200250 g) of e i t h e r sex were k i l l e d by a blow to the head. The kidneys were removed through a midline i n c i s i o n , cleared of f a t and connective t i s s u e , and placed on ice. The renal cortex was dissected from the rest of the kidney at the cortico-medullary border using a scalpel blade. The pooled c o r t i c a l t i s s u e was forced through nylon mesh (212 um diameter) which disrupted most of the tubular structures but l e f t qlomeruli i n t a c t . The minced t i s s u e was suspended in 50 ml ice-cold normal saline (NaCI 0.9% w/v) and 5 ml of t h i s suspension taken as the control ( F I ) . The rest was centrifuged at 100 q for 1 min in a MSE Mistral 4 l_ centrifuqe. A 5 ml aliquot of the supernatant was collected to represent the 'qlomerulus free' f r a c t i o n (F2). The p e l l e t was resuspended in 50 ml of normal saline (4°C) and gently passed through 149 um nylon mesh. This retained undisrupted clumps of cortex but allowed qlomeruli to pass through (rat glomeruli diameter approximately 120 um). Material l e f t on top of the mesh was discarded. The suspension was then recentrifuged and washed in normal saline a f u r t h e r three times before pouring through a ~5 um nylon mesh which retained the glomeruli. Microscopic examination of the preparation (F3) showed glomeruli with r e l a t i v e l y l i t t l e tubular contamination. Each f r a c t i o n (FI, F2, F3) was made up to 20 ml with normal saline and homogenised in an U l t r a t u r r a x TR50 tissue homogeniser (30 sec. f u l l speed). The suspension was then centrifuged at 45,000 g for i0 min in an IEC B20A centrifuge. The p e l l e t mass was determined and the p e l l e t resuspended in 20 volumes of ice-cold Krebs phosphate b u f f e r (containing in mM NaCI, 119; KCI, 4.8; MgS04, 1.2; NaH2P04, i0; CaCI2, 1.27; pH 7.4) and recentrifuged. A f i n a l membrane suspension was made by resuspending the p e l l e t in 50 volumes of Krebs phosphate b u f f e r . The protein concentration of each f r a c t i o n was determined (18) using BSA as the standard. Mean protein concentrations were 0.64, 0.82 and 0.91 mg/ml f o r F3, F? and F1 respect i vel y. Saturation Studies: One ml aliquots of membrane suspension were added to an equal volume of Kreb§ phosphate buffer containing EDTA ( 0 . I mM) and increasing amounts of [°H]dihydroalprenolol ([aH]DHA - f i n a l concentration 0.05 - 5 nM). At each concentration of radioligand, non-specific bindinq was determined using ( - ) i s o p r e n a l i n e (~OI) uM). Membranes were incubated f o r 30 rain at 37% and all determinations were performed in duplicate. At liqand concentrations between 0.1 and 5 nM the percentage of specific to t o t a l binding decreased from 63 to 36 in FI, 54.9 to 23 in F2 and 76.2 to 49.7 in F3 membrane f r a c t i o n s . Competition Studies: Competition studies were performed to assess the a ~ i l i t y of s e l e c t i v e 6-adrenoceptor agonists and antagonists to displace [~H]DHA binding from membranes prepared from a p u r i f i e d glomerulus preparation (F3). 0.5 ml aliquots of membrane suspension were combined with an equal volume of Krebs phosphate b u f f e r containing I nM of [aH]DHA
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81-adrenoceptors in Rat Renal Glomeruli
with or without 6-8 concentrations of displacing drug. Me~ranes were incubated for 30 rain at 37% before f i l t r a t i o n and washing Association and Dissociation of ~3H]DHA: The association rate of [3H]DHA to membranes prepared from the F3 fraction was determined at 0.5, 1, 2, 4, 6, 8, 10 and 20 rain in duplicate samples. Non-specific binding was determined in duplicate at each time point using 200 ~M (-) isoprenaline. Dissociation rates were determined by adding 200 uM (-) isoprenaline to samples after incubation to equilibrium (30 rain) and continuinq the incubation for 0.5, I , 2, 4, 6, 8, 10 and 20 min before rapid f i l t r a t i o n and washing. The components of the dissociation curve were separated using the polyexponential curve f i t t i n g programme, ESTRIP (19). Sample Counting and Calculation of Results: After incubation, bound and free ligand were separated by f i l t r a t i o n through Whatman GF/B f i l t e r s and washed with 3 x 5 ml aliquots of the ice cold buffer. Filters were l e f t under partial vacuum for five minutes, transferred to scintillation vials and the radioactivity eluted by 3 ml of 2-methoxyethanol followed by 10 ml of the s c i n t i l l a t i o n cocktail (PPO, 0.4%; POPOP, 0.01% w/v in toluene). Samples were counted at ~45% efficiency in a Searle Delta 300 liquid s c i n t i l l a t i o n counter. P r e l i m i n a r y estimates of binding constants were obtained f o r s a t u r a t i o n data by Scatchard and H i l l a n a l y s i s and f o r drug displacement experiments using an i t e r a t i v e curve f i t t i n g programme and the Cheng and Prusoff equation (20) as p r e v i o u s l y described (6, 21). The n o n - l i n e a r curve f i t t i n g programme 'LIGAND' (22) was used to obtain f i n a l parameter est i mat es.
