Muscarinic binding sites on bovine pulmonary arterial endothelial cells in culture.

We have investigated the presence and nature of muscarinic binding sites on membranes from cultured bovine pulmonary arterial endothelial cells (BPAE). BPAE were harvested and subcultured nonenzymatically; experiments were performed 3-5 days postconfluence and between 10 and 25 passage numbers. Utilizing radioligand binding techniques with the muscarinic receptor antagonists [3H]3-quinuclidinyl benzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]MS) as probes, we identified a small population of atropine-sensitive muscarinic sites (1,800-2,000 sites/cell or 7-8 fmol/mg protein). Muscarinic binding sites on BPAE membranes resembled classical muscarinic receptors in that (a) the binding of 2 nM [3H]QNB was inhibited by muscarinic agonists and antagonists, (b) [3H]QNB binding was 30 times more sensitive to R(-)- than to S(+)-QNB, (c) binding of the muscarinic receptor agonist carbamylcholine involved high and low affinity components, (d) the stable GTP analog, Gpp(NH)p (100 microM) shifted agonist binding curves to the right by a factor of three, and (e) the high affinity binding of the agonist [3H]oxotremorine-M to muscarinic receptors was depressed by Gpp(NH)p. On the other hand, gallamine, which allosterically regulates muscarinic receptor binding in other tissues, did not affect the rates of dissociation of [3H]QNB, [3H]MS or [3H]oxotremorine-M from BPAE binding sites. We concluded that BPAE in culture exhibit muscarinic binding sites which possess many but not all of the properties associated with classical muscarinic receptors.     

Modulation of agonist binding by guanine nucleotides in CHO cells expressing muscarinic m1 – m5 receptors

In membranes prepared from CHO-m2 cells, inhibition of [³H]-N-methylscopolamine ([³H]NMS) binding by several muscarinic agonists resulted in competition curves with Hill slopes significantly different from unity. Addition of 5-guanylylimidodiphosphate (Gpp(NH)p) led to an increase in the IC₅₀ value of the agonists with significant steepening of the inhibition curves. The shift in potency induced by Gpp(NH)p differed among the agonists with a rank order of oxotremorine-M = carbachol > oxotremorine > McN-A-343 = pilocarpine. In CHO-m4 membranes, Gpp(NH)p was less efficacious than in CHO-m2 membranes whereas no effect of the guanine nucleotide was found in membranes prepared from CHO-m1, -m3, and-m5 cells. No major differences in the effect of Gpp(NH)p among agonists were found in CHO-m4 cells. Atropine binding was not affected by the guanine nucleotide. Together, these results indicate that coupling of G-proteins to muscarinic receptors linked to inhibition of cyclic adenosine monophosphate (cAMP) (m2 and m4) but not of those linked to phosphoinositol turnover (m1, m3 and m5) can be perturbed by Gpp(NH)p. The differential effects observed with Gpp(NH)p between agonist binding to m2 and m4 receptors appear to be receptor-specific and may reflect differences in the G proteins activated by these receptors in CHO cells.     

MgCl2和Gpp（NH）p对腺苷A1受体激动剂和拮抗剂结合的作用比较

AIM: To investigate modulation of antagonist and agonist binding to adenosine A1 receptors by MgCI2 and 5'-guanylimidodiphosphate (Gpp(NH)p) using rat brain membranes and the A1 antagonist [^3H]-8-cyclopentyl-1,3-dipropylxanthine ([^3H]DPCPX) and the A1 agonist [^3H]-2-chloro-N^6-cyclopentyladenosine ([^3H]CCPA). METHODS:Parallel saturation and inhibition studies were performed using well-characterised radioligand binding assays and aBrandel Cell Harvester. RESULTS: MgCI2 produced a concentration-dependent decrease (44%), whereasGpp(NH)p increased [^3H]DPCPX binding (19%). In [^3H]DPCPX competition studies, agonist affinity was 1.5-14.6-fold higher and 4.6-10-fold lower in the presence of l0 mmol/L MgCl2 and l0μmol/L Gpp(NH)p respectively;antagonist affinity was unaffected. The decrease in agonist affinity with increasing Gpp(NH)p concentrations was due to a reduction in the proportion of binding to the high affinity receptor state. In contrast to [^3H]DPCPX, MgCl2produced a concentration-dependent increase (72%) and Gpp(NH)p a decrease (85%) in [^3H]CCPA binding.Using [^3H]CCPA, agonist affinities were 5-17-fold higher than those for [^3H]DPCPX, consistent with binding onlyto the high affinity receptor state. Agonist affinity was 1.3-10.5-fold higher and 2.4-4.7-fold lower on addingMgCl2 or Gpp(NH)p respectively; antagonist affinities were as for [^3H]DPCPX. CONCLUSION: The inconsistencies surrounding the effects of MgCl2 and guanine nucleotides on radioligand binding to adenosine A1 receptorswere systematically examined. The effects of MgCl2 and Gpp(NH)p on agonist binding to A1 receptors are consistent with their roles in stimulating GTP-hydrolysis at the G-protein α-subunit and in blocking formation of the highaffinity agonist-receptor-G protein complex.     

Temperature effect on the detection of muscarinic receptor-G protein interactions in ligand binding assays

The ability of guanine nucleotides to lower agonist binding affinity provides a convenient indication of receptor-G protein coupling: guanine nucleotides convert muscarinic receptors from high-affinity states for agonists to low-affinity states. We studied the influence of assay temperature on the demonstration of this coupling in rat brainstem and atrium. Agonist affinity of brainstem receptors increased as temperature was lowered, reflecting a greater proportion of receptors in high-affinity conformations. The influence of 5'-guanylylimidodiphosphate, a stable analog of GTP, on agonist binding, determined directly (using [3H]oxotremorine-M) or indirectly (in [3H]N-methylscopolamine/carbamylcholine competition studies), was greatest from 16 to 20 degrees. Guanine nucleotide sensitivity was much reduced at 0-4 degrees and 37 degrees. Brainstem and atrial muscarinic receptors were similarly affected by temperature. We suggest that high-affinity receptor-G protein complexes are unstable at high temperatures, thereby decreasing agonist affinity and masking the guanine nucleotide effect. At low temperatures, the receptor-G protein complex is stabilized and fails to dissociate in the presence of guanine nucleotides. The optimum temperature for monitoring receptor-G protein interactions in binding assays was 16-20 degrees.     

Diethyl ether effects on muscarinic acetylcholine receptor complexes in rat brainstem

The influence of diethyl ether on muscarinic acetylcholine receptor-G protein interactions was studied using membranes isolated from rat brainstem. Membranes were equilibrated with diethyl ether (0.5 to 10%) for 20 min before, and then during, the binding assay. The affinity, but not the number, of [3H]N-methylscopolamine [( 3H]MS) binding sites was increased in the presence of diethyl ether (KD in air = 0.41 nM, KD in 2% diethyl ether = 0.21 nM). This increase in affinity reflected a decrease in the rapid dissociation rate constant (air k-1 = 13 X 10(-3) min-1, 2% diethyl ether k-1 = 7 X 10(-4) min-1) rather than a change in the association rate constant. Diethyl ether had no effect on the binding affinity of the muscarinic agonist carbamylcholine. However, the binding of a radiolabeled muscarinic agonist, [3H]oxotremorine-M [( 3H]Oxo-M), to high affinity binding sites decreased about 25% in the presence of 2% diethyl ether. The ability of a guanine nucleotide to depress the high affinity binding of both carbamylcholine and [3H]Oxo-M was decreased or eliminated by diethyl ether. Diethyl ether appears to interfere with muscarinic receptor-G protein interactions, perhaps by stabilizing receptor-G protein complexes or inhibiting the binding of guanine nucleotides.     

