Abstract
Opiate binding sites on cultured neurons derived from 6-day-old (E6) chick embryo cerebral hemispheres (CH), shown to be cholinergic by choline acetyltransferase immunostaining, were labeledd with [ 3H]etorphine (μ and δ opiate receptors expression) and [ 3H]morphine (mostly μ). When examined by light microscope autoradiography, opiate receptors were found to be expressed by most neurons and were distributed predominantly on neuronal perikarya. Muscarnic and opiate receptors in E6CH cultured neurons were found to be functionally coupled when the effects of opiate receptor occupancy on the inositol phosphate-linked muscarinic receptors was studied. Carbachol stimulated the release of [ 3H]inositol phosphates (InsP) from cultures preincubated with [ 3H]inositol and LiCl, in a dose-dependent manner, and the functional expression of muscarinic receptors peaked in number at day 7 in culture, declining thereafter. Short-term (<1h) treatment of E6 neuronal cultures with 1 μM opioid peptides such as morphiceptin or d-Ala 2- d-Leu 5-enkephalin (DADLE) did not not inhibit the release of inositol phosphates in response to 1 mM carbachol whereas forskolin, which also activates adenylate cyclase and raises cAMP levels, inhibited InsP release by about 25%. In contrast, long-term (48 h) opioid treatment with either morphiceptin or DADLE (1–10 μM) inhibited the carbachol-stimulated inositol phosphate release by ⩾50%. Prolonged treatment with morphiceptin also inhibited the bradykinin-mediated release of InsP from E6CH cells. In both cases, the inhibition was blocked by the continuous presence of naloxone, suggesting that the inhibition was mediated through opiate receptors. When E6CH cells were depolarized by 30 mM K +, allowing [Ca 2+]i to increase, the inhibition of inositol phosphate formation caused by long-term morphiceptin exposure was partially relieved. This 48 h (chronic) exposure to opioids may involve some depletion of available [Ca 2+]i in cholinergic neurons. We propose that these interactions between the opiate and muscarinic receptor transducing systems may represent the molecular basis of the neuromodulating activity of opioids upon the cholinergic system early in development.
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