MU-OPIOID RECEPTOR-INDUCED CA2- MORPHINE INHIBITS DNA-SYNTHESIS AND STIMULATES CELLULAR HYPERTROPHY THROUGH A CA2+-DEPENDENT MECHANISM( MOBILIZATION AND ASTROGLIAL DEVELOPMENT )

Morphine, a preferential mu-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellula r differentiation. Although morphine affects cellular differentiation through a Ca2+-dependent mechanism, few studies have examined whether Ca2+ mediates the effect of opioids on cell proliferation, or whether a particular Ca2+ signal transduction pathway mediates opioid actions. Moreover, it is uncertain whether one or more opioid receptor types m ediates the developmental effects of opioids. To address these questio ns, the present study examined the role of mu-opioid receptors and Ca2 + mobilization in morphine-induced astrocyte development. Morphine (1 mu M) and non-morphine exposed cultures enriched in murine astrocytes were incubated in Ca2+-free media supplemented with < 0.005, 0.3, 1.0, or 3.0 mM Ca2+ ([Ca2+](o)), or in unmodified media containing Ca2+ io nophore (A23187), nifedipine (1 mu M), dantrolene (10 mu M), thapsigar gin (100 nM), or L-glutamate (100 mu M) for 0-72 h. mu-Opioid receptor expression was examined immunocytochemically using specific (MOR1) an tibodies. Intracellular Ca2+ ([Ca2+](i)) was measured by microfluorome tric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillar y acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating mu-opioid rece ptors. Astrocytes expressed MORI immunoreactivity and morphine's actio ns were mimicked by the selective mu agonist PL017. In addition, morph ine inhibited DNA synthesis by mobilizing [Ca2+](i) in developing astr oglia. At normal [Ca2+](o), morphine attenuated DNA synthesis by incre asing [Ca2+](i); low [Ca2+](o) (0.3 mM) blocked this effect, while tre atment with Ca2+ ionophore or glutamate mimicked morphine's actions. A t extremely low [Ca2+](o) (< 0.005 mM), morphine paradoxically increas ed BrdU incorporation. Although opioids can increase [Ca2+](i) in astr ocytes through several pathways, not all affect DNA synthesis or cellu lar morphology. Nifedipine (which blocks L-type Ca2+ channels) did not prevent morphine-induced reductions in BrdU incorporation or cellular differentiation, while thapsigargin (which depletes IP3-sensitive Ca2 + stores) severely affected inhibited DNA synthesis and cellular diffe rentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca2+-dependent Ca2+ release) selectively blocked the eff ects of morphine. Collectively, the findings suggest that opioids supp ress astroglial DNA synthesis and promote cellular hypertrophy by inhi biting Ca2+-dependent Ca2+ release from dantrolene-sensitive intracell ular stores. This implies a fundamental mechanism by which opioids aff ect central nervous system maturation.