Halothane effects on muscarinic acetylcholine receptor complexes in rat brain

Muscarinic acetylcholine receptors in membranes from rat cerebral cortex or brainstem were equilibrated with halothane (0.5 to 5%). Halothane did not affect the number of [3H]methylscopolamine [( 3H]MS) binding sites. [3H]MS binding affinity, however, was increased in the presence of halothane (KD, air = 0.41 nM; KD, 2% halothane = 0.26 nM). This increase reflected a decrease in the dissociation rate constant (from 13 X 10(-3) min-1 to 6.5 X 10(-3) min-1) rather than a change in the bimolecular rate constant of association (1.8 and 1.9 X 10(7) M-1 min-1 in the absence and presence of 2% halothane respectively). Carbamylcholine affinity for brainstem or cortical muscarinic receptors was not affected by halothane. The ability of a guanine nucleotide to lower carbamylcholine affinity for brainstem receptors, however, was eliminated after equilibration with 2% halothane.     

Agonist interactions with cardiac muscarinic receptors. Effects of Mg2+, guanine nucleotides, and monovalent cations

Analysis of [3H]quinuclidinyl benzilate/acetylcholine competition curves indicated that the agonist acetylcholine bound with three different affinities to chick heart muscarinic receptors. The estimated KD values for acetylcholine were 2.7, 240, and 4000 nM. Mg2+ increased and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p) decreased the proportion of the receptors in the highest affinity state without altering the KD values. Monovalent cations increased the KD values of the three affinity states and obscured the detection of the highest affinity state. The nature of the three affinity states and the sites of action of Mg2+, guanine nucleotides, and monovalent cations were probed with three experimental protocols. Treatments with N-ethylmaleimide or pertussis toxin eliminated both the highest affinity state and the sensitivity to Gpp(NH)p. In contrast, partial effects of Mg2+ were retained after either of these treatments. The effects of monovalent cations on the affinity of the receptor for agonists were unaffected by both treatments. Solubilization of the receptors with digitonin-cholate yielded preparations displaying only the low affinity state for agonist. Agonist binding to the solubilized receptors was insensitive to Mg2+ and guanine nucleotides but retained sensitivity to monovalent cations. The results indicate that chick heart muscarinic receptors can exist in vitro in three agonist affinity states and that the entire population of receptors can be interconverted from one state to another by Mg2+ and guanine nucleotides. Guanine nucleotides presumably act via the inhibitory guanine nucleotide-binding regulatory (Ni) protein, whereas there appear to be at least two distinct sites of action of Mg2+. One site is associated with Ni. Another is distinguishable from Ni but does not appear to be on the receptor itself. The effect of monovalent cations on the interaction of agonists with cardiac muscarinic receptors is qualitatively different and mediated at distinct sites from the effects of Mg2+ and guanine nucleotides.     

Alpha 1-adrenergic receptor-linked guanine nucleotide-binding protein in muscle and kidney epithelial cells

We have studied the interaction of guanine nucleotides with alpha 1-adrenergic receptors of two cloned cell lines, the Madin Darby canine kidney (MDCK-D1) cells and BC3H-1 muscle cells. Although guanylylimidodiphosphate, Gpp(NH)p, had no effect on the affinity or the total number of [3H]prazosin-binding sites in membranes prepared from these cells, the nucleotide decreased the apparent affinity of the agonists (-)-epinephrine and (-)-norepinephrine in competing for [3H]prazosin-binding sites in both cell types. A maximal effect of Gpp(NH)p occurred at 10 microM. Guanine nucleotides were significantly more effective in shifting agonist affinity for the alpha 1 receptor than adenine nucleotides, and Mg2+ was required to observe a maximal effect. Binding of agonist to alpha 1-adrenergic receptors activated phosphatidylinositol (PI) hydrolysis in both cell types but had no effect on membrane adenylate cyclase activity. Incubation of MDCK cells for 19 hr with 100 ng/ml pertussis toxin, which eliminated the ability of pertussis toxin added to membranes to ADP-ribosylate 39-41-KDa substrate(s), failed to alter binding of agonists to alpha 1-adrenergic receptors, the ability of Gpp(NH)p to regulate agonist binding to these receptors, or epinephrine-stimulated PI hydrolysis and prostaglandin E2 production. Incubation of BC3H1 cells with pertussis toxin had no effect on the ability of epinephrine to stimulate PI turnover. These results show that binding of agonists to alpha 1-adrenergic receptors in mammalian kidney and muscle cells is regulated by guanine nucleotides, presumably by interaction with a guanine nucleotide-binding (G) protein. The failure of the G-protein to regulate adenylate cyclase activity and the lack of effect of pertussis toxin to alter receptor-mediated binding or functional activity suggests that a G-protein other than Gs, Gi, or Go interacts with alpha 1-adrenergic receptors in kidney and smooth muscle.     

Agonist binding to M1 muscarinic receptors is sensitive to guanine nucleotides

Putative M1 (high-affinity pirenzepine) muscarinic receptors in rabbit hippocampal membranes, treated with 0.1 mM N-ethylmaleimide (NEM), were selectively labeled with [3H]pirenzepine. A single class of binding sites was labeled with a Kd of 3.4 nM, consistent with the pharmacologically-defined M1 subtype of muscarinic receptors. While full muscarinic agonists bound to high- and low-affinity states of [3H]pirenzepine-labeled M1 sites with a KL/KH ratio of approximately 100, the ratio for partial muscarinic agonists was approximately 10. The high-affinity binding of all agonists tested required divalent cations, and was interconverted to low-affinity binding in the presence of the non-hydrolyzable GTP analogue, guanylyl imidodiphosphate (GppNHp). Direct labeling of the high-affinity agonist state of M1 receptors was achieved with 5 nM [3H]oxotremorine-M by selectively uncoupling the high-affinity agonist state of M2 (low-affinity pirenzepine) receptors with NEM. The rate of dissociation of [3H]Pxotremorine-M from M1 receptors was accelerated 6-fold by GppNHp. These results provide further evidence which suggests that putative M1 muscarinic receptors activate second messenger systems by coupling to NEM-insensitive guanine nucleotide-binding proteins.     

Effect of pertussis toxin on the heart acetylcholine muscarinic receptor affinity

The effect of pertussis toxin on the affinity for agonists and antagonists of the heart muscarine acetylcholine receptor was studied using the radiolabeled antagonist [3H]quinuclidinyl benzylate ([3H]QNB). In cardiac membranes from control rats the displacement of [3H]QNB by carbachol was consistent with two classes of binding sites, kDH 25 +/- 10 nM and kDL 3,006 +/- 869 nM. The proportion of sites in the high and low affinity state for agonists was 55 and 45% respectively. In the presence of 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), only the low affinity state for agonists was observed (kDL 3,804 +/- 759 nM). In cardiac membranes from pertussis toxin-treated rats, two classes of binding sites with affinities similar to those seen in the controls were also observed in the absence of guanine nucleotide (kDs 39 +/- 12 and 3,315 +/- 845 nM) but the proportion of sites were 20 and 80% for high and low affinity respectively. Gpp(NH)p shifted the remaining 20% of sites from the high affinity to the low affinity state (KD 4,093 +/- 744 nM). The receptor KD for antagonists was decreased by pretreatment with pertussis toxin from 83 +/- 7 to 56 +/- 5 pM (P less than 0.01); Gpp(NH)p induced a further change in the affinity for the antagonist in membranes from both control and pertussis toxin-treated rats. The change suggested positive cooperativity. The total number of sites was not modified significantly by either pertussis toxin treatment or guanine nucleotides. These results are consistent with a possible reciprocal modulation of the affinity for agonists and antagonists of the cardiac muscarine receptor.     

Guanine nucleotides and alpha1 adrenergic receptors in the heart

Guanine nucleotides such as GTP and Gpp(NH)p are known to regulate the affinity of beta and alpha₂ adrenergic receptors for agonists as assessed by radioligand binding techniques. Recent studies in the rat heart using the radioligand [³H]WB4101, which reportedly labels alpha₁ adrenergic receptors, have suggested that the affinity of alpha₁ adrenergic receptors for epinephrine is altered by guanine nucleotides. To assess the possible role of guanine nucleotides in alpha₁ adrenergic function, we have constructed (?)epinephrine competition curves in the absence and presence of guanine nucleotides using the highly alpha₁ subtype selective agents [³H]prazosin and [¹²⁵I]BE2254 in rat cardiac membranes. Epinephrine competition curves in the absence of guanine nucleotides were steep and uniphasic in character, and the addition of Gpp(NH)p (10^?4 M) had no effect on the ability of epinephrine to compete for either [³H]prazosin or [¹²⁵I]BE2254 binding sites. In this same membrane preparation, the ability of (?)isoproterenol to compete with [³H]dihydroalprenolol for beta adrenergic receptors binding sites was decreased significantly by Gpp(NH)p. These findings demonstrate that, under conditions where guanine nucleotide regulation of agonist-receptor binding in these membranes can be observed, no nucleotide regulation of agonist-alpha₁ receptor interactions was evidenced using subtype selective radioligands. These results suggest that previous reports of agonist-alpha₁ receptor regulation by guanine nucleotides may represent a manifestation of the anomalous binding characteristics of [³H]WB4101 as compared with [³H]prazosin and [¹²⁵I]BE2254.     

Two distinct, divalent cation-sensitive, antagonist binding states of heart muscarinic receptors: differential modulation by guanine nucleotide

1. The binding of [3H]quinuclidinylbenzilate (QNB), a muscarinic antagonist, to cardiac muscarinic receptors was investigated in two subcellular fractions (washed particles and microsomes) isolated from rat heart atria and ventricles. 2. 5'-guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM), increased (2-3-fold) the binding to washed particles, but not to microsomes, whereas Mg2+ (1-20 mM) increased (up to 5-fold) the binding to microsomes, but not to washed particles. Gpp(NH)p modestly increased the affinity while Mg2+ decreased the affinity towards the radiolabelled antagonist. 3. Treatment with N-ethylmaleimide (NEM, 2 mM) increased the antagonist binding to either fraction. The stimulatory effect of Gpp(NH)p was not evident while that of Mg2+ survived in the NEM-treated fractions. 4. The treatment of fractions with divalent cations chelators (EDTA, EGTA; 10 mM) augmented the stimulatory effect of Mg2+ on [3H]QNB binding to microsomes while that of Gpp(NH)p on the washed particle [3H]QNB binding was decreased. Such treatment further revealed an inhibitory action (about 40%) of Mg2+ on the washed particle binding.     

Differential sensitivity of 3H-agonist binding to pre- and postsynaptic 5-HT1A receptors in bovine brain.

1. The full and weak partial 5-HT1A agonist ligands [3H]-8-OH-DPAT and [3H]-BMY-7378 were used to characterize the binding parameters of pre- and postsynaptic 5-HT1A binding sites in bovine dorsal raphe and hippocampal membranes, respectively. The Kd and Bmax values for the individual radioligands were indistinguisable across the regions tested, as were the Ki values generated by a series of agents acting at 5-hydroxytryptamine (5-HT) receptors. 2. The concentration-dependent allosteric attenuation of [3H]-8-OH-DPAT and [3H]-BMY-7378 binding produced by the nonhydrolyzable guanyl nucleotide, Gpp(NH)p, generated similar IC50 values within a particular region; however, these were significantly different between regions. While the maximal attenuation of [3H]-8-OH-DPAT and [3H]-BMY-7378 binding was similar in dorsal raphe, Gpp(NH)p produced a significantly greater attenuation of [3H]-8-OH-DPAT binding in hippocampal membranes when compared to [3H]-BMY-7378. The maximal attenuation of [3H]-8-OH-DPAT binding by Gpp(NHp) in hippocampus was also significantly greater than that seen with either radioligand in dorsal raphe. 3. Although exposure to Gpp(NH)p had no effect on the affinity constants of either radioligand in either region, it produced a concentration-dependent reduction in the maximal number of binding sites for both radioligands in both regions. While the percentage reduction in Bmax values were similar for both radioligands in the dorsal raphe, Gpp(NH)p reduced the Bmax of [3H]-8-OH-DPAT in hippocampus significantly more than that of [3H]-BMY-7378. 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute in vivo amphetamine produces a homologous desensitization of dopamine receptor-coupled adenylate cyclase activities and decreases agonist binding to the D1 site

We have previously reported that, 30 min after a single injection of 7.5 mg/kg d-amphetamine sulfate, there was a significant 25% decrease in the apparent Vmax for stimulation of adenylate cyclase activity by the D1 receptor-selective partial agonist SKF 38393 in rat striatal membranes, as compared with saline-injected controls. This desensitization was seen in the striatal but not the mesolimbic forebrain. In the present study this desensitization was further characterized by using various ligands that interact with the three components of the D1 receptor-coupled adenylate cyclase complex to determine the site of modification that resulted in the desensitization. The desensitization was not associated with a change in the stimulation of adenylate cyclase at the level of the catalytic subunit or the guanyl nucleotide-regulatory protein Ns. Receptor number, as assessed by the binding of the D1 selective antagonist [3H]SCH 23390, was unaltered in the desensitized state. In contrast, the number of high affinity binding sites, as measured with the agonist [3H]dopamine was decreased 30% by acute amphetamine exposure. This suggests that the amphetamine-induced desensitization may be the result of an uncoupling of the receptor from Ns. In order to further assess the effects of amphetamine on receptor/Ns coupling, we measured the ability of the guanyl nucleotide guanosine-5'-(beta,gamma-imido)triphosphate [Gpp(NH)p] to decrease high affinity [3H]dopamine binding to striatal membranes. The inclusion of 100 microM Gpp(NH)p in the assay decreased the number of receptors in the high affinity state by 40% and 52% in membranes from saline- and amphetamine-pretreated rats, respectively. These results imply that amphetamine treatment does not modify the ability of Gpp(NH)p to decrease high affinity agonist binding. It is possible that amphetamine treatment decreases the number of receptors that can couple to Ns but the remaining receptors can still form a high affinity complex and are sensitive to the effects of Gpp(NH)p. We also report that maximal D2 dopamine receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity was decreased in striatal membranes prepared from amphetamine-treated rats as compared with saline-injected controls, implying that the D2 pathway was desensitized by amphetamine treatment. Conversely, acute amphetamine injection did not alter the ability of either the adenosine A2 receptor to stimulate or the muscarinic cholinergic receptor to inhibit adenylate cyclase activity in the rat striatum. These results suggest that acute amphetamine treatment produces a dopamine receptor-specific or homologous desensitization.     

Cardiac M2 receptors consist of two different types, both regulated by GTP

Cardiac muscarinic receptors are predominantly M2 receptors, and have three agonist binding sites (super-high(SH), high(H) and low(L) affinity agonist binding sites). Treatment of cardiac membranes with 50 nM propylbenzilyl choline mustard (PrBCM) caused 88% loss of binding sites for [3H]QNB. Carbamyl choline (CCh) inhibits this alkylation dose dependently and, theoretically, generates uneven alkylation of multiple agonist binding sites. Pretreatment of the membranes with 50 nM PrBCM and 0.5 mM CCh resulted in almost complete disappearance of L sites with similar degrees of conservation of H sites and SH sites. In these pretreated membranes, guanine nucleotide and sulfhydryl reagent caused a change in the ratio of residual SH and H sites but not of L sites though previous studies showed that, in intact membranes, these reagents affected the ratio of SH and L sites without significantly changing that of the H site. These results indicate the existence of two equilibria regulated by guanine nucleotide and sulfhydryl reagent in cardiac muscarinic receptors: one between SH and H sites and the other between H and L sites. The participation of GTP binding protein(s) in all cardiac muscarinic responses is suggested.     

