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.     

Fatty acid incorporation increases the affinity of muscarinic cholinergic receptors for agonists

Incorporation of unsaturated fatty acids into membrane fragments from rat brain cortex and medulla pons selectively increased the affinity of the muscarinic agonist, carbamylcholine. The affinity and number of binding sites for the labeled antagonist, N-[3H]methyl-4-piperidyl benzilate was unchanged. The effect on agonist binding was most prominent in the cortex, in which carbamylcholine IC50 values were decreased up to 5-fold. Selectivity of the effect was observed with fatty acids of chain length 18-20 carbons, unsaturation in position 11-12, and a cis conformation of the double bond being most effective. The effects of fatty acids on agonist binding were due primarily to alterations in the affinity constants for the binding reaction, with minor increases in the proportion of high-affinity sites. Transition metals selectively increased the percentage of high-affinity sites in the cortex, but in cis-vaccenic-acid-treated membranes more than additive effects of the metal were observed; both were reversed by GTP. GTP also reversed binding parameters in cis-vaccenic-acid-treated medulla membranes to control level. We conclude that the primary effect of the active fatty acids is to alter the thermodynamic properties of muscarinic agonist binding without markedly inducing interconversion.     

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.     

Characterization of the alpha adrenergic receptor population in hippocampus up regulated by serotonergic raphe deafferentiation

Serotonergic raphe deafferentiation elicits an up regulation of a nM (3H)WB-4101 binding site in rat hippocampus for which norepinephrine displays high affinity and prazosin displays low affinity. Guanine nucleotide affects the nM binding to hippocampal alpha-1 adrenergic receptors. Firstly, Gpp(NH)p, a nonhydrolyzable analog of GTP, inhibits (3H)WB-4101 binding at 3 nM concentration of the radioligand, the ligand concentration labelling the lower affinity, nM, binding site. Secondly, the addition of Gpp(NH)p causes recovery of the heterogeneity of binding sites lost upon preincubation of the membranes with 100 microM epinephrine, apparently by decreasing the affinity of the nM (3H)WB-4101 binding site for the adrenergic receptors. The phenomenon was still observed in the presence of saturating concentrations of the alpha-2 antagonist, yohimbine, and the beta antagonist, propranolol. The results imply that Gpp(NH)p regulates ligand binding to hippocampal alpha-1 agonist sites. It is likely that agonist and antagonist binding sites for the alpha-1 receptor exist in hippocampus with the agonist site being modulated by serotonin.     

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)     

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.     

Effects of low density lipoproteins and mevinolin on cholesterol content and muscarinic cholinergic responsiveness in cultured chick atrial cells. Regulation of levels of muscarinic receptors and guanine nucleotide regulatory proteins

Cultures of myocytes from embryonic chick atria grown in medium supplemented with fetal calf serum from which lipoproteins had been removed demonstrated a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared to cells grown with control serum. This effect was reversed by growth of cells in medium supplemented with lipoprotein-depleted serum (LPDS) reconstituted with the low density lipoprotein fraction from fetal calf serum. In cells grown in LPDS, total cell cholesterol was increased 32% over control levels and returned to control levels in cells grown with LPDS reconstituted with low density lipoprotein. Growth of cells in LPDS plus mevinolin, an inhibitor of endogenous cholesterol synthesis, also reversed the effects of LPDS on cholesterol content and sensitivity of beating to carbamylcholine. The ability of mevinolin (30 microM) to reverse the effect of LPDS on sensitivity of beating to carbamylcholine was inhibited by mevalonic acid, a metabolic precursor to cholesterol, with an IC50 of 7 x 10(-5) M. These data suggest that mevinolin reverses the effects of LPDS by altering cellular cholesterol levels. Enhanced responsiveness of embryonic chick heart cells to muscarinic stimulation was associated with a 2-fold increase in the number of muscarinic receptors with high affinity for agonist from 82 +/- 10 fmol/mg protein in media containing fetal calf serum to 175 +/- 12 fmol/mg protein in cells grown in the presence of LPDS. The distribution of receptors between high affinity (RH) and low affinity (RL) forms changed from 41% RH and 59% RL in cells grown in control serum to 66.5% RH and 33.5% RL in cells grown in LPDS. Quantitation of the effect of growth in LPDS on the levels of guanine nucleotide regulatory proteins No and Ni which couple the muscarinic receptor to a physiologic response, demonstrated that the relative levels of the 39-kDa alpha subunits of No and 41-kDa alpha subunits of Ni determined by ADP ribosylation with pertussis toxin and immunoblotting increased 2-fold compared to control cells grown with fetal calf serum. Growth of cells with medium supplemented with LPDS plus mevinolin reduced the levels of alpha 39 and alpha 41 to below the levels in control cells. Levels of the beta subunit of No and Ni were unaffected by growth with LPDS.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential interactions of muscarinic drugs with binding sites of [3H]pirenzepine and [3H]quinuclidinyl benzilate in rat brain tissue