Drugs and Chemicals: (-) [3H]Dihydroalprenolol (Spec. Act. 41.8 Ci/mmol New England Nuclear); (-) and (+) isoprenaline bitartrate (Sigma and Sterling Winthrop); (-)propranolol hydrochloride, (+)propranolol hydrochloride, atenolol, practolol, ICI 118,551 hydrochloride (Imperial Chemical Industries); betaxolol (SL 75212 Synthelabo). All other chemicals were of analytical grade. Results Kinetics of [3H]DHA BindiQ9 in Membranes from Rat Isolated Glomeruli The association of [oI4]DHA to sites in membranes prepared from rat qlomeruli was rapid and reversible. Binding had reached 50% of its final BOUND(dpm/1000) B
FIG. 1 Association of [3H]DHA to membranes prepared from rat renal glomeruli in a reDresentati ve experiment. M~mbranes were incubated with
i
15
TIMEIMin)
i
30
[~H] DHA (~ 1 nM) f o r 1-30 min at 37°C. The points shown are the means of d u p l i c a t e dete rmi nat i o ns.
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value within I min of the start of incubation and reached plateau levels within 4 tR 10. mins as shown in Figure 1. The association rate constant K1 (2.48 x 10° M-I min-I) was derived from the equation KI = (Kobs - ._ I)/([3H]DHA conc.) where Kobs is the slope of the plot In (Beq/(Beq-Bt)) vs. time, where B~n is the specific bindinq at equilibrium and Bt binding at time t , taken fPom the computed line of best f i t . [3H]DHA dissociated from its binding site at two different rates as shown in Fiqure 2. The rate constant (K_ I) determined by the polyexponential curve f i t t i n g programme (19) fo~ the high a f f i n i t y s i t e (slowly dissociating phase) was 0.2 ± 0.03 mi~ - I and for the low a f f i n i t y s i t e ( f a s t dissociating phase) 7.7 ± 2.2 rain- . The K0 (dissociation rate constant) calculated from the equation KD = K 1/KI values of 0.80 nM for the high a f f i n i t y s i t e and 31 nM f o r the low a f f i n i t y s i t e . % BOUNO 100
FIG. 2
30
i
5 TIME(Min)
i
10
Dissociation of [3H]DHA from membranes prepared from rat renal glomeruli in a representati ve experiment. M~mbranes were incubated with [~H]DHA (~ 1 riM) for 30 rain at 37°C. Dissociation of the liqand is shown at various times a f t e r addition of 200 uM (-) isoprenaline. The points shown are the means of duplicate determination and the line is the computed line of best f i t (19).
Binding of [3H]DHA to Tissue Fractions From Rat Kidney Cortex Three fractions were prepared from rat renal cortex, F1 (whole cortex), F2 (~raction with qlomeruli removed) and F3 (qlomerulus enriched fraction). [ H]DHA bound with hiqh a f f i n i t y to sites present in each of the three regions. Analys~s of data by 'LIGAND' indicated that over the concentration range used [~H]DHA was binding to a single population of sites in each f r a c t i o n , and t h i s is also shown by the l i n e a r i t y of the Scatchard plots (Figure 3). The apparent dissociation constants (KD) obtained in each region 1.58 ± 0.19, 1.28 ± 0.07 and 1.77 ± 0.04 nM for FI, F2 and F3 were not s i g n i f i c a n t l y d i f f e r e n t from each other (n=3). They also showed good agreement with the value obtained f o r the high a f f i n i t y s i t e (0.8 nM) in the kinetic experiments. There were however marked differences in the density of binding sites obtained i~ each f r a c t i o n which were 74.6 ± 3.9, 43.2 ± 10.3 and 280 _+ 24 fmoles mq-~ protein f o r FI, F2 and F3 respectively. The decrease in bindinq sites in F2 (most qlomeruli removed) and the increase in F3 (qlomeruli enriched) were s i g n i f i c a n t l y (P < 0.02 and P < 0.005 respectively~ d i f f e r e n t from control (n=3, paired t - t e s t ) . The H i l l c o e f f i c i e n t s in all three fractions were not s i g n i f i c a n t l y d i f f e r e n t from
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Bl-adrenoceptors in Rat Renal Glomeruli
unity, indicating a lack of cooperati~ity in binding. These studies indicate a marked enrichment of (-) [OH]DHA binding sites in membranes prepared from the glomerulus enriched f r a c t i o n . BIF
0:12
\\
FIG. 3 Scatchard plots obtained by addition of increasing amounts of [~H]DHA (0.05-5 nM) to membranes prepared from renal cortex (o), cortex with qlomeruli removed (A) or glomeruli (e). Incubation was for 30 rain at 37°C and non specific binding was defined by (-) isoprenaline (200 uM).
