Dopamine-dependent cyclic AMP generating system in chick retina and its relation to melatonin biosynthesis

Stimulation of a D₄-like dopamine (DA) receptors inhibits a cAMP-dependent increase in serotonin N-acetyltransferase activity and melatonin biosynthesis in the chick retina. In order to gain more insight into the molecular mechanisms underlying this suppressive action of DA, the effects of selective stimulation of the D2-family of DA receptors (including the D₄-subtype) on cAMP formation were examined in chick retina using two experimental approaches: measurements of adenylyl cyclase activity in retinal homogenates, and cAMP accumulation in eye cup preparations prelabeled with [³H]adenine. The DA-sensitive adenylyl cyclase system is well expressed in chick retina. DA increased both basal and forskolin-stimulated adenylyl cyclase activity. This effect of DA was antagonized by SCH 23390 (a blocker of D1-family of DA receptors) and not affected by sulpiride (a D2-family blocker). Incubation of retinal homogenates with quinpirole (a predominant agonist of D₃/D₄ DA receptor subtypes) did not produce any major changes in adenylyl cyclase activity. On the other hand, activation of D₄-like DA receptor subtype by quinpirole decreased forskolin-stimulated cAMP formation in intact chick retinas maintained in “eye-cup” preparations. It is suggested that D₄-like DA receptors regulating melatonin biosynthesis in chick retina may be indirectly linked to the cAMP generating system.     

Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

Syntheses and molecular structures of 3-N,N-di-n-Propylamino-2-chromanones as new analogues of dopamine

A series of 3-N,N-di-n-propylamino-2-chromanones were synthesized as dopamine analogues. The lactone ring was introduced as a means to reduce their propensity to cross the blood-brain barrier and to avoid central side effects, rendering these compounds potentially useful for the treatment of glaucoma. Pharmacological activities were determined in vitro on rat striatum, by examining their capacity to displace the specific binding of the labeled dopaminergic ligand ³H sulpiride or ³H spiperone and ³H SCH 23390 for D₂ and D₁ sites, respectively. Compound 6a showed a weak dopaminergic activity on D₂-receptors and no affinity for D₁-receptors, which can be explained, at least in part, by a weak pKa and the presence of an internal hydrogen bonding. Furthermore, computer molecular modelling studies showed that the aromatic ring of 6a was negatively charged in contrast to the classical D₂-agonists aminotetralin derivatives, hampering a possible interaction with a negatively charged area of the D₂-receptor. These results, taken together, can account for the moderate dopaminergic activities exhibited by these lactone derivatives.     

Effect of In Vivo Modulation of Membrane Docosahexaenoic Acid Levels on the Dopamine-Dependent Adenylate Cyclase Activity in the Rat Retina

Abstract: We have studied the effect of a dietary deprivation of n-3 fatty acids on the activity of the dopamine (DA)-de-pendent adenylate cyclase in the rat retina. Experiments were conducted in 6-month-old rats raised on semipurified diets containing either safflower oil (n-3 deficient diet) or soybean oil (control diet). The levels of docosahexaenoic acid [22:6 (n-3)] in retinal phospholipids were significantly decreased in n-3 deficient rats (35–42% of control levels). This was compensated by a rise in 22:5 (n-6), the total content of poly-unsaturated fatty acids (PUFA) remaining approximately constant. Adenylate cyclase activity was measured in retinal membrane preparations from dark-adapted or light-exposed rats. The enzyme activity was stimulated by DA and SKF 38393 in a light-dependent fashion. The activation was lower in rats exposed to light than in dark-adapted animals, suggesting a down-regulation of the DI DA receptors by light. The activation by guanine nucleotides and forskolin was also decreased in light-exposed rats. There was no significant effect of the dietary regimen on the various adenylate cyclase activities and their response to light. Furthermore, the guanine nucleotide- and DA-dependent adenylate cyclase activities of retinal membranes were found to be relatively resistant to changes in membrane fluidity induced in vitro by benzyl alcohol. The results indicate that in the absence of changes in total PUFA content, a decreased ratio of n-3 to n-6 fatty acids in membrane phospholipids does not significantly affect the properties of adenylate cyclase in the rat retina.     

INTRASTRIATAL ADMINISTRATION OF AN OLIGODEOXYNUCLEOTIDE ANTISENSE TO THE D2 DOPAMINE RECEPTOR mRNA INHIBITS D2 DOPAMINE RECEPTOR-MEDIATED BEHAVIOR AND D2 DOPAMINE RECEPTORS IN NORMAL MICE AND IN MICE LESIONED WITH 6-HYDROXYDOPAMINE

Previous studies have shown that the intracerebroventricular injection of antisense oligodeoxynucleotides targeted to the mRNAs encoding the different subtypes of dopamine receptors inhibited behaviors mediated by these receptors. The present studies were designed to determine whether such antisense oligodeoxynucleotides could produce similar effects when injected into a discrete brain area. A D₂ dopamine receptor antisense oligodeoxynucleotide (D₂ antisense) was repeatedly injected into one corpus striatum of either normal mice or mice with unilateral lesions of the striatum induced by 6-hydroxydopamine. In the latter, intrastriatal injection of D₂ antisense blocked the contralateral rotational behavior induced by the parenteral administration of the D₂ dopamine receptor agonist quinpirole. The inhibitory effect of D₂ antisense was dose- and time-related and was reversed upon cessation of D₂ antisense treatment. This inhibitory effect was also selective in that D₂ antisense treatment inhibited the rotational behavior induced by quinpirole but not that induced by the D₁ dopamine receptor agonist SKF 38393 or by the muscarinic cholinergic agonist oxotremorine. Following repeated intrastriatal injections of D₂ antisense into normal mice, parenteral administration of quinpirole caused rotational behavior ipsilateral to the side in which the D₂ antisense was injected. No such rotational behavior was seen when similarly treated mice were challenged with SKF 38393 or oxotremorine. The quinpirole-induced rotational behavior in mice given intrastriatal injections of D₂ antisense disappeared upon cessation of D₂ antisense treatment. Repeated intrastriatal administration of D₂ antisense also caused a significant reduction in the levels of D₂, but not D₁, dopamine receptors in striatum, as determined by receptor autoradiography. The levels of D₂ dopamine receptors returned to normal upon cessation of D₂ antisense treatment. Intrastriatal administration of an oligodeoxynucleotide with randomly placed nucleotides failed to alter the rotational response to quinpirole in either 6-hydroxydopamine-lesioned or normal mice and failed to alter the levels of D₂ dopamine receptors in striatum. These results show that selective inhibition of behavioral responses mediated by D₂ dopamine receptors can be achieved by the direct injection of a D₂ antisense oligodeoxynucleotide into a discrete brain area. Copyright © 1996 Elsevier Science Ltd     

Forskolin Potentiates the Stimulation of Rat Striatal Adenylate Cyclase Mediated by D-1 Dopamine Receptors, Guanine Nucleotides, and Sodium Fluoride

We report here that forskolin acts in a synergistic manner with dopaminergic agonists, guanine nucleotides, or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase mediated by these reagents. In the presence of 100 microM GTP, 100 microM guanyl-5'-yl imidodiphosphate [Gpp(NH)p], or 10 mM NaF, there is a greater than additive increase in forskolin-stimulated enzyme activity as well as a concomitant decrease (two- to fourfold) in the EC50 value for forskolin stimulation of striatal enzyme activity. In the presence of various concentrations of forskolin (10 nM-100 microM), the stimulation of adenylate cyclase elicited by GTP, Gpp(NH)p, and NaF is potentiated 194-1,825%, 122-1,141%, and 208-938%, respectively, compared with the stimulation by these agents above basal activity in the absence of forskolin. With respect to 3,4-dihydroxyphenylethylamine (dopamine) receptor-mediated stimulation of striatal enzyme activity, the stimulation of enzyme activity by dopaminergic agonists, in the absence or presence of forskolin, was GTP-dependent and could be antagonized by the selective D-1 antagonist SCH23390 (100 nM), indicating that these effects are mediated by D-1 dopamine receptors. In the presence of 100 microM GTP, forskolin at various concentrations markedly potentiates the stimulation elicited by submaximal as well as a maximally effective concentrations of dopamine (100 microM) and SKF38393 (1 microM). At higher concentrations of forskolin (10-100 microM) the stimulation elicited by the partial agonist SKF38393 is comparable to that of the full agonist dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Dopamine D1 and D2 Receptors and Their Signal System Present in Coated Vesicles Prepared from Bovine Striatal Tissue

