Muscarinic receptor-induced calcium responses in astroglia.
BACKGROUND
The objective of this study was to characterize and quantitate the calcium responses to cholinergic stimulation in individual primary rat cortical astrocytes and human 132 1N1 astrocytoma cells. Materials and Methods The fluorescent calcium probe Indo-1 AM and an attached cell analysis and sorting (ACAS) instrument were used to quantitate calcium responses in these cells.


RESULTS
A concentration-dependent response to carbachol was seen in both cell types. However, carbachol was more potent and efficacious, and the response was more homogeneous in the cell line. The calcium response was mediated by the M3 subtype of muscarinic receptors. Experiments in the absence of extracellular calcium and with EGTA demonstrated that the initial calcium spike was due to calcium release from intracellular calcium stores, whereas the sustained elevation and oscillations were dependent on calcium influx. Protein kinase C exerts a feedback inhibition of these calcium responses, and appears to be involved in maintaining the elevated calcium concentration and oscillations.


CONCLUSIONS
This study provided a detailed quantitation of the changes in intracellular calcium evoked in individual astroglial cells by activation of M3 muscarinic receptors. This will allow for the study of pharmacological and toxicological agents on this response.
Catlin
M C
MC
Department of Environmental Health, University of Washington, Seattle, Washington 98105, USA.
Kavanagh
T J
TJ
Costa
L G
LG
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001254:Astrocytoma / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D018679:Cholinergic Agonists / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D011247:Pregnancy; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D051381:Rats; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002614:Chelating Agents; D018679:Cholinergic Agonists; D007211:Indoles; D018727:Muscarinic Antagonists; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C059587:Indo-1 pentaacetoxymethyl ester; D004533:Egtazic Acid; C016658:phorbolol myristate acetate; D002217:Carbachol; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium