Kinetic evidence for differential agonist and antagonist binding to bovine hippocampal synaptic membrane opioid receptors

To examine the kinetics of opioid receptor binding, the agonists [D-Ala2-D-Leu5]enkephalin (DADL) and [D-Ala2-MePhe4-Gly-ol5]enkephalin (DAGO) and the antagonists diprenorphine and naltrexone were used with bovine hippocampal synaptic plasma membranes. By computer modeling of equilibrium binding displacement curves utilizing the LIGAND program, we found opioid peptides bind with high affinity to single populations of synaptic plasma membranes receptors, whereas opiate alkaloids bind to multiple sites. Initial kinetic experiments revealed that agonist rates of association were radioligand concentration-independent. Pseudo first-order rate constants for DADL, DAGO, diprenorphine, and naltrexone association were estimated to be 5.63 X 10(5), 5.08 X 10(5), 4.60 X 10(6), and 2.3 X 10(6) mol-1 X s-1, respectively. After preincubation of 0.2-1 nM radioligand for variable time intervals, dissociation was initiated by addition of 1 microM unlabeled ligand. If saturation binding was achieved before dissociation was initiated, then nearly monophasic dissociation of DADL, DAGO, and diprenorphine and a biphasic off-rate for naltrexone were observed. When association times were reduced to pre-equilibrium intervals, the kinetics of dissociation of agonists became biphasic and association time-dependent, but that for antagonists did not change significantly. Comparisons by both graphical methods and computerized nonlinear regression analyses of rate constants revealed that the fraction of the rapid component of agonist dissociation decreases and that of the slow component is elevated with increasing receptor occupancy. In the presence of 100 mM NaCl, DADL dissociation became association time-independent. These data are consistent with the idea that the Na+ effect is brought about by a change of receptor to an antagonist-like conformation. On the basis of both association and dissociation kinetic data, opioid agonists appear to interact in a multistep process in which a rapid, reversible association is followed by the formation of a more tightly bound complex.     

Guanine nucleotide regulation of B2 kinin receptors. Time-dependent formation of a guanine nucleotide-sensitive receptor state from which [3H]bradykinin dissociates slowly

We have examined the binding of [3H]bradykinin to bovine myometrial membranes and assessed its sensitivity to guanine nucleotides. Total binding displayed a typical B2 kinin receptor specificity. However, saturation binding isotherms were resolved into at least two components with KD values of 8 pM (45%) and 378 pM (55%). Low affinity binding exhibited relatively rapid rates of association (kobs = 1.40 x 10(-2) s-1) and dissociation (k-1 = 3.82 x 10(-3) s-1), while high affinity binding exhibited considerably slower rates (kobs = 9.52 x 10(-4) s-1 and k-1 = 4.43 x 10(-5) s-1). Pre-equilibrium dissociation kinetics revealed that formation of high affinity binding was characterized as a time-dependent accumulation of the slow dissociation rate at the expense of at least one other more rapid dissociation rate. In the presence of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), at least two binding components were resolved with KD values of 37 pM (12%) and 444 pM (88%). Gpp(NH)p apparently specifically perturbed high affinity binding by completely preventing the accumulation of the slow dissociation phase. Instead, two more rapid dissociation rates (k-1 = 8.53 x 10(-3) s-1 and 4.43 x 10(-4) s-1) were observed. These results suggest that [3H]bradykinin interacts with at least two B2 kinin receptor-like binding sites in bovine myometrial membranes. A three-state model for the guanine nucleotide-sensitive agonist interaction with the high affinity binding sites is proposed.     

