Adenylate cyclase from sea urchin eggs is positively and negatively regulated by D-1 and D-2 dopamine receptors

The adenylate cyclase present in membranes prepared from sea urchin eggs is sensitive to dopamine stimulation. The receptor sites coupled to sea urchin adenylate cyclase were characterized by means of specific agonists and antagonists. The D-1 dopamine agonist SKF-38393 was able to stimulate enzyme activity, while the two D-1 dopamine antagonists, SCH-23390 and SKF-83566, suppressed the stimulatory effect of dopamine. In addition, the D-2 dopamine agonists, PPHT and metergoline, brought about a dose-dependent inhibition of dopamine-stimulated adenylate cyclase activity. These data show that: (i) in sea urchin eggs adenylate cyclase is regulated by dopamine receptors; (ii) these receptors share characteristics with D-1 and D-2 dopamine receptors present in the mammalian brain.     

3H-SCH 23390 binding sites in the rat substantia nigra: evidence for a presynaptic localization and innervation by dopamine

Chronic treatment with SCH 23390, a selective D-1 dopamine receptor antagonist, elicited a 32% increase in the density of 3H-SCH 23390 binding sites in nigral membrane preparations but failed to change the apparent KD of the ligand for its binding sites. Haloperidol, a D-2 dopamine receptor antagonist which blocks the dopamine-sensitive adenylate cyclase and (-) sulpiride, a selective D-2 dopamine receptor blocker, which does not block the dopamine-sensitive adenylate cyclase, failed to change both the Bmax and KD of 3H-SCH 23390 binding. Finally, the intrastriatal injection of kainic acid produced a marked decrease of both GAD activity and GABA content and 3H-SCH 23390 binding sites (65%) in the homolateral substantia nigra. The results show that in the rat substantia nigra most of the 3H-SCH 23390 binding sites have a presynaptic localization on the striato-nigral GABAergic afferent terminals and suggest that dopamine released from nigral dendrites exerts a tonic influence on these presynaptic D-1 dopamine receptors.     

Evidence for the presence of both D-1 and D-2 dopamine receptors in human esophagus.

Clinical and pharmacological evidence suggested that dopamine is involved in the control of esophageal motility. The present study was designed to determine whether or not dopamine receptors are present in human esophagus. With this aim we measured adenylate cyclase activity as a biochemical index of dopamine receptor function in esophageal specimens taken from five patients during surgery for upper esophageal carcinoma. The selective D-1 agonist fenoldopam stimulated cAMP formation in the lower esophageal sphincter, but not in the esophageal body; this effect was prevented by the selective D-1 antagonist SCH 23390 and by d-butaclamol. Bromocriptine, a selective D-2 stimulator, inhibited adenylate cyclase activity in the lower esophageal sphincter, an effect blocked by the D-2 antagonist (-)sulpiride. No effects of bromocriptine were found in the esophageal body. These data indicate that both D-1 and D-2 receptors are present in the lower esophageal sphincter, but not in esophageal body and emphasize the role of dopamine in the regulation of esophageal function.     

6-Hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors

Dopamine-sensitive adenylate cyclase and 3H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and/or dendrites of dopaminergic neurons; striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers.     

Interactions Between Vasoactive Intestinal Peptide and Dopamine in the Rabbit Retina: Stimulation of a Common Adenylate Cyclase

Although 3,4-dihydroxyphenylethylamine (dopamine, DA) and vasoactive intestinal peptide (VIP) have been reported to stimulate adenylate cyclase activity in the rabbit retina, possible interactions between VIP-sensitive and DA-sensitive adenylate cyclase systems have not been previously investigated. To elucidate the interactions between these two putative transmitter-stimulated cyclase systems, the effects of VIP, DA, and VIP + DA on the conversion of [alpha-32P]ATP to [32P]cyclic AMP in rabbit retinal homogenates were measured. VIP stimulated adenylate cyclase activity in a biphasic manner, suggesting that two classes of VIP receptors may be involved in the induction of cyclic AMP formation. DA was less potent than VIP, and stimulated cyclase activity with a monophasic dose-response curve. When assayed together, these stimulations were partially nonadditive, implying the existence of a common adenylate cyclase pool that may be stimulated by both putative neurotransmitters. The dopaminergic antagonist (+)-butaclamol completely blocked dopaminergic stimulation, but had no significant effect on VIP-induced stimulation, indicating that VIP interacts with specific VIP receptor sites, which are distinct from the dopaminergic receptor sites. Furthermore, the specific D-2 dopaminergic receptor agonist LY141865 demonstrated no inhibitory effect on adenylate cyclase activity, suggesting that the interaction between the VIP- and DA-sensitive adenylate cyclase systems does not result from a D-2 receptor-mediated cyclase inhibition in the rabbit retina. Finally, at maximally effective concentrations, DA and VIP were less potent than fluoride or forskolin in the stimulation of cyclic AMP formation, suggesting that adenylate cyclase pools that are not sensitive to DA and VIP may also be present in this retina.(ABSTRACT TRUNCATED AT 250 WORDS)     

Somatostatin acts through G-proteins on dopaminergic adenylate cyclase in the caudate-putamen of the rat

Somatostatin was incubated in an adenylate cyclase assay of a particulate fraction of caudateputamen tissue of the rat in order to examine the effect of the peptide on D-1 receptor coupled adenylate cyclase in vitro. Somatostatin was able to enhance cyclic AMP formation in the presence of guanylylimidodiphosphate and guanosine-triphosphate. In contrast to this, somatostatin inhibited both dopamine and forskolin-stimulated cyclic AMP accumulation. Pertussis toxin and cholera toxin also depressed forskolin-induced stimulation. Somatostatin was found to antagonize these inhibitory effects of pertussis toxin and cholera toxin. The results suggest that somatostatin acts through a stimulatory as well as an inhibitory guanine nucleotide regulatory protein subtype to affect dopaminergic adenylate cyclase activity.     

Stimulation of brain adenylate cyclase activity by the undecapeptide substance P and its modulation by the calcium ion

Synthetic substance P stimulated adenylate cyclase activity in particulate preparations from rat and human brain.The concentration of substance P for half maximal stimulation in rat brain was 1.8 · 10⁻⁷ M.The stimulatory effect of substance P on the rat brain adenylate cyclase activity was 88% compared with 48% by noradrenalin, 163% by prostaglandin E₁ and 184% by prostaglandin E₂.Both the basal and substance P-stimulated adenylate cyclase activity in rat brain were inhibited by concentration of Ca²⁺ above 10⁻⁶ M.The chelating agent ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid at a concentration of 0.1 mM reduced the basal adenylate cyclase activity by 64% and eliminated the substance P-stimulated activity.The inhibition by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid was completely reversed by increasing concentrations of Ca²⁺.     

Vasopressin affects adenylate cyclase activity in rat brain: a possible neuromodulator

The effect of vasopressin on adenylate cyclase activity was measured in the homogenates of selected rat brain regions. Adenylate cyclase activity in homogenate of the caudate nucleus did not change significantly with various concentrations of vasopressin. Furthermore, vasopressin did not reliably alter adenylate cyclase activity in various brain regions. Vasopressin in low concentrations significantly enhanced the activation of caudate adenylate cyclase activity by dopamine. This effect of vasopressin was dose dependent. Maximal enhancement by vasopressin occurred at 100 microM vasopressin. These results indicate that vasopressin may not have a direct effect on brain adenylate cyclase activity but appears to modulate the action of dopamine on brain adenylate cyclase.     

D-1 Dopaminergic and β-Adrenergic Stimulation of Adenylate Cyclase in a Clone Derived from the Human Astrocytoma Cell Line G-CCM

Clones have been isolated from the human astrocytoma cell line G-CCM. Homogenates of clone D384 contain an adenylate cyclase that is stimulated by 3,4-dihydroxyphenylethylamine (dopamine), noradrenaline, and isoprenaline with Ka apparent values of 4, 56, and 2.7 microM, respectively. The Ka apparent value for dopamine was increased by the D-1 antagonist cis-flupenthixol, 25 and 100 nM, to 23 and 190 microM, respectively, but was unaffected by propranolol (1 microM). Noradrenaline stimulation of adenylate cyclase was only partially inhibited by either propranolol (10 microM) or cis-flupenthixol (1 microM). Propranolol (10 microM), but not cis-flupenthixol (1 microM), prevented stimulation by isoprenaline. The stimulation of adenylate cyclase by dopamine and noradrenaline remained unchanged in the presence of phentolamine (1 microM) and sulpiride (1 microM). These results suggest that clone D384 contains both D-1 dopaminergic and beta-adrenergic receptors coupled to adenylate cyclase. Dopamine stimulates D384 adenylate cyclase through D-1 receptors, isoprenaline via beta-receptors, and noradrenaline through both receptors.     

