Modulation of agonist and antagonist interactions at kidney α1-adrenoceptors by nucleotides and metal ions

European Journal of Pharmacology, 133 (1987) 165-176

165

Elsevier EJP 00615

Modulation of agonist and antagonist interactions at kidney cq-adrenoceptors by nucleotides and metal ions Paul E m s b e r g e r * a n d D a v i d C. U ' P r i c h a r d Neuroscience Program and Department of Pharmacology, Northwestern University Medical School, 303 East Chicago Avenue, Chicago, IL 60611, U.S.A.

Received 30 July 1986, revised MS received 30 September 1986, accepted 14 October 1986

In order to characterize putative high- and low-affinity states of the renal al-adrenoceptor , binding sites for the selective antagonist radioligand [ 3H]prazosin were examined in washed membranes prepared from rat renal cortex and medulla. Norepinephrine competition curves at [3H]prazosin sites were biphasic and were best fit by a two-site model. Na ÷ and GTP selectively decreased the proportion of sites exhibiting a high affinity for norepinephrine. In contrast, Mg 2+ facilitated high-affinity interactions of norepinephrine at the renal al-receptor. Guanine nucleotides and Na + increased the affinity of some antagonists ([3H]prazosin, WB-4101), but not others (phentolamine). Mg 2+ again had opposite effects. The effects of ions and nucleotides on both agonist and antagonist interactions were concentrationdependent. The order of potencies for monovalent cations (Na + > Li + >> K+), divalent cations (Mn 2+ > Mg 2+) and nucleotides (Gpp (NH)p, GTP >> GMP, ATP) were similar to those reported for cyclase-coupled receptor systems. However, unlike other divalent cations C a 2+ decreased both agonist and antagonist binding, possibly due to a Ca2+-sensitive proteinase. Receptor binding properties were similar in renal cortex and medulla. Renal al-receptor sites appear to display high- and low-affinity states with respect to agonists, and the equilibrium between these states may be modulated by guanine nucleotides and mono-' and divalent metal ions. Some antagonists appear to bind preferentially to sites with low agonist affinity, and this effect is probably independent of retained endogenous catecholamines. al-Adrenoceptor; Affinity states; [3H]Prazosin; Kidney; GTP; Na+; Mg 2÷

1. Introduction

Agonist binding affinity at many receptors coupled to adenylate cyclase, including fl- and a 2adrenoceptors, is decreased by monovalent cations and guanine nucleotides and increased by divalent cations (Hoffman and Lefkowitz, 1980). These receptors may be coupled to the catalytic moiety of adenylate cyclase by a nucleotide-binding regulatory protein (N) (Hoffman and Lefkowitz, 1980; Rodbell, 1980; Limbird, 1981). Receptor (R) and * To whom all correspondence should be addressed: Laboratory of Neurobiology, Cornell University Medical College, 411 E. 69th St., New York, NY 10021, U.S.A.

N proteins may interact to generate an R.N complex, the formation of which is necessary for signal transduction. The R.N complex may be equivalent to .a high-affinity state of the receptor with respect to agonist binding (Smith and Limbird, 1981; Michel et al., 1981). Although it was originally postulated that antagonists bind to complexed and uncomplexed forms of the receptor with equal affinity (Hoffman and Lefkowitz, 1980), it has been proposed that some antagonists may interact preferentially with the low-affinity state (Glossman and Hornung, 1980a; Salama et al., 1982; Bylund and U'Prichard, 1983; Asakura et al., 1985). Several authors have proposed modifications of the ternary

0014-2999/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

166

complex model wherein antagonists destabilize the interaction of N proteins with the receptor while agonists, in contrast, serve to stabilize this coupling. These modified models of receptor-effector coupling have been proposed for dopamine D 2 (DeLean et al., 1982; Wreggett and DeLean, 1984) and muscarinic acetylcholine (Burgisser et al., 1982) receptors, as well as a 2- (Bylund and U'Prichard, 1983; Asakura et al., 1985), and fl(Lang and Lemmer, 1985) adrenoceptors. Guanine nucleotides and monovalent cations appear to shift the equilibrium states of the receptor in favor of a low-affinity form, resulting in decreased agonist affinities and increased antagonist affinities. Divalent cations such as Mg 2+ and Mn 2+, in contrast, appear to favor high-affinity state and thus increase agonist and decrease antagonist affinities. The al-receptor is believed to be coupled to inositol lipid hydrolysis rather than to adenylate cyclase (Fain and Garcia-Sainz, 1980; Tyroler et al., 1986). The affinity of agonists or antagonists for the al-receptor would therefore not be expected to be sensitive to guanine nucleotides or mono- and divalent cations. However, recent studies have suggested that a guanine-nucleotide binding protein may couple receptors to inositol lipid hydrolysis (Litosch et al., 1985; Smith et al., 1986). If renal a~-receptors are able to form a complex with N analogous to the proposed R.N complex of cyclase-coupled receptors, then the ion and nucleotide effects observed in the a2-receptor system should also be observed for al-receptors. In support of this hypothesis, Na + has been shown to decrease the affinity of norepinephrine and other full agonists at brain a~-adrenoceptors selectively labeled with the antagonist radioligand [3H]prazosin (Glossman and Hornung, 1980b). Furthermore, guanine nucleotide decrease the affinity of epinephrine at heart al-receptor sites labeled with [3H]WB-4101 (Yamada et al., 1980). a~-Antagonist binding can also be modulated, since Na + directly increases the affinity of [125I]2-fl(4-hydroxyphenyl-ethylaminomethyl)-tetralone ([125I]HEAT) at brain al-receptors (Glossman et al., 1981). Divalent cation effects at al-adrenoce ptors have not been previously reported. By analogy to other receptor systems sensitive to guanine nucleotides and Na +, divalent cations should have

