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.     

Chemical lesion and drug induced supersentivity and subsensitivity of caudate dopamine receptors

In order to elucidate the mechanism of denervation supersensitivity, the effects of 6-hydroxydopamine lesion, placed in the substantia nigra, were examined on rat brain caudate adenylate cyclase and ³H-haloperidol binding to membrane dopamine receptors. In addition, the effects of chronic administration of L-DOPA, bromocriptine and piribedil were also investigated on ³H-haloperidol binding and dopamine, K⁺ isoproterenol (IPNE) and 2-Cl-adenosine stimulated formation of cyclic AMP in caudate slices. 6-Hydroxydopamine lesions resulted in significantly greater stimulation of adenylate cyclase by dopamine at various concentrations tested. The haloperidol binding sites were increased by 28% on lesioned side caudate without changes in dissociation constants (K_D). Three weeks after treatment with L-DOPA, bromocriptine or piribedil, the ³H-haloperidol binding sites were decreased by 40% with no change in K_D. The stimulatory effect of dopamine on cyclic AMP formation was also abolished, although there was no change in IPNE, K⁺, or 2-Cl-adenosine stimulated cyclic AMP formation in caudate slices, suggesting a specific effect of dopamine agonists on dopamine receptors. The results of these studies suggest a close relationship between at least some populations of dopamine receptors and adenylate cyclase in the caudate nucleus.     

Reconstitution of dopamine-sensitive adenylate cyclase from dissociated components in canine caudate nucleus

The catalytic component of adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were solubilized with 2% Lubrol PX in the presence of NaF from the synaptic membranes of canine caudate nucleus and were separated into distinct fractions by gel exclusion chromatography on a Sephadex G-200 column. The dissociated adenylate cyclase was no longer responsive to dopamine but was considerably stimulated by 10 mm NaF. Dissociated [${}^3\text{H}$]-dopamine binding protein possessed the apparent dissociation constant of 3.2 μm for dopamine, almost identical to that of the particulate preparations. The affinities of [${}^3\text{H}$]-dopamine binding protein to catecholamines and neuroleptics were also very similar to those of particulate preparations. After the adenylate cyclase and [${}^3\text{H}$]dopamine binding protein were preincubated together at 4 °C for 30 min, the cyclase activity displayed a dose-dependent increase by dopamine with the K_a of 1.6 μm, the concentration of dopamine to stimulate half-maximally. Stimulation of the reconstituted adenylate cyclase by dopamine was maximally 2.7-fold and was strongly inhibited by neuroleptics such as chlorpromazine and haloperidol. These results suggest that [${}^3\text{H}$]dopamine binding protein is identical to the regulatory subunit of dopamine-sensitive adenylate cyclase in the synaptic membranes of canine caudate nucleus.     

Effect of neuropeptide-EI on the binding of [3H]SCH 23390 to the dopamine D1 receptor in rat striatal membranes

We have previously demonstrated that neuropeptide-EI, at high doses, stimulates the production of cAMP, in caudate putamen, through the activation of adenylate cyclase coupled to specific D₁ receptors. The aim of the present work was to find evidences for a probable interaction between this neuropeptide and the dopamine D₁ receptor in the mammalian central nervous system. The present data show that neuropeptide-EI, at high concentrations, affected both the maximum binding and the apparent affinity of [n-methyl-³H] (R)-(+)-8 chloro-2,3,4,5- tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hemimaleate to the dopamine D₁ receptor in a concentration-dependent manner.     

D2 Receptor Antagonists Do Not Modify Guanine Nucleotide-Sensitive Interactions Between Dopamine and D1 Dopamine Receptors Under In Vitro Conditions

Abstract: This study investigated possible D₁/D₂ interactions in rat and bovine striatal tissue by examining the effects of D₂ antagonists on the action of dopamine at D₁ dopamine receptors. In addition, the extent to which D₂ antagonists may induce an agonist low-affinity state of the D₁ receptor was evaluated in comparison with the effects of the guanine nucleotide analogue 5′-guanylylimidodiphosphate [Gpp(NH)p]. In saturation experiments dopamine caused a dose-dependent decrease in rat striatal and bovine caudate D₁ receptor density. This effect of dopamine, which has been shown to be sensitive to Gpp(NH)p, was not altered by pretreatment with either of the selective D₂ antagonists eticlopride (200 nM) or domperidone (200 nM). Results from displacement experiments show that the affinity of dopamine for D₁ receptors and the proportion of receptors in an agonist high-affinity state, are reduced by Gpp(NH)p (100 µM) but not by eticlopride. A molar excess of dopamine (100 µM) promotes the dissociation of (±)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol ([³H]SCH 23390) from rat striatal D₁ receptors at a rate that is significantly slower than when dissociation is initiated using 1 µM piflutixol. After pretreatment with Gpp(NH)p, [³H]SCH 23390 dissociation induced by dopamine occurred at an even slower rate. Pretreatment with eticlopride had no effect on the dopamine-induced rate of [³H]SCH 23390 dissociation. These results indicate that all experimental approaches detected dopamine effects at D₁ receptors that are Gpp(NH)p sensitive and D₂ antagonist insensitive and provide no evidence to support a D₁/D₂ link operating at the receptor level.     

Dopamine receptors in the goldfish retina: 3H-spiroperidol and 3H-domperidone binding; and dopamine-stimulated adenylate cyclase activity

Dopamine receptors in the goldfish retina have been examined by binding studies using ³H-spiroperidol and ³H-domperidone as specific ligands, and by measuring retinal adenylate cyclase activities in the presence and absence of dopamine. Our results indicate that washed membranes from goldfish retinal homogenate bind a variety of dopamine agonists and antagonists with high affinities and with characteristics similar to those reported for the brain, with the exception that in this retina there is virtually no binding of the specific D₂ receptor antagonist, ³H-domperidone. In addition, there is a very low basal activity of adenylate cyclase which can be greatly stimulated by dopamine, possibly reflecting a high degree of coupling between this enzyme and the dopamine receptor. Taken together, our findings indicate that the goldfish retina contains a high density of D₁ type dopamine receptors and few, if any, D₂ type receptors.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Receptor-mediated inhibition of octopamine-stimulated adenylate cyclase in the optic lobe of Octopus vulgaris

1. Inhibition of octopamine-stimulated adenylate cyclase was studied in the optic lobe of Octopus vulgaris.2. The octopamine antagonist, mianserin, and the dopamine D₂ agonists, PPHT and metergoline, induced dose-dependent inhibition of octopamine-stimulated adenylate cyclase activity.3. The binding of the tritiated benzazepine neuroleptic YM-09151-2 to octopus membranes and the displacement of [³H]YM-09151-2 by PPHT, metergoline and spiperone were consistent with the presence of a D₂-like dopamine receptor in the octopus optic lobe.4. The conclusion is drawn that octopamine-stimulated adenylate cyclase in the octopus is negatively regulated by a dopamine D₂-like receptor.     

Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate

A general feature of membrane-bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane-bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane-bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane-bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by P_i or PP_i, and (4) and apparent loss of the Me²⁺-reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane-bound form.     

Dopamine inhibition of anterior pituitary adenylate cyclase is mediated through the high-affinity state of the D2 receptor

The diterpinoid forskolin stimulated adenylate cyclase activity (measured by conversion of [³H]-ATP to [³H]-cAMP) in anterior pituitary from male and female rats. Inhibition of stimulated adenylate cyclase activity by potent dopaminergic agonists was demonstrable only in female anterior pituitary. The inhibition of adenylate cyclase activity displayed a typically dopaminergic rank order of agonist potencies and could be completely reversed by a specific dopamine receptor antagonist. The IC₅₀ values of dopamine agonist inhibition of adenylate cyclase activity correlated with equal molarity with the dissociation constant of the high-affinity dopamine agonist-detected receptor binding site and with the IC₅₀ values for inhibition of prolactin secretion. These findings support the hypothesis that it is the high-affinity form of the D₂ dopamine receptor in anterior pituitary which is responsible for mediating the dopaminergic function of attenuating adenylate cyclase activity.     

Effect of Dopamine on Activation of Rat Striatal Adenylate Cyclase by Free Mg2+ and Guanyl Nucleotides1

Abstract: Stimulation of rat striatal adenylate cyclase by guanyl nucleotides was examined utilizing either MgATP or magnesium 5′-adenylylimidodiphos-phate (MgApp(NH) p) as substrate. GTP and 5′- guanylylimidodiphosphate (Gpp(NH) p) stimulate adenylate cyclase under conditions where the guanyl nucleotide is not degraded. The apparent stimulation of adenylate cyclase by GDP is due to an ATP-dependent transphosphorylase present in the tissue which converts GDP to GTP. We conclude that GTP is the physiological guanyl nucleotide responsible for stimulation of striatal adenylate cyclase. Dopamine lowers the K_a for Gpp(NH) p stimulation twofold, from 2.4 μM to 1.2 μM and increases maximal velocity 60%. The kinetics of Gpp(NH) p stimulation indicate no homotropic interactions between Gpp(NH) p sites and are consistent with one nonessential Gpp(NH) p activator site per catalytic site. Double reciprocal plots of the activation by free Mg²⁺ were concave downward, indicating either two sets of sites with different affinities or negative cooperativity (Hill coefficient = 0.3, K_0.5= 23 mM). The data conform well to a model for two sets of independent sites and dopamine lowers the K_a for free Mg²⁺ at the high-affinity site threefold, from 0.21 mM to 0.07 mM. The antipsy-chotic drug fluphenazine blocks this shift in K_a due to dopamine. Dopamine does not appreciably affect the affinity of adenylate cyclase for the substrate, MgApp(NH) p. Therefore, dopamine stimulates striatal adenylate cyclase by increasing the affinity for free Mg²⁺ and guanyl nucleotide and by increasing maximal velocity.     

5'guanylyl-imidodiphosphate actions on adenylate cyclase in homogenates of rat cerebral cortex plus neuronal and capillary fractions

A variety of neurohumoral agents activate adenylate cyclase in homogenates of rat frontal cortex (norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 4-Me-histamine and prostaglandins E₁, E₂ and A₂). The enzyme in homogenates of isolated cortical neurons is likewise sensitive to norepinephrine, isoproterenol, dopamine, apomorphine, histamine, 2-Me- and 4-Me-histamine, and prostaglandin F_2α. Capillary-enriched fractions from the cortex possess an enzyme that is activated by norepinephrine, isoproterenol and dopamine. Addition of 5′-guanylyl-imidodiphosphate (Gpp(NH)p) to the cortical homogenates and neuronal fractions resulted in enhanced enzyme responses to norepinephrine, isoproterenol, dopamine, 2-Me- and 4-Me-histamine and the prostaglandins E₁ and E₂. The actions of histamine and apomorphine were not increased by the GTP analog. The sensitivity of the catecholamine-induced adenylate cyclase activation in cortical capillaries was augmented by Gpp(NH)p. Thus various cellular types within the cerebral cortex may possess different receptor characteristics with respect to stimulation of adenylate cyclase by neurohormones.     

Differential effects of guanine nucleotides on the first step of VIP and glucagon action in membranes from liver cells

Despite the presence of a similar number of glucagon and VIP receptors in liver membranes, VIP induces a negligeable stimulation of adenylate cyclase when compared with glucagon effect. In order to elucidate these discrepancies, the effects of guanine nucleotides on the VIP and glucagon-responsive adenylate cyclase of liver were compared using pure ATP as substrate. 10^?8 M VIP accounted for a 1.5-fold increase of basal activity. In the presence of GTP or Gpp(NH)p (10^?9 to 10^?5 M), the level of cAMP production induced by VIP was no more than additive. In contrast, Gpp(NH)p potentiated the effect of glucagon on liver adenylate cyclase. These discrepancies are not explained by a difference in the peptide binding process. These data suggest that, in liver membranes, a GTP-binding protein N₂ is associated with the glucagon-sensitive adenylate cyclase, but is not detected for VIP. It is suggested that N₂ appears to be specific for the peptidic receptor.     

