Cyclic AMP Stimulates Serotonin N-Acetyltransferase Activity in Xenopus Retina In Vitro

The possible involvement of cyclic AMP in the regulation of retinal serotonin N-acetyltransferase (NAT) activity was investigated using eye cups of Xenopus laevis cultured in a defined medium. Addition of dibutyrylcyclic AMP (dbcAMP) increased retinal NAT activity in eye cups cultured in light. Addition of adenosine or 5'-AMP under identical conditions was without effect. 3-Isobutylmethylxanthine (IBMX) increased both retinal cyclic AMP levels and NAT activity in light-exposed eye cups. Forskolin also increased the concentration of cyclic AMP and the activity of NAT, and the effect of forskolin on both of these parameters was synergistically enhanced by IBMX. The effects of forskolin and of dbcAMP did not require the addition of calcium to the medium; thus, Ca2+ -dependent synaptic transmission does not appear to be required for the response to these drugs. Incubation conditions that activate cyclic AMP-dependent protein kinase in retinal homogenates had no effect on NAT activity, suggesting that direct phosphorylation of NAT was probably not involved in the response to elevating cyclic AMP in situ. The effect of dbcAMP was blocked by protein synthesis inhibitors. These results suggest that cyclic AMP increases retinal NAT activity by a mechanism that involves protein synthesis, and support a role for cyclic AMP in the nocturnal increase of NAT activity in darkness.     

Diurnal Variations in Cyclic AMP and Melatonin Content of Golden Hamster Retina

Abstract: The diurnal variations and photic regulation of cyclic AMP and melatonin content in golden hamster retina were studied. Both parameters showed significant diurnal variations with maximal values at night. Light exposure during the night inhibited retinal cyclic AMP and melatonin levels, whereas exposure to darkness during the day significantly increased cyclic AMP and melatonin content. Incubation with melatonin of retinas excised at different intervals indicated that the methoxyindole inhibited cyclic AMP accumulation in a time-dependent manner. The inhibitory effect of melatonin at 2400 h and at noon showed a threshold concentration of 1 and 10 pM, respectively. At 0400 h melatonin did not affect cyclic AMP accumulation. The results indicate a diurnal variability of retinal cyclic AMP and melatonin content in hamsters, mainly influenced by a photic stimulus. Cyclic AMP could be a putative second messenger for melatonin action in golden hamster retina.     

Effects of Vasoactive Intestinal Peptide and Other Peptides on Cyclic AMP Accumulation in Intact Pieces and Isolated Horizontal Cells of the Teleost Retina

The effects of vasoactive intestinal peptide (VIP) and several other peptides have been examined on cyclic AMP accumulation in intact pieces and isolated horizontal cells of the teleost (carp) retina. VIP was the most effective peptide examined, inducing a dose-related response, and an approximately fivefold increase in cyclic AMP production when used at a concentration of 10 microM. Porcine histidine isoleucine-containing peptide and secretin, peptides structurally related to VIP, also stimulated cyclic AMP accumulation, but at concentrations of 10 microM induced responses which were only approximately 40% and 10%, respectively, of the response observed with 10 microM VIP. In contrast, several other peptides, including glucagon, neurotensin, somatostatin, luteinizing hormone-releasing hormone, alpha-melanocyte-stimulating hormone, cholecystokinin octapeptide26-33, gastrin-releasing peptide, thyrotropin-releasing hormone, and VIP10-28 were totally inactive. The response to 10 microM VIP was not antagonized by several dopamine antagonists, indicating the presence of a population of specific VIP receptors coupled to adenylate cyclase, distinct from the population of dopamine receptors coupled to adenylate cyclase also known to be present in this tissue. Finally, experiments involving the use of fractions of isolated horizontal cells indicate that these neurons possess a population of VIP receptors coupled to cyclic AMP production which would appear to share a common pool of adenylate cyclase with a population of similarly coupled dopamine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Melatonin Receptor-Mediated Inhibition of Cyclic AMP Accumulation in Chick Retinal Cell Cultures