actions opposing those of the former two agents. In order to characterize putative high- and low-affinity states of the a~-adrenoceptor, the present study examined the effects of guanine nucleotides and mono- and divalent cations on agonist and antagonist interactions at al-receptor sites in renal cortex and medullary membranes. The results of this study have been previously reported in abstract form (Ernsberger and U'Prichard, 1982). 2. Materials and methods

2.1. Membrane preparation Kidneys were removed from male and female young adult Sprague-Dawley rats either under pentobarbital anesthesia (60 mg/kg) or following decapitation. Tissue obtained by either killing procedure yielded indistinguishable results in binding experiments. Kidneys were decapsulated, chilled on ice, and dissected into cortical and medullary regions. Tissue samples were minced and then homogenized using a Polytron (Brinkman; setting 6) for 30 s in 20 volumes of ice-cold 50 mM Tris-HCl buffer (pH 7.7 at 25°C) containing 5.0 mM EDTA (Tris-EDTA). Preliminary studies indicated that EDTA pretreatment enhanced specific binding of three different a-adrenoceptor ligands. Homogenates were centrifuged at 300 × g for 10 min at 4°C, the pellet was resuspended in TrisEDTA, and the supernatant and resuspended pellet were separately recentrifuged at 300 x g for 10 min. The pooled supernatants were centrifuged at 50000 × g for 10 min, and the pellet washed twice by centrifugation, once in Tris-EDTA, and again in EDTA-free Tris-HC1. The final pellet was flash-frozen in a dry ice-acetone bath, and stored at - 7 0 ° C for later use. Freezing and thawing of either whole tissue or membrane suspensions had no significant effect on binding characteristics. Tissue or membrane pellets could be stored as long as 6 months at - 7 0 ° C with no appreciable loss of binding.

2.2. [ 3H]Prazosin binding assays For [3H]prazosin binding assays, washed membrane pellets were slowly thawed, resuspended in

167

100 volumes of 50 mM Tris-HC1 buffer, and incubated at 25°C for 30 min. Equilibrium binding was reached by 20 min (data not shown). Nonspecific binding was defined by parallel incubations with 10 #M phentolamine. Assays contained 0.2-0.4 mg protein in a volume of 1.0 ml. All drugs were added in a volume of 10 #1. In catecholamine competition experiments, all samples included 0.001% ascorbic acid. Incubation was terminated by vacuum filtration over Whatman G F / B filters, which were rinsed three times with 5.0 ml ice-cold Tris-HC1 buffer. Filters were placed in glass vials, dried overnight at 40°C, covered with 4.0 ml of scintillation cocktail (Research Products International), and counted at 45% efficiency. Protein was measured by the method of Lowry et al. (1951). In studies of metal ion effects on [3H]prazosin binding, 50 mM Tris-HC1 buffer was the fundamental ionic medium and constituted the 'no ion' control. The chloride salts of all ions were used. Ion solutions were made up in distilled water and added to the assay tubes in either a 10/~1 volume (when a single concentration was used) or a 40/~1 volume (when multiple concentrations were compared). Nucleotides were dissolved in Tris-HCl buffer and added to the assay in a 10 ~1 volume.

Hofstee plots which includes an explicit model of data weighting (Zivin and Waud, 1982).

