κ-Opioid receptor expression defines a phenotypically distinct subpopulation of astroglia: Relationship to Ca²⁺ mobilization, development, and the antiproliferative effect of opioids

To assess the role of κ-opioid receptors in astrocyte development, the effect of κ-agonists on the growth of astroglia derived from 1-2-day-old mouse cerebra was examined in vitro. κ-Opioid receptor expression was assessed immunocytochemically (using KA8 and KOR1 antibodies), as well as functionally by examining the effect of κ-receptor activation on intracellular calcium ([Ca²⁺](i)) homeostasis and DNA synthesis. On days 6-7, as many as 50% of the astrocytes displayed κ-receptor (KA8) immunoreactivity or exhibited increases in [Ca²⁺](i) in response to κ-agonist treatment (U69,593 or U50,488H). Exposure to U69,593 (100 nM) for 72 h caused a significant reduction in number and proportion of glial fibrillary acidic protein-immunoreactive astrocytes incorporating bromodeoxyuridine (BrdU) that could be prevented by co-administering the κ-antagonist, nor-binaltorphimine (300 nM). In contrast, on day 14, only 5 or 14%, respectively, of the astrocytes were κ-opioid receptor (KA8) immunoreactive or displayed functional increases in [Ca²⁺](i). Furthermore, U69,593 (100 nM) treatment failed to inhibit BrdU incorporation at 9 days in vitro. Experimental manipulations showed that κ-receptor activation increases astroglial [Ca²⁺](i) both through influx via L-type channels and through mobilization of intracellular stores (which is an important Ca²⁺ signaling pathway in cell division). Collectively, these results indicate that a subpopulation of developing astrocytes express κ-opioid receptors in vitro, and suggest that the activation of κ-receptors mobilizes [Ca²⁺](i) and inhibits cell proliferation. Moreover, the proportion of astrocytes expressing κ-receptors was greatest during a period of rapid cell growth suggesting that they are preferentially expressed by proliferating astrocytes.