Abstract: Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina. Cultured cells contained high-affinity 2-[¹²⁵I]iodomelatonin binding sites (K_D = 41.6 pM), similar to those in intact retina. The effects of melatonin and related indoles on cyclic AMP accumulation were examined. Melatonin (10^?7M) had no effect on basal or K⁺-stimulated cyclic AMP accumulation, but inhibited forskolin-stimulated cyclic AMP accumulation by approximately 50%. Melatonin inhibited forskolin-stimulated cyclic AMP accumulation in the presence or absence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, suggesting an effect on cyclic AMP synthesis rather than degradation. Half-maximal inhibition was observed at 5.9 × 10^?10M melatonin. The relative order of potency among melatonin analogues was 2-iodomelatonin > melatonin ≈ 6-chloromelatonin ≥ 6-hydroxymelatonin > N-acetylserotonin ≈ 5-methoxytryptophol > serotonin. The EC₅₀ value for inhibition of cyclic AMP accumulation by 2-iodomelatonin (36.7 pM) was comparable to the K_D value for binding of the radioligand, suggesting that the binding sites represent functional receptors. The inhibitory effect of melatonin was antagonized by the putative melatonin antagonists luzindole, N-acetyltryptamine, and N-(2,4-dinitrophenyl)-5-methoxytryptamine, with estimated K_B values of 0.12, 0.17, and 1 µM, respectively. At a concentration of 10 µM, N-(2,4-dinitrophenyl)-5-methoxytryptamine significantly inhibited forskolin-stimulated cyclic AMP accumulation when added alone; at 30 µM, luzindole and N-acetyltryptamine also had significant inhibitory effects. The inhibitory effect of melatonin was blocked by pretreatment with pertussis toxin. The results of this study indicate that melatonin receptors on retinal cells are coupled via inhibitory G proteins to cyclic AMP accumulation. Thus, some of the effects of melatonin on retinal physiology may be related to regulation of cyclic nucleotide metabolism.     

Increased Levels of 3'',5''-Cyclic Adenosine Monophosphate Induced by Cobaltous Ion or 3-Isobutylmethylxanthine in the Incubated Mouse Retina: Evidence Concerning Location and Response to Ions and Light

Dark levels of 3',5'-cyclic adenosine monophosphate (cyclic AMP) of mouse retinas incubated in Earle's medium were elevated by 3-isobutyl-methylxanthine (IBMX) and/or Co2+ or Mn2+, but not by Cd2+, methylverapamil, or excess Mg2+ of Ca2+. Light reduced elevated dark levels of cyclic AMP in the presence of agents known to block the light modulation of post-receptoral neurons (aspartate, Co2+, high Mg2+), a finding consistent with a cyclic AMP metabolism in photoreceptors. Co2+-elevated cyclic AMP levels were not less light-sensitive than cyclic GMP levels. Ouabain substantially increased IBMX-elevated cyclic AMP with a persistent light response, but reduced the dark action of Co2+. IBMX, but not Co2+, also increased cyclic AMP in receptorless (rd/rd) retinas; haloperidol partly reduced this IBMX effect. In normal retinas in Co2+ medium, progressively replacing Na+ by K+ (but not choline+) from 1--50 mM caused a progressive fall in dark, light-sensitive cyclic AMP levels, but from 50 to 100 mM-K+ there appeared haloperidol-preventable increases in both the dark- and light-insensitive levels of cyclic AMP. In IBMX-aspartate medium a haloperidol-preventable, light-insensitive increase in cyclic AMP appeared from 20 mM-K+ upwards. Haloperidol-preventable increases in cyclic AMP as induced by high K+ required Co2+ in normal retinas, but not in receptorless retinas, and 5 nM-Co2+ greatly increased the response to dopamine in receptorless retinas. The post-dopaminergic neurons, which are 4th-order neurons, may have become hypersensitive to dopamine in receptorless retinas consequent to the absent signal from the 1st-order photoreceptors, or directly, as an effect of the same gene underlying the dystrophy.     

Galanin Inhibits Noradrenaline-Induced Accumulation of Cyclic AMP in the Rat Cerebral Cortex

The effect of galanin on noradrenaline (NA)-induced accumulation of cyclic AMP was investigated in slices of rat cerebral cortex. NA (10(-4)M) increased cyclic AMP levels during a 20-min observation period. Galanin (3 X 10(-7)M) significantly inhibited this response at all time points examined, although it did not change the basal levels of cyclic AMP. Galanin (10(-8)-3 X 10(-6)M) inhibited the cyclic AMP response to NA (10(-4)M) in a dose-dependent manner, with an IC50 of approximately 5.6 X 10(-8)M and a maximum inhibition of 59%. These results suggest that galanin, devoid of any detectable effects by itself, modulates the cyclic AMP response to NA in the rat cerebral cortex.     