3. Results

3.1. Ion and nucleotide modulation of [3H]prazosin binding at renal a t-receptor sites

[3H]Prazosin binding to rat renal membranes was saturable and high-affinity, yielding Scatchard plots best fit by a one-site model (fig. 1). Iterative non-linear curve-fitting (Munson and Rodbard, 1980) yielded an alternative two-site fit which included a low-affinity component accounting for no more than 15% of the total binding sites, but inclusion of this second site into the analytical model did not improve the goodness-of-fit. The binding characteristics of the single-site model were similar to those previously reported for rat kidney membranes (McPherson and Summers, 1981; Schmitz et al., 1981). Specific binding defined by 10 #M phentolamine represented ap8O(3

2.3. Drugs and reagents x\O

[3 H]Prazosin (specific activity 33 Ci/mmol) was obtained as a gift from Amersham International, stored at - 2 0 ° C in a 1:1 ethanol/water mixture, and diluted prior to assay in 10 - 6 N HC1. The following drugs were generous gifts from the indicated sources: phentolamine (Ciba-Geigy, Summit, N J) and WB-4101, (Ward Blenkinsop Ltd., Wembley, England). All other drugs and chemicals were purchased commercially (Sigma Chemical Co., St. Louis, MO). 2.4. Data analysis

Data were analyzed using computerized curve fitting (Munson and Rodbard, 1980; McPherson, 1983), except when saturation and competition curves were best fit by a single site model. Monophasic data were analyzed by a linear regression of

4OO ~ en

, "-..

0

~oo 3H-pRAZ bound, fmol / mcj

200

Fig. 1. Sc~itchard plots of eqmlibrium saturation isotherms of

[3H]prazosin specific binding to rat renal cortex membranes in the absence or presence of Na +, Mg 2+ or GTP. Rat renal cortex membranes from the same preparations (0.3-0.4 mg protein suspended in 50 mM Tris-HC1 buffer) were incubated for 30 min at 25°C with various concentrations of [3H]prazosin (0.05-5.0 nM), either in the absence of added ion or nucleotide (squares, solid line), or in the presence of 100 mM Na + (circles, dashed line), or 5.0 mM Mg 2÷ (triangles, dotted line). Non-specific binding was determined in the presence of 10 /~M phentolamine, and was not significantly affected by ions and nucleotides. Each regression line represents the best fit as determined from Eadie-Hofstee plots using the method of Zivin and Wand (1982).

168

proximately 80% of the total at a concentration of [3H]prazosin close to its K o. We examined the effects of ions and nucleotides on specific binding of the antagonist radioligand [3H]prazosin (fig. 1, table 1). N a +, Mg 2÷, and G T P had little or no effect the number of [3H]prazosin sites, but selectively modified [3H] prazosin affinity. N a ÷ (100 mM) increased the affinity of [3H]prazosin for renal al-receptors, an effect similar to that previously obtained with another al-antagonist, [125I]HEAT in rat cerebral cortex (Glossman et al., 1981). In contrast, Mg 2+ (5.0 mM) decreased the affinity of [3H]prazosin. Ca 2÷ (5.0 mM), unlike other metal ions, caused a loss of nearly half of the available binding sites (table 1). At a 0.1 mM concentration, G T P did not increase [3H]prazosin affinity. In the presence of N a +, G T P still had no effect. However, in the presence of Mg 2÷, G T P did increase radioligand affinity, since the K D of [3H]prazosin was reduced by 39%. In the presence of both Mg 2+ and N a +, G T P induced a similar fall in K D (30%). Thus, N a + increased while Mg 2÷ decreased the affinity of the antagonist radioligand [3H]prazosin, while Ca 2÷ causes an apparent loss of binding sites. G T P effects were relatively modest and depended on the presence of metal ions. In order to determine whether the effects of

metal ions on cq-antagonist binding were concentration-dependent, the specific binding of a fixed concentration of [3H]prazosin (0.2 nM) was examined in the presence of increasing concentrations of metal ion. Na + increased the specific binding of [3H]prazosin to renal cortex membranes in a concentration-dependent manner (fig. 2A) with an EDs0 for Na + of approximately 10 m M and a maximum effect at 50 mM. G T P over a wide concentration range had no apparent effect on [3H]prazosin binding (fig. 2C). Both Mg 2+ and Ca 2+ decreased binding in a concentration-related manner (fig. 2B,D).

3.2. 1on and nucleotide effects on the inhibition of [SH]prazosin binding by adrenoceptor agents In order to characterize putative high- and low-affinity states of the renal al-receptor, we examined ion and nucleotide modulation of the interactions of unlabeled agonist and antagonist competitors at [3H]prazosin sites (table 2, fig. 3). The direct effects of ions and G T P on [3H]prazo_ sin binding were taken into account in the determination of affinity constants.

