Role of protein kinases in regulation of apical secretion and basal-lateral membrane recycling traffic in reconstituted rabbit lacrimal gland acini.

Abstract

Cholinergic stimulation activates at least two different membrane traffic functions in lacrimal gland acinar cells: (i) exocytic release of stored secretory proteins across the apical plasma membranes, which is followed by retrieval and recycling of secretory vesicle constituents, and (ii) recycling traffic between basal-lateral plasma membranes and endomembrane compartments, e.g., membranes involved in translocation of neurotransmitter and neuropeptide receptors and of Na,K-ATPase and other ion transport proteins. The signal transduction and effector mechanisms involved in regulated protein secretion have been studied most extensively in rat extraorbital lacrimal glands.1–4 Work with this preparation has produced considerable evidence implicating both branches of the phosphoinositide cascade, including Ca2+-mediated activation of Ca2+/calmodulin-dependent protein kinase and diacylglycerol-mediated activation of protein kinase C.5,6 A role for phospholipase D has also been suggested.7 In contrast, relatively little is known about regulation of the basal-lateral plasma membrane-endomembrane recycling traffic. Therefore, the goals of the present study were to assess the effects of protein kinase activators and inhibitors on secretion of β-hexosaminidase, a marker for function of the apical secretory pathway,8 and on endocytosis of Lucifer Yellow, a marker for function of the basal-lateral plasma membrane-endomembrane recycling pathway, in rabbit lacrimal gland acinar cells.9,10 Accordingly, we employed the phorbol ester, phorbol-12-myristate-13-acetate (PMA); the Ca2+-ionophore, A23187; the protein kinase C inhibitor, calphostin c; and the Ca2+/calmodulin-dependent protein kinase (CaM kinase II) inhibitor, KN62.