Mechanisms of opioid receptor-induced elevation in intracellular calcium by confocal laser scanning microscopy

OBJECTIVE: To determine the acute and chronic effects of opioid receptor agonists on the intracellular free calcium concentration ([Ca2+]i) in NG-LNCXiNOS cells, stably expressing iNOS gene, and regulation of G-protein on opioid-induced response in [Ca2+]i. METHODS: A single cell [Ca2+]i is measured by confocal laser scanning microscopy using Ca(2+)-sensitive dye Fluo-3 as an new calcium fluorescent probe. RESULTS: DPDPE(D-Pen2, D-Pen5-enkephalin), a delta-opioid receptor agonist, and morphine acutely induced the increase in [Ca2+]i of NG-LNCXiNOS cells. The elevation in [Ca2+]i by DPDPE could be abolished with naloxone. Pretreatment of the cells with pertussis toxin (PTX) at 100 ng/ml for 24 hours almost completely blocked morphine-evoked response. In contrast to acute effect of opioid agonists on [Ca2+]i, the cells exposed to 1 mumol/L DPDPE or 10 mumol/L morphine for 48 hours also appeared to raise [Ca2+]i. However, the elevation in [Ca2+]i was not greater than that caused by acute effect of DPDPE or morphine. After cell "withdrawal" was precipitated by the addition of 10 mumol/L naloxone, the increase in [Ca2+]i could further be intensified. CONCLUSIONS: The opioid agonist-induced increase in [Ca2+]i is mediated by opioid receptor and regulated though PTX-sensitive G-protein. The attenuation of this response in chronically treated cells with opioid agonist is associated with receptor desensitization.