Guanine Nucleotides Modulate Cortical S2 Serotonin Receptors

Abstract

Many radiolabelled receptors coupled to intracellular adenylate cyclase activity have been found to be modulated by physiological modulators such as GTP (guanosine triphosphate) and Gpp(NH)p (guanosine-imido-diphosphate). In particular, the apparent affinity of agonists competing for the binding of ³H-antagonist-labelled receptors is reduced in the presence of GTP and Gpp(NH)p. We report herein the agonist-specific effects of GTP and Gpp(NH)p on rat brain cortical S² serotonin receptors. The agonists serotonin, 5-methoxytryptamine, bufotenine, and tryptamine display threefold lower affinities for S₂ serotonin receptors in the presence of 10^-4M GTP or Gpp(NH)p than in the absence of the nucleotides. The antagonists spiperone, cinanserin, cyproheptadine and methysergide are unaffected by the guanine nucleotides. The Hill coefficients of the agonists increase from between 0.70–0.80 to 0.90–1.00 due to guanine nucleotides. ATP, ADP, and GDP have little or no effect. This pattern of guanine nucleotide effects has been found with receptors which are modulated by a guanine nucleotide regulatory protein and may indicate that the S₂ serotonin receptor may be coupled to intracellular adenylate cyclase activity.     

Properties of detergent-dispersed myocardial adenylate cyclase

Adenylate cyclase from rabbit ventricle was solubilized in 30 to 50% yield by the nonionic detergent Lubrol PX. The detergent, when present in the assay at concentrations above 0.05%, rapidly inactivated the enzyme in assays conducted above 26 °C; assays were valid only when conducted below this temperature. The solubilized enzyme was eluted from diethylaminoethyl (DEAE)-Bio-Gel A (DEAE-agarose) with 100 mm NaCl in a yield of 25% and was free of detergent. Several properties of the solubilized detergent-free enzyme were similar to properties of the native membrane-bound species. The K_m for substrate was 0.1 mm, the K_a for Mg²⁺ was 2.5 mm, and ATP in excess of Mg²⁺ was inhibitory. The enzyme was activated by F^? and guanyl-5′-yl imidodiphosphate [Gpp(NH)p] in a time- and temperature-dependent manner, and activation by the latter was persistent. Activation by F^? and Gpp(NH)p reduced the K_a for Mg²⁺. Activation by Gpp(NH)p was increased by Mg²⁺; the apparent K_a for activation was 0.1 μm. Multiple binding sites for Gpp(NH)p were present: one class with a K_d value of 0.11 μm was probably associated with activation of the enzyme. The soluble enzyme was insensitive to catecholamines, in both the presence and the absence of Gpp(NH)p. Sensitivity to catecholamines was not restored by the addition of phospholipids, particularly phosphatidyl inositol, in either the presence or the absence of Gpp(NH)p, and this phospholipid did not increase the sensitivity of the membrane-bound enzyme to epinephrine. Catecholamine binding sites were present, and their association with adenylate cyclase was seemingly not affected by phospholipids.     

Dopamine receptor binding: differentiation of agonist and antagonist states with 3H-dopamine and 3H-haloperidol.

