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 astrocyte proliferation and cause premature differentiation. The emerging importance of Ca²⁺ in astrocyte function prompted us to explore whether opiates might affect astrocyte development by altering Ca²⁺ homeostasis. Astrocyte-enriched cultures were derived from newborn ICR mouse cerebra. 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 μM morphine significantly increased [Ca²⁺]_i in flat, polyhedral, glial fibrillary acidic protein (GFAP) immunoreactive astrocytes at 2 and 6 min, and at 72 h. Co-administration of 3 μM naloxone blocked morphine-dependent increases in [Ca²⁺]_i. Treatment with 1 μM concentrations of the κ-opioid receptor agonist, U69,593, but not equimolar amounts of μ ([d-Ala²,MePhe⁴,Gly(ol)⁵]enkephalin)- or δ ([d-Pen²,d-Pen⁵]enkephalin)-opioid receptor agonists, significantly increased [Ca²⁺]_i in astrocytes. To assess the role of Ca²⁺ in morphine-induced astrocyte differentiation, untreated and 1 μM morphine-treated astrocyte cultures were incubated for 5 days in <0.01, 0.3, 1.0, or 3.0 mM extracellular Ca²⁺ ([Ca²⁺]_o), or incubated with 1.0 mM [Ca²⁺]_o in the presence of 1 μM of the Ca²⁺ ionophore, A23187. The areas of single astrocytes were measured and there was a positive correlation between astrocyte area and [Ca²⁺]_o. Morphine had an additive effect on area and form factor measures when [Ca²⁺]_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 presence of 3.0 mM Ca²⁺. Morphine also had no effect in the presence of low (<0.01 mM) Ca²⁺ or 1 μM A23187. 1 μM A23187 alone mimicked the effects of morphine. Collectively, these findings suggest that Ca²⁺ normally affects the morphology of developing astrocytes, and that morphine-induced changes in the morphology of flat, polyhedral astrocytes are mediated through changes in [Ca²⁺]_i. Furthermore, κ-, but not μ- or δ-, opioid receptor agonists increase [Ca²⁺]_i in astrocytes, suggesting that 1 μM morphine may increase [Ca²⁺]_i by stimulating κ-opioid receptors in flat, polyhedral astrocytes.