Desensitization of the D-2 dopamine receptor and the β2-adrenoceptor in the intermediate lobe of the rat pituitary gland

A D-2 dopamine receptor and a β₂-adrenoceptor occur in the intermediate lobe of the rat pituitary gland (IL). Exposure of intact IL tissue to a D-2 agonist diminished the ability of dopaminergic agonists [but not 5′-guanylyl imidodiphosphate (Gpp(NH)p)] to inhibit adenylate cyclase activity. Conversely, exposure of intact IL tissue to a β-adrenergic agonist diminished the ability of a β-adrenergic agonist (but not forskolin) to stimulate adenylate cyclase activity. Treatment of ovariectomized rats with 17β-estradiol desensitizes the β₂-adrenoceptor but not the D-2 receptor. Desensitization of the IL catecholamine receptors is discussed within the framework of a previously published “working model” of these receptors.     

Agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland

The mechanism of agonist-induced desensitization of the D-2 dopamine receptor in the intermediate lobe (IL) of the rat pituitary gland was investigated. Exposure of neurointermediate lobe to 60 microM (-)apomorphine (APO) for 60 min altered the binding of [125I]-N-(p-aminophenethyl)spiperone (NAPS), a D-2 receptor-specific ligand. The capacity of the tissue to bind the ligand (Bmax) was not significantly altered by the exposure to (-)APO but the affinity for [125I]NAPS was decreased 3.6-fold in (-)APO-exposed tissue. The molar potency of YM-09151-2, a D-2 receptor-specific antagonist, showed a minimal difference between in control and (-)-APO-exposed tissue. However, the molar potency of (-)APO towards the D-2 receptor was diminished. The loss of [125I]NAPS binding in (-)APO-exposed tissue was reversed by the addition of guanyl nucleotide. These data suggest that exposure to agonist causes a persistent occupancy of the high affinity state of the receptor. Exposure to (-)APO had no effect on either basal or forskolin-activated adenylate cyclase activity of the intermediate lobe. However, the inhibitory effect of (-)APO upon adenylate cyclase activity of IL homogenates was diminished when the tissue was exposed to (-)APO before homogenization. Furthermore, the ability of GTP but not 5'-guanylyl imidodiphosphate [Gpp(NH)p] to inhibit enzyme activity diminished in the (-)APO-exposed tissue. These data suggest that an agonist-induced desensitization of D-2 receptor in rat IL is thought to occur by uncoupling the receptor from the inhibitory guanyl nucleotide binding protein (Gi) or potentiating the hydrolysis of GTP by Gi.     

Iron inhibits D-1 dopamine receptor coupled adenylate cyclase via G-proteins in the caudate nucleus of the rat.

The effects of iron ions (Fe(II)sulfate) on basal, forskolin, and dopamine-stimulated activity of adenylate cyclase in membrane preparations from caudate-putamen of the rat have been studied. Iron dose-dependently inhibited both basal and activated adenylate cyclase activity. In contrast to guanylylimidodiphosphate (Gpp(NH)p), guanosine triphosphate (GTP) was found to enhance this inhibitory effect of iron ions. In addition, cholera toxin was able to antagonize the inhibitory effect of iron on forskolin-activated adenylate cyclase. In our preliminary study we suggest an interaction between iron and the guanine nucleotide regulatory subunit. However, further studies are necessary.     

Regulation of agonist and antagonist binding to striatal D-1 dopamine receptors: studies using the selective D-1 antagonist [3H]SK&F R-83566

The potent and D-1 versus D-2 selective dopamine receptor antagonist, SK&F R-83566, was radiolabelled with tritium and was used as a radioligand for examination of D-1 receptors in rat striatum. Binding of the radioligand was stereoselective, saturable and reversible. In homogenates of rat striatum, nonspecific binding of the radioligand was less than 5% of total binding, the KD was 1.1 +/- 0.2 nM and the Bmax was 1130 +/- 70 fmoles/mg protein. Results of competition binding analyses yielded a pharmacological profile that was characteristic of dopamine D-1 receptor interaction. Competition studies of dopamine agonists against the potent antagonist radioligand indicated multiple affinities of agonist binding to the D-1 receptor. Displacement was best fit to a two-site model of ligand binding and high and low affinities were subject to regulation by guanine, but not adenine, nucleotides. Antagonist binding was not complex and was unaffected by guanine nucleotides. The role of monovalent cations in regulating D-1 receptor binding was evaluated by comparing effects of Na+, Li+, and K+ on binding of the antagonist [3H]SK&F R-83566 and the agonist [3H]fenoldopam (SK&F 82526). Whereas agonist binding was reduced in a concentration dependent fashion by monovalent cations with a ranking of potency Li+ greater than Na+ greater than K+, antagonist binding was enhanced by the cation Na+ but little affected by Li+ or K+. This effect of relatively low concentrations of Na+ to decrease agonist binding and increase antagonist binding suggests similarities between the D-1 receptor which is positively-coupled to adenylate cyclase and other receptors, e.g. alpha 2 adrenergic receptors, which are negatively-coupled to adenylate cyclase.     

Sulfhydryl group(s) in the ligand binding site of the D-1 dopamine receptor: specific protection by agonist and antagonist

An iodinated compound, [125I]-8-iodo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin -7-ol, has been recently reported [Sidhu, A., & Kebabian, J.W. (1985) Eur. J. Pharmacol. 113, 437-440] to be a specific ligand for the D-1 dopamine receptor. Due to its high affinity and specific activity, this ligand was chosen for the biochemical characterization of the D-1 receptor. Alkylation of particulate fractions of rat caudate nucleus by N-ethylmaleimide (NEM) caused an inactivation of the D-1 receptor, as measured by diminished binding of the radioligand to the receptor. The inactivation of the receptor sites by NEM was rapid and irreversible, resulting in a 70% net loss of binding sites. On the basis of Scatchard analysis of binding to NEM-treated tissue, the loss in binding sites was due to a net decrease in the receptor number with a 2-fold decrease in the affinity of the receptor for the radioligand. Receptor occupancy by either a D-1 specific agonist or antagonist protected the ligand binding sites from NEM-mediated inactivation. NEM treatment of the receptor in the absence or presence of protective compound abolished the agonist high-affinity state of the receptor as well as membrane adenylate cyclase activity. The above-treated striatal membranes were fused with HeLa membranes and assayed for dopamine-stimulated adenylate cyclase activity. When the sources of D-1 receptors were from agonist-protected membranes, the receptors retained their ability to functionally couple to the HeLa adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of isomers of apomorphines on dopamine receptors in striatal and limbic tissue of rat brain

The optical isomers of apomorphine (APO) and N-propylnorapomorphine (NPA) were interacted with three biochemical indices of dopamine (DA) receptors in extrapyramidal and limbic preparations of rat brain tissue. There were consistent isomeric preferences for the R(-) configuration of both DA analogs in stimulating adenylate cyclase (D-1 sites) and in competing for high affinity binding of 3H-spiroperidol (D-2 sites) and of 3H-ADTN (DA agonist binding sites) in striatal tissue, with lesser isomeric differences in the limbic tissue. The S(+) apomorphines did not inhibit stimulation of adenylate cyclase by DA. The tendency for greater activity or higher apparent affinity of R(-) apomorphines in striatum may reflect the evidently greater abundance of receptor sites in that region. There were only small regional differences in interactions of the apomorphine isomers with all three receptor sites, except for a strong preference of (-)NPA for striatal D-2 sites. These results do not parallel our recent observations indicating potent and selective antidopaminergic actions of S(+) apomorphines in the rat limbic system. They suggest caution in assuming close parallels between current biochemical and functional, especially behavioral, methods of evaluating dopamine receptors of mammalian brain.     

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.     

Heterologous desensitization by 1,25-dihydroxyvitamin D-3 of cyclic AMP response to parathyroid hormone in osteoblast-like cells and the role of the stimulatory guanine nucleotide regulatory protein

The influence of 1,25-dihydroxyvitamin D-3 on the cAMP response to parathyroid hormone was studied in the osteoblast-like rat osteosarcoma cells ROS 17/2.8. The stimulation by parathyroid hormone of cAMP production in intact cells and of adenylate cyclase activity in isolated plasma membranes was attenuated by 1,25-dihydroxyvitamin D-3 treatment. This was associated with a reduction of the stimulatory guanine nucleotide regulatory protein, as demonstrated by a lower response to NaF and guanosine 5'-[beta, gamma-imido]triphosphate, and by a lower activity of solubilized plasma membrane extracts in the reconstitution assay. 1,25-dihydroxyvitamin D-3 blunted also the cAMP response to parathyroid hormone in cells incubated with the glucocorticoid dexamethasone, where a higher activity of the adenylate cyclase catalytic unit was observed. Thus, the two steroids appear to affect distinct levels of the adenylate cyclase system. Furthermore, the two hormones also showed an antagonistic effect upon the production of osteocalcin, an osteoblast-specific extracellular matrix protein. The release of this non-collagenous matrix protein by ROS 17/2.8 cells was increased by 1,25-dihydroxyvitamin D-3 and decreased by dexamethasone.     

Specificity of the dopamine sensitive adenylate cyclase for antipsychotic antagonists

Chlorpromazine, haloperidol and clozapine are approximately equipotent in antagonizing dopamine sensitive adenylate cyclase activity in homogenates of rat brain striatum, in contrast to the differences in clinical antipsychotic potencies reported by others. The antagonism appeared to occur at a structurally specific dopamine site, as inhibition by a series of chlorpromazine analogues of similar hydrophobicity exhibited a structural specificity similar to that found for their neuroleptic and cataleptic activities. Sulpiride, a dopamine antagonist with antipsychotic activity, and metoclopramide, a structurally related central dopamine antagonist, failed to inhibit the dopamine sensitive adenylate cyclase. Pre-treatment of rats with haloperidol (3 mg/kg per day) for 6 or 28 days did not induce a supersensitive response of the adenylate cyclase to stimulation by dopamine or apomorphine or inhibition by clozapine. It was concluded that the dopamine sensitive adenylate cyclase may not be the site of action of all anti-psychotic agents.     

Activation of adenylate cyclase from rat striatum and tuberculum olfactorium by adenosine

Adenosine caused a dose-dependent stimulation of adenylate cyclase in homogenates from rat striatum and tuberculum olfactorium (200 and 300% stimulation by 100 muM adenosine). The effect of adenosine was not antagonized by haloperidol. Subcellular fractionation suggested that adenosine stimulates a different adenylate cyclase than dopamine. Basal adenylate cyclase activity in freshly prepared homogenates was reduced by dialysis and by the addition of adenosine deaminase. Basal adenylate cyclase activity was enchanced by papaverine and dipyridamole, but reduced by theophylline and isobutylmethylxanthine. The results are compatible with the opinion that endogenous adenosine is capable of activating adenylate cyclase in these areas of the rat brain.     

Adenylate Cyclase Activity in the Superior Cervical Ganglion of the Rat

Abstract: Adenylate cyclase activity in cell-free homogenates of the rat superior cervical ganglion (SCG) was assayed under a variety of experimental conditions. Adenylate cyclase activity was decreased by approximately one-half when 1 m M EGTA was included in the homogenization buffer and assay mixture, indicating the presence of a Ca²⁺-sensitive adenylate cyclase in the ganglion. In the presence of EGTA, basal adenylate cyclase activity in homogenates of the SCG was 12.9 ± 0.6 pmol cyclic AMP/ganglion/10 min. Enzyme activity was stimulated three- to fourfold by 10 m M NaF or 10 m M MnCl₂, Both GTP and its nonhydrolyzable analog guanylylimidodiphosphate (GppNHp) stimulated adenylate cyclase in a concentration-dependent manner over the range of 0.1–10.0 μ M . Stimulation by GppNHp was five to six times greater than that produced by GTP at all concentrations tested. Decentralization of the ganglion had no effect on basal or stimulated adenylate cyclase activity. Receptor-linked stimulation of adenylate cyclase was not obtained with any of the following: isoproterenol, epi-nephrine, histamine, dopamine, prostaglandin E₂, or va-soactive intestinal peptide. Thus the receptor-linked regulation of adenylate cyclase activity appears to be lost in homogenates of the ganglion.