Affinity labeling of delta-opiate receptors using [D-Ala2,Leu5,Cys6]enkephalin. Covalent attachment via thiol-disulfide exchange

[D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is a synthetic enkephalin analog which contains a sulfhydryl group. DALCE binds with high affinity to delta-receptors, with moderate affinity to mu-receptors, and with negligible affinity to kappa-receptors. Pretreatment of rat brain membranes with DALCE resulted in concentration-dependent loss of delta-binding sites. Using 2 nM [3H][D-Pen2,D-Pen5]enkephalin (where Pen represents penicillamine) to label delta-sites, 50% loss of sites occurred at about 3 microM DALCE. Loss of sites was not reversed by subsequent incubation in buffer containing 250 mM NaCl and 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), conditions which cause dissociation of opiate agonists. By contrast, the enkephalin analogs [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, [D-Pen2,D-Pen5]enkephalin, and [D-Ala2,D-Leu5,Lys6]enkephalin were readily dissociated by NaCl and Gpp(NH)p, producing negligible loss at 3 microM. This suggests that DALCE binds covalently to the receptors. Pretreatment of membranes with the reducing agents dithiothreitol and beta-mercaptoethanol had no effect on opiate binding. Thus, loss of sites required both specific recognition by opiate receptors and a thiol group. The irreversible effect of DALCE was completely selective for delta-receptors. Pretreatment with DALCE had no effect on binding of ligands to mu- or kappa-receptors. The effect of DALCE on delta-binding was: 1) markedly attenuated by inclusion of dithiothreitol in the preincubation buffer, 2) partially reversed by subsequent incubation with dithiothreitol, 3) slightly enhanced when converted to the disulfide-linked dimer, and 4) prevented by blocking the DALCE sulfhydryl group with N-ethylmaleimide or iodoacetamide. These results indicate that DALCE binds covalently to delta-receptors by forming a disulfide bond with a sulfhydryl group in the binding site. The mechanism may involve a thiol-disulfide exchange reaction.     

Binding of [3H]forskolin to human platelet membranes. Regulation by guanyl-5'-yl imidodiphosphate, NaF, and prostaglandins E1 and D2

[3H]Forskolin binds to human platelet membranes in the presence of 5 mM MgCl2 with a Bmax of 125 fmol/mg of protein and a Kd of 20 nM. The Bmax for [3H]forskolin binding is increased to 455 and 425 fmol/mg of protein in the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and 10 mM NaF, respectively. The increase in the Bmax for [3H]forskolin in the presence of Gpp(NH)p or NaF is not observed in the absence of MgCl2. The EC50 values for the increase in the number of binding sites for [3H]forskolin by Gpp(NH)p and NaF are 600 nM and 4 mM, respectively. The EC50 value for Gpp(NH)p to increase the number of [3H]forskolin binding sites is reduced to 35 mM and 150 nM in the presence of 50 microM PGE1 or PGD2, respectively. The increase in the number of [3H]forskolin binding sites observed in the presence of NaF is unaffected by prostaglandins. The binding of [3H]forskolin to membranes that are preincubated with Gpp(NH)p for 120 min or assayed in the presence of PGE1 reaches equilibrium within 15 min. In contrast, a slow linear increase in [3H]forskolin binding is observed over a period of 60 min when Gpp(NH)p and [3H]forskolin are added simultaneously to membranes. A slow linear increase in adenylate cyclase activity is also observed as a result of preincubating membranes with Gpp(NH)p. In human platelet membranes, agents that activate adenylate cyclase via the guanine nucleotide stimulatory protein (Ns) increase the number of binding sites for [3H]forskolin in a magnesium-dependent manner. This is consistent with the high affinity binding sites for [3H]forskolin being associated with the formation of an activated complex of the Ns protein and adenylate cyclase. This state of the adenylate cyclase may be representative of that formed by a synergistic combination of hormones and forskolin.     

Differential regulation of high-affinity agonist binding to muscarinic sites in the rat heart, cerebellum, and cerebral cortex

The muscarinic agonist [3H]cismethyldioxolane ([3H]CD) was used to characterize the effects of regulators upon high-affinity agonist binding sites of the rat heart, cerebral cortex and cerebellum. Comparative studies with sodium ions (Na+), magnesium ions (Mg++), N-ethylmaleimide (NEM) and the guanine nucleotide Gpp(NH)p revealed tissue-specific effects. Mg++ preferentially enhanced while Gpp(NH)p and NEM reduced high-affinity [3H]CD binding in the heart and cerebellum. By comparison NEM enhanced high-affinity agonist binding in the cerebral cortex while Gpp(NH)p and Mg++ had little or no effect. Kinetic studies support an allosteric mechanism for these effects and provide further evidence for muscarinic receptor subtypes in mammalian tissues.     

