Agonist Binding to Mammalin Beta1 and Beta2 Adrenoceptors: Modulation by Guanine Nucleotides and Magnesium

Abstract

Agonist interaction with beta₁ and beta₂ adrenoceptors in rat rabbit lung has been examined using ligand binding techniques and the results are discussed in relation to current models of beta-adrenoceptor-adenylate cyclase coupling. Agonist binding has been assessed by examining the ability of isoprenaline or salbutamol to displace the labelled antagonist ³H-dihydroalprenolol (³H-DHA) from specific receptor sites. beta₁ and beta₂ adrenoceptors, even within the same tissue, exhibited different ion and temperature requirements for guanine nucleotide modulation of agonist binding. Thus, in contrast to the situation at beta₂ sites, agonist binding to beta₁ adrenoceptors was only sensitive to GTP if incubations were performed at physiological temperatures in the presence of Mg++ ions. These findings suggest that there may be different receptor-effector coupling relationships between the two beta-adrenoceptor subtypes.     

Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

Isoproterenol-stimulated adenylate cyclase activity in membrane preparations of reticulocyte-rich (90%) erythrocytes from phenylhydrazine-treated rats is 9 times greater than in untreated animals (1% reticulocytes); basal and fluoride-stimulated activities are also enhanced 2 and 4-fold respectively. In contrast, the number of β-adrenergic receptor sites detected by the binding of ¹²⁵I-hydroxybenzylpindolol (¹²⁵I-HYP) is increased only 40% in these same preparations. The dissociation constant (K_D) of ¹²⁵I-HYP and the IC₅₀ of (-)-isoproterenol for receptor binding sites are unchanged, as is the EC₅₀ of (-)-isoproterenol for activation of adenylate cyclase. The disproportionately large increase in the activity of the isoproterenol-sensitive adenylate cyclase, compared with the small increase in the number of ¹²⁵I-HYP binding sites indicates that the functions of catecholamine recognition and consequent adenylate cyclase response can vary independently and suggest that the receptor and the cyclase may be autonomous molecular entities.     

Adenosine receptor-mediated stimulation of GTP hydrolysis in adipocyte membranes

The adenosine derivative, N⁶-phenylisopropyladenosine (PIA), which inhibits adenylate cyclase in adipocyte membranes by a GTP-dependent and sodium-amplified process, was studied on GTPase activity in hamster adipocyte ghosts. PIA stimulated a high affinity GTPase without apparent lag phase. Both unstimulated and PIA-stimulated GTPases exhibited very similar K_m values of about 0.2 μM GTP. PIA-induced low K_m GTPase stimulation was amplified by sodium ions and was half-maximal and maximal at about 0.02 and 0.1 μM PIA, respectively. Stimulations of the low K_m GTPase by PIA and PGE₁, both inhibiting adipocyte adenylate cyclase, were not additive. Similar to PIA-induced adenylate cyclase inhibition, stimulation of the GTPase by PIA but not by PGE₁ was prevented by the adenosine receptor antagonist, 3-isobutyl-1-methylxanthine. The data suggest that PIA-induced stimulation of a high affinity GTPase is an essential mechanism of adenosine receptor-mediated adipocyte adenylate cyclase inhibition.     

Effects of adenosine and adenosine-analogs on adenylate cyclase activity in the rat adipocyte plasma membrane: comparison of the properties of the enzyme with Mn2+ and Mg2+ as divalent cations

Summary We investigated the influence of Mg²⁺ and Mn²⁺ on the effects of adenosine and some derivatives on basal adenylate cyclase activity in rat fat cell membranes as well as on enzyme activity stimulated by isoprenaline or sodium fluoride. Adenosine and derivatives modified in the ribose function were inhibitory, irrespective of the stimulant used, both in the presence of MgCl₂ or MnCl₂. Inhibition of basal and sodium fluoride stimulated adenylate cyclase activity was more pronounced in the presence of MnCl₂ than in the presence of MgCl₂. N⁶-substituted adenosine analogs proved to be inhibitory in the presence of 5 MM MgCl₂, but in the presence of 1 mM MnCl₂ the fluoride stimulated adenylate cyclase activity was potentiated, while basal and isoprenaline stimulated activity were not significantly inhibited. These effects of adenosine and derivatives could not be blocked by theophylline with or without guanyl nucleotides.The potentiating effect of N⁶-substituted adenosine derivatives on sodium fluoride activated adenylate cyclase is dependent on the structure of the N₆-substitutent and consists of an enhancement of Vrnax in combination with a small decrease of the K_m for MnATP^2–, indicative of an allosteric effect on adenylate cyclase. No potentiation by N⁶-phenylisopropyladeno sine of sodium fluoride stimulated cyclase was found on digitonin solubilized cyclase, while the inhibitory effect of adenosine was retained. The relevance of these findings is discussed in connection with the current hypothesis concerning the presence of two adenosinesensitive sites on rat fat cell membranes.     

[3H]norepinephrine binding by rat glial cells in culture lack of correlation between binding and adenylate cyclase activation

Subcellular fractions prepared from rat glial cells in culture (clonal line C₆) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [³H]norepinephrine binding and enzyme activation were measured under identical experimental conditions.Binding sites for norepinephrine could be detected; their main characteristics were: apparent K_m : 4 · 10⁻⁶ M, maximal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrenergic agonist) and the blocking agent propranolol were unable to compete with [³H]norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [³H]norepinephrine binding.As in several other systems so far studied, the presence on the glial cell's membrane of a large number of “catechol-binding sites” makes it difficult to characterize the β-adrenergic receptor.     

Differential Calcitonin Gene-Related Peptide (CGRP) and Amylin Binding Sites in Nucleus Accumbens and Lung: Potential Models for Studying CGRP/Amylin Receptor Subtypes

Abstract: Calcitonin gene-related peptide (CGRP), a 37-amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin. These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism. The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-[¹²⁵I]iodohistidyl¹⁰)humanCGRPα ([¹²⁵I]hCGRPα) binding (IC₅₀ = 0.4–7.7 nM), which was displaced by hCGRP_8–37α with equally high affinity (IC₅₀ = 0.4–7.3 nM). High-affinity binding for [¹²⁵I]Bolton-Hunter human amylin ([¹²⁵I]BH-h-amylin) was also observed in these tissues (IC₅₀ = 0.2–6.0 nM). In membranes from the nucleus accumbens of both species, salmon calcitonin competed for amylin binding sites with high affinity (IC₅₀ = 0.1 nM) but was poor in competing for amylin binding in lung membranes. Rat amylin_8–37 competed for [¹²⁵I]hCGRPα binding with higher affinity (IC₅₀ = 5.4 nM) compared with [¹²⁵I]BH-h-amylin binding (IC₅₀ = 200 nM) in porcine nucleus accumbens, whereas in guinea pig nucleus accumbens, the IC₅₀ values for rat amylin_8–37 were 117 and 12 nM against [¹²⁵I]hCGRPα and [¹²⁵I]BH-h-amylin, respectively. Also, functional studies evaluating the activation of adenylate cyclase and generation of cyclic AMP in response to these agonists indicated that hCGRPα (EC₅₀ = 0.3 nM), h-amylin (EC₅₀ = 150 nM), and salmon calcitonin (EC₅₀ = 1,000 nM) activated adenylate cyclase, resulting in increased cyclic AMP production in porcine lung membranes that was antagonized by hCGRP_8–37α. The affinity of hCGRP_8–37α was similar for all three peptides. The cyclic AMP responses to amylin and salmon calcitonin were significantly (p < 0.05) lower than that of hCGRPα and not additive, suggesting that they are acting as partial agonists at the same CGRP1-type receptor in porcine lung membranes. Similar observations were made for guinea pig lung membranes. However, human amylin and salmon calcitonin were weaker than hCGRPα in activating lung adenylate cyclase. None of these peptides activated adenylate cyclase in membranes prepared from the nucleus accumbens of both species. The data from these studies demonstrate both species and tissue differences in the existence of distinct CGRP and amylin binding sites and present a potential opportunity to study further CGRP and amylin receptor subtypes.     

The hamster adipocyte adenylate cyclase system II. Regulation of enzyme stimulation and inhibition by monovalent cations

In hamster adipocyte ghosts, ACTH and β-adrenergic agonists stimulate adenylate cyclase by a GTP-dependent process; in contrast, inhibition of the enzyme by hormonal factors requires both GTP and sodium ions. The interaction of various monovalent cations and guanine nucleotides was studied on basal, stimulated and inhibited adenylate cyclase activities. In the presence of GTP (0.03–10 μM), which reduced basal activity by up to 90%, monovalent cations (10–500 mM, added as chloride salts) increased the enzyme activity by up to about 8-fold. The potency order obtained was Na⁺>Li⁺>K⁺>choline. The stable GTP analogue, guanylyl-5′-imidodiphosphate, which like GTP was capable of decreasing basal activity, diminished the cation-induced activation. The stimulatory effects of ACTH and isoproterenol on adipocyte adenylate cyclase activity were impaired by the cations in the potency order, Na⁺>Li⁺>K⁺>choline. Additionally, NaCl shifted the concentration-response for ACTH to the right and caused an increase in the maximal activation by the hormone. Similar to basal activity, fluoride-stimulated activity was increased by NaCl, when GTP was present. The inhibitory effect of prostaglandin E₁ on basal adipocyte adenylate cyclase activity was revealed by the cations in the above mentioned potency order by an apparent reversal of the cation-induced activation. In the presence of NaCl, the ACTH- or fluoride-stimulated activities were also reduced by prostaglandin E₁, but the inhibitory hormonal factor did not reverse the NaCl-induced shift in the concentration-response curve for ACTH. Guanylyl-5′-imidodiphosphate completely prevented hormonal inhibition. The data suggest that monovalent cations interact with the guanine nucleotide-binding regulatory component of the adipocyte adenylate cylase system and that this interaction somehow changes the properties of this component, now revealing hormone-induced inhibition partially impairing hormone-induced stimulation.     

N-Bromoacetyl-amino-cyanopindolol: a highly potent beta-adrenergic affinity label blocks irreversibly a non-protein component tightly associated with the receptor

A new chemical affinity label for the beta-adrenergic receptor, based on the structure of pindolol, has been synthesized and iodinated with 125I. The compound, N-bromoacetylamino-cyanopindolol (BAM-CYP), has an apparent dissociation constant of 44 +/- 7 pM towards the turkey erythrocyte membranes. This compound blocks irreversibly both the ability of beta-adrenergic receptors to bind 125I-cyanopindolol and the ability of beta-receptors to activate adenylate cyclase in the presence of beta-agonists. Furthermore, the irreversible binding of BAM-CYP to half of the beta-receptor sites abolishes the ligand binding activity of all the sites. These findings suggest that the beta-receptor is oligomeric in its native state. Although 125I-BAM-CYP blocks irreversibly and specifically the beta-adrenergic receptor, it does so by labeling a non-protein component, most probably a water-soluble lipid. The labeling is stereospecific since it is prevented by l-propranolol and not by d-propranolol. It is suggested that this lipid is tightly associated with the receptor in close proximity to the binding site. It is also suggested that this water-soluble lipid fraction may prove crucial for the optimal interaction between the beta-adrenergic receptor and the components of adenylate cyclase.     

Reconstitution of the Turkey erythrocyte adenylate cyclase sensitivity to l-epinephrine upon re-insertion of the Lubrol solubilized components into phospholipid vesicles

Turkey erythrocyte adenylate cyclase was activated by GppNHp and l-epinephrine to its stable, highly active form. In this form the enzyme could be solubilized by Lubrol-PX and subsequently re-inserted into phospholipid vesicles concomitantly with the removal of up to 99.3% of the Lubrol. The ability of GTP and l-epinephrine to reverse the GppNHp/epinephrine activated state was taken as a measure for the reappearance of hormone sensitivity in the reconstituted vesicles. An incomplete but significant reappearance of hormone sensitivity in the reconstituted adenylate cyclase was achieved. This hormone sensitivity was found to be stereospecific for (?)epinephrine. The ¹²⁵I-cyanopindolol binding properties of the reconstituted β-receptor depend on the nature of the detergent and the phospholipids used in the reconstitution.     

Beta-adrenergic receptor in the brain: comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates.

The properties of specific ³H-dihydroalprenolol binding sites resemble the properties of the beta-receptor regulating hormone-sensitive adenylyl cyclase activity in an homogenate of rabbit cerebellum. The rabbit cerebellum has 5 to 6 pmole per gm (wet weight) of high affinity (K_D=1.3 nM) specific binding sites for ³H-dihydroalprenolol. the interaction of several beta-adrenergic agonists and antagonists with the specific binding sites is rapid, reversible, and demonstrates stereospecificity which parallels the properties of the beta receptor. Beta-adrenergic agonists show a similar potency as agonists upon adenylyl cyclase activity and as inhibitors of ³H-dihydroalprenolol binding: i.e. l-isoproterenol > l-epinephrine > l-norepinephrine (suggesting a beta₂ adrenergic receptor). The binding affinities of several beta-adrenergic agonists and antagonists for the specific binding sites approximate the affinities of these compounds for the stimulation of adenylyl cyclase. Thus, the ³H-dihydroalprenolol binding sites have properties similar to the beta-adrenergic receptor regulating adenylyl cyclase activity in a rabbit cerebellar homogenate.     

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 × g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was β-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein- and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted in the second peak of the DEAE chromatogram was sensitive to alkaline phosphatase, resistant to trypsin, completely dialyzable and contained both GTP (30 μM) and GDP (10 μM) in significant concentrations. Neither the crude supernatant not its two active components affected the binding of [¹²⁵I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.     

Apparent “negative cooperativity” kinetics in the absence of a nonlinear Scatchard plot of thyrotropin-receptor interaction in a human thyroid adenoma

A human thyroid adenoma (benign nodule) was identified which exhibited a linear Scatchard plot of ¹²⁵I-TSH binding, characteristic of a single class of binding site with high affinity (K_d = 0.5±0.1 nM) and low binding capacity (0.8±0.2 pmol/mg protein). In contrast, Scatchard analysis of binding to adjacent normal thyroid was nonlinear, suggesting the presence of high and low-affinity binding sites with K_d's of 0.4±0.2 and of 27.9±11.0 nM and capacities of 0.7±0.3 and 1.8±1.0 pmol/mg protein, respectively. Dissociation of bound ¹²⁵I-TSH from membranes of both adenoma and normal tissue revealed identical enhancement of dissociation in the presence of excess native hormone, thought to be evidence for the “negative cooperativity” model of hormone-receptor interaction. Furthermore, adenylate cyclase from both tissues was equally responsive to TSH. Thus, a thyroid adenoma which contains TSH-responsive adenylate cyclase still exhibited enhanced dissociation by native hormone, even though Scatchard analysis yielded a single, non-cooperative class of binding sites. This suggests that enhanced dissociation of bound hormone does not provide a demonstration of negatively-cooperative site-site interaction. Furthermore, nonlinear Scatchard plots, typical of TSH binding in normal thyroid, represent two classes of binding sites, of which the high affinity type is responsible for stimulation of adenylate cyclase.     

Effects of neuroleptics on 3H-haloperidol and 3H-cis(Z)-flupenthixol binding and on adenylate cyclase activity in vitro.

The subcellular localization of dopamine-sensitive adenylate cyclase was studied in rat brain striatum and compared to the distribution of dopamine binding sites. The highest specific activity of adenylate cyclase activities sensitive to dopamine was associated almost exclusively with synaptic membranes (mithchondrial fraction; P₂). Using [³H] haloperidol and [³H] apomorphine as markers for the dopamine receptor, specific binding was observed in both the mitochondrial (P₂) and microsomal (P₃) fractions. Data for the mitochondrial fraction revealed a heterogeneity of binding sites. Two saturable sites for [³H] haloperidol were observed with K_d values of 2.5nM and 12.5nM respectively. Overall, the localization of multiple binding sites in the crude synaptosomal fraction correlates well with the localization of dopamine-sensitive adenylate cyclase in this fraction.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of ¹²⁵I-labeled calmodulin to bovine cerebellar membranes have been determined and correlted with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca²⁺, calmodulin binds to a finite number of specific membrane sites with a dissociation constant (K_d) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca²⁺-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be infered from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca²⁺ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca²⁺ atoms. The concentration of the active calmodulin ·Ca²⁺ species required for half-maximal activation of adenylate cyclase is very similar to the K_d of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca²⁺-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

Isolation of plasma membranes from bovine corpus luteum possessing adenylate cyclase, 125I-hCG binding and NaK-ATPase activities

Plasma membranes from bovine corpora lutea have been purified by sucrose density gradient centrifugation. The purified membranes, in addition to binding ¹²⁵I-hCG, also possess hCG-stimulated adenylate cyclase and NaK-ATPase. The relative purification of ¹²⁵I-hCG binding, adenylate cyclase and NaK-ATPase on the basis of the specific activities in the whole homogenate were 7.8, 6.4 and 2.6, respectively. The presence of both the hormone sensitive adenylate cyclase and ¹²⁵I-hCG binding activities suggest that these plasma membranes might possess the ‘receptor’ for gonadotropin.     

Homologous Regulation of the MSH Receptor in Melanoma Cells

Abstract

We have examined the mechanism of homologous regulation of MSH receptor binding and receptor-mediated adenylate cyclase activation in three human and two mouse melanoma cell lines. Pretreatment with α-MSH resulted in a time- and dose-dependent up-regulation of MSH receptors in human D10 and 205 melanoma cells whereas in human HBL and in mouse B16–F1 and Cloudman S91 cells α-MSH induced receptor down-regulation. Up-regulation of receptors was maximal after a 24–h incubation period and an α-MSH concentration of 100 nM (EC₅₀ = 2.4 nM). The increase in α-MSH binding was independent of adenylate cyclase activation and protein synthesis and appeared to be caused by recruitment of spare receptors. The structural requirements of the peptide for triggering this process differed from those found in receptor-binding analyses. Receptor down-regulation was maximal after 12 h and hence more rapid than up-regulation. In B16–F1 cells, 10 nM α-MSH caused the disappearance of 85–90% of the MSH receptors, the EC₅₀ of 0.23 nM lying exactly between that for α-MSH-induced melanogenesis (0.027 nM) and the dissociation constant of receptor binding (1.31 nM). Down-regulation in B16–F1 cells appears to be the consequence of receptor internalization following MSH binding and seems to be initiated during an early step in MSH signalling, preceding the activation of adenylate cyclase and the cAMP signal. Receptor up- and down- regulation were not accompanied by an alteration in affinity to a-MSH, as demonstrated by Scatchard analysis of the binding curves.     

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.     

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.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Adrenocorticotropin/α-Melanocyte-Stimulating Hormone (ACTH/MSH)-Like Peptides Modulate Adenylate Cyclase Activity in Rat Brain Slices: Evidence for an ACTH/MSH Receptor-Coupled Mechanism

Abstract: The regulation of adenylate cyclase activity by adrenocorticotropin/α-melanocyte–stimulating hormone (ACTH/MSH)-like peptides was investigated in rat brain slices using a superfusion method. Adenylate cyclase activity was concentration-dependently increased by ACTH-(1–24), α-MSH (EC₅₀ values 16 and 6 nM, respectively), and [Nle⁴,D-Phe⁷]α-MSH (EC₅₀ value 1.6 nM), in the presence of forskolin (1 μM, optimal concentration). 1-9-Dideoxy-forskolin did not augment the response of adenylate cyclase to ACTH-(1–24). Various peptide fragments were tested for their ability to enhance [³H]cyclic AMP production. [Nle⁴,D-Phe⁷]α-MSH increased [³H]cyclic AMP formation with a maximal effect of 30% and was more potent than ACTH-(1–24), ACTH-(1–16)-NH₂, α-MSH, ACTH-(1–13)-NH₂, [MetO⁴]α-MSH, [MetO₂⁴,D-Lys⁸,Phe⁹]ACTH-(4–9), ACTH-(7–16)-NH₂, ACTH-(1–10), and ACTH-(11–24), in order of potency. This structure–activity relationship resembles that found for the previously described peptide-induced display of excessive grooming. ACTH-(1–24) stimulated adenylate cyclase activity in both striatal (maximal effect, ?20%) and septal slices (maximal effect, ?40%), but not in hippocampal or cortical slices. Lesioning of the dopaminergic projections to the striatum did not result in a diminished effect of [Nle⁴,D-Phe⁷]α-MSH on [³H]cyclic AMP accumulation, which indicates that the ACTH/MSH receptor–stimulated adenylate cyclase is not located on striatal dopaminergic terminals. ACTH-(1–24) did not affect the dopamine D₁ or D₂ receptor–mediated modulation of adenylate cyclase activity. Based on the present data, we suggest that the binding of endogenous ACTH or α-MSH to a putative ACTH/MSH receptor in certain brain regions leads to the activation of a signal transduction pathway using cyclic AMP as a second messenger.