3.2.1. Inhibition by agonists Pseudo-Hill coefficients (nil) were approxi-

TABLE 1 Ion and nucleotide effects on renal [3H]prazosin saturation characteristics. Values represent the means of 2-9 experiments in renal cortex, and 3 experiments in renal medulla, with the S.E.M. N = number of experiments. * Differs from control (No addition) tested in parallel incubations, P < 0.05 by paired t-test. Rat renal cortex or medulla membranes were incubated with one of 4-7 concentrations of [3H]prazosin, ranging from 0.05 to 5.0 nM, for 30 min at 25°C. The incubation medium consisted of 50 mM Tris-HCl, pH 7.7, with either no addition, 100 mM Na +, 5.0 mM Mg 2÷, 0.1 mM GTP (Na salt), or 5.0 mM Ca 2+. K D and Bmax values were calculated from Eadie-Hofstee plots using the method of Zivin and Waud (1982). N

No addition Na4,100mM Mg 2+, 5.0 mM GTP, 0.1 mM Na~ and Mg 2÷ Na + and GTP Mg 2+ and GTP Mg 2+, GTP and Na + Ca2+,5.0 mM

9 6 4 3 4 3 2 2 3

Cortex

Medulla

K D (pM)

Bm~~ ( f m o l / mg protein)

K D (pM)

Bmax ( f m o l / mg protein)

251 -+ 15 180-+11 345-+69 247-+40 278+58 210-+32 210 194 165-+44

122_+ 6 118+ 9 107_+14 154+ 16 98_+11 142_+16 104 113 62-+ 4 *

176+26 116_+35 * 340+67 * 245 +_61 191+47 106-+36 *

75+ 5 66_+16 73+ 5 98 + 24 58-+ 6 * 68+15

* *

*

*

169

1.6 a

1.4

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60 [Na] mM

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Fig. 2. Dose-dependent effects of Na ÷, GTP, Mg 2+ and Ca 2÷ upon [3H]prazosin binding and its inhibition by norepinephrine. [3 H]Prazosin (0.2 nM) was incubated at 25 o C for 30 min with rat renal cortex or medulla membranes (suspended in 50 mM Tris-HCl buffer) in the presence or absence of increasing concentrations of ions or nucleotide. Agonist inhibition of binding was determined by incubating parallel samples with 1.0 ~M norepinephrine. Non-specific binding was determined in the presence of 10 /~M phentolamine, and was not affected by additions to the buffer. Filled symbols i'epresent B / B 0, or the specific binding (B) expressed as a fraction of specific binding in the absence of additions to the medium (Bo). Open symbols represent I / I 0 , or inhibition of specific binding by 1.0/~M norepinephrine (I), expressed as a fraction of the inhibition of specific binding by norepinephrine in the absence of additions to the medium (I0). I 0 was approximately 50% in all cases. The effects are shown of increasing concentrations of (A) Na ÷, renal cortex; (B) Mg 2÷, renal cortex (circles) and renal medulla (squares); (C) GTP, renal cortex; (D) Ca 2÷, renal cortex.

mately 0.5 for norepinephrine competition curves in Tris-HCl buffer alone, indicating a heterogeneous interaction of the agonist at [3H]prazosin sites in both renal cortex and medulla (table 2). Na ÷ (100 mM) decreased the apparent affinity of norepinephrine at [3 H]prazosin sites and increased the n H value, indicating a reduced heterogeneity of norepinephrine binding to the az-receptor. These effects are reflected in the steepening of norepinephrine competition curves in the presence of Na ÷ (fig. 3, panels A and B). Similar effects were observed in the presence of G T P (0.1 mM). Na ÷ and G T P effects did not appear to be additive. However, either 100 mM Na ÷ or 0.1 mM

G T P appeared to be sufficient to shift [3H]prazosin sites into a nearly homogeneous population of low-affinity sites. Na ÷ or G T P produced nearmaximal effects and the combination was not more effective. Mg 2÷ (5.0 mM) increased the apparent affinity of norepinephrine for [3H]prazosin sites in the renal cortex, but not in the medulla (table 2). This represented the only regional difference in [3 H]prazosin binding properties that could be detected. In both renal cortex and medulla, Mg 2÷ and Na ÷ effects were not additive, with the effect of Na + predominating. Iterative non-linear curve-fitting analysis (Mun-

170 TABLE 2 Ion and nucleotide effects on norepinephrine competition at [3H]prazosin binding sites. * Differs from control (No. addition) P < 0.05, paired t-test. K ] / K ] o , proportionate change in K I relative to the 'No addition' control (Klo). Values were determined by dividing the K I obtained under the various experimental conditions by the control Kj. Values are in nM and represent the mean of 4-8 experiments in the renal cortex, and 3-4 experiments in the renal medulla, with the S.E.M. Rat renal cortex or medulla membranes were incubated with 0.2 nM [3 H]prazosin and 5-11 concentrations of norepinephrine. K l values were calculated using the Cheng and Prusoff equation, utilizing the K D value determined under identical conditions (table 1). IC50 and % sites for renal cortex data were determined using non-linear iterative curve fitting with no contraint on % sites. (Site I + Site 2) was normalized to 100%. Actual (Site 1 + Site2) values ranged between 82 and 91%. Combined analysis of data from renal cortex and medulla yielded similar results (data not shown). Cortex

