Purpose: To further understand the regulation of microtubules and their function in the lacrimal gland, we investigated the effects of two serine/threonine phosphatase inhibitors, okadaic acid (300 nM-1 microM) and calyculin A (20-100 nM), on microtubules and stimulated secretion in lacrimal acini.
Methods: Primary rabbit lacrimal acini cultured for two days were utilized. Microtubule structure was probed using biochemical analysis and confocal fluorescence microscopy. Carbachol-stimulated and basal protein secretion were determined by measurement of released protein or, for pulse-chase studies, [(35)S]-protein.
Results: Biochemical analysis and confocal fluorescence microscopy showed that both inhibitors caused a major loss of cellular microtubules and also of acetylated (stable) microtubules. However, calyculin A was more potent than okadaic acid in causing microtubule loss. Because changes in microtubules can partially impair stimulated protein secretion in lacrimal acini, the effects of inhibitors on protein secretion were also evaluated. Both inhibitors caused a comparable dose-dependent and significant (p </= 0.05) inhibition of carbachol-stimulated (1 mM) but not basal protein secretion. These agents also significantly inhibited protein synthesis, although pulse-chase experiments suggested that the effects on secretion were elicited post-synthetically.
Conclusions: Interference with normal cycles of protein phosphorylation and dephosphorylation in lacrimal acini impairs the stimulated secretory response. Although microtubules were clearly affected by protein phosphatase inhibition, changes in this array were not directly correlated with the reduced secretory response, suggesting that the inhibitory effects on secretion may proceed through microtubule-independent as well as microtubule-dependent mechanisms.