Abstract:In this study the subunits of the dihydropyridine-sensitive L-type Ca2~channels (L-channels) expressed in rat pinealocytes were characterized using reverse transcription (RT)-PCR analysis, and the modulation of these channels by adrenergic agonists and by pituitary adenylate cyclase-activating polypeptide (PA-CAP) was studied using the patch-clamp technique. RT-PCR analysis showed that rat pinealocytes expressed a2b, /32, and /34 Ca2-channel subunit mRNAs. Other a 1 subunit transcripts were either not expressed or present at very low levels, indicating that the pinealocytes express predominantly aID L-channels. Electrophysiologcal studies confirmed that the pineal expressed a single population of L-channels. The L-channel currents were inhibited by two agonists that elevate cyclic AMP: the /3-adrenergic agonist isoproterenol and PACAP. Similar inhibition was observed with a cyclic AMP analogue, 8-bromo-cyclic AMP. The presence of a cyclic AMP antagonist, Rp -adenosine 3',5 '-cyclic monophosphorothioate, blocked the inhibition by isoproterenol and PACAP. Norepinephrine, a mixed a-and f3-adrenergic agonist, also inhibited the L-channel currents, but the inhibition was smaller. The smaller inhibition by norepinephrine was secondary to the simultaneous activation of a-and /3-adrenergic receptors. These results indicatethat (a) pinealocytes express predominantly a~L-channels, and (b) the /3-adrenergic agonist isoproterenol and PACAP inhibit the L-channel currents through elevation of cyclic AMP. However, an a-adrenergic-mediated mechanism also appears to be involved in the effect of norepinephrine on the L-channel currents. Key Words: Ca 2~channel-Cyclic AMP-Norepinephrine-PACAP.
Atvstract Exposure of neuronal cells to the chronic presence of opiates leads to a complex series of biochemical events which reflect the changes that result in tolerance and dependence in animals. To achieve a better understanding of the molecular mechanisms underlying these processes, we have examined the effect of agonist efficacy on the regulation of the fi-opioid receptor mRNA in NGI08-15 cells. Incubation with various opiates decreased receptor numbers in the order of their efficacy. Northern blot analysis showed that there are 4 size classes of mRNA coding for the 6-opioid receptor in NGI08-15 cells even though only one known protein species is found. Moreover, the amount of each transcript is coordinately decreased by long-term etorphine treatment, but not necessarily to the same extent. The etorphine-indnced decrease in receptor mRNA was found to be slow in onset, whereas a much more rapid loss of receptor number was observed. This disparity suggests that the down-regulation induced by etorphine can occur both at the levels of receptor protein modification and receptor gene expression, and that the mechanisms of the two processes may be different.
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