mu-opioid receptor-induced Ca2+ mobilization and astroglial development: Morphine inhibits DNA synthesis and stimulates cellular hypertrophy through a Ca2+-dependent mechanism

Morphine, a preferential mu-opioid receptor agonist, alters astroglial development by inhibiting cell proliferation and by promoting cellular 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 mediates the developmental effects of opioids. To address these questions, 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+ ionophore (A23187), nifedipine (1 mu M), dantrolene (10 mu M), thapsigargin (100 nM), or L-glutamate (100 mu M) for 0-72 h. mu-Opioid receptor expression was examined immunocytochemically using specific (MOR1) antibodies. Intracellular Ca2+ ([Ca2+](i)) was measured by microfluorometric analysis using fura-2. Astrocyte morphology and bromodeoxyuridine (BrdU) incorporation (DNA synthesis) were assessed in glial fibrillary acidic protein (GFAP) immunoreactive astrocytes. The results showed that morphine inhibited astroglial growth by activating mu-opioid receptors. Astrocytes expressed MORI immunoreactivity and morphine's actions were mimicked by the selective mu agonist PL017. In addition, morphine inhibited DNA synthesis by mobilizing [Ca2+](i) in developing astroglia. At normal [Ca2+](o), morphine attenuated DNA synthesis by increasing [Ca2+](i); low [Ca2+](o) (0.3 mM) blocked this effect, while treatment with Ca2+ ionophore or glutamate mimicked morphine's actions. At extremely low [Ca2+](o) (< 0.005 mM), morphine paradoxically increased BrdU incorporation. Although opioids can increase [Ca2+](i) in astrocytes through several pathways, not all affect DNA synthesis or cellular 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 differentiation-irrespective of morphine treatment. However, dantrolene (an inhibitor of Ca2+-dependent Ca2+ release) selectively blocked the effects of morphine. Collectively, the findings suggest that opioids suppress astroglial DNA synthesis and promote cellular hypertrophy by inhibiting Ca2+-dependent Ca2+ release from dantrolene-sensitive intracellular stores. This implies a fundamental mechanism by which opioids affect central nervous system maturation.