Effects of pertussis toxin-catalyzed ADP-ribosylation on interactions of transducin and the inhibitory GTP-binding protein of adenylate cyclase with guanyl nucleotides

Release of bound [3H]Gpp(NH)p from NG108-15 cell membranes was induced by carbamylcholine, enkephalinamide, and norepinephrine, all of which inhibit adenylate cyclase. Release was blocked by antagonist, was greater with multiple agonists than with one, and required guanyl nucleotides. With membranes from pertussis toxin-treated cells, both total [3H] Gpp(NH)p binding and agonist-induced [3H]Gpp(NH)p release was decreased. ADP-ribosylation by toxin of transducin, the retinal GTP-binding protein which is similar in structure and function to that in cyclase, decreased [3H]Gpp(NH)p binding. Thus, the inability to demonstrate agonist-induced [3H]Gpp(NH)p release from toxin-treated NG108-15 membranes may result in part from absence of bound [3H]Gpp(NH)p.     

Supersensitivity of beta-adrenoceptor coupled adenylate cyclase in pulmonary tissue of the spontaneously hypertensive rat

Basal adenylate cyclase activity was similar in plasma membranes prepared from the lungs of 12 week old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). However, sensitivity to Gpp[NH]p, isoproterenol plus GTP or Gpp[NH]p was significantly greater in the SHR. Beta-receptor density measured by [3H]DHA binding was unaltered. The dissociation constant, Kd, revealed a significantly greater binding affinity of the radioligand in the SHR (6.23 +/- 0.45 nM) compared with the WKY (8.53 +/- 0.82 nM). Activity of Gs was assessed by complementing S49 cyc- acceptor membranes with lung cholate extract. Basal activity of the reconstituted system was decreased 43% in the SHR. However, sensitivity to NaF, Gpp[NH]p, and isoproterenol plus Gpp[NH]p was significantly elevated. These data suggest that desensitization of the adenylate cyclase complex is not a generalized response to chronic hypertension. A tissue specific increase in sympathetic drive appears to be responsible for the lowered concentration of cardiac beta-adrenoceptors in the SHR. In contrast, both indirect and direct evidence indicate an enhanced functional sensitivity of pulmonary Gs in the hypertensive rats.     

Coupling of adrenal medullary opioid receptors to islet-activating protein-sensitive GTP-binding proteins

Possible coupling of bovine adrenal medullary opioid receptors to islet-activating protein (IAP, pertussis toxin)-sensitive GTP-binding proteins was investigated by studying effects of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and IAP treatment of membranes on opioid binding. Gpp(NH)p inhibited [3H]D-Ala2-D-Leu5-enkephalin ([3H]DADLE) binding by increasing the dissociation constant of [3H]DADLE and membranes, and enhanced slightly [3H]diprenorphine binding. IAP treatment of membranes reduced [3H]DADLE binding and abolished almost completely the Gpp(NH)p inhibition of [3H]DADLE binding. Treatment of membranes with IAP and [32P]NAD resulted in radio-labeling of membrane proteins of approximately 39,000 dalton. DADLE inhibited adenylate cyclase activity in rat brain caudate nucleus. However, DADLE, beta-endorphin, levorphanol and dynorphin A(1-13) did not show any significant inhibitory action on bovine adrenal medullary adenylate cyclase activity. These results suggest that bovine adrenal medullary opioid (DADLE) receptors are linked to IAP-sensitive GTP-binding proteins which are not directly coupled to adenylate cyclase.     

Irreversible inactivation of the beta-adrenoreceptor by a partial agonist. Evidence for selective loss of the agonist high affinity binding sites

The catecholamine derivatives aminomenthylnorepinephrine (compound 1) and bromoacetylaminomenthylnorepinephrine (compound 2) were synthesized and their interaction with the rat lung beta-adrenoreceptor was characterized. Compared to (-)-isoproterenol, compounds 1 and 2 were 10 and 280 times less potent, respectively, at inhibiting (-)-[3H]dihydroalprenolol binding. At pH 7.4, all 3 compounds induced a loss of receptors (40-60%) which could be recovered by treatment with guanyl-5'-yl imidodiphosphate (Gpp(NH)p). However, at pH 8.1 Gpp(NH)p treatment did not recover those receptors lost by compound 2 only. The compound 2-induced receptor loss at pH 8.1 was time-dependent, prevented by propranolol but unaffected by Gpp(NH)p or after membrane heating at 50 degrees C which prevented the formation of the agonist high affinity binding state. Although, the maximal receptor loss as measured by [3H]dihydroalprenolol was 40-60%, more than 80% of the receptors were lost when measured by direct agonist binding, and the receptors left showed little agonist high affinity binding state formation. In rat reticulocyte membranes, compounds 1 and 2 stimulated adenylate cyclase activity with intrinsic activities of 0.55 and 0.31, respectively. However, at pH 8.1, compound 2 initially stimulated the enzyme followed by a blockade. These data indicated that both compounds 1 and 2 were partial beta-adrenoreceptor agonists and, at pH 8.1, compound 2 appeared to bind irreversibly only to those lung receptors able to form the agonist high affinity binding state. Furthermore, after irreversible binding, compound 2 appeared to act as an antagonist.     

Microsomal Opiate Receptors: Characterization of Smooth Microsomal and Synaptic Membrane Opiate Receptors

In continuing studies on smooth microsomal and synaptic membranes from rat forebrain, we compared the binding properties of opiate receptors in these two discrete subcellular populations. Receptors in both preparations were saturable and stereospecific. Scatchard and Hill plots of [3H]naloxone binding to microsomes and synaptic membranes were similar to plots for crude membranes. Both synaptic membranes and smooth microsomes contained similar enrichments of low- and high-affinity [3H]naloxone binding sites. No change in the affinity of the receptors was observed. When [3H]D-ala2-D-leu5-enkephalin was used as ligand, microsomes possessed 60% fewer high-affinity sites than did synaptic membranes, and a large number of low-affinity sites. In competition binding experiments microsomal opiate receptors lacked the sensitivity to (guanyl-5'-yl)imidodiphosphate [Gpp(NH)p] shown by synaptic and crude membrane preparations. In this respect microsomal opiate receptors resembled membranes that were experimentally guanosine triphosphate (GTP)-uncoupled with N-ethylmaleimide (NEM). Agonist binding to microsomal and synaptic membrane opiate receptors was decreased by 100 mM NaCl. Like NEM-treated crude membranes, microsomal receptors were capable of differentiating agonist and antagonists in the presence of 100 mM NaCl. MnCl2 (50-100 microM) reversed the effects of 100 mM NaCl and 50 microM GTP on binding of the mu-specific agonist [3H]dihydromorphine in both membrane populations. Since microsomal receptors are unable to distinguish agonists from antagonists in the presence of Gpp(NH)p, they are a convenient source of guanine nucleotide-uncoupled opiate receptors.     

Alkylation of sulfhydryl groups on Galpha(s/olf) subunits by N-ethylmaleimide: regulation by guanine nucleotides