Expression of A1 Adenosine Receptors Modulating Dopamine-Dependent Cyclic AMP Accumulation in the Chick Embryo Retina

Dopamine and 2-chloroadenosine independently promoted the accumulation of cyclic AMP in retinas from 16-day-old chick embryos. The two compounds added together either in saturating or subsaturating concentrations were not additive for the accumulation of the cyclic nucleotide in the tissue. This fact was shown to be due to the existence of an adenosine receptor that mediates the inhibition of the dopamine-dependent cyclic AMP accumulation in the retina. Adenosine inhibited, in a dose-dependent fashion, the accumulation of cyclic AMP induced by dopamine in 12-day-old chick embryo retinas, with an IC50 of approximately 1 microM. This effect was not blocked by dipyridamole. N6-(l-Phenylisopropyl)adenosine, (l-PIA) was the most potent adenosine analog tested, showing an IC50 of 0.1 microM which was two orders of magnitude lower than its stereoisomer d-PIA (10 microM). The maximal inhibition of the dopamine-elicited cyclic AMP accumulation by adenosine and related analogs was 70%. The inhibitory effect promoted by adenosine was blocked by 3-isobutyl-1-methylxanthine (IBMX) or by adenosine deaminase. Adenine was not effective; whereas ATP and AMP promoted the inhibition of the dopamine effect only at very high concentrations. Apomorphine was only 30% as effective as dopamine in promoting the cyclic AMP accumulation in retinas from 11- to 12-day-old embryos and 2-chloroadenosine did not interfere with the apomorphine-mediated shift in cyclic AMP levels. In the retinas from 5-day-old posthatched chickens dopamine and apomorphine were equally effective in eliciting the accumulation of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscarinic Receptor Stimulation Increases Inositol-Phospholipid Metabolism and Inhibits Cyclic AMP Accumulation in PC 12 Cells

Muscarinic receptor stimulation increased the accumulation of 3H-inositol phosphates in PC12 cells whose phospholipids had been prelabeled with [3H]inositol. Muscarine also inhibited the increase in cyclic AMP (cAMP) accumulation caused by 5'-N-ethylcarboxamide adenosine or by vasoactive intestinal peptide. This effect of muscarine was apparently due to the inhibition of adenylate cyclase rather than to a stimulation of a cAMP specific phosphodiesterase. The muscarinic receptor antagonist pirenzepine inhibited both the stimulation of inositol-phospholipid metabolism and the inhibition of cAMP production with Ki values of 0.34 microM and 0.36 microM, respectively. PC12 cells contained a single class of N-[3H]methylscopolamine ([3H]NMS) binding sites. Competition studies with muscarine (KD, 15 microM) and pirenzepine (Ki, 0.12 microM) revealed no evidence for multiple muscarinic receptors. The Ki of pirenzepine for the inhibition of [3H]NMS binding and the inhibition of muscarinic actions is consistent with the possibility that this is not an M1 receptor. Muscarine inhibited cAMP accumulation in cells made deficient in protein kinase C; therefore, this protein kinase is probably not involved in mediating the inhibitory effect of muscarine. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate also inhibited cAMP accumulation in PC12 cells but the mechanism of this effect differed from that of muscarine. Bradykinin caused a large increase in the accumulation of 3H-inositol phosphates and [3H]diacylglycerol relative to muscarine but did not inhibit cAMP production. Oxotremorine inhibited cAMP accumulation but it did not stimulate inositol-phospholipid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP Accumulation Induces a Rapid Desensitization of the Cyclic AMP-Dependent Protein Kinase in Mouse Striatal Neurons

