A novel analogue of clonidine with opiate-receptor agonist activity

A new analogue of the α₂-adrenergic receptor ligand clonidine, N-(4-hydroxphenacetyl)-4-aminoclonidine, was synthesized. The analogue possesses opiate-receptor agonist activity in addition to α-adrenergic partial agonist activity. The analogue elicits inhibition of adenylate cyclase of NG108-15 neuroblastoma × glioma hybrid cells; most of the inhibition is reversed by the opiate-receptor antagonist naloxone. The analogue also inhibits the binding of [³H]D-Ala²-Met⁵-enkephalinamide and [³H]dihydromorphine to rat brain opiate receptors. The structure of the analogue suggests common elements in the ligand binding sites of α- and opiate receptors and may lead to a new class of opiate analgesics.     

Studies on histamine H2 receptors coupled to cardiac adenylate cyclase. Effects of guanylnucleotides and structural requirements for agonist activity.

In particulate preparations from guinea-pig ventricle, histamine in the concentration range 10(-6)--10(-3) M caused a 3--5fold stimulation of adenylate cyclase activity which was dependent on the presence of GTP. The effects of fourteen analogs of histamine were examined on this cyclase preparation. Five of the compounds studied proved to be partial agonists relative to histamine while nine others had essentially the same intrinsic activity as histamine. The intrinsic activities of the partial agonists were increased by GppNHp to the extent that dimaprit, which was a partial agonist in the presence of GTP, became a full agonist in the presence of GppNHp. The relative potencies of the full agonists as activators of the cyclase were found to correlate with the relative potencies on physiologically defined H2 receptor systems. Activation of the cyclase by histamine, as well as by several of the agonist analogs, including dimaprit and tolazoline, was completely blocked by the H2 antagonist cimetidine, but was not affected by pharmacologically relevant concentrations of the H1 antagonist mepyramine, the beta-blocker alprenolol, or the alpha-blocker phentolamine. The results suggest that all the agonists studied probably interact with a common H2 receptor site on the cardiac muscle cell leading to activation of adenylate cyclase. The accompanying increase in cyclic AMP is presumably responsible for the chronotropic and inotropic effects of histamine and related compounds on cardiac muscle.     

Ethanol Does Not Modify Opiate-Mediated Inhibition of Striatal Adenylate Cyclase

Ethanol increases the activity of "basal," guanine nucleotide- and dopamine-stimulated adenylate cyclase in mouse striatum. In contrast, ethanol, in vitro, did not modify the inhibition of striatal adenylate cyclase activity by opiates (morphine or [D-Ala2,D-Leu5] enkephalin). Following chronic in vivo ethanol treatment of mice, there was also no change in the character of opiate inhibition of striatal adenylate cyclase activity. Since ethanol, in vitro, does decrease striatal opiate receptor binding, the results suggest that the changes in affinity detected by ligand binding studies are not relevant for receptor-coupled adenylate cyclase activity, or that opiate receptor binding and opiate regulation of adenylate cyclase can be modulated independently. The selective effects of ethanol on systems that modulate adenylate cyclase activity may produce imbalances in neuronal function during in vivo ethanol exposure.     

Adenylate cyclase inhibition by hormones. The Mg2+ hypothesis

In washed anterior pituitary membranes, there is enough GTP to occupy Ns and therefore to obtain activation of adenylate cyclase by vasointestinal peptide. GTP concentrations needed to obtain adenylate cyclase inhibition by dopamine (above 5 X 10- M) stimulate the adenylate cyclase. The dopamine effect is a blockade of this stimulation. We propose that at least in this system, Ni does not inhibit but stimulates the adenylate cyclase and that inhibitory hormones block this stimulation. We also demonstrate in several adenylate cyclase systems that hormones produced adenylate cyclase inhibition by lowering their Mg affinity A general model for adenylate cyclase activation and inhibition is proposed.     

