The possible interaction of melatonin receptors and D1 dopamine receptors was investigated in neural cells prepared from embryonic day 8 chick retinas and cultured for 6 d. Dopamine stimulated cAMP accumulation in cultured retinal cells. This effect of dopamine was antagonized by addition of dopamine receptor antagonists (haloperidol and SCH23390) or melatonin receptor agonists (melatonin, 2- iodomelatonin, and 6-chloromelatonin). The inhibition of dopamine- stimulated cAMP accumulation by melatonin was concentration dependent, with half-maximal inhibition at approximately 160 pM. Melatonin inhibited the effect of dopamine at all dopamine concentrations, suppressing the maximal response to the neurotransmitter by approximately 70%. Melatonin also inhibited the stimulation of cAMP accumulation by SKF 82958, a selective D1 dopamine receptor agonist. Pretreatment of cultures with pertussis toxin had no significant effect on dopamine-stimulated cAMP accumulation, but inhibited the response to melatonin. In contrast to its effect on cAMP accumulation, melatonin had no effect on dopamine-stimulated inositol phosphate accumulation. These results suggest that melatonin receptors are coupled to dopamine receptor-regulated adenylate cyclase via an inhibitory G protein, and demonstrate another mechanism, in addition to inhibition of dopamine release, through which melatonin can modulate dopaminergic neurotransmission.
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.
Melatonin receptors were characterized in cultured neurons and photoreceptors prepared from chick embryo retina. Cultured cells contained high‐affinity 2‐[125I]iodomelatonin binding sites (KD = 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 KD 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 KB 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.
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 nM for melatonin and 7.2 nM 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 Ki value (0.8 nM) similar to the EC50 for inhibition of cyclic AMP formation. In contrast, 5‐methoxytryptamine only inhibited 2‐[125I]iodomelatonin binding at very high concentrations (Ki = 650 nM). 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.
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