No addition Na+,100mM Mg 2 + , 5 m M GTP, 0 . 1 m M Ca 2+, 5 mM Na ÷ and Mg 2÷ Na + and GTP

Medulla

K]

nH

Kt/KIo

385 + 51 885_+ 1 2 6 " 239_+ 7 1 " 645_+ 8 0 " 4417_+2100 911+ 143 * 6 8 1 _ + 185 *

0.56 + 0.05 0.84_+0.06* 0.54_+0.13 0.70_+0.07 0.51 -+0.02 0.65-+0.18 0.68_+0.09

2.32+0.03 0.62_+0.14 2.01+0.51 8.04-+4.54 2.12-+0.33 1.55_+0.18

Site 1

Site 2

Kl

%sites

KI

%sites

39 15 41

31 0 39 11

1378 1242 1478 1407

69 100 61 89

K1

nH

KI/KIO

457_+ 90 900+ 4* 507_+ 60 2210+1197"

0.53 +_0.05 0.75+0.03* 0.73_+0.21 0.61+0.05

1.56+0.26 1.01_+0.10 3.48-+1.44

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-8

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Fig. 3. Effects of Na ÷, Mg 2+ and GTP on norepinephrine inhibition of [3H]prazosin binding. [3H]Prazosin (0.2 nM) was incubated for 30 rain at 25°C with renal cortex membranes and 11 concentrations of norepinephrine (30 nM-30 #M), (A) in the absence of additions to the medium, (B) in the presence of 100 mM Na +, (C) in the presence of 0.1 mM GTP and (D) in the presence of 5.0 Mg 2+. Ordinate: percent inhibition of binding. Abscissa: Log molar concentration of norepinephrine. The continuous line represents the best fit by a non-linear, iterative curve-fitting procedure (Munson and Rodbard, 1980). Values represent the means of 3 experiments, each performed in triplicate.

171 son and Rodbard, 1980) of competition curves revealed two different interactions for norepinephrine at [3H]prazosin sites, a high-affinity c o m p o n e n t with a K I of 15-40 n M and a low-affinity c o m p o n e n t with a K x of 1200-1 500 n M (table 2). In the absence of added ion or nucleotide, there was an approximately 2 : 1 ratio of lowto high-affinity states (fig. 3A). In the presence of 100 m M N a +, only the low-affinity site was detected (fig. 3B). With the addition of 0.1 m M GTP, the ratio of low- to high-affinity sites increased to 8 : 1 (fig. 3C). In contrast, this ratio fell to 1.5 : 1 when 5.0 m M Mg 2÷ was included in the incubation m e d i u m (fig. 3D). Thus, N a ÷ and G T P appear to facilitate the conversion of sites with a high-affinity for agonists into low-affinity sites, while Mg 2÷ has opposite effects. In order to determine whether ion and nucleotide modulation of agonist affinity at al-receptors is concentration-dependent, the proportion of [3H]prazosin (0.2 nM) binding inhibited by a fixed concentration of norepinephrine ( 1 . 0 / t M ) was determined in the presence of increasing concentrations of metal ions or GTP, and expressed as a ratio to control inhibition obtained in the absence of additions to the medium. N a ÷ produced a concentration-dependent decrease in the ability of norepinephrine to displace specific [3H]prazosin binding, with an EDs0 for N a ÷ of 2 m M and a m a x i m u m effect at 10 m M (fig. 2A). In contrast, Mg 2+ facilitated norepinephrine inhibition of [3H]prazosin binding to renal cortex membranes, with an EDs0 of approximately 0.5 m M and a maximally effective concentration of 2 m M (fig. 2B). However, Mg 2÷ had no effect on norepi-

nephrine inhibition of medullary [3H]prazosin binding (fig. 2B). This finding is consistent with the lack of effect, in the renal medulla, of 5.0 m M Mg 2+ on the K i of norepinephrine (table 2). Like N a ÷, G T P decreased the ability of norepinephrine to inhibit [3 H]prazosin binding in a concentrationrelated m a n n e r (fig. 2C). Ca z+ caused a progressive decrease in the ability norepinephrine to inhibit [3H]prazosin binding through a 5.0 m M concentration (fig. 2D). Thus, ion and G T P modulation of the affinity of norepinephrine at al-receptors is concentration-dependent. Furthermore, modulation of binding affinity occurs at concentrations k n o w n to occur in physiological systems.

