Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells

Item Type:	Article
Title:	Inhibition of voltage-dependent Ca2+ currents and activation of pertussis toxin-sensitive G-proteins via muscarinic receptors in GH3 cells
Creators Name:	Offermanns, S. and Gollasch, M. and Hescheler, J. and Spicher, K. and Schmidt, A. and Schultz, G. and Rosenthal, W.
Abstract:	In the rat pituitary cell line GH3, carbachol inhibits PRL secretion in a pertussis toxin-sensitive manner. For elucidation of the underlying mechanisms, we studied the effect of carbachol on voltage-dependent Ca2+ currents. Under voltage-clamp conditions, carbachol inhibited whole-cell Ca2+ currents by about 25%. This inhibitory action of carbachol was not observed in cells treated with pertussis toxin, indicating the involvement of a pertussis toxin-sensitive G-protein. In membranes of GH3 cells, carbachol stimulated a pertussis toxin-sensitive high-affinity GTPase. In immunoblot experiments with peptide antisera, we identified two forms of the Gi alpha-subunit (41 and 40 kDa) and two forms of the Go alpha-subunit (40 and 39 kDa). The 40-kDa Gi alpha-subunit was recognized by an antibody specific for the Gi2 alpha-subunit, and the 39-kDa Go alpha-subunit was detected by an antibody specific for the Go2 alpha-subunit. Incubation of membranes with the photoreactive GTP analog [alpha-32P]GTP azidoanilide resulted in photo-labelling of 40- and 39-kDa pertussis toxin substrates comigrating with G-protein alpha-subunits of the corresponding molecular masses. Carbachol dose-dependently stimulated incorporation of the photoreactive GTP analog into the 39-kDa pertussis toxin substrate and, to a lesser extent, into 40-kDa pertussis toxin substrates. The data indicate that muscarinic receptors of GH3 cells couple preferentially to Go, which is likely to be involved in the inhibition of secretion, possibly by conferring an inhibitory effect to voltage-dependent Ca2+ channels.
Keywords:	Affinity Labels, Azides, Calcium Channels, Carbachol, Cell Line, Dihydropyridines, Electric Conductivity, GTP-Binding Proteins, Guanosine Triphosphate, Molecular Weight, Pertussis Toxin, Photochemistry, Pituitary Gland, Prolactin, Muscarinic Receptors, Bordetella Virulence Factors, Animals, Rats
Source:	Molecular Endocrinology
ISSN:	0888-8809
Publisher:	Endocrine Society
Volume:	5
Number:	7
Page Range:	995-1002
Date:	July 1991
Official Publication:	http://mend.endojournals.org/cgi/content/abstract/5/7/995
PubMed:	View item in PubMed

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