\ 00~
i
i
IO0
200
3OO
Bound { fmol rncj-4prot )
Bi ndi nq Recovery The total binding possible in sieved whole cortex {FI) could be calculated from the density of binding sites and the total protein Dresent before i s o l a t i o n of the glomeruli. The amount of protein recovered in the F2 f r a c t i o n (glomeruli free) was 65.0 +_ 6.5% and that in the F3 f r a c t i o n (glomeruli) 8.0 _+ 1.7% of that in the s t a r t i n g material. The total binding in F2 membranes was 36.7 ± 8.8% and in F3 membranes 30.7 ± 8.7% of that in the s t a r t i n g material. No correction has been made f o r the 27% of protein discarded during the i n i t i a l sieving procedures or with the subsequent washes. Thus in the F3 f r a c t i o n some 30.7% of the binding was to 8.0% of the total protein. Charac~erisation of [3H]DHA Bindin$ in Membranes from Isolated Glomeruli [ HI DHA binding was displaced by drugs known to act at B adrenoceptors. All compgunds tested were capable of completely displacing all s p e c i f i c a l l y bound [JH]DHA and the rank order of potency f o r a series of antagonists was (-) propranolol > betaxolol > ICI 118,551 > (+) propranolol > atenolol > practolol (Table I ) . Kn values f o r each antaqonist were obtained using computer a s s i s t e d - i t e r a t i v e curve f i t t i n g (22). In all cases the minimum residual variance was obtained by assuming a single s i t e model although some deviation from ideal behaviour was noted at high concentrations of competitor probably related to the influence of the low a f f i n i t y s i t e observed in the dissociation studies. (-) Isoprenaline also displaced with a Ki of 84 nM a l b e i t with a low pseudo H i l l c o e f f i c i e n t (0.63). S t e r e o - s e l e c t i v i t y was shown to both aoonist and antagonist competitors with the (+) isomers of isoprenaline and propranolol being 14 and 90 times less effective than t h e i r corresponding (-) isomers. _Drugs acting on other receptor systems were without significant effect on [3H] DHA binding. These included phentolamine and rauwolscine (~adrenoceptors) ; atropi ne and pempidi ne (acetylcholi ne receptors) ; parqyli ne (MAO i nhibitor) ; dopamine and d-butaclamol (dopamine receptors) ; serotonin, histamine and the opiate antagonist naloxone. All these agents had Ki values in excess of 10 uM.
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Table I Displacement of [3H] (-) DHA Binding from Membranes Prepared from Rat Isolated Renal Glomeruli by F~ Adrenoceptor Antagonists.
DRUG
KD nM
(-) propranolol betaxolol ICI 118,551 (+) propranolol atenolol practolol
2.65 7.81 llO 240 310 622
_+ 0.27 _+ 0.12 _+ 36 _+ 72 _+ I I 0 ± 84
Displacement curves were obtained using #-8 concentrations of displacing druq and approximately I nM [°H] (-) DHA. Values qiven are the mean ~ s.e.m, f o r 3-4 experiments, conducted in duplicate. K0 values were obtained usinq 'LIGAND' (22). Best f i t s (residual variance range 3-F~9) were obtained by adopting a single s i t e model. Discussion The results from t h i s study suqqest that [3H]DHA bindinq is markedly enriched in membranes prepared from rat isolated qlomeruli compared to cortex. A preparation similar to that used here has been previously characterized both morphologically and in terms of renin content (23). Morphologically three types of glomeruli are seen and most lack Bowmans capsule. Glomeruli with no apparent attachments were most common, then qlomeruli with a single a r t e r i o l a r attachment and f i n a l l y and least commonly, qlomeruli with both a r t e r i o l e s attached. Microscopical examination of the qlomeruli obtained in the present study confirmed these observations. Only 14% of the renin present in renal cortex is recovered in the qlomeruli (23) i n d i c a t i n g e i t h e r that only 14% of the qlomeruli retain renin containing cells or that all renin containing cells are present but r e t a i n only 14% of t h e i r o r i g i n a l content. Most probably the proportion of qlomeruli with renin containing c e l l s s t i l l attached would f a l l between these two extremes. There are severa~ cell types w i t h i n the qlomerulus which could be responsible for the [°H]DHA binding including the c a p i l l a r i e s and the JGA. The B-adrenoceptors associated with the JGA a l t e r renin release. Stimulation of the sympathetic nerve supply to the kidney increases renin release (I0, 12) and at least part of t h i s response is due to stimulation of intrarenal 8-adrenoceptors by noradrenaline released from the nerves. In isolated perfused rat kidney (24), renal slices (25) or isolated glomeruli (23), noradrenaline, adrenaline and isoprenaline increase the rate of renin release and t h i s e f f e c t is blocked s t e r e o s e l e c t i v e l y hy propranolol (24, 25). The evidence suggests that these adrenoceptors are of the (31-subtype in rat (12, 13, 26, 27), dog (28, 29) cat (30), and human ( f o r ~eferences see 12). Radioliqands have been used in only ~ few studies to characterise 8adrenoceptors in rat kidney (7, 8, 9). [JH]DHA binding was s t e r e o s e l e c t i v e l y displaced by propranolol, isoprenaline, adrenaline and
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B1-adrenoceptors in Rat Renal Glomeruli
noradrenaline (7, 8) and i t was claimed that these adrenoceptors are of the BI type and localized to kidney tubular cell membranes (7). In membranes prepared from whole rat kidney the use of selective antagonists allows both B and B~ adrenoceptors to be characterised and in competition studies blst f i t ~ are obtained usinq a two site model (9). The present studies confirm the presence of B adrenoceptors in the kidney and indicate that in the rat a significant proportion of these are associated with g~omeruli. The kinetic studies indicated that both high and low a f f i n i t y [aH]DHA binding sites are present but these were not seen in the saturation studies where relativeIv low (< 5 nM) concentrations of liqand were used. The low a f f i n i t y sites probably represent binding associated with the lipophilic character of the ligand (34). The use of selective antagonists to characterise the B adrenoceptor subtypes present indicated that the receptor in glomerular membranes is of the B1 subtype. This conclusion is based f i r s t l y on the observation that the best computer curve f i t s were obtained assuming a single site model even for highly selective displacers and secondly~on the relative potency of a series of antagonists in displacing [JH]DHA binding. The f i r s t point can be challenged on the basis that owing to the small amounts of membrane material recovered only 6-8 points were used to construct displacement curves but the low a f f i n i t y of IC1118,551 (KD 110 nM) and the high a f f i n i t y of betaxolol (KF) 7.8 nM) strongly suggest that the receptor is of the BI subtype. I t is likely that at least some of these receptors control renin release. The KD values for the antagonists (-) propranolol, betaxolol, atenolol and practolol were similar to those found at el-receptors in guinea pig lunq and l e f t ventricle (31). Betaxolol ~as been shown to be some 100 times less potent at ~p-adrenoceptors (32). Althouqh thg Bp selective antagonist IC1118,551 was ~ relatively potent displacer of [~H]t)HA binding in qlomerular membranes the KD value of 110 nM is considerably less than aqainst B2adrenoceptors in cerebral microvessels (Culvenor and Jarrott, personal communication) or rat erythrocytes (32). This potency ratio is also observed in pharmacoloqical studies (33~. I t is also clear that the B]adrenoceptors in rat kidney are not confined to tubular membranes since a~ much as 30% of total hindinq in kidney cortex was to the glomerular fraction. The tubular membranes as prepared in previous studies (7) may well have contained significant glomerular contamination since no attempt was made to remove these organelles durinq preparation. In conclusion, therefore, [3H]DHA binding is markedly enriched in membranes from rat isolated glomeruli. Competition studies with selective agents suggest that the binding site has recognition characteristics of a Bl-adrenoceptor. At least a proportion of these receptors are probably B1adrenoceptors known from pharmacological studies to be directly associate~ with the JGA and controlling renin release. Ac k now I ed 9ement S
The authors wish to thank the National Health and Medical Research Council of Australia of which R.J.S. is a Senior Research Fellow for a grant-in-aid, ICI Ltd for gifts of propranolol isomers and IC1118,551, and Synthelabo for a q i f t of betaxolol. Refere nces I. 2. 3.