3.2.2. Inhibition by antagonists N a +, Mg 2+ and G T P had no effect on phentolamine inhibition of [3H]prazosin binding to renal cortex al-receptors (table 3). However, N a + and G T P increased the affinity of the antagonist WB-4101 for eq-receptor sites labeled by [3H] prazosin. This finding resembles previous results at brain a2-adrenoceptors , where the affinity of WB-4101 but not phentolamine is regulated by metal ions (Salama et al., 1982).

3.3. Specificity of ion and nucleotide effects In order to determine the specificity of ion and nucleotide modulation of agonist and antagonist interactions at renal al-adrenoceptors, the actions of a series of m o n o - and divalent cations and nucleotides were examined. The non-hydrolyzable G T P analog guanyl-5'-yl imidophosphate ( G p p -

TABLE 3 Ion and nucleotide effects on the inhibition of [3H]prazosin binding by antagonist drugs in rat renal cortex membranes. Mean of 3 experiments, each conducted in triplicate. * P < 0.05, ratio differs from unity (t-test). For details, see legend to table 2. Phentolamine

No addition Na +, 100 mM Mg 2+, 5.0 mM GTP, 0.1 mM

WB-4101

K 1 (nM)

nH

19.3 + 7.3 15.3+4.6 21.9 ± 6.9 15.1 ± 4.6

0.82 + 0.06 0.83 ± 0 . 0 8 0.78 ± 0.07 0.99 ± 0.28

Kj K l (control) 0.89±0.12 1.24 i 0.16 0.80 ± 0.11

K t (nM)

nH

1.0 ± 0.2 0.44+__0.12 0.91 ± 0.21 0.65 ± 0.24

0.61 ± 0.09 0.73+.+_0.11 0.91 ± 0.12 0.74 ± 0.05

Kj K I (control) 0.41 +0.05 * 0.88 ± 0.06 0.61 ± 0.15 *

172 (NH)p), at a 0.1 m M concentration, decreased the affinity of norepinephrine at renal cortex a~-receptor sites relative to that observed in the absence of additions to the medium, and this effect was similar in magnitude to that produced by G T P ( K i / K i ( c o n t r o l ) = 2.36 _+ 0.31). The simultaneous inclusion of 100 m M Na + had no additional effect ( K i / K i ( c o n t r o l ) = 2.54 _+ 0.40). G M P or ATP (0.1 mM) had no effect on norepinephrine affinity. The potency order G M P PMP, G T P >> GMP, ATP in reducing agonist affinity at renal oq-receptors is similar to that observed at m a n y adenylate cyclase-coupled receptor systems. Li + (100 mM) decreased the affinity of norepinephrine for cortex [3H]prazosin sites ( K i / K i ( c o n t r o l ) - - 1.46), although to a lesser extent than the same concentration of Na +. In addition, Li ÷, like N a +, increased the affinity of [3H]prazosin for the al-receptor. K + (100 raM) had no effect on norepinephrine or [3H]prazosin al-receptor interactions. The potency order N a + > Li + >> K + is similar to that for agonist and antagonist interactions at the kidney ct2-adrenoceptor (Ernsberger and U'Prichard, 1983). Mn 2+ (1.0 mM), like 5.0 m M Mg 2+, increased the affinity of norepinephrine for cortex ([3H] prazosin sites. Ca 2+, however, acted in a manner opposite to that of Mg 2+ and Mn 2+, decreasing the affinity of norepinephrine for [3H]prazosin sites (fig. 2D). The potency order Mn 2+ > Mg 2+ >> Ca 2+ is similar that observed for agonist interactions at the ot2-receptor (Rouot et al., 1980).

4. Discussion

The present study demonstrates that Na + and G T P decrease agonist affinities and increase some antagonist affinities at kidney cq-adrenoceptors, while the divalent cations Mg 2+ and Mn 2+, in contrast, increase agonist and decrease antagonist affinities. Norepinephrine competition curves at [3H]prazosin sites were heterogeneous, and were best fit by a two-site model. These data suggest the existence of high- and low-affinity states of kidney al-receptor sites, which possess differential

