Year-end Sale % OFF 
Abstract
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.
Highlights
** Recipient of a postdoctoral award from the Alberta Heritage Foundation during the course of these studies
D2 dopamine receptors expressed in mouse L cells and C6 glioma cells activate Naϩ/Hϩ exchangers (NHEs) [8], as do ␣2-adrenergic, muscarinic, and ␦-opiate receptors expressed in NG108-15 hybridoma cells [9], and all act through pertussis toxin-insensitive mechanisms
Such measures include hypertonic stress, phorbol 12-myristate, 13acetate acting through protein kinase C, maneuvers that increase intracellular Ca2ϩ levels, and growth factors such as fibroblast growth factors (FGF) and thrombin [41,42,43,44]
Summary
** Recipient of a postdoctoral award from the Alberta Heritage Foundation during the course of these studies. That notion has been supported by recent studies in which constitutively active G protein ␣-subunits were transiently expressed in mammalian host cells Those studies showed clearly that Gq␣, G12␣, and G13␣ can increase NHE activity, the former two through a protein kinase C-dependent (phorbol ester-sensitive) pathway, and the latter through a protein kinase C-independent (phorbol ester-insensitive) pathway (10 –12). D3 and D4 dopamine receptors, have been shown to stimulate NHE activity in CHO cells through pertussis toxin-sensitive G proteins [13, 14]. We chose CHO-K1 fibroblast cells, because they have previously proven useful in elucidating some of the pathways involved in plateletderived growth factor receptor [4, 19]- and dopamine receptor [13, 14]-stimulated NHE-1 activity. Because most cells endogenously express two or more subtypes of pertussis toxinsensitive G proteins, it has been very difficult to assign specific downstream regulatory functions to individual ␣-subunit types
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