Muscarinic cholinergic receptors in the embryonic chick heart: interaction of agonist, receptor, and guanine nucleotides studied by an improved assay for direct binding of the muscarinic agonist [3H]cismethyldioxolane

Muscarinic agonist binding has been studied by the indirect method of competition between binding of agonist and 3H-antagonist. Studies of 3H-agonist binding have either been complicated by high levels of nonspecific binding or have been carried out at low concentrations of agonist, which bind only to high affinity sites. A new assay for binding of the muscarinic agonist, [3H]cismethyldioxolane, allows measurement of binding at concentrations up to 1000 nM with a high degree of specificity (92% at 18 nM, 50% at 1000 microM). Using this new binding method, it is possible not only to directly establish the validity of those observations made using competition binding studies, but also to obtain new insight into the mechanism of interaction of receptor agonist and guanine nucleotide. Specifically, data have been presented which demonstrate: 1) that binding of agonist to the high and low affinity forms of the muscarinic receptor involves two independent parallel reactions, and, in the absence of guanine nucleotides, agonist binding alone does not mediate the interconversion of the receptor from one affinity state to another, thus suggesting that high affinity receptors are present in chick heart membranes and do not require agonist binding for their formation; 2) that the interaction of agonist and guanine nucleotide with the receptor-guanine nucleotide-binding protein complex involves formation of an intermediate state in which both agonist and guanine nucleotide are bound to the receptor-guanine nucleotide-binding protein complex; and, finally, 3) that the order of binding of agonist and guanine nucleotide during formation of this intermediate state is random.     

Activation of adrenal adenylate cyclase by guanine nucleotides. Promotion of nucleotide binding by calcium but not by adrenocorticotropic hormone

Calcium ion is essential for normal stimulation of adrenal cortical adenylate cyclase by adrenocorticotropic hormone (ACTH). Both ACTH and Ca2+ act to promote the activation of adenylate cyclase by guanine nucleotides such as guanyl-5'-yl imidodiphosphate [Gpp(NH)p]. To define further the mechanisms by which Ca2+ and ACTH interact with guanine nucleotides, we have correlated the binding of [3H]Gpp(NH)p to adrenal membranes and solubilized membrane proteins with activation of membrane-bound and solubilized adenylate cyclase. Ca2+ increases both the rate of reversible nucleotide binding and the rate of adenylate cyclase activation by nucleotide. This effect is accompanied by the appearance of binding sites having an 8- to 10-fold higher affinity for [3H]Gpp(NH)p. In contrast to Ca2+, ACTH increases the rate of enzyme activation but has no significant effect on nucleotide binding. In Ca2+-depleted membranes, measured nucleotide binding is low, and ACTH has no effect on enzyme activation. Once nucleotide is initially bound, both divalent cations and hormone can promote the transition of the enzyme to an activated state. Mg2+ is more effective than Ca2+ in promoting this transition, while Ca2+ is more effective than Mg2+ in promoting initial nucleotide binding. When membranes containing bound [3H]Gpp(NH)p are solubilized with Lubrol PX, adenylate cyclase activity elutes on Sepharose 4B with an apparent molecular weight of 160,000. The major fraction containing bound nucleotide elutes with an apparent molecular weight of 40,000-50,000. Nucleotide bound to this fraction is increased by pretreatment of the membranes with Ca2+ but is not affected by pretreatment with ACTH. Nucleotide bound to solubilized membrane components dissociates after treatment with EDTA. These findings suggest that Ca2+ promotes the initial binding of Gpp(NH)p to a biologically effective site that may involve a guanine nucleotide regulatory protein. ACTH activates adenylate cyclase by promoting a step subsequent to the binding of guanine nucleotide.     

N-ethylmaleimide blocks the modulatory effects of divalent cations and guanine nucleotides on the brain substance P receptor

The binding of [3H]physalaemin ([3H]PHY) to rat brain substance P receptors is modulated by cations and guanine nucleotides. [3H]PHY binding in the presence of either monovalent or divalent cations (125 mM Na2SO4 or 2.5 mM MnCl2) shows a KD of 5.9 and 5.5 nM and a Bmax of 44.4 and 63.9 fmol/mg protein respectively. In the presence of both, there is a 2-fold increase in the affinity (KD 2.8 nM) and a 25-80% increase in the Bmax (81.6 fmol/mg protein). Addition of 100 microM GTP or Gpp(NH)p in either 125 mM Na2SO4 or 2.5 mM MnCl2 or both decreases the Bmax by 25-55%. However, the receptor affinity for [3H]PHY is not significantly altered by guanine nucleotides. N-Ethylmaleimide (NEM) irreversibly inhibits the receptor binding with an IC50 of 1.0 mM, demonstrating that SH groups play a critical role in the interaction of the ligand with the receptor. If the SP receptors are protected with 1 microM PHY, NEM irreversibly inhibits the effect of divalent cations and guanine nucleotides. Analysis of [3H]PHY binding in 125 mM Na2SO4, 2.5 mM MnCl2 on membranes that were protected with 1 microM PHY and then preincubated with NEM demonstrates a variable decline in receptor number and a 2-fold decrease in the affinity (KD, from 2.8 to 6.9 nM). These observations indicate the existence of a second class of SH groups that are essential for the interaction of divalent cations and guanine nucleotides with the receptor. The blockade of the modulatory effects of divalent cations and guanine nucleotides by NEM treatment further suggests that brain SP receptors are coupled to a guanine nucleotide binding regulatory protein.     

[3H]-Domperidone labels only a single population of receptors which convert from high to low affinity for dopamine in rat brain

Summary Dopamine recognized and competed for a single population of [³H]-domperidone-binding sites in rat striatum and olfactory tubercle when tested in the presence of sodium ions and guanine nucleotide [Gpp(NH)p]. In the absence of Na⁺ and Gpp(NH)p, however, dopamine recognized two components of [³H]-domperidone binding. Thus, [³H]-domperidone labelled only a single population of dopamine receptors (type D₂) which fully converted from high to low affinity for dopamine. These results agree with those found previously using [³H]-spiperone and [³H]-YM-09151-2.     

Interaction of [3H]spiperone with rat striatal dopamine D-2 receptors: kinetic evidence for antagonist-induced formation of ternary complex

The characteristics of [3H]spiperone interactions with rat striatal dopamine D-2 receptor were investigated. Although the association of [3H]spiperone occurred monoexponentially, the pseudo-first order rate constant of association showed a hyperbolic dependence on ligand concentration. The data were therefore analyzed with the assumption of a two-step binding reaction leading to ligand-induced receptor isomerization. For the first equilibrium, the dissociation constant (KD) was 1.2 nM, while for the second equilibrium, the association and the dissociation rate constants were 71.6 X 10(-3) sec-1 and 0.9 X 10(-3) sec-1, respectively. The dissociation rate constant of the overall binding reaction, as determined by inducing the dissociation of [3H]spiperone from its binding sites by 1 microM (+)-butaclamol, was 0.92 X 10(-3) sec-1. However, the kinetically derived KD (15 pM) of the binding reaction differed significantly from the KD (218 pM) obtained from equilibrium binding experiments. This inconsistency between the two KD values appeared to have arisen from using different receptor concentrations in deriving kinetic and equilibrium data. The KD of the equilibrium binding reaction indeed showed significant variation with the receptor concentrations in an inverse way, implicating the involvement of a third component in the two-step binding reaction to form a high affinity ternary complex rather than a simple ligand induced receptor isomerization. Pretreatment of the membrane with 0.1 mM guanosine 5'-imidodiphosphate [Gpp(NH)p] reduced the affinity of the equilibrium binding reaction to a value (KD = 1.2 nM) which corresponded to the kinetically derived KD of the first step of the binding reaction, indicating the involvement of a guanine nucleotide-binding protein or G protein in inducing the formation of the high affinity ternary complex. The affinity of the binding reaction in Gpp(NH)p-pretreated membranes, however, increased with the duration of incubation, indicating that the ligand receptor complex still can couple with the G protein even in the presence of Gpp(NH)p. Pretreatment of the membrane with pertussis toxin irreversibly decreased the affinity of the binding reaction without significantly affecting the total number of binding sites, implying the involvement of the Gi subclass of G protein in the interaction of [3H]spiperone with D-2 receptors. Inhibition of the [3H]spiperone binding by a dopamine receptor agonist, bromocriptine, also yielded a monophasic dose response curve both in the presence and in the absence of Gpp(NH)p.(ABSTRACT TRUNCATED AT 400 WORDS)