Protein phosphatase inhibitors alter cellular microtubules and reduce carbachol-dependent protein secretion in lacrimal acini

Purpose. To further understand the regulation of microtubules and their fun ction in the lacrimal gland, we investigated the effects of two serine/thre onine phosphatase inhibitors, okadaic acid (300 nM-1 mu M) 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 f luorescence microscopy. Carbachol-stimulated and basal protein secretion we re determined by measurement of released protein or, for pulse-chase studie s, [S-35]-protein. Results. Biochemical analysis and confocal fluorescence microscopy showed t hat both inhibitors caused a major loss of cellular microtubules and also o f acetylated (stable) microtubules. However, calyculin A was more potent th an okadaic acid in causing microtubule loss. Because changes in microtubule s can partially impair stimulated protein secretion in lacrimal acini, the effects of inhibitors on protein secretion were also evaluated. Both inhibi tors caused a comparable dose-dependent and significant (p less than or equ al to 0.05) inhibition of carbachol-stimulated (1 mM) but not basal protein secretion. These agents also significantly inhibited protein synthesis, al though 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 respo nse. Although microtubules were clearly affected by protein phosphatase inh ibition, changes in this array were not directly correlated with the reduce d secretory response, suggesting that the inhibitory effects on secretion m ay proceed through microtubule-independent as well as microtubule-dependent mechanisms.