Studies on histamine H2 receptors coupled to cardiac adenylate cyclase Effects of guanylnucleotides and structural requirements for agonist activity

In particular preparations from guinea-pig ventricle, histamine in the concentration range 10^?6–10^?3 M caused a 3–5-fold stimulation of adenylate cyclase activity which was dependent on the presence of GTP. The effects of fourteen analogs of histamine were examined on this cyclase preparation. Five of the compounds studied proved to be partial agonists relative to histamine while nine others had essentially the same intrinsic activity as histamine. The intrinsic activities of the partial agonists were increased by GppNHP to the extent that dimaprit, which was a partial agonist in the presence of GTP, became a full agonist in the presence of GppNHp. The relative potencies of the full agonists as activators of the cyclase were found to correlate with the relative potencies on physiologically defined H₂ receptor systems. Activation of the cyclase by histamine, as well as by several of the agonist analogs, including dimaprit and tolazoline, was completely blocked by the H₂ antagonist cimetidine, but was not affected by pharmacologically relevant concentrations of the H₁ antagonist mepyramine, the β-blocker alprenolol, or the α-blocker phentolamine. The results suggest that all the agonists studied probably interact with a common H₂ receptor site on the cardiac muscle cell leading to activation of adenylate cyclase. The accompanying increase in cyclic AMP is presumably responsible for the chronotropic and inotropic effects of histamine and related compounds on cardiac muscle.     

The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes

Extracellular cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum cells. Conditions for both stimulation and inhibition of adenylate cyclase by guanine nucleotides in membranes are reported. Stimulation and inhibition were induced by GTP and non-hydrolysable guanosine triphosphates. GDP and non-hydrolysable guanosine diphosphates were antagonists. Stimulation was maximally twofold, required a cytosolic factor and was observed only at temperatures below 10 degrees C. An agonist of the cAMP-receptor-activated basal and GTP-stimulated adenylate cyclase 1.3-fold. Adenylate cyclase in mutant N7 could not be activated by cAMP in vivo; in vitro adenylate cyclase was activated by guanine nucleotides in the presence of the cytosolic factor of wild-type but of not mutant cells. Preincubation of membranes under phosphorylation conditions has been shown to alter the interaction between cAMP receptor and G protein [Van Haastert (1986) J. Biol. Chem. in the press]. These phosphorylation conditions converted stimulation to inhibition of adenylate cyclase by guanine nucleotides. Inhibition was maximally 30% and was not affected by the cytosolic factor involved in stimulation. In membranes obtained from cells that were treated with pertussis toxin, adenylate cyclase stimulation by guanine nucleotides was as in control cells, whereas inhibition by guanine nucleotides was lost. When cells were desensitized by exposure to cAMP agonists for 15 min, and adenylate cyclase was measured in isolated membranes, stimulation by guanine nucleotides was lost while inhibition was retained. These results suggest that Dictyostelium discoideum adenylate cyclase may be regulated by Gs-like and Gi-like activities, and that the action of Gs but not Gi is lost during desensitization in vivo and by phosphorylation conditions in vitro.     

Interaction of clonidine and clonidine analogs with human platelet alpha 2-adrenergic receptors

Several new clonidine analogs were synthesized and their ability to inhibit [3H]phentolamine binding to human platelet alpha 2-adrenergic receptors was tested. The order of potency and calculated dissociation constants for clonidine and its analogs were as follows: clonidine (0.020 +/- 0.005 microM) greater than p-aminoclonidine (0.100 +/- 0.010 microM) greater than hydroxy-phenacetyl-aminoclonidine (0.20 +/- 0.03 microM) greater than p-dansyl clonidine (1.00 +/- 0.20 microM) greater than t-boc-tyrosine clonidine (1.80 +/- 0.60 microM). Thus, p-amino substitution reduces alpha 2-adrenergic affinity in the platelet system. The effects of clonidine and its p-amino analogs on platelet adenylate cyclase were also evaluated. This enzyme is inhibited by epinephrine acting via alpha 2-adrenergic receptors. Both clonidine and p-aminoclonidine cause slight inhibition of basal adenylate cyclase and reverse the inhibition induced by epinephrine. These observations indicate that clonidine is a partial agonist for platelet alpha 2-adrenergic receptors.     

Vasopressin-sensitive adenylate cyclase. Reversibility of hormonal activation

The reversibility of adenylate cyclase activation induced by vasopressin was studied by reducing the concentration of active peptide in contact with kidney medullo-papillary membranes. Reversibility of hormonal activation was only partial. The use of antagonists failed to demonstrate the reversibility of an adenylate cyclase activation induced by high affinity agonists. When antagonist was added after the agonist to membranes, a non-competitive inhibition was apparent. Active peptide was also eliminated from the incubation medium by treatment with agents capable of reducing the disulfide bridge of the hormonal molecule. Direct effects of reducers on adenylate cyclase activity were measured on enzyme activation induced by peptides lacking a disulfide bridge. There was no apparent correlation between the abilities of different reducers to inactivate free peptide in solution and their abilities to promote the reversibility of hormone-induced enzyme activation. Upon the addition of dithiothreitol, enzyme activity could be lowered to basal value and adenylate cyclase was again fully stimulatable. However, when dithiothreitol addition to stiumlated enzyme was combined with a 60-fold dilution of the incubation medium, no reversibility of hormonal activation occurred. These results illustrate that the processes involved in adenylate cyclase activation are only partially reversible.     

Sensitization of adenylate cyclase by short-term activation of 5-HT1A receptors

Long-term (18 h) activation of 5-HT1A receptors alters 5-HT1A receptor-G protein coupling and leads to heterologous sensitization of adenylate cyclase. In contrast, the effects of short-term (2 h) 5-HT1A receptor activation on subsequent adenylate cyclase activity have not been determined. The present study examined and characterized 5-HT1A receptor-induced heterologous sensitization following short-term activation in CHO-5-HT1A cells. Short-term activation of 5-HT1A receptors with full agonists, as well as the partial agonist, buspirone, markedly enhanced subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization was evident after 30 min treatment with 5HT and appeared to be near maximal following 2 h agonist treatment. Sensitization was characterized by a dose-dependent increase in forskolin-stimulated cyclic AMP accumulation and was prevented by WAY 100635 or by pertussis toxin treatment. The ability of the 5-HT1A agonists to induce heterologous sensitization was not significantly altered by agents shown previously to modulate 5-HT1A-mediated inhibition of cyclic AMP accumulation.     

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.     

The regulatory GTPase cycle of turkey erythrocyte adenylate cyclase.

It has recently been suggested that adenylate cyclase activity is controlled by a regulatory cycle consisting of two reactions: a hormone induced formation of the active adenylate cyclase-GTP complex, and a subsequent turn-off reaction in which hydrolysis of the bound nucleotide reverts the system to the inactive state. To test this model each of the two reactions was measured separately and their rate constants were used to estimate the steady state adenylate cyclase and GTPase activities. The first order rate constants were kon = 3 min-1 for the activation reaction and koff = 15 min-1 for the turn-off reaction. Substitution of these rate constants in the steady state equation of the regulatory cycle gave values of hormone stimulated adenylate cyclase and GTPase activities similar to those determined by direct measurements. Treatment of the adenylate cyclase with cholera toxin caused a decrease of 96% in the rate constant of the turn-off reaction. In this case too the activities calculated from the steady state equation were in good agreement with those determined directly.     

Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for receptor-regulated phosphotransfer reactions involved in activation of the adenylate cyclase inhibitory G protein in human platelet membranes

The activity of the adenylate cyclase inhibitory guanine-nucleotide-binding regulatory protein (Gi), measured as inhibition of forskolin-stimulated cyclic AMP formation, and its regulation by various nucleotides and the inhibitory alpha 2-adrenoreceptor agonist epinephrine was studied in membranes of human platelets. When adenylate cyclase activity was measured with ATP as substrate and in the absence of a nucleoside-triphosphate-regenerating system, GTP (0.1-10 microM) by itself potently and efficiently inhibited the enzyme. GDP was almost as potent and as effective as GTP. In the additional presence of epinephrine, the potencies of both GTP and GDP were increased about threefold, while maximal inhibition by these nucleotides was only slightly increased by the receptor agonist. In contrast to GTP and GDP, the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate, had only a very small effect, suggesting that GDP but not its stable analog is converted to the active GTP. Addition of UDP (1 mM), used to block the GDP to GTP conversion reaction, completely suppressed the inhibitory effect of GDP, while that caused by GTP was not affected. Most important, the inhibitory receptor agonist epinephrine counteracted the suppressive effect of UDP on GDP's action, suggesting that, while UDP inhibits the formation of GTP from GDP, the activated receptor stimulates this conversion reaction. In the presence of a complete nucleoside-triphosphate-regenerating system, which by itself had no influence on control forskolin-stimulated adenylate cyclase activity, GTP alone, at concentrations up to 10 microM, did not decrease enzyme activity, but required the presence of an inhibitory receptor agonist (epinephrine) to activate the Gi protein. Addition of the regenerating system creatine phosphate plus creatine kinase not only abolished adenylate cyclase inhibition by GTP alone, but also largely reduced both the potency and efficiency of epinephrine to activate the Gi protein in the presence of GTP. Furthermore, the nucleoside-triphosphate-regenerating system also largely delayed the onset of adenylate cyclase inhibition by the GTP analog, guanosine-5'-[beta-thio]triphosphate (10 nM), which was accelerated by epinephrine, and it also decreased the final enzyme inhibition caused by this GTP analog.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sodium Regulation of Hormone-Sensitive Adenylate Cyclase

