OPIATES SELECTIVELY INCREASE INTRACELLULAR CALCIUM IN DEVELOPING TYPE-1 ASTROCYTES - ROLE OF CALCIUM IN MORPHINE-INDUCED MORPHOLOGIC DIFFERENTIATION

Endogenous opioids and opiate drugs inhibit nervous system maturation, in part, by affecting the growth of astrocytes. Opiates inhibit astro cyte proliferation and cause premature differentiation. The emerging i mportance of Ca2+ in astrocyte function prompted us to explore whether opiates might affect astrocyte development by altering Ca2+ homeostas is. Astrocyte-enriched cultures were derived from newborn ICR mouse ce rebra. Quantitative fluorescent measurements of intracellular free Ca2 + ([Ca2+](i)) using Fura-2 as well as fluo-3 and computer-aided image analysis showed that 1 mu M morphine significantly increased [Ca2+](i) in flat, polyhedral, glial fibrillary acidic protein (GFAP) immunorea ctive astrocytes at 2 and 6 min, and at 72 h. Co-administration of 3 m u M naloxone blocked morphine-dependent increases in [Ca2+](i). Treatm ent with 1 mu M concentrations of the K-opioid receptor agonist, U69,5 93, but not equimolar amounts of mu ([D-Ala(2),MePhe(4),Gly(ol)(5)]enk ephalin)- or delta ([D-Pen(2),D-Pen(5)]enkephalin)-opioid receptor ago nists, significantly increased [Ca2+](i) in astrocytes. To assess the role of Ca2+ in morphine-induced astrocyte differentiation, untreated and 1 mu M morphine-treated astrocyte cultures were incubated for 5 da ys in < 0.01, 0.3, 1.0, or 3.0 mM extracellular Ca2+ ([Ca2+](o)), or i ncubated with 1.0 mM [Ca2+](o) in the presence of 1 mu M of the Ca2+ i onophore, A23187. The areas of single astrocytes were measured and the re was a positive correlation between astrocyte area and [Ca2+](o). Mo rphine had an additive effect on area and form factor measures when [C a2+](o) was 1.0 mM. High [Ca2+](o) (3.0 mM) alone mimicked the action of morphine. Morphine alone had no effect on astrocyte area in the pre sence of 3.0 mM Ca2+. Morphine also had no effect in the presence of l ow (< 0.01 mM) Ca2+ or 1 mu M A23187. 1 mu M A23187 alone mimicked the effects of morphine. Collectively, these findings suggest that Ca2+ n ormally affects the morphology of developing astrocytes, and that morp hine-induced changes in the morphology of flat, polyhedral astrocytes are mediated through changes in [Ca2+](i). Furthermore, kappa-, but no t mu- or delta-, opioid receptor agonists increase [Ca2+](i) in astroc ytes, suggesting that 1 mu M morphine may increase [Ca2+](i) by stimul ating kappa-opioid receptors in flat, polyhedral astrocytes.