affinity for agonists, and some antagonists as well. The results are consistent with the notion that monovalent cations and G T P shift an equilibrium of eta-receptor binding states in favor of a low-affinity state with respect to agonists, while divalent cations shift the equilibrium in favor of a high-affinity state. These effects resemble those observed in fl- and az-adrenoceptor systems (Rodbell, 1980; Limbird, 1981; H o f f m a n and Lefkowitz, 1980; Bylund and U'Prichard, 1983). N a + and G T P were able to reduce or eliminate the high-affinity component of norepinephrine interactions at renal oq-receptor sites, while facilitating the interactions of the antagonists [3H]prazosin or WB-4101. In contrast, Mg 2+ enhanced high-affinity agonist interactions at eq-receptor sites in the renal cortex. Mg 2+ failed to affect agonist interactions at [3H]prazosin sites in the renal medulla, while antagonist interactions were inhibited in a manner identical to that observed in the cortex. The functional significance of this regional difference within the kidney is presently unclear, although involvement of ion transport sites is possible. Metal ion effects on binding are unlikely to reflect changes in ionic strength, since monovalent ion effects were specific for N a +, and divalent ion effects were specific for Mg 2+ or Mn 2+. Furthermore, monovalent cations generally exerted effects on binding opposite to those of divalent cations, yet both increase ionic strength. Ca 2+ induced a concentration-dependent loss of [3H]prazosin binding sites accompanied by a decrease in norepinephrine affinity and an increase in [3H]prazosin affinity at the remaining binding sites. In contrast, other divalent cations (Mg 2+ or Mn 2+) increased agonist and decreased antagonist affinity, while not affecting the total number of sites. The opposing actions of Ca z+ relative to other divalent cations contrasts with previous findings that Ca 2+ and Mg 2+ have similar effects on agonist interactions at the az-receptor (Rouot et al., 1980). The unique effects of C a 2+ o n renal [3H]prazosin binding may be accounted for by the actions of an endogenous Ca2+-sensitive proteinase which degrades the threceptor (Lynch et al., 1986). Partially degraded receptors reportedly do not exhibit high-affinity

173 agonist binding, which may explain the selective loss of high-affinity sites in the presence of Ca 2÷. The binding of some antagonists to the renal al-adrenoceptor was affected by ions and nucleotides, but in a direction opposite to that of the agonist norepinephrine. This suggests that some antagonists may bind preferentially to the low-affinity agonist state. In a growing number of receptor systems, treatments that decrease agonist affinities have been shown to increase antagonist affinities, while treatments increasing agonist affinities have been shown to decrease antagonist affinities. Na ÷ has been shown to enhance antagonist interactions at a 2- (Bylund and U'Prichard, 1983) opiate (Pert and Snyder, 1974), histamine (Chang and Snyder, 1980), muscarinic acetylcholine (Ehlert et al., 1980) and dopamine D 2 (Usdin et al., 1980) receptors. Guanine nucleotides have been shown to increase antagonist affinities at a 2(Bylund and U'Prichard, 1983; Asakura et al., 1985) and /3-adrenoceptors (Wolfe and Harden, 1981; Lang and Lemmer, 1985), as well as muscarinic acetylcholine (Burgisser et al., 1982) and dopamine D 2 (Wreggett and De Lean, 1984) receptors. Mg 2÷ or Mn2+ decrease antagonist affinites at a 2- (Bylund and U'Prichard, 1983; Asakura et al., 1985) and dopamine D 2 (DeLean et al., 1982; Wreggett and De Lean, 1984) receptors. Several authors have proposed modifications to the ternary complex model of Hoffman and Lefkowitz (1980) which take into account differential interactions of antagonists with complexed and uncomplexed receptors (Burgisser et al., 1982; Bylund and U'Prichard, 1983; Wreggett and De Lean, 1984; Asakura et al., 1985). The present findings are consistent with these models and suggest that they are applicable to renal a~-receptors. The selective binding of antagonists to the low-affinity state of adrenoceptors has been questioned by Cheung et al. (1984), who demonstrated that when tissue is homogenized and washed in hypertonic buffer, endogenous norepinephrine is retained, presumably in intact synaptic vesicles. Under these conditions, endogenous norepinephrine inhibits antagonist radioligand binding. Guanine nucleotides reverse this inhibition by decreasing the affinity of norepinephrine, leading to apparent regulation of antagonist binding by

