Delta-opioid receptors on astroglial cells in primary culture: mobilization of intracellular free calcium via a pertussis sensitive G protein.
Astrocytes in primary culture from rat cerebral cortex were probed concerning the expression of delta-opioid receptors and their coupling to changes in intracellular free calcium concentrations ([Ca2+]i). Fluo-3 or fura-2 based microspectrofluorometry was used for [Ca2+]i measurements on single astrocytes in a mixed astroglial-neuronal culture. Application of the selective delta-opioid receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE), at concentrations ranging from 10 nM to 100 microM, induced concentration-dependent increases in [Ca2+]i (EC50 = 114 nM). The responses could be divided into two phases, with an initial spike in [Ca2+]i followed by either oscillations or a sustained elevation of [Ca2+]i. These effects were blocked by the selective delta-opioid receptor antagonist ICI 174864 (10 microM). The expression of delta-opioid receptors on astroglial cells was further verified immunohistochemically, using specific antibodies, and by Western blot analyses. Pre-treatment of the cells with pertussis toxin (100 ng/ml, 24 h) blocked the effects of delta-opioid receptor activation, consistent with a Gi- or Go-mediated response. The sustained elevation of [Ca2+]i was not observed in low extracellular Ca2+ and was partly blocked by nifedipine (1 microM), indicating the involvement of L-type Ca2+ channels. Stimulating neurons with DPDPE resulted in a decrease in [Ca2+]i, which may be consistent with the closure of the plasma membrane Ca2+ channels on these cells. The current results suggest a role for astrocytes in the response of the brain to delta-opioid peptides and that these opioid effects in part involve altered astrocytic intracellular Ca2+ homeostasis.
Institute of Neurobiology, Department of Neurobiology, Göteborg University, Sweden. thorleif.thorlin@anatcell.gu.se
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Thorlin
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Eriksson
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Persson
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Aberg
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Hansson
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Rönnbäck
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007150:Immunohistochemistry; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D037342:Pertussis Toxin; D051381:Rats; D017207:Rats, Sprague-Dawley; D017465:Receptors, Opioid, delta / Q000819:agonists / Q000502:physiology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015220:Calcium Channels; D004745:Enkephalins; D017465:Receptors, Opioid, delta; D010566:Virulence Factors, Bordetella; D020881:Enkephalin, D-Penicillamine (2,5)-; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium