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Am. J. Respir. Cell Mol. Biol., Volume 25, Number 3, September, 2001 306-315

Secretion in Alveolar Type II Cells at the Interface of Constitutive and Regulated Exocytosis

Manfred Frick, Stephan Eschertzhuber, Thomas Haller, Norbert Mair, and Paul Dietl

Department of Physiology, University of Innsbruck, Innsbruck, Austria

Long-term, simultaneous, measurements of cytoplasmic free Ca2+ concentrations and single exocytotic fusion events in surfactant-secreting type II cells were performed. All fusion (constitutive, phorbol ester-induced, and agonist-induced) was Ca2+-dependent. Kinetic analysis revealed that agonist (adenosine triphosphate [ATP])-induced fusion exhibited a kinetic pattern that correlated well with the Ca2+ signal. The effects of Ca2+ release from intracellular stores (early) and Ca2+ entry (late) could be demonstrated for the first time by dissecting the slow (10-to-15-min) fusion response to ATP into these two components. Bath Ba2+ or Sr2+ could replace Ca2+ to elicit a fusion response in thapsigargin-pretreated cells lacking ATP-induced Ca2+ release from stores. Although the late response was partially inhibited by interrupting the phospholipase D-protein kinase C axis, a high Ca2+ dependence of the entire secretory course was demonstrated by a significant correlation between the integrated Ca2+ signal and the fusion response. There was also a highly significant correlation between constitutive and ATP-stimulated fusion activity in individual cells. We propose a common mechanistic model for all types of fusion in this slow secretory cell, in which constitutive and regulated forms of exocytosis are subject to the same principles of regulation.

Abbreviations: acetoxymethylester, AM; adenosine triphosphate, ATP; [1,2,bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester], BAPTA-AM; 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]- 3-(1H-indol-3-yl)-maleimide, BIS I; 2,3-bis(1H-indol-3-yl)-N-methylmaleimide, BIS V; intracellular free Ca2+ concentration, [Ca2+]i; 1,2-dioctanoyl-3-(2-nitrobenzyl)-sn-glycerol, diacylglycerol, DAG; ethyleneglycol-bis-(beta -aminoethyl ether)-N,N'-tetraacetic acid, EGTA; FM1-43 fluorescence, FFM1-43; phosphatidic acid, PA; protein kinase, PK; phospholipase, PL; phorbol 12-myristate 13-acetate, PMA; standard error of the mean, SEM.




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