Abstract

The influence of sodium was studied on hormone and guanine nucleotide-induced stimulation and inhibition of adenylate cyclase and on ß-adrenoceptor binding in various membrane systems. Sodium exerted almost identical effects on stimulation and inhibition of adenylate cyclase by various stimulatory and inhibitory hormones in all of the systems studied. The potencies of the hormones and of GTP to increase or to decrease the enzyme activity were reduced by sodium ions, without changing the maximal degree of adenylate cyclase stimulation or inhibition. Stimulation and inhibition of adenylate cyclase by the stable GTP analog, GTPγS, was affected in an identical manner by sodium, causing a retardation in the onset without a change in final stimulation or inhibition by the analog. Similar to the well-known reduction in α₂-adrenoceptor affinity for agonists, sodium also reduced the apparent affinity of ß-ad-renoceptors for the agonist, isoproterenol. It is concluded that sodium exerts identical effects on N_s and N_i, inhibiting the activation process of these two coupling components of the adenylate cyclase.     

Inhibition of parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by transforming growth factor alpha and epidermal growth factor

The effects of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TFG-alpha and EGF incubated with UMR-106 cells for 48 h each produced concentration-dependent inhibition of PTH-responsive adenylate cyclase, with maximal inhibition of 38-44% at 1-3 ng/ml of either growth factor. TGF-alpha and EGF also inhibited beta-adrenergic agonist (isoproterenol)-stimulated adenylate cyclase by 32%, but neither growth factor affected enzyme response to prostaglandin or basal (unstimulated) activity. Nonreceptor-mediated activation of adenylate cyclase by forskolin and cholera toxin was inhibited 18-20% by TGF-alpha and EGF. Pertussis toxin augmented PTH-stimulated adenylate cyclase, suggesting modulation of PTH response by a functional inhibitory guanine nucleotide-binding regulatory component of the enzyme. However, pertussis toxin had no effect on TGF-alpha inhibition of PTH response. Growth factor inhibition of PTH response was time-dependent, with maximal inhibition by 4-12 h of TGF-alpha exposure, and was reduced by prior treatment of UMR-106 cells with cycloheximide. TGF-alpha was not mitogenic for UMR-106 cells. The results indicate that TGF-alpha and EGF selectively impair PTH- and beta-adrenergic agonist-responsive adenylate cyclase of osteoblast-like cells. Growth factor inhibition of adenylate cyclase may be exerted at the receptor for stimulatory agonist and at nonreceptor components excluding pertussis toxin-sensitive guanine nucleotide-binding regulatory proteins. The inhibitory action of growth factors may also require protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protease inhibitors block hormonal activation of adenylate cyclase

To investigate the mechanism of serine protease stimulation of rat ovarian adenylate cyclase, a variety of synthetic protease inhibitors were used. These inhibitors blocked trypsin, chymotrypsin and hCG stimulation of adenylate cyclase in nearly the same manner. The inhibition of hormone stimulated adnylate cyclase could not be explained by a loss of [¹²⁵I]hCG binding. Cholera toxin and epinephrine stimulation of adenylate cyclase were similarly inhibited, whereas basal and fluoride-stimulated activities were only affected by higher doses of the inhibitors. The results suggest that adenylate cyclase in the ovary may be regulated by membrane protease activity.     

Pertussis Toxin Attenuates 5-Hydroxytryptamine1A Receptor-Mediated Inhibition of Forskolin-Stimulated Adenylate Cyclase Activity in Rat Hippocampal Membranes

The inhibition of forskolin-stimulated adenylate cyclase activity by 5-hydroxytryptamine (5-HT) receptor agonists was measured in rat hippocampal membranes isolated from animals treated with vehicle or islet-activating protein (IAP; pertussis toxin). In vehicle-treated animals, 5-HT, 8-hydroxy-2-(di-n-propylamino)tetralin, buspirone, and gepirone were potent in inhibiting forskolin-stimulated adenylate cyclase activity with EC50 values of 60, 76, 376, and 530 nM, respectively. IAP treatment reduced by 30-55% the 5-HT1A agonist inhibition of adenylate cyclase activity via 5-HT1A receptors. The data indicate that the inhibitory guanine nucleotide-binding protein or Go (a similar GTP-binding protein of unknown function purified from brain) mediates the 5-HT1A agonist inhibition of hippocampal adenylate cyclase.     

Differential effects of cholera toxin on guanine nucleotide regulation of beta-adrenergic agonist high affinity binding and adenylate cyclase activation in frog erythrocyte membranes

The guanine nucleotide regulatory protein(s) regulates both adenylate cyclase activity and the affinity of adenylate cyclase-coupled receptors for hormones or agonist drugs. Cholera toxin catalyzes the covalent modification of the nucleotide regulatory protein of adenylate cyclase systems. Incubation of frog erythrocyte membranes with cholera toxin and NAD+ did not substantially alter the dose dependency for guanine nucleotide activation of adenylate cyclase activity. In contrast, toxin treated membranes demonstrated a 10 fold increase in the concentrations of guanine nucleotide required for a half maximal effect in regulating beta-adrenergic receptor affinity for the agonist (+/-) [3H]hydroxybenzylisoproterenol. The data emphasize the bifunctional nature of the guanine nucleotide regulatory protein and suggest that distinct structural domains of the guanine nucleotide regulatory protein may mediate the distinct regulatory effects on adenylate cyclase and receptor affinity for agonists.     

Alpha-adrenergic inhibition of renal cortical adenylate cyclase

Adenylate cyclase in homogenates of rat renal cortex was inhibited by alpha-adrenergic agonists. Inhibition required sodium ion and GTP. A maximum inhibition of 17.8 +/- 1.4% (S.E.M.) was produced by l-epinephrine in the presence of 0.2 M NaCl, 10 microM GTP and 10 microM propranolol. Similar inhibition was produced by l-norepinephrine and alpha-methylnorepinephrine. The EC50 values for l-epinephrine, l-norepinephrine and alpha-methylnorepinephrine were respectively 1.9 +/- 0.7 microM, 2.3 +/- 1.6 microM and 5.1 +/- 1.8 microM. Clonidine was a partial agonist causing 50% as much inhibition as epinephrine. Phenylephrine and methoxamine did not inhibit at concentrations up to 100 microM. Micromolar concentrations of phentolamine and yohimbine prevented the inhibition of adenylate cyclase by epinephrine. However, prazosin was ineffective. Thus the adenylate cyclase coupled alpha-receptors have alpha-2 specificity. Inhibition of adenylate cyclase by alpha-adrenergic agonists was not observed in homogenates of renal medulla.     

Presence of a functional inhibitory GTP-binding regulatory component, Gi, linked to adenylate cyclase in adipocytes of ob/ob mice

It has been reported recently (Begin-Heick, N. (1985) J. Biol. Chem. 260, 6187-6193) that adipocytes from the obese mouse strain (ob/ob), unlike normal mice (+/+), lack functional Gi, a GTP-regulated protein complex that mediates inhibition of adenylate cyclase. In contrast, we have found functional Gi linked to inhibition of adenylate cyclase in adipocyte membranes from both ob/ob and +/+ mice. This conclusion is based on observation of: 1) GTP-dependent inhibition of adenylate cyclase by antilipolytic agents, such as prostaglandin E2, nicotinic acid, and the adenosine receptor agonist, phenylisopropyladenosine (PIA); 2) classical biphasic GTP kinetics, with stimulation by low and inhibition by high concentrations of GTP; and 3) elimination of cyclase inhibition by antilipolytic agents upon treatment of ob/ob adipocytes with pertussis toxin. Upon treatment with pertussis toxin and [32P] NAD, purified adipocyte membranes from ob/ob mice incorporated twice as much radioactivity per unit membrane protein than those from +/+ mice in the 40,000-42,000 region. The inhibitory actions of PIA on adenylate cyclase were blocked by the adenosine receptor antagonists, theophylline and isobutylmethylxanthine. However, in contrast to other known inhibitory adenosine receptors, relatively high (100 nM) PIA concentrations were required for half-maximal inhibition of adenylate cyclases from both +/+ and ob/ob adipocytes. The adipocyte adenylate cyclase from both mouse strains were approximately equally susceptible to inhibition by nicotinic acid and prostaglandin E2. However, the ob/ob cyclase was inhibited by 47% with PIA, whereas the enzyme from the +/+ mouse was inhibited by only 27% (p less than 0.01). This greater inhibition by adenosine may contribute to abnormal fat metabolism in adipocytes from ob/ob mice.