In rat striatum A(2A) adenosine receptors activate adenylyl cyclase through coupling to G(s)-like proteins, mainly G(olf) that is expressed at high levels in this brain region. In this study we report that the sulfhydryl alkylating reagent, N-ethylmaleimide (NEM), causes a concentration- and time-dependent inhibition of [3H] 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamido adenosine ([3H]CGS21680) binding to rat striatal membranes. Membrane treatment with [14C]N-ethylmaleimide ([14C]NEM) labels numerous proteins while addition of 5'-guanylylimidodiphosphate (Gpp(NH)p) reduces labeling of only three protein bands that migrate in SDS-polyacrylamide gel electrophoresis with apparent molecular masses of approximately 52, 45 and 39 kDa, respectively. The 52- and 45-kDa labeled bands show electrophoretic motilities as Galpha(s)-long and Galpha(s)-short/Galpha(olf) subunits. An anti-Galpha(s/olf) antiserum immunoprecipitates two 14C labeled bands of 44 and 39 kDa. The band density decreases by 21-26% when membranes are treated with NEM in the presence of Gpp(NH)p. An anti-A(2A) receptor antibody also immunoprecipitates two 14C labeled bands of 40 and 38 kDa, respectively. However, such protein bands do not show any decrease of their density upon membrane treatment with NEM plus Gpp(NH)p. These results indicate that in rat striatal membranes NEM alkylates sulfhydryl groups of both Galpha(s/olf) subunits and A(2A) adenosine receptors. In addition, cysteine residues of Galpha(s/olf) are easily accessible to modification when the subunit is in the GDP-bound form. The 39- and 38-kDa labeled proteins may represent proteolytic fragments of Galpha(s/olf) and A(2A) adenosine receptor, respectively.     

Characteristics of an adenosine A1 binding site in human placental membranes

Binding sites were solubilized from human placental membrane using 1.5% sodium cholate and were assayed using polyethylene glycol precipitation. These soluble binding sites had properties of an adenosine A1 binding site. 2-[3H]Chloroadenosine and N-[3H]-ethylcarboxamidoadenosine (NECA) binding were time dependent and reversible. Scatchard plots indicate two classes of binding sites with Kd values of 6 and 357 nM for 2-chloro[8-3H]adenosine and 0.1 and 26 nM with [3H]NECA. The specificity of [3H]NECA binding was assessed by the ability of adenosine analogs to complete for binding sites. Using this approach the estimated IC50 values were 60 nM for (R-PIA), 160 nM for S-PIA, 80 nM for NECA, and 20 nM for 2-chloroadenosine. Binding of [3H]NECA to the soluble sites is inhibited to 48% of the control value by 100 microM guanylyl-5'-imidodiphosphate (Gpp(NH)p). The IC50 value for NECA binding to the soluble binding site was increased from 80 nM to 1500 by Gpp(NH)p. There was a shift of binding affinity from a mixture of high and low affinity to only low affinity with 100 microM Gpp(NH)p. Despite these alterations a NECA prelabeled molecular species of 150 kDa did not decrease in molecular weight upon the addition of 100 microM Gpp(NH)p during high-performance liquid chromatography on a Superose 12 column. Other evidence to support the concept of preferential solubilization and assay of a small population of A1 binding sites was obtained. Following solubilization adenosine A2-like binding sites could be detected only in reconstituted vesicles. The existence of small amounts of A1 binding sites in intact human placental membranes was directly demonstrated using the A1 agonist ligand N6-[3H]cyclohexyladenosine and the A1 antagonist ligand 8-[3H]cyclopentyl-1,3-dipropylxanthine. JAR choriocarcinoma cells have "A2-like" membrane binding sites. In contrast to placental membranes, only A2-like binding sites could be solubilized from JAR choriocarcinoma cells. These observations indicate that human placental membranes contain adenosine A1 binding sites in addition to A2-like binding sites. These sites are guanine nucleotide sensitive, but do not shift to a lower molecular weight form upon assumption of a low affinity state.     

Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H] CD). Characterization studies on rat forebrain homogenates showed that [³H] CD binding was linear with tissue concentration and was unaffected by a change in pH from 5.5 to 8.0. The regional variation in [³H] CD binding in the rat brain correlated generally with [³H] (?)3-quinuclidinyl benzilate ([³H] (?)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], caused a 43–77% inhibition of [³H] CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H] CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H] CD binding, Gpp(NH)p caused an increase in [³H] (?)QNB binding in the heart heart and ileum and no change in [³H] (?)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H] (?)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100 μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies on NEM treated forebrain homogenates revealed an enhancement of [³H] CD binding by NEM.     