nucleotide. Several lines of evidence suggest that the present observations of ion and nucleotide modulation of antagonist binding are not merely an artifact of retained endogenous catecholamines: (a) When tissue is homogenized and washed in hypotonic Tris-HC1 buffer containing 5.0 mM EDTA, as was done in the present study, endogenous norepinephrine is effectively removed (Cheung et al., 1984). (b) A radioenzymatic assay for catecholamine content indicated that endogenous norepinephrine was present in the binding assay at a final concentration of 0.3-0.6 nM, while epinephrine was below the limit of detection (< 0.01 nM) (A. Sved, personal communication). The concentration of norepinephrine is more than three orders of magnitude below the IC50 of norepinephrine at [3H]prazosin sites. (c) The addition of a preincubation step, which would decrease endogenous agonist levels still further, had no effect on [3H]prazosin binding (data not shown). (d) By reducing the availability of binding sites, retained endogenous agonist should decrease the affinity of all antagonists to an equal extent. Instead, ions and nucleotides affected the binding of some antagonists ([3H]prazosin, WB-4101) but not others (phentolamine). (e) Previous studies using membranes prepared in isotonic or hypertonic buffers, which allows retention of endogenous agonist, have reported unusually low radioligand affinity, receptor density, or both (Cheung et al., 1984). In contrast, radioligand affinities and receptor densities in the present study are slightly higher than average values in the literature (Bylund and U'Prichard, 1983). Therefore, the retention of endogenous agonist cannot account for ion and nucleotide modulation of antagonist binding in the present study. Renal al-receptors may be localized on both vascular and tubular elements, mediating vasoconstriction (Schmitz et al., 1981) or gluconeogenesis (McPherson and Summers, 1982), presumably via a Ca2+-dependent mechanism involving phosphatidyl inositol metabolism (Tyroler et al., 1986). The present study describes multiple affinity states of the al-receptor site, and the modulation of these apparent affinity states by ions and nucleotides. The characteristics of al-receptor affinity states are similar to those described for a2-rece p-

174 tors (Kahn et al., 1982; Bylund and U'Prichard, 1983; Asakura et al., 1985), and other receptor systems in which signal transduction is mediated by a protein-protein interaction between the receptor and an additional membrane element (N). These findings support the hypothesis that a membrane protein sensitive to guanine nucleotides and, possibly, mono- and divalent cations may participate in the coupling of cq-receptors to phosphoinositide metabolism (Litosch et al., 1985). The characteristics of [3H]prazosin binding observed in the present study are similar to those previously described in rat renal cortex membranes (McPherson and Summers, 1981; Schmitz et al., 1981), although the K D and agonist K i values were four-fold less and the Bma~ two-fold greater than that described in the latter study. The lower apparent affinity reported by Schmitz et al. (1981) may reflect the routine inclusion 10 m M Mg 2÷ in the incubation medium. The present findings are consistent with previous reports localizing renal a~-receptors predominantly to the cortex (McPherson and Summers, 1981; 1982). A recent report has described a decrease in epinephrine affinity at renal cortex [3H]prazosin sites in the presence of N a ÷ or guanine nucleotides (Snavely and Insel, 1983). However, these experiments were uniformly carried out in the presence of 10 m M Mg 2÷. Thus, divalent cation effects were not described, and the interaction of guanine nucleotide and monovalent ion effects with the effects of 10 m M Mg 2÷ were described, rather than the effects of guanine nucleotides and monovalent ions in an 'ion-free' medium. When similar assay conditions were used, however, comparable results were obtained. Snavely and Insel (1983) reported that 100 mM Na + had no effect of the K D of [3H]prazosin saturation binding; this effect did not occur in the presence of Mg 2+ in the present study. Epinephrine competed at two sites labeled by [3H]prazosin, and Na + shifted the proportion of sites in favor of the low-affinity component. The relative proportion of the two sites and their relative IC50 values agree closely with results obtained in the present study in the presence of Mg 2+. However, guanine nucleotides did not shift the proportion of sites, but rather decreased the affinity of the low-affinity site

(Snavely and Insel, 1983), while in our hands G T P and Na + had similar effects. The present study presents a detailed characterization of ion and nucleotide modulation of possible high- and low-affinity states of the ax-receptor site, confirming and extending previous reports (Glossman and Hornung, 1980b; Yamada et al., 1980; Glossman et al., 1981; Snavely and Insel, 1983). As previously suggested (Glossman and Hornung, 1980b; Snavely and Insel, 1983) a nucleotide binding protein may participate in signal transduction in the al-receptor system, just as such a protein participates in the negative coupling of a2-receptors to adenylate cyclase (Smith and Limbird, 1982). This possibility is supported by recent evidence that a protein sensitive to guanine nucleotides could participate in a link between al-receptor occupation and phosphoinositide metabolism (Litosch et al., 1985; Smith et al., 1986). Preliminary evidence suggests that the multiple affinity states of the cq-adrenoceptor may be functionally relevant. Chronic antidepressant treatment increases agonist affinity at brain a~-receptors without affecting receptor density (Menkes et al., 1983). Furthermore, it has been proposed that the effects of Na + on cq-receptor affinity states may pertain to hypertension, since excess dietary Na + may exert its pressor effect in part by potentiating aa-adrenoceptor function (Ernsberger and U'Prichard, 1983; Insel and Motulsky, 1984).

Acknowledgements Supported by an NSF Predoctoral Fellowship to P. Ernsberger, a grant from Nova Pharmaceuticals, and USPHS NIH36083.

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