Exocytosis in bovine chromaffin cells: studies with patch-clamp capacitance and FM1-43 fluorescence.
In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.
Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. gordan.kilic@uchsc.edu
Gordan
G
Kilic
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D005089:Exocytosis; D005456:Fluorescent Dyes / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods*; D011726:Pyridinium Compounds / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D002118:Calcium