B. JARROTT and R.J. SUMMERS,Br. J. Pharmacol., 64, 418-419P (1978). B. JARROTT, W.J. L(IUIS and R.J. SUMMERS,B r i t . J. Pharmacol. 65, 663670 (1979). D.C. U'PRICHARD and S.H. SNYDER, Life Sci. 2_99, 79-88 (1979).
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W. SCOTT YOUNG 111 and M.J. KUHAR, Eur. J. Phamac., 6_7_7,493-495 (1980). G.A. McPHERSON and R.J. SUMMERS, Biochem. Pharmac., 3]_1, 583-587 (1982). G.A. McPHERSON and R.J. SUMMERS, Br. J. Pharmacol., 7_]_7, 177-184 (1982). S. GAVENDO, S. KAPULER, I . SERBAN, A. IAINA, E. BEN-DAVID and H. ELIAHOU, Kidney I n t . , 1_7_7,764-770 (1980). E.A. WOODCOCKand C.I. JOHNSTON, Biochim. Biophys. Acta., 631, 317-326 (1980). M.D. SNAVELY, H.J. MOTULSKY, E. MOUSTAFFA, L.C. MAHAN and P.A. INSEL., Circ. Res., 5__I_I,504-513 (1982). J.A. JOHNSON, J.O., DAVIS, R.W. GOTSWALL, T.E. LOHMEIER, J.L. DAVIS, B. BRAVERMANand G.E. TEMPLE, Am. J. Physiol., 230, 410-418 (1976). M.S. TAHER, L.G. MclAIN, K.M. McDONALD and R.Wo SCHRIER, J. Clin. Invest., 57, 459-465 (1976). T.K. KEET~ and W.B. CAMPBELL, Pharmacol. Rev., 32, 81-227 (1980). H.F. OATES, L.M. STOKER, J.C. MONAGHANand G.S. STOKES, Archs.lnt. Pharmacodyn., 234, 205-213 (1978). I.A. REID, B.J. MORRIS and W.F. GANONG, Ann. Rev. Physiol., 4(], 337410 (1978). E. BELLO-REUSS, Am. J. Physiol., 23__88,F347-F352 (1980). G.F. DI BONA, In New Trends in Arterial Hypertension. Inserm Symposium No. 17, Ed. M. Worcel. (1981). F. MOREL, Am. J. Physiol., 240, F159-164 (1981). O.H. LOWRY, N.J. ROSEBROUGFF;-'D-.L. FARR and R.J. RANDALL, J. Biol. Chem., 193 , 265-275 (1951). R.D. BROWN and J.E. MANNO, J. Pharmaceutical Sci., 6_7_7,1687-1691 (1978). Y. CHENG and W.H. PRUSOFF, Biochem. Pharmacol., 22, 3099-3108 (1973). G.A. McPHERSON, Comput. Prog. Biomed. in press. P.J. MUNSONand D. RODBARD, Anal. Biochem., 197, 220-239, (1980). B.J. MORRIS, R.L. NIXON and C.I. JOHNSTON, Clin. Exp. Pharmacol. Physiol., 3, 37-47 (1976). R. VANDONGEN,W.S. PEART and G.W. BOYD, Circ. Res., 3_22, 290-296 (1973). M. WEINBERGER, W. AOI and C. GRIM, Circ. Res., 37, 318-324 (1975). E. DESAULLES, F. MIESCH and J. SCHWARTZ, Dr. J.~harmacol., 6__3, 421425 (1978). G. HAEUSLER, Naunyn-Schmiedeberq's Arc. Pharmacol., 302, R52 (1978). N. HIMORI, A. IZUMI and T. ISHIMORI, Am. J. Physiol., 238, F387-F393 (1980). U. KOPP, M. AURELL, I.M. NILSSON and I3. ABLAD, Pfluqers Arch., 4387, 107-113 (1980). E.J. JOHNS, Dr. J. Pharmacol., 73, 749-754 (1981). G. ENGEL, D. HOYER, R. BERTHOLD and H. WAGNER, Naunyn-Schmiedberq's Arch. Pharmacol., 317, 277-285 (1981). K. DICKINSON, A. RICHARDSON and S.R. NAHORSKI, MoI. Pharmacol., 19, 194-204 (1981). S.R. O'DONNELL and J.C. WANSTALL, Life Sci., 27, 671-677 (1980). E.M. DAX and J.S. PARTILLA, MoI. Pharmacol., 2_~, 5-7 (1982).