Abstract: Coated vesicles (CVs) isolated from bovine striatal tissue were examined to determine whether they are associated with dopamine signal systems consisting of dopamine D₁ and D₂ receptors, G proteins, and adenylate cyclase. Dopamine receptors in CVs were characterized by a dopamine D₁ receptor antagonist, [³H]SCH 23390, and a dopamine D₂ receptor antagonist, [³H]-spiroperidol. The bindings of both ligands were specifically saturable and reversible with a dissociation constant ( K _D) of 0.65 and 0.5 n M , respectively. Dopaminergic antagonists and agonists inhibited the specific bindings of [³H]SCH 23390 and [³H]spiroperidol in a stereoselective and concentration-dependent manner with an appropriate rank order potency for dopamine D₁ or D₂ receptors. The regulations of the agonist binding by guanyl-5-ylimidodiphosphate were observed. ADP ribosylation of the CVs with [³²P]NAD demonstrated predominant labeling of bands of M_r 47,000–52,000, 42,000–45,000, and 40,000-39,000, which corresponded to the known molecular weights of the α subunits of G_s and G_i proteins. The presence of α and β subunits of G proteins in the CVs was also confirmed by immunoblotting assay. Adenylate cyclase activity, which was stimulated by SKF 38393 and inhibited by dopamine D₂ receptor agonists, was present in the CVs. These findings suggest that the dopamine D₁ and D₂ receptors in the CVs couple with adenylate cyclase via G_s/G_i protein.     

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.     

Mouse neuroblastoma adenylate cyclase. Adenosine and adenosine analogues as potent effectors of adenylate cyclase activity.

1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity.     

Regulation of adenylate cyclase in cultured cardiocytes from neonatal rats by adenosine and other agonists

The presence of adenosine receptors coupled to adenylate cyclase in cultured cardiocytes from atria and ventricles from neonatal rats is demonstrated in these studies. N-Ethylcarboxamideadenosine (NECA), l-N⁶-phenylisopropyladenosine (PIA), and 2-chloroadenosine (2-cl-Ado) stimulated adenylate cyclase in a concentration-dependent manner in both cultured atrial and ventricular cells. The order of potency of stimulation was NECA > PIA > 2-cl-Ado. The stimulation of adenylate cyclase by NECA was enhanced by guanine nucleotides and was blocked by 3-isobutyl-1-methylxanthine in both these cells. Other agonists such as epinephrine, norepinephrine, dopamine, F^?, and forskolin were also able to stimulate adenylate cyclase, although the extent of stimulation by these agents was higher in ventricular than in atrial cells. The stimulation of adenylate cyclase by epinephrine and norepinephrine was inhibited by propranolol but not by phentolamine. On the other hand, phentolamine, propranolol, and haloperidol inhibited dopamine-stimulated adenylate cyclase activity to the same extent. Forskolin, at its maximal concentration, potentiated the stimulatory effect of epinephrine, norepinephrine, and dopamine on adenylate cyclase in both atrial and ventricular cardiocytes, but the interaction of NECA with epinephrine, norepinephrine, or dopamine was different in atrial and ventricular cells. The stimulation by an optimal concentration of NECA was additive with maximal stimulation by the catecholamines in atrial cells but not in ventricular cells. The data suggest the existence of adenosine “Ra” and catecholamine receptors in cultured atrial and ventricular cardiocytes. It can be postulated that adenosine in addition to its role as a potent vasodilator might regulate cardiac performance through its interaction with “Ra” receptors associated with adenylate cyclase. The difference in the mode of interaction of adenosine with catecholamines in atrial and ventricular cells suggests that the mechanism by which these agents activate adenylate cyclase may be different in these cells.     

Pertussis Toxin Attenuates D2 Inhibition and Enhances D1 Stimulation of Adenylate Cyclase by Dopamine in Rat Striatum

The response of adenylate cyclase to GTP and to dopamine (DA) was investigated in synaptic plasma membranes isolated from rat striatum injected with pertussis toxin, which inactivates the inhibitory guanine nucleotide-binding regulatory protein (Ni) of adenylate cyclase. Pertussis toxin treatment reverted the inhibitory effects on the enzyme activity elicited by micromolar concentrations of GTP and reduced by 50% the DA inhibition of cyclase activity via D2 receptors. The toxin treatment enhanced the net stimulation of enzyme activity by DA in the presence of micromolar concentrations of GTP. However, the stimulatory effect of the selective D1 receptor agonist SKF 38393 was not significantly affected. The data indicate that Ni mediates D2 inhibition of striatal adenylate cyclase and participates in the modulation of D1 stimulation of the enzyme activity by DA.     

Modulation of muscarinic receptor-mediated adenylate cyclase and phospholipase C responses in rat retina

1. Agonist activation of rat retina muscarinic receptors results in suppression of cyclic AMP (cAMP) generation and enhanced phosphoinositide hydrolysis. 2. Pharmacological manipulations that elevate cAMP or stable analogues of cAMP attenuate the acetylcholine (ACh)-induced enhancement of phosphoinositide hydrolysis. We postulate that cross-talk between adenylate cyclase and phospholipase C signal transducing systems probably exists in rat retina, as has been described for other systems. 3. Intraocular administration of pertussis toxin attenuated the response of both adenylate cyclase and phospholipase C to muscarinic stimulation, suggesting that some retinal muscarinic receptors are apparently coupled to their effector systems via pertussis toxin sensitive G proteins.     

Catecholamine-Sensitive Adenylate Cyclase in the White Perch (Roccus americanus) Retina: Evidence for β-Adrenergic and Dopamine Receptors Linked to Adenylate Cyclase

We have examined the catecholamine-sensitive adenylate cyclase in the retina of the white perch (Roccus americanus). Both dopamine and the beta-adrenergic agonist isoproterenol stimulate cyclic AMP accumulation in this retina, but serotonin, an indoleamine, and phenylephrine, an alpha-adrenergic agonist, had no effect. The stimulation of adenylate cyclase by isoproterenol is more potent and effective than that of dopamine. The effects of dopamine and isoproterenol are mediated via independent dopamine and beta-adrenergic receptors. Haloperidol, a dopamine antagonist, blocks the stimulatory effect of dopamine but not of isoproterenol. Conversely, propranolol, a beta-adrenergic antagonist, blocks the stimulatory effect of isoproterenol but not of dopamine. The effects of dopamine and isoproterenol are not additive. In fractions of purified horizontal cells we found evidence for dopamine receptors linked to adenylate cyclase but did not find evidence for the presence of cyclase coupled beta-adrenergic receptors. The cellular location of the beta-adrenergic receptors is unknown. Our findings demonstrate the existence of both beta-adrenergic and dopamine receptors coupled to adenylate cyclase in the white perch retina. However, we did not find either epinephrine or norepinephrine, endogenous ligands of the beta-receptor, to be present in retinal extracts subjected to HPLC.     

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.     

Studies on a Capillary-Rich Fraction Isolated from Brain: Histaminic Components and Characterization of the Histamine Receptors Linked to Adenylate Cyclase

Abstract: A fraction enriched in capillaries has been prepared from the guinea pig cerebral cortex. The purity of this fraction was checked by light- and electron-microscopic examination and by its high enrichment in alkaline phosphatase and γ-glutamyl transpeptidase. In the capillary-rich fraction, the endogenous level of histamine was 1.9%'of that measured in the initial hornogenate. The histamine-synthesizing enzyme, I-histidine decarboxylase, and the metabolizing enzyme, histamine-N-methyltransferase, were barely detectable. In addition, histamine elicits a twofold stimulation in the accumulation of cyclic AMP in this capillary fraction with an EC₅₀ of 5 γM. Agonists and antagonists of the two types of histamine receptors (H₁ and H₂) were used for the characterization of the receptors mediating this action: H₂-receptor agonists were able to activate the adenylate cyclase with "relative potencies" similar to that found on typical H₂-receptors, and cimetidine, a specific H₂-receptor antagonist, competitively inhibited the response to histamine with a K₁ value reflecting its interaction with a single population of H₂-receptors. On the contrary, data obtained with H₁-receptor agonists and antagonists reflect their interaction with H₂-receptors rather than H₁-receptors. Thus H₂-receptors are involved in the activation of adenylate cyclase of the capillary fraction.     

Accumulation of 3H-Phosphoinositides Mediated by Muscarinic Receptors in the Developing Chick Retina: Inhibition of Carbachol-Induced Response by Glutamate Receptors

Abstract: In the present work we show the development of carbachol-induced accumulation of ³H-inositol phosphates (³H-InsPs) in the chick embryonic retina and its regulation by glutamate receptors. Although basal levels of ³H-InsPs increased during development, the retinal response to carbachol was high in the early developing stages and decreased after synaptogenesis in the retina. Eserine also stimulated the turnover of phosphoinositides in the embryonic but not in the mature retina. The effect of eserine could be blocked by atropine, suggesting that acetylcholine could be released from developing retina cells and further stimulate the turnover of InsPs in the embryonic tissue. Our data also show that muscarinic stimulation of turnover of ³H-InsPs could be blocked by stimulation of glutamatergic ionotropic receptors. Moreover, the effect of glutamate agonists did not seem to be mediated by the release of other neurotransmitters such as GABA, glycine, adenosine, or dopamine from the tissue because these neurotransmitters did not interfere with the retinal response to carbachol. These results suggest that muscarinic activation of phosphoinositide turnover occurs mainly in the embryonic retina and that activation of glutamate receptors can inhibit directly the muscarinic stimulation of hydrolysis of ³H-InsPs in this tissue.     

5-HT1a receptors positively coupled to C-AMP formation in the rabbit retina

Serotonin, 8-OH-DPAT, buspirone, ipsapirone, RU-24969, dopamine and isoproterenol all stimulate C-AMP production in rabbit retinal homogenates. The dopamine and isoproterenol responses are specifically antagonized by haloperidol and propranolol, respectively. In contrast the effects produced by the other substances are specifically nullified by spiroxatrine, a known 5-HT_1a antagonist. Since 5-HT₂ antagonists (methysergide, kentanserin), 5-HT_1b antagonist (propranolol), 5-HT₄ antagonist (ICS-205930), 5-HT₃ antagonist (MDL-72222, ICS-205930) or the primarily ₂-antagonist mianserin, had no influence on the stimulation of C-AMP caused by serotonin or 8-OH-DPAT, it is concluded that 5-HT_1a receptors, positively coupled to C-AMP production exist in the retina.     

Stimulation of Distinct D2 Dopaminergic and α2-Adrenergic Receptors Induces Light-Adaptive Pigment Dispersion in Teleost Retinal Pigment Epithelium

In the retinal pigment epithelium (RPE) of lower vertebrates, melanin pigment granules aggregate and disperse in response to changes in light conditions. Pigment granules aggregate into the RPE cell body in the dark and disperse into the long apical projections in the light. Pigment granule movement retains its light sensitivity in vitro only if RPE is explanted together with neural retina. In the absence of retina, RPE pigment granules no longer move in response to light onset or offset. Using a preparation of mechanically isolated fragments of RPE from green sunfish, Lepomis cyanellus, we investigated the effects of catecholamines on pigment migration. We report here that 3,4-dihydoxyphenylethylamine (dopamine) and clonidine each mimic the effect of light in vivo by inducing pigment granule dispersion. Dopamine had a half-maximal effect at approximately 2 nM; clonidine, at 1 microM. Dopamine-induced dispersion was inhibited by the D2 dopaminergic antagonist sulpiride but not by D1 or alpha-adrenergic antagonists. Furthermore, a D2 dopaminergic agonist (LY 171555) but not a D1 dopaminergic agonist (SKF 38393) mimicked the effect of dopamine. Clonidine-induced dispersion was inhibited by the alpha 2-adrenergic antagonist yohimbine but not by sulpiride. These results suggest that teleost RPE cells possess distinct D2 dopaminergic and alpha 2-adrenergic receptors, and that stimulation of either receptor type is sufficient to induce pigment granule dispersion. In addition, forskolin, an activator of adenylate cyclase, induced pigment granule movement in the opposite direction, i.e., dark-adaptive pigment aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)