³H-Dopamine and ³H-haloperidol bind with high affinity and selectivity to synaptic dopamine receptors in membrane preparations of the calf caudate. Binding of both ligands shows marked regional variations with greatest density in caudate, putamen, globus pallidus, nucleus accumbens and olfactory tubercle, areas rich in dopamine nerve terminals. The rank-order of phenothiazines and related agents as well as catecholamines in displacing both dopamine and haloperidol binding closely parallels their pharmacological potencies and affinities for the dopamine-sensitive adenylate cyclase. Dopamine's affinity for specific ³H-dopamine binding sites is 100 times its apparent affinity for the dopamine sensitive adenylate cyclase. Agonists have about 50 times more affinity for dopamine than haloperidol sites, whereas antagonists display about 100 times greater affinity for haloperidol than dopamine sites.     

Dopamine sensitive adenylate cyclase in planaria Dugesia gonocephala

1. The adenylate cyclase activity present in the particulate fraction of planaria homogenates has been characterized.2. The enzyme requires divalent cations (Mg²⁺), and a K_m for ATP of 0.58 at 30°C was measured.3. GTP and Gpp(NH)p, in an optimal range of 10⁻⁴–10⁻⁵M, increase the enzymatic activity.4. In the presence of GTP, dopamine stimulates the adenylate cyclase and its action is inhibited by dopaminergic antagonist.5. Both D-1 and D-2 selective dopaminergic agonists stimulate the enzymatic activity and their action is selectively antagonized by D-1 and D-2 antagonists.6. The high concentrations required for some D-1 and D-2 agents to be effective, suggest an only partial consistency with mammalian dopaminergic receptors.     

Molecular Characterization of Helodermin-Preferring VIP Receptors in SUP T1 Lymphoma Cells: Evidence for Receptor Glycosylation

Abstract

Cross-linking of [¹²⁵I]helodermin to human SUP-T1 lymphoblasts with bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) revealed a 63 K binding protein. This cross-linking was inhibited by helodermin and VIP. In cells submitted for 3–4 days to 0.2 μg/ml tunicamycin, the Mr of an increasing proportion of helodermin-preferring receptors was reduced to 50 K and the total number of receptors was decreased by about 50%, without alteration in binding affinity and specificity. In parallel, the VIP-mediated adenylate cyclase stimulation was reduced by 30% with no change in NaF-, Gpp[NH]p-, and PGE₁-stimulations. We conclude that a proper N-glycosylation of helodermin-preferring VIP receptors is required for normal receptor targeting and turnover but not for ligand binding and adenylate cyclase coupling.     

A simple test for enhanced guanyl nucleotide exchange in brain adenylate cyclase systems activated by neurotransmitters

The mechanism of receptor-induced activation of adenylate cyclase has been proposed to involve an enhanced exchange of GDP for GTP. The kinetics of this process have not been investigated so far in the brain due to a spontaneous activation of the enzyme by guanyl nucleotides, which precludes the ability to follow receptor-dependent events. We show that it is possible to investigate the mechanism of receptor action in such systems by using a combination of guanosine 5'-(beta-gamma-imino)triphosphate (Gpp(NH)p) and guanosine 5'-(2-O-thio)diphosphate (GDP beta S). In pineal membranes, beta-adrenergic agonists increase the rate of adenylate cyclase activation by 10 or 100 microM Gpp(NH)p about 40-fold (0.023-0.9 min-1 kact) and decrease the inhibitory potency of GDP beta S nearly 1000-fold. As a result, 100 microM GDP beta S which blocks 90% of the activation by 10 microM Gpp(NH)p has no inhibitory effect in the presence of 10 microM Gpp(NH)p and 10 microM noradrenaline or isoproterenol. In caudate nucleus, dopamine does not appear to increase the rate of activation of adenylate cyclase by 10 microM Gpp(NH)p. Nevertheless, 100 microM GDP beta S blocks 90% of the activation by 10 microM Gpp(NH)p but has no inhibitory effects in the presence of dopamine. Thus, one can demonstrate that even weakly activating receptors have the capacity to facilitate a functional exchange of GDP beta S for Gpp(NH)p and measure the efficacy of the interaction between the receptor and the functionally linked guanyl nucleotide subunit.     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.