Striatal neurons from the mouse brain embryo grown in primary culture express high levels of cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. To study the modulation of PKA in intact neurons, a rapid method based on Zn(2+)-protein precipitation was developed. This strategy allowed analysis of the stimulation of PKA under conditions of intracellular cAMP concentration increases. Whereas increases up to 1 microM lead to an activation, large and sustained accumulations of cAMP result in a loss of the enzyme activity. With 8-bromo-cAMP (8-Br-cAMP) at 100 microM, the PKA refractoriness occurs within 2 min. It is rapidly reversible because incubation of treated neurons in fresh medium leads to a complete recovery of PKA activity within 30 min. The decrease in assayable PKA does not involve an activation of phosphatases because the histone dephosphorylation rate is not affected by 8-Br-cAMP treatment. Finally, not only 8-Br-cAMP- but also forskolin- and vasoactive intestinal peptide-induced increases in intracellular cAMP concentration can lead to the PKA desensitization. Altogether, these data unravel a new mechanism of PKA regulation.     

Histamine Stimulation of Cyclic AMP Accumulation in Astrocyte-Enriched and Neuronal Primary Cultures from Rat Brain

Histamine stimulates cyclic AMP accumulation in astrocyte-enriched and neuronal primary cultures from rat brain in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. The response in the astrocyte cultures (Emax = 304 +/- 44% over basal, EC50 = 43 +/- 5 microM) was much higher than in neuronal cultures (Emax = 24 +/- 2%, EC50 = 14 +/- 7 microM). The histamine effect in astrocytes was competitively inhibited by the H2 antagonists cimetidine (Ki = 1.1 +/- 0.2 microM) and ranitidine (Ki = 46 +/- 10 nM) but was insensitive to the H1 antagonist mepyramine (1 microM). The two selective H2 agonists impromidine and dimaprit behaved as partial agonists and showed relative potencies (139 and 0.5, respectively) consistent with an interaction with H2 receptors. The more selective H1 agonist 2-thiazolylethylamine (0.01-1 mM) did not potentiate the response to impromidine (10 microM). Thus, in contrast to what is generally observed in intact cell preparations from brain, the histamine-induced cyclic AMP accumulation in astroglial cells is mediated solely by H2 receptors. The small effect shown in neuronal cultures also appears to be mediated by H2 receptors.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

Adrenocortical Steroids Modify Neurotransmitter-Stimulated Cyclic AMP Accumulation in the Hippocampus and Limbic Brain of the Rat

Glucocorticoid hormones are known to affect limbic system structures that have high levels of specific receptors for glucocorticoids, especially the hippocampus (HIPP). To understand how glucocorticoids may affect synaptic transmission, we have tested the effects of adrenal removal and glucocorticoid replacement on neurotransmitter-stimulated cyclic AMP accumulation in brain slices from the rat limbic system. Adrenalectomy (ADX) caused an enhancement of vasoactive intestinal peptide (VIP)-stimulated cyclic AMP accumulation in HIPP, amygdala (AMYG), and septum (SEP). In HIPP, ADX increased the cyclic AMP response to isoproterenol (ISOP) and decreased the response to histamine (HIST). In the AMYG and SEP, ADX did not affect significantly the action of ISOP, but ADX did decrease the response to HIST in AMYG. Administration of dexamethasone or corticosterone reversed the effects of ADX on cyclic AMP accumulation in the HIPP. The dexamethasone action on VIP-stimulated cyclic AMP accumulation takes place within 48 h and is most apparent in the mid-range of the VIP dose-response curve. These results demonstrate that glucocorticoids regulate neurotransmitter-stimulated cyclic AMP generation in a fashion that is specific, both for the neurotransmitter involved and for the brain regions affected.     

Cannabinoid Receptors and Modulation of Cyclic AMP Accumulation in the Rat Brain

The mechanism by which cannabinoid compounds produce their effects in the rat brain was evaluated in this investigation. Cannabinoid receptors, quantitated by [3H]CP-55,940 binding, were found in greatest abundance in the rat cortex, cerebellum, hippocampus, and striatum, with smaller but significant binding also found in the hypothalamus, brainstem, and spinal cord. Using rat brain slice preparations, we evaluated the effect of desacetyllevonantradol on basal and forskolin-stimulated cyclic AMP accumulation in the regions exhibiting the greatest cannabinoid receptor density. Desacetyllevonantradol (10 microM) reduced cyclic AMP levels in the hippocampus, frontal cortex, and striatum. In the cerebellum, however, the response to desacetyllevonantradol was biphasic with cyclic AMP accumulation being decreased at lower and increased at higher concentrations. Desacetyllevonantradol reduced cyclic AMP accumulation in isoproterenol-stimulated slices in the cortex and cerebellum, but not in the hippocampus. Cells that responded to vasoactive intestinal peptide with an increase in cyclic AMP accumulation in the hippocampus and cortex also responded to desacetyllevonantradol. The modulation of cyclic AMP accumulation by desacetyllevonantradol could be attenuated following stereotaxic implantation of pertussis toxin, supporting the involvement of a G protein in the cannabinoid response in the brain. However, other actions of cannabinoid compounds may also affect the cyclic AMP levels in brain slice preparations.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

K+-Evoked Depolarization Stimulates Cyclic AMP Accumulation in Photoreceptor-Enriched Retinal Cell Cultures: Role of Calcium Influx Through Dihydropyridine-Sensitive Calcium Channels

The effect of membrane depolarization on cyclic AMP synthesis was studied in glia-free, low-density, monolayer cultures of chick retinal photoreceptors and neurons. In photoreceptor-enriched cultures prepared from embryonic day 6 retinas and cultured for 6 days, elevated K+ concentrations increased the intracellular concentration of cyclic AMP and stimulated the conversion of [3H]adenine to [3H]cyclic AMP. The K(+)-evoked increase of cyclic AMP accumulation was blocked by omitting CaCl2 from the incubation medium, indicating a requirement for extracellular Ca2+. Stimulation of cyclic AMP accumulation was also inhibited by nifedipine, methoxyverapamil, Cd2+, Co2+, and Mg2+, and was enhanced by the dihydropyridine Ca2+ channel agonist Bay K 8644. The enhancement of K(+)-evoked cyclic AMP accumulation by Bay K 8644 was antagonized by nifedipine. Thus, Ca2+ influx through dihydropyridine-sensitive channel is required for depolarization-evoked stimulation of cyclic AMP accumulation in photoreceptor-enriched cultures.     

Forskolin Stimulation of Cyclic AMP Accumulation in Rat Brain Cortex Slices Is Markedly Enhanced by Endogenous Adenosine

Stimulation of cyclic AMP (cAMP) accumulation in rat cortex slices by 1 microM forskolin (F) was markedly reduced (96%) by treatment with adenosine deaminase (ADA). The effect of ADA was progressively less at higher concentrations of F, but still inhibited the response by 50% at 100 microM F. ADA-mediated inhibition of the cAMP response to 1 microM F was completely reversed by 5 microM 2-chloroadenosine (CA), an ADA-resistant analogue. Stimulation by F (controls) and F plus CA (ADA treated) in cortex slices was significantly inhibited by 200 microM caffeine (CAF) and by 10 microM 8-phenyltheophylline. cAMP accumulation in ADA-treated cortex slices stimulated with CA at concentrations from 5 to 100 microM was markedly enhanced by 1 microM F. Neither ADA treatment nor 200 microM CAF significantly affected cAMP accumulation in slices stimulated by 1 microM vasoactive intestinal polypeptide or adenylate cyclase in membranes stimulated by 1 microM F. CAF (1 mM) did not significantly increase basal cAMP levels in cortex slices, whereas 1 mM 3-isobutyl-1-methylxanthine caused a significant 80% increase and 100 microM rolipram enhanced cAMP levels by 4.5-fold. F-stimulated cAMP accumulation (1 microM) in cortex slices was inhibited 98% by 1 mM CAF and 49% by 1 mM 3-isobutyl-1-methylxanthine, and was enhanced 2.5-fold by 100 microM rolipram. These data have been interpreted to indicate that the stimulation of cAMP accumulation in rat cortex slices by 1 microM F is predominantly due to synergistic interaction with endogenous adenosine and that the inhibition of this response by CAF is largely due to blockade of adenosine receptors.     