Interaction of l-Prolyl-l-Leucyl Glycinamide with Dopamine D2 Receptor: Evidence for Modulation of Agonist Affinity States in Bovine Striatal Membranes

The role of the hypothalamic tripeptide L-prolyl-L-leucyl-glycinamide (PLG) in modulating the agonist binding to bovine striatal dopamine D2 receptor was investigated using a selective high-affinity agonist, n-propylnorapomorphine (NPA). PLG caused an enhancement in [3H]NPA binding in striatal membranes in a dose-dependent manner, the maximum effect being observed at 10(-7)-10(-6) M concentration of the tripeptide. The Scatchard analysis of [3H]NPA binding to membranes preincubated with 10(-6) M PLG revealed a significant increase in the affinity of the agonist binding sites. In contrast, there was no effect of PLG on the binding pattern of the antagonist [3H]spiroperidol. The antagonist versus agonist competition curves analyzed for agonist high- and low-affinity states of the receptor displayed an increase in the population and affinity of the high-affinity form of the receptor with PLG treatment. The low-affinity sites concomitantly decreased with relatively small change in the affinity for the agonists. Almost similar results were obtained when either NPA or apomorphine was used in the competition experiments. A partial antagonistic effect of PLG on 5'-guanylylimidodiphosphate [Gpp(NH)p]-induced inhibition of high-affinity agonist binding was also observed, as the ratio of high- to low-affinity forms of the receptor was significantly higher in the PLG-treated membranes compared to the controls. Direct [3H]NPA binding experiments demonstrated that PLG attenuated the Gpp(NH)p-induced inhibition of agonist binding by increasing the EC50 of the nucleotide (concentration that inhibits 50% of the specific binding). No effect of PLG on high-affinity [3H]NPA binding, however, could be observed when the striatal membranes were preincubated with Gpp(NH)p.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of radiolabeled agonist binding to beta-adrenergic receptors in mammalian tissues

Recent evidence suggests that the molecular interactions of agonists with beta-adrenergic receptors differ from those of antagonists. Most of this evidence has come from studies of agonist inhibition of radiolabeled antagonist binding. We have examined agonist binding directly in rat lung membranes using radiolabeled hydroxybenzylisoproterenol (3H-HBI). Specific binding of 3H-HBI was stereoselective and was inhibited by catecholamines with a potency order characteristic of beta 2-adrenergic receptors. Gpp(NH)p increased the rates of association and dissociation of 3H-HBI from the receptor. In the absence of Gpp(NH)p, Scatchard plots were curvilinear suggesting a complex interaction of the agonist with the receptor. The total number of 3H-HBI binding sites was similar to that of 125I-IHYP binding sites. In the presence of increasing concentrations of Gpp(NH)p, the affinity of 3H-HBI was decreased and Scatchard plots became linear. Sodium chloride mimicked the effect of Gpp(NH)p in lowering the affinity of the receptor for 3H-HBI. Magnesium chloride had the opposite effect in that it promoted high affinity binding. The effect of sodium chloride was largely overcome by the presence of magnesium chloride.     

Multiple affinity states of opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy

The existence of multiple affinity states for the opiate receptor in neuroblastoma x glioma NG108-15 hybrid cells has been demonstrated by competition binding studies with tritiated diprenorphine and [D-Ala2, D-Leu5]enkephalin (DADLE). In the presence of 10 mM Mg2+, all receptors exist in a high affinity state with Kd = 1.88 +/- 0.16 nM. Addition of 10 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p) decreased the affinity of DADLE to Kd = 8.08 +/- 0.93 nM. However, in the presence of 100 mM Na+, which is required for opiate inhibition of adenylate cyclase activity, analysis of competition binding data revealed three sites: the first, consisting of 17.5% of total receptor population has a Kd = 0.38 +/- 0.18 nM; the second, 50.6% of the population, has a Kd = 6.8 +/- 2.2 nM; and the third, 31.9% of the population, has a Kd of 410 +/- 110 nM. Thus, in the presence of sodium, a high affinity complex between receptor (R), GTP binding component (Ni), and ligand (L) was formed which was different from that formed in the absence of sodium. These multiple affinity states of receptor in the hybrid cells are agonist-specific, and the percentage of total opiate receptor in high affinity state is relatively constant in various concentrations of Na+. Multiple affinity states of opiate receptor can be demonstrated further by Scatchard analysis of saturation binding studies with [3H]DADLE. In the presence of Mg2+, or Gpp(NH)p, analysis of [3H]DADLE binding demonstrates that opiate receptor can exist in a single affinity state, with apparent Kd values of [3H]DADLE in 10 mM Mg2+ = 1.75 +/- 0.28 nM and in 10 microM Gpp(NH)p = 0.85 +/- 0.12 nM. There is a reduction of Bmax value from 0.19 +/- 0.02 nM in the presence of Mg2+ to 0.14 +/- 0.03 nM in the presence of Gpp(NH)p. In the presence of 100 mM Na+, Scatchard analysis of saturation binding of [3H]DADLE reveals nonlinear plots; two-site analysis of the curves yields Kd = 0.43 +/- 0.09 and 7.9 +/- 3.2 nM. These Kd values are analogous to that obtained with competition binding studies. Again, this conversion of single site binding Scatchard plots to multiple sites binding plots in the presence of Na+ is restricted to 3H-agonist binding only.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sodium and guanine nucleotide regulation of dopamine receptor agonist and antagonist binding sites in MtTW15 pituitary tumors

The modulation of the dopamine receptor in MtTW15 tumors was investigated. The antagonist dopaminergic binding site in MtTW15 tumors labelled with [3H]spiperone remains unchanged at 25 degrees C in the presence or absence of sodium or guanine nucleotides (Gpp(NH)p); by contrast at 37 degrees C sodium increases the affinity while Gpp(NH)p decreases it slightly. The dopamine receptor in this tumor, such as the intact adenohypophysis, exists in a high and low affinity state for dopamine agonists. These agonist affinity states evaluated with apomorphine competition for [3H]spiperone binding show similar affinities as those of intact tissue but have a lower proportion of the high affinity state. At 25 degrees C, a partial conversion of the high into the low affinity state is obtained in the presence of both sodium and Gpp(NH)p, while at 37 degrees C a complete conversion is observed. These data show differences in the modulation of antagonist and agonist dopaminergic binding sites in MtTW15 pituitary tumors compared with the intact pituitary.     

MgCl2-sensitive and Gpp(NH)p-sensitive antagonist binding states of rat heart muscarinic receptors: preferential detection at ambient temperature assay and location in two subcellular fractions

Summary Some novel observations dealing with antagonist binding to cardiac particulate muscarinic receptors are described. Gpp(NH)p increased (2–3 fold) the specific binding of [³H]-QNB or [³H]-NMS, both potent muscarinic antagonists, to washed particles (WP), but not microsomes (MIC), when the binding was conducted at 30°C. Magnesium, on the other hand, increased (2–3 fold) the binding of these antagonists to MIC, but not to WP, under the same condition. The treatment of subcellular fractions with 0.2 mM N-ethylmaleimide (NEM), a sulfhydryl reagent, failed to significantly modify the respective stimulatory actions of either Gpp(NH)p on WP binding or of magnesium on MIC binding of these antagonists; treatment with dithiothreitol (1 mM) was also ineffective in this regard. Gpp(NH)p decreased K_d (WP) while magnesium increased K_d (MIC) for [³H]-QNB. Repeated freezing/thawing of isolated subcellular fractions abolished the stimulatory effect of magnesium on onist binding to MIC but not of Gpp(NH)p on WP antagonist binding; the freeze/thaw procedure per se increased MIC binding but not WP binding of these antagonists. When the binding was conducted at 4°C (24 hr), the stimulatory effect of Gpp(NH)p on [³H]-QNB binding was enhanced (6-fold) in the case of WP and was detectable (80%) in the case of MIC. Under this condition, the stimulatory effect of magnesium on [³H]-QNB binding was also enhanced (5-fold) in the case of MIC and became evident (200%) in the case of WP. The results of this work support the following views: (a) antagonist-occupied cardiac muscarinic receptors are capable of interaction with guanine nucleotide binding proteins (G protein like G₁,G_o) and such interaction influences antagonist binding properties (e.g. increased affinity) of the cardiac membrane-associated muscarinic receptors (b) magnesium influences (decreased affinity) antagonist binding properties by interacting with multiple sites of which some are likely associated with components other than G proteins of the particulate fractions (c) a pool of NEM-sensitive sulfhydryls involved in the regulation of Gpp(NH)p-sensitive agonist binding to cardiac muscarinic receptors is not involved in the regulation by either Gpp(NH)p or magnesium of antagonist binding in these subcellular fractions and (d) membrane fluidity and microenvironment surrounding the receptor and G proteins contribute to the actions of Gpp(NH)p and magnesium on antagonist binding.     

Regulation of thyrotropin-releasing hormone binding by monovalent cations and guanyl nucleotides

Binding of thyrotropin-releasing hormone (TRH) to specific receptors on membranes isolated from GH4C1 pituitary cells was inhibited by monovalent cations and guanyl nucleotides. NaCl and LiCl inhibited TRH binding by 70%, with half-maximal inhibition at 30 mM; RbCl and KCl inhibited only 10% at concentrations up to 150 mM. NaCl decreased both the apparent number and the affinity of TRH receptors and increased the rate of dissociation of TRH from both membrane and Triton X-100-solubilized receptors. Guanyl nucleotides inhibited TRH binding up to 80%, with guanyl-5'-yl imidodiphosphate (Gpp(NH)p) approximately GTP much greater than GDP approximately ATP greater than GMP. GTP and Gpp(NH)p exerted half-maximal effects at 0.3 microM and decreased receptor affinity to one-third of control but did not change receptor number. Gpp(NH)p accelerated the dissociation of TRH from membranes but not from solubilized receptors. The effects of NaCl were independent of temperature, while GTP and Gpp(NH)p were much more inhibitory at 22 degrees C (70%) than at 0 degrees C (10%). Inhibition by NaCl could be reversed by washing the membranes, and inhibition by GTP was reversed if membranes were chilled to 0 degrees C. The inhibitory effects of low concentrations of NaCl and Gpp(NH)p were additive. Neither monovalent cations nor GTP prevented the TRH-receptor complex from undergoing transformation from a state with rapid dissociation kinetics to a slower dissociating form. The results suggest that sodium ion regulates TRH binding by interacting with a site on the receptor, while guanyl nucleotides regulate TRH binding indirectly.     

Solubilization and separation of the glucagon receptor and adenylate cyclase in guanine nucleotide-sensitive states.

Adenylate cyclase in liver membranes was solubilized with Lubrol PX and partially purified by gel filtration. The partially purified enzyme was susceptible to activation by guanyl-5'-yl imidodiphosphate (Gpp(NH)p). Studies on the binding of [3H]Gpp(NH)p to various fractions eluted from the gels revealed that an upper limit of 1% of the Gpp(NH)p binding sites is associated with adenylate cyclase activity stimulated by the nucleotide. The glucagon receptor, pretagged with 125I-glucagon in the membranes, solubilized with Lubrol PX, and fractionated on the same gel columns, eluted in a peak fraction that overlaps with, but is separate from, adenylate cyclase in its Gpp(NH)p-stimulated form. Addition of GTP to the solubilized glucagon-receptor complex caused complete dissociation of the complex, as has been shown with the membrane-bound form of the complex. Since the GTP-sensitive form of the glucagon receptor complex separates from the Gpp(NH)p-sensitive form of adenylate cyclase, it is concluded that the receptor and the enzyme are separate molecules, each associated with a distinct nucleotide regulatory site or component. These findings are discussed in terms of the possible structure of the hormone-sensitive state of adenylate cyclase.