Some atypical muscarinic drugs were compared with classical drugs with respect to inhibition of specific binding of [3H]pirenzepine ([3H]PZ) and [3H]quinuclidinyl benzilate ([3H]QNB) to membrane preparations of rat brain. The interactions of the agonists McN-A343 and carbachol with [3H]QNB at muscarinic sites in brain stem preparations were differently modulated in the presence of an excess of PZ. Moreover, McN-A343 exhibited a preferential affinity for [3H]PZ sites in whole brain membranes whereas carbachol bound with high affinity to [3H]QNB sites in brain stem preparations. Various muscarinic agonists and antagonists displayed different affinity patterns in the [3H]PZ and [3H]QNB binding. These data are indicative of two populations of pharmacologically distinguishable binding sites and support the concept of muscarinic receptor heterogeneity in rat brain.     

Attenuation of chick heart adenylate cyclase by muscarinic receptors after pertussis toxin treatment

Pertussis toxin selectively modifies the function of Ni, the inhibitory guanine nucleotide binding protein of the adenylate cyclase complex. In chick heart membranes, guanine nucleotide activation of Ni resulted in a decrease in the apparent affinity of the muscarinic receptor for the agonist oxotremorine, inhibition of basal adenylate cyclase activity, and the attenuation of adenylate cyclase by oxotremorine. Treatment of chicks with pertussis toxin caused the covalent modification of 80-85% of cardiac Ni. After this treatment Gpp(NH)p had no effect on muscarinic receptor affinity and GTP stimulated basal adenylate cyclase activity. In contrast, the GTP-dependent attenuation of adenylate cyclase caused by muscarinic receptors was unaffected.     

Characterization of ion movements and their relationship to muscarinic receptor binding and excitation-contraction coupling in guinea pig ileal longitudinal smooth muscle

The relationship between ion movements (sodium uptake and potassium release) and agonist-induced contractile responses or muscarinic receptor binding was investigated in the guinea pig ileal longitudinal muscle (GPLM). Sodium uptake and potassium release were agonist-dependent, concentration-dependent, and stereoselective, with the following rank order of maximum ion movement: muscarinic agonists greater than histamine greater than substance P = serotonin. Potassium depolarization did not initiate sodium uptake or potassium release. Sodium uptake was rapid and monophasic, preceding potassium release which was biphasic in nature. Full muscarinic agonists produced equal maximal increases in sodium uptake, while maximal potassium release varied for all muscarinic agonists and in addition differed from sodium uptake in the following ways: time course, stereoselectivity, sensitivity to calcium antagonists, modulation by the guanylyl nucleotide derivative, 5'-guanylylimidodiphosphate (Gpp(NH)p), and inhibition by muscarinic receptor blockade with benzilylcholine mustard. The calcium ionophores A23187 and ionomycin (SQ23377) did not produce any sodium uptake; A23187 but not ionomycin produced potassium release comparable to that evoked by muscarinic agonists. Ion movement in response to combinations of agonists were not additive. Muscarinic agonist binding as measured by competition for [3H]quinuclidinyl benzilate ([3H]QNB) binding, was best described by multiple sites and was regulated by Gpp(NH)p. Excellent correlations were observed between the dissociation constants for binding and sodium uptake, potassium release, and contraction. The best correlations were those between the pharmacologic responses and the high affinity binding site in the absence, and the low affinity site in the presence, of Gpp(NH)p, respectively. Furthermore, the potencies of muscarinic agonists to evoke ion movements and to inhibit [3H]QNB binding were similar, and from one to two orders of magnitude less than those for contraction. It is suggested that contraction and potassium release were mediated by the high affinity, and sodium uptake by the low and average affinity muscarinic agonist binding sites, respectively. These findings suggest an agonist-activated receptor-effector coupling model in GPLM that leads to the activation of sodium uptake, potassium release, and subsequently, contraction.     

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.     

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.     

Effect of urea-treatment on agonist binding affinity of the muscarinic acetylcholine receptor

Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of ³H-quinuclidinyl benzilate (³H-QNB). This effect was similar to that of Gpp(NH)p. The effects of urea-treatment and Gpp(NH)p were not additive. On the other hand, treatment of the microsome fraction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of urea-treatment and Gpp(NH)p, when these treatments were combined with DTNB.     

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.     

Rat brain and heart muscarinic receptors: modification with tetranitromethane

Tetranitromethane at a concentration of 50 microM modifies the muscarinic receptors in membrane preparations from rat striatum, hippocampus and heart atrium, but not from the rat brain stem. While the binding of antagonists is only slightly altered, the modified receptor possesses an increased affinity of up to 8-fold for [3H]-acetylcholine binding to the high affinity state. This effect is absent if the nitration is carried out in the presence of an antagonist, but not in the presence of an agonist. The affinity for carbamylcholine is increased for both the high and the low affinity state of the receptor, as is evident from its ability to compete with a labeled antagonist. In addition, the proportion of binding sites (alpha) exhibiting the high affinity state for [3H]-acetylcholine or for carbamylcholine is increased upon nitration. This increase cannot be protected against by an antagonist, and is enhanced when nitration takes place in the presence of an agonist. With the agonists oxotremorine and [3H]-oxotremorine-M only the latter effect (i.e., increase in alpha) is observed following nitration, while their dissociation constants for the receptor are unchanged. Data are discussed with respect to the proposed existence of subtypes of muscarinic receptors, as well as the importance of the agonist chosen for studies of ligand-receptor interactions.     

Effects of trypsin-treatment on agonist binding affinity to the muscarinic acetylcholine receptor

Trypsin-treatment of the microsome fraction of the ileum and the synaptic membrane fraction of the cerebral cortex of guinea-pig caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of ³H-quinuclidinyl benzilate (³H-QNB). This effect was similar to that of Gpp(NH)p. The effects of trypsin and Gpp(NH)p were not additive. On the other hand, treatment of these fractions with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of trypsin and Gpp(NH)p, when the fractions were treated with two reagents simultaneously.     

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.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.     

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.     

Interactions between the muscarinic receptors, sodium channels, and guanine nucleotide-binding protein(s) in rat atria

The effects of the voltage-sensitive sodium channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors were studied in homogenates of rat atria. Also studied were the effects of muscarinic ligands on the binding of tritium-labeled batrachotoxin ([3H]BTX) to the same preparation. BTX (1 microM), which induces an open state in sodium channels, enhanced the affinity of binding of several agonists to the muscarinic receptors. Analysis of the data indicated that the effect of BTX was to increase the affinity of the agonists toward the high-affinity sites. Binding of antagonists was not affected by BTX. At higher concentrations of toxin, the density of the high affinity muscarinic sites was also affected. The binding of agonists (but not of antagonists) to muscarinic receptors in turn enhanced the specific binding of [3H]BTX to sodium channels. These effects on the muscarinic receptors and on the sodium channels were inhibited in the presence of Gpp(NH)p at concentrations lower than those bringing about conversion of binding sites from the high affinity to the low affinity conformation. On the basis of these findings we suggest that the opening of sodium channels and the binding of agonists to muscarinic receptors in rat atrial membranes are coupled events which are mediated by guanine nucleotide-binding protein(s). Such a hypothesis is consistent with previously proposed models for signal transduction in the membrane.