Differences and Similarities in Muscarinic Receptors of Rat Heart and Retina: Effects of Agonists, Guanine Nucleotides, and N-Ethylmaleimide

Muscarinic receptor stimulation inhibits cyclic AMP formation in rat atria but not in retina. We compared the properties of the muscarinic receptors in rat atrial and retinal membranes using the antagonist [3H]quinuclidinyl benzilate. In both atria and retina there is a single binding site for antagonists, while agonists appear to interact at two classes of binding sites. Muscarinic receptors in atria and retina have the same apparent affinities for several antagonists and for a series of muscarinic agonists. In both tissues N-ethylmaleimide decreases agonist affinity for the high-affinity binding sites. Muscarinic receptors in atria and retina differ, however, in several properties relating to the proportions of high- and low-affinity agonist sites. First, guanine nucleotides markedly increase the proportion of low-affinity binding sites in atria, but not in retina. Second, for all agonists there are fewer high-affinity binding sites in retina. Third, the "partial agonist" pilocarpine appears to interact with two classes of binding sites in atria, but with only a single class of sites in retina. Our data suggest that muscarinic receptors that inhibit cyclic AMP formation and those that do not share common properties that determine receptor affinity for agonists and classic antagonists. The differences between these receptors are manifest, however, in the effects of guanine nucleotides and the ability of agonists, especially those of low efficacy, to affect the proportion of high- and low-affinity sites and to effect a biological response.     

Melatonin Biosynthesis in Cultured Chick Retinal Photoreceptor Cells: Calcium and Cyclic AMP Protect Serotonin N-Acetyltransferase from Inactivation in Cycloheximide-Treated Cells

Abstract: The aim of the present study was to examine the roles of membrane depolarization, calcium influx, and cyclic AMP synthesis in regulating the stability and inactivation of serotonin N -acetyltransferase activity (NAT) in cultured chick photoreceptor cells. NAT activity was induced by pretreating cells for 6 h with 1 µ M forskolin. Cycloheximide was subsequently added, and the rate of loss of enzyme activity (inactivation) was determined. After induction, in the presence of cycloheximide, NAT activity declined with a half-life of ∼30 min. The rate of inactivation was greatly reduced when depolarizing concentrations of K⁺, forskolin, 8-bromoadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine were added together with cycloheximide. The apparent increase in NAT stability caused by K⁺ was abolished by addition of EGTA or nifedipine and potentiated by Bay K 8644, indicating the involvement of Ca²⁺ influx through dihydropyridine-sensitive channels. MDL-12330A, an inhibitor of K⁺-stimulated cyclic AMP formation, blocked the effect of depolarizing concentrations of K⁺. This result suggests that the effect of Ca²⁺ influx on the stability of NAT is at least partially mediated by increased levels of cyclic AMP. Thus, depolarization-evoked Ca²⁺ influx and cyclic AMP formation have two roles in the regulation of NAT activity in chick photoreceptor cells. First, they stimulate the de novo synthesis of NAT or a regulatory protein required for NAT activity. Second, they increase the half-life of the enzyme, presumably by regulating the turnover of existing enzyme molecules.     

Potentiation of Agonist-Stimulated Cyclic AMP Accumulation by Tyrosine Kinase Inhibitors in Rat Pinealocytes

Abstract: To study cross-talk mechanisms in rat pinealocytes, the role of tyrosine kinase or kinases in the regulation of adrenergic-stimulated cyclic AMP production was investigated. Both norepinephrine- and isoproterenol-stimulated cyclic AMP accumulation were increased by two distinct tyrosine kinase inhibitors, genistein or erbstatin, in a concentration-dependent manner. A similar increase was observed with two other inhibitors, tyrphostin B44 and herbimycin. In contrast, daidzein, an inactive analogue of genistein, was ineffective; whereas vanadate, a phosphotyrosine phosphatase inhibitor, reduced the adrenergic-stimulated cyclic AMP accumulation. The tyrosine kinase inhibitors were effective in potentiating the cholera toxin-or forskolin-stimulated cyclic AMP accumulation, indicating that their sites of action are at the postreceptor level. Neither an activator nor inhibitors of protein kinase C influenced the potentiation of the cyclic AMP responses by genistein, suggesting that the potentiation effect by tyrosine kinase inhibitors does not involve the phospholipase C/protein kinase C pathway. However, when the phosphodiesterase was inhibited by isobutylmethylxanthine, genistein failed to potentiate and vanadate did not inhibit the adrenergic-stimulated cyclic AMP accumulation, indicating that the phosphodiesterase is a probable site of action for these inhibitors. These results suggest that cyclic AMP metabolism in the pinealocytes is tonically inhibited by tyrosine kinase acting on the cyclic AMP phosphodiesterase.