G protein betagamma subunits modulate the number and nature of exocytotic fusion events in adrenal chromaffin cells independent of calcium entry.
G-protein-coupled receptors (GPCR) play important roles in controlling neurotransmitter and hormone release. Inhibition of voltage-gated Ca(2+) channels (Ca(2+) channels) by G protein betagamma subunits (Gbetagamma) is one prominent mechanism, but there is evidence for additional effects distinct from those on calcium entry. However, relatively few studies have investigated the Ca(2+)-channel-independent effects of Gbetagamma on transmitter release, so the impact of this mechanism remains unclear. We used carbon fiber amperometry to analyze catecholamine release from individual vesicles in bovine adrenal chromaffin cells, a widely used neurosecretory model. To bypass the effects of Gbetagamma on Ca(2+) entry, we stimulated secretion using ionomycin (a Ca(2+) ionophore) or direct intracellular application of Ca(2+) through a patch pipette. Activation of endogenous GPCR or transient transfection with exogenous Gbetagamma significantly reduced the number of amperometric spikes (the number of vesicular fusion events). The charge ("quantal size") and amplitude of the amperometric spikes were also significantly reduced by GPCR/Gbetagamma. We conclude that independent from effects on calcium entry, Gbetagamma can regulate both the number of vesicles that undergo exocytosis and the amount of catecholamine released per fusion event. We discuss possible mechanisms by which Gbetagamma might exert these novel effects including interaction with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex.
Yoon
Eun-Ja
EJ
Dept. of Pharmacology, Vanderbilt University Medical Center, T-4202 Medical Center North, 1161 21st Ave. South, Nashville, TN 37232-2520, USA.
Hamm
Heidi E
HE
Currie
Kevin P M
KP
D000200:Action Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000311:Adrenal Glands / Q000166:cytology*; D000701:Analgesics, Opioid / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology*; D004307:Dose-Response Relationship, Radiation; D004563:Electrochemistry / Q000379:methods; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism*; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D014162:Transfection / Q000379:methods
D000701:Analgesics, Opioid; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D049452:Green Fluorescent Proteins; D015759:Ionomycin; D000255:Adenosine Triphosphate; D002118:Calcium