5-HT1A receptor activates Na+/H+ exchange in CHO-K1 cells through Gialpha2 and Gialpha3.
5-HT1A receptors couple to many signaling pathways in CHO-K1 cells through pertussis toxin-sensitive G proteins. The purpose of this study was to determine which members of the Gi/o/z family mediate 5-HT1A receptor-activated Na+/H+ exchange as measured by microphysiometry of cell monolayers. The method was extensively validated, showing that proton efflux was sodium-dependent, inhibited by amiloride analogs, and activated by growth factors, phorbol ester, calcium ionophore, and hypertonic stress. 5-HT and the specific agonist (+/-)-8-hydroxy-2-(di-N-propylamino)tetralin hydrobromide rapidly stimulated proton efflux that was blocked by a specific receptor antagonist, amiloride analogs or pertussis toxin. The activation by 5-HT depended upon extracellular sodium and could be demonstrated under conditions of imposed intracellular acid load, as well as in the presence and absence of glycolytic substrate. Acceleration of proton efflux was not inhibited by sequestration of G protein betagamma-subunits, a maneuver that blocked 5-HT1A receptor activation of mitogen-activated protein kinase. Transfection of Gzalpha and pertussis toxin-resistant mutants of Goalpha and Gialpha1 did not reverse the blockade induced by pertussis toxin. In contrast, pertussis toxin-resistant mutants of Gialpha2 and Gialpha3 "rescued" the ability of 5-HT to increase proton efflux after pertussis toxin treatment. These experiments demonstrate clearly that Gialpha2 and Gialpha3 can specifically mediate rapid agonist-induced acceleration of Na+/H+ exchange.
Garnovskaya
M N
MN
Department of Medicine, Medical University of South Carolina and Veterans Affairs Medical Centers, Charleston, South Carolina 29425, USA.
Gettys
T W
TW
van Biesen
T
T
Prpic
V
V
Chuprun
J K
JK
Raymond
J R
JR
D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D019204:GTP-Binding Proteins / Q000378:metabolism*; D037342:Pertussis Toxin; D011485:Protein Binding; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins / Q000378:metabolism; D017366:Serotonin Receptor Agonists / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D011985:Receptors, Serotonin; D044263:Receptors, Serotonin, 5-HT1; D011994:Recombinant Proteins; D017366:Serotonin Receptor Agonists; D017923:Sodium-Hydrogen Exchangers; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins