Receptor-stimulated Adenylate Cyclase Research Articles

μ‐Opioid Receptors Mediate the Inhibitory Effect of Opioids on Dopamine‐Sensitive Adenylate Cyclase in Primary Cultures of Rat Neostriatal Neurons

The receptors mediating the inhibition of D1 dopamine receptor-stimulated adenylate cyclase by opioids were examined in primary cultures of rat neostriatal neurons. Adenylate cyclase activity was dose-dependently increased by the selective D1 dopamine receptor agonist SKF 38393 (EC50 = 0.05 microM). This stimulation was fully antagonized by the selective D1 dopamine receptor antagonist SCH 23390 (1 microM). SKF 38393 (1 microM)-stimulated adenylate cyclase activity was strongly reduced (by almost 60%) by the highly selective mu-agonist [D-Ala2, MePhe4, Gly-ol5]-enkephalin (DAGO; EC50 = 0.006 microM) and high concentrations of the selective delta-agonist [D-Ser2(O-tert-butyl), Leu5]-enkephalyl-Thr6 (DSTBU-LET; EC50 = 0.13 microM) but not by the selective delta-agonist [D-penicillamine2, D-penicillamine5]enkephalin (DPDPE). D1 dopamine receptor-stimulated adenylate cyclase activity was also slightly reduced (by approximately 20%) by high concentrations of the kappa-agonist U50,488 (EC50 = 0.63 microM). The inhibitory effects of submaximally effective concentrations of DAGO, DSTBULET, and U50,488 were equally well antagonized by the mu-opioid receptor-selective antagonist naloxone (EC50 of approximately 0.1 microM). Neither the irreversible delta-ligand fentanyl isothiocyanate (1 microM) nor the reversible delta-antagonist ICI 174864 (1 microM) reversed the inhibitory effects of DSTBULET. The inhibitory effects of DAGO and U50,488 were equally well reversed by high concentrations (greater than 0.1 microM) of the kappa-opioid receptor-selective antagonist norbinaltorphimine. The effect of DAGO (1 microM) was already detectable after 1 day in culture, whereas DPDPE (1 microM) had no effect even after 28 days in culture. These data indicate that an homogeneous population of mu-opioid receptors coupled as inhibitors to D1 dopamine receptor-stimulated adenylate cyclase is expressed in rat neostriatal neurons in primary culture.

Ca2+‐Independent Stimulation of Adenylate Cyclase Activity by Muscarinic Receptors in Rat Olfactory Bulb

In rat olfactory bulb homogenate, carbachol stimulated adenylate cyclase activity in a concentration-dependent manner (EC50 = 1.1 microM). The carbachol stimulation occurred fully in membranes that had been prepared in the presence of 1 mM EGTA and incubated in a Ca2(+)-free enzyme reaction medium. Under these conditions, exogenous calmodulin (1 microM) failed to stimulate adenylate cyclase activity. In miniprisms of olfactory bulb, carbachol (1 mM) increased accumulation of inositol phosphates, but this response was markedly reduced in a Ca2(+)-free medium. Moreover, the carbachol stimulation of adenylate cyclase activity was not affected by staurosporine at a concentration (1 microM) that completely blocked the stimulatory effect of phorbol 12-myristate 13-acetate, an activator of Ca2+/phospholipid-dependent protein kinase. Quinacrine, a nonselective phospholipase A2 inhibitor, reduced the carbachol stimulation of adenylate cyclase activity, but this inhibition appeared to be competitive with a Ki of 0.2 microM. Nordihydroguaiaretic acid and indomethacin, two inhibitors of arachidonic acid metabolism, failed to affect the carbachol response. These results indicate that in rat olfactory bulb, muscarinic receptors stimulate adenylate cyclase activity through a mechanism that is independent of Ca2+ and phospholipid hydrolysis.

Ontogeny of μ-, δ- and κ-opioid receptors mediating inhibition of neurotransmitter release and adenylate cyclase activity in rat brain

The ontogeny was examined of functional opioid receptors mediating presynaptic inhibition of neurotransmitter release and inhibition of dopamine (DA)-sensitive adenylate cyclase in the rat brain, using highly selective agonists for μ-, δ- and κ-receptors. On gestational day 17 (E17) strong inhibitory effects of the selective μ-agonist DAGO on the electrically evoked release of [ 3H] noradrenaline from cortical slices and of the selective κ-agonist U-50,488 on the electrically evoked release of [ 3H] DA from striatal slices were found. Electrically evoked release of [ 3H] acetylcholine from striatal slices was not detectable before postnatal day 7 (P7), but on that day it was already strongly inhibited by the selective δ-agonist DPDPE. Although μ- and δ-opioid receptors coupled to DA-sensitive adenylate cyclase in the striatum are likely to be physically associated in an opioid receptor complex in the adult, they were found to develop asynchronously. Whereas selective activation of μ-receptors with DAGO resulted in an inhibition of D 1 dopamine receptor-stimulated adenylate cyclase activity on E17, activation of δ-receptors with DPDPE was not effective until P14. This study confirms the early appearance of μ- and κ-opioid receptors and the relatively late development of δ-opioid receptors in the rat brain. Most importantly, it shows that in an early stage of development opioids are already able to mediate modulation of noradrenergic (via activation of μ-receptors) and dopaminergic (via activation of μ- and κ-receptors) neurotransmission processes. Therefore, these opioid receptor types could play a role in brain development and/or developmental disturbances.

Intrinsic sympathomimetic activity of β-adrenoceptor antagonists: down-regulation of cardiac β1- and β2-adrenoceptors

Prolonged treatment of cultured rat heart muscle cells containing β 1- and non-muscle cells containing β 2-adrenoceptors with β-adrenoceptor antagonists devoid of intrinsic sympathomimetic activity had no effect on β-adrenoceptor density. In contrast, antagonists with intrinsic sympathomimetic activity decreased β-adrenoceptor density and response (adenylate cyclase stimulation) in both heart muscle ( β 1) and non-muscle cells ( β 2) by a maximum of about 50%. An even larger down-regulation of β-adrenoceptors and loss of receptor-stimulated adenylate cyclase activity was induced by the full endogenous agonist, noradrenaline, with the β-adrenoceptors of heart muscle cells ( β 1) being much more sensitive to the β 1-selective noradrenaline than the heart non-muscle cell β 2-adrenoceptors. When combined with noradrenaline, the antagonists with intrinsic sympathomimetic activity prevented the action of noradrenaline at both β 1- and β 2-adrenoceptors, thereby leading to an apparent up-regulation of receptor density and response. This apparent reversal from an agonist to an antagonist action was observed at much lower concentrations of noradrenaline at β 1- than at β 2-adrenoceptors. The data presented indicate that the β-adrenoceptor antagonists with intrinsic sympathomimetic activity, but not those without, upon prolonged treatment decrease the density and responsiveness of both β 1- and β 2-adrenoceptors in cultured rat heart cells. This suggests that the intrinsic sympathomimetic activity of these agents is not a subtype-selective component. Furthermore, the agonist and antagonist activity of these agents apparently depends on the concomitant presence of an endogenous full agonist and an its own affinity and that of the partial agonist for the β-adrenoceptor subtype.

Beta-adrenergic receptor number and adenylate cyclase function in denervated transplanted and cardiomyopathic human hearts.

To test the hypothesis that there is up-regulation of beta-adrenergic receptor density or supersensitivity of beta-adrenergic receptor-stimulated adenylate cyclase in the denervated transplanted human heart, we studied myocardium from transplanted, normal, and failing hearts. Myocardium was obtained from 10 patients 9 +/- 3 months after cardiac transplantation, from 10 patients without cardiac disease, and from eight patients with symptomatic congestive heart failure due to idiopathic cardiomyopathy. beta-Adrenergic receptor density in transplanted myocardium (15 +/- 3 fmol/mg protein, 1.20 +/- 0.14 fmol/mg DNA) was not different from that in normal myocardium (22 +/- 3 fmol/mg protein, 1.46 +/- 0.13 fmol/mg DNA; p = NS for both). In myocardium from cardiomyopathic hearts, beta-adrenergic receptor density was markedly reduced (8 +/- 2 fmol/mg protein, 0.84 +/- 0.13 fmol/mg DNA; p less than 0.05 and p less than 0.01 vs. normal myocardium, respectively). Likewise, the response of adenylate cyclase to isoproterenol in transplanted myocardium was not significantly different from that in normal myocardium, but the response was markedly depressed in cardiomyopathic myocardium. Although forskolin-stimulated adenylate cyclase activity was similar in all three groups, guanine nucleotide-stimulated adenylate cyclase activity was markedly reduced in transplanted myocardium (20 +/- 17 vs. 78 +/- 13 pmol/mg/min for normal myocardium, p less than 0.01) and to a lesser degree in cardiomyopathic myocardium (39 +/- 14 pmol/mg/min, p less than 0.03 vs. normal myocardium). Thus, there is no evidence of beta-adrenergic receptor up-regulation or supersensitivity in denervated transplanted human myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

The rapid desensitization of glucagon-stimulated adenylate cyclase is a cyclic AMP-independent process that can be mimicked by hormones which stimulate inositol phospholipid metabolism.

Treatment of intact hepatocytes with glucagon, TH-glucagon [( 1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), angiotensin or vasopressin led to a rapid time- and dose-dependent loss of the glucagon-stimulated response of the adenylate cyclase activity seen in membrane fractions isolated from these cells. Intracellular cyclic AMP concentrations were only elevated with glucagon. All ligands were capable of causing both desensitization/loss of glucagon-stimulated adenylate cyclase activity and stimulation of inositol phospholipid metabolism in the intact hepatocytes. Maximally effective doses of angiotensin precluded any further inhibition/desensitizing action when either glucagon or TH-glucagon was subsequently added to these intact cells, as has been shown previously for the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) [Heyworth, Wilson, Gawler & Houslay (1985) FEBS Lett. 187, 196-200]. Treatment of intact hepatocytes with these various ligands caused a selective loss of the glucagon-stimulated adenylate cyclase activity in a washed membrane fraction and did not alter the basal, GTP-, NaF- and forskolin-stimulated responses. Angiotensin failed to inhibit glucagon-stimulated adenylate cyclase activity when added directly to a washed membrane fraction from control cells. Glucagon GR2 receptor-stimulated adenylate cyclase is suggested to undergo desensitization/uncoupling through a cyclic AMP-independent process, which involves the stimulation of inositol phospholipid metabolism by glucagon acting through GR1 receptors. This action can be mimicked by other hormones which act on the liver to stimulate inositol phospholipid metabolism. As the phorbol ester TPA also mimics this process, it is proposed that protein kinase C activation plays a pivotal role in the molecular mechanism of desensitization of glucagon-stimulated adenylate cyclase. The site of the lesion in desensitization is shown to be at the level of coupling between the glucagon receptor and the stimulatory guanine nucleotide regulatory protein Gs, and it is suggested that one or both of these components may provide a target for phosphorylation by protein kinase C.

Inhibition of dopamine-sensitive adenylate cyclase by opioids: possible involvement of physically associated mu- and delta-opioid receptors.

D-1 dopamine receptor-stimulated cyclic AMP efflux from rat neostriatal slices (induced by 30 microM dopamine + 10 microM (-)sulpiride) was concentration-dependently reduced by morphine, [D-Ala-D-Leu]-enkephalin (DADLE), [D-Pen-D-Pen]enkephalin (DPDPE) and bremazocine. Naloxone (0.1 microM) selectively antagonized the inhibitory effect of (a submaximally effective concentration of) morphine, whereas ICI 174864 (0.75 microM) completely blocked the inhibitory effects of DADLE, DPDPE and bremazocine without affecting that of morphine, indicating a role of mu- as well as delta-opioid receptors. Upon simultaneous activation of D-1 dopamine receptors and delta-opioid receptors the (mu-receptor-mediated) inhibitory effect of morphine was abolished, while it was not changed following simultaneous activation of D-1 and (inhibitory) D-2 dopamine receptors. Cyclic AMP efflux induced by isoprenaline or adenosine was not affected by the opioids and that induced by vasoactive intestinal peptide (VIP) was inhibited by morphine and DADLE only. In the latter case naloxone, but not ICI 174864, antagonized the inhibitory effects. These data show that D-1 dopamine receptor-stimulated adenylate cyclase activity in rat neostriatum, but not that stimulated through other receptors, is inhibited by two pharmacologically distinct opioid receptor subtypes. It is speculated that these mu- and delta-opioid receptors share a common inhibitory guanine nucleotide binding protein and may represent closely associated recognition sites of a functional opioid receptor complex.

P-24 - Desensitization of Renal Vasopressin Receptor and Vasopressin Stimulated Adenylate Cyclase Following DDAVP Administration In Vivo. Partial Prevention of Desensitization by A Vasopressin Antagonist, d(CH2)5Tyr(Me)AVP

P-24 - Desensitization of Renal Vasopressin Receptor and Vasopressin Stimulated Adenylate Cyclase Following DDAVP Administration In Vivo. Partial Prevention of Desensitization by A Vasopressin Antagonist, d(CH2)5Tyr(Me)AVP

Biochemical and behavioral studies of D1 dopamine receptors utilizing SCH 23390.

Previous research has demonstrated the existence of two distinct dopamine receptor subtypes (Kebabian & Calne, 1979; Creese et al., 1983), possessing unique pharmacologic and biochemical properties. D1 dopamine receptors stimulate adenylate cyclase activity (Hyttel, 1978), while D2 dopamine receptors inhibit this enzyme (Stoof and Kebabian, 1981; Onali et al., 1984; Battaglia et al., 1985). However, both receptor subtypes co-exist in many tissues making the determination of their respective physiological and behavioral roles difficult. All neuroleptics, commonly used drugs in the treatment of schizophrenia, have been shown to be either mixed D1/D2 dopamine receptor antagonists or selective D2 dopamine receptor antagonists. Thus, D2 dopamine receptors have been implicated as the site mediating the antipsychotic and antidopaminergic activity of neuroleptics (Creese et al., 1976; Seeman et al., 1976). By inference, D2 dopamine receptors have been considered to mediate dopaminergic agonists’ behavioral effects as well (Seeman, 1981).

Inhibitory adenosine receptors in the heart: Characterization by ligand binding studies and effects on β-adrenergic receptor stimulated adenylate cyclase and membrane protein phosphorylation

Adenosine causes negative chronotropic and inotropic effects on cardiac tissue. We have investigated the nature of the cardiac adenosine receptor and its effector mechanisms in preparations of newborn chick heart. The adenosine analog [3H]N6 (L-phenylisopropyl) adenosine (L-PIA), an agonist at R-type adenosine receptors, bound with high affinity to receptors in crude and highly-purified membrane preparations. The KD was 3-5 nM. The receptor density was low in crude membranes (10 fmol/mg protein) but significantly enriched in purified sarcolemma (164 fmol/mg protein). Competition studies showed that N-ethylcarboxamide adenosine and N6(D-phenylisopropyl)adenosine were less potent than N6(L-phenylisopropyl)adenosine at the chick heart adenosine receptor, as expected for an Ri-type adenosine receptor. Gpp(NH)p decreased the binding of [3H]N6(L-phenylisopropyl)adenosine to chick heart membranes, suggesting that the guanine nucleotide converted the receptor to a lower affinity state. N6(L-phenylisopropyl)adenosine inhibited beta-adrenergic receptor stimulated adenylate cyclase activity. The IC50 for cyclase attenuation by N6(L-phenylisopropyl)adenosine was 1 microM. N6(L-phenylisopropyl)adenosine reversed the effect of the beta-receptor agonist isoproterenol on phospholamban phosphorylation in 32P-labelled slices of newborn chick hearts. This effect of N6(L-phenylisopropyl)adenosine was evident by 2 min, had an IC50 of 200 nM, and was prevented by the adenosine receptor antagonist 8-phenyltheophylline. Taken together, the results suggest that the antiadrenergic effects of adenosine on cardiac tissue are mediated by a decrease in membrane protein phosphorylation signalled by activation of Ri-adenosine receptors. The coupling mechanism between receptor activation and protein phosphorylation may be an attenuation of adenylate cyclase.

Regulation of dopamine- and adenosine-dependent adenylate cyclase systems of chicken embryo retina cells in culture.

We have obtained evidence that receptor-stimulated adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] is regulated physiologically in both embryonic and mature neurons. In a series of experiments using cultured retina cells from chicken embryos, we found that dopamine-sensitive adenylate cyclase activity spontaneously desensitized as cultures differentiated. The cellular response to dopamine reached a maximum after 5 days in culture and then decreased to 40% during the next 5 days. This spontaneous desensitization appeared to be caused by functional dopaminergic transmission because it could be blocked by the dopamine antagonist haloperidol. The ability of added dopamine at 100 microM to cause near-complete desensitization is consistent with this conclusion. Pharmacologically induced desensitization required 31 hr for maximal effect and was half-maximal at 1-10 microM dopamine. Analogous desensitization of the adenosine-dependent adenylate cyclase system also was noted. When dopamine was removed from the medium of chronically treated cultures, cells resensitized to subsequent stimulation at a very slow rate. Resensitization likely depended on replacement of dopamine receptors because chronic dopamine treatment caused the disappearance of binding sites for the ligand [3H]spiroperidol. In a second series of experiments, using hatched animals, we found that similar regulation of dopamine receptor binding sites and activity could be elicited by manipulation of environmental light, a treatment thought to influence dopaminergic transmission. Retinas from animals in constant light had less specific [3H]spiroperidol binding (35 fmol/mg of protein) than did retinas from animals in constant darkness (66 fmol/mg of protein) and made less cAMP in response to added dopamine. Our results indicate that regulation of the dopamine receptor system begins early in development and continues to function in mature synapses.

Antibodies raised against β-adrenergic receptors stimulate adenylate cyclase

Antibodies raised in mice against β-adrenergic receptors purified from turkey erythrocyte membranes, specifically bind to cells which possess a β-adrenergic receptor and immunoprecipitate radiolabelled purified receptor. These antibodies stimulate the adenylate cyclase activity of the turkey erythrocytes, although they do not compete with the catecholamine hormones for binding to the β-adrenergic receptor. Thus the receptor-antibody interaction, although occuring at another site than the receptor-hormone interaction, may still trigger the enzymatic activity.