R W Lambert

Carbachol-induced increase of Na+/H+ antiport and recruitment of Na+,K(+)-ATPase in rabbit lacrimal acini.

Parallel arrays of Na+/H+ and Cl-/HCO3- antiporters are believed to catalyze the first step of transepithelial electrolyte secretion in lacrimal glands by coupling Na+ and Cl- influxes across acinar cell basolateral membranes. Tracer uptake methods were used to confirm the presence of Na+/H+ antiport activity in membrane vesicles isolated from rabbit lacrimal gland fragments. Outwardly-directed H+ gradients accelerated 22Na+ uptake, and amiloride inhibited 96% of the H+ gradient-dependent 22Na+ flux. Amiloride-sensitive 22Na+ influx was half-maximal at an extravesicular Na+ concentration of 14 mM. In vitro stimulation of isolated lacrimal acini with 10 microM carbachol for 30 min increased Na+/H+ antiport activity of a subsequently isolated basolateral membrane sample 2.5-fold, but it did not significantly affect Na+/H+ antiport activity measured in intracellular membrane samples. The same treatment increased basolateral membrane Na+,K(+)-ATPase activity 1.4-fold; this increase could be accounted for by decreases in the Na+,K(+)-ATPase activities of intracellular membranes. Thus, it appears that cholinergic stimulation causes recruitment of additional Na+,K(+)-ATPase pump units to the acinar cell basolateral plasma membrane. The mechanistic basis of the increase in basolateral membrane Na+/H+ antiport activity remains unclear.

Stimulation-Associated Redistribution of Na,K-ATPase in Rat Lacrimal Gland

SummaryTo test the possibility that stimulation of secretion leads Na,K-ATPase to be recruited from cytoplasmic pools and inserted into basal-lateral plasma membranes, we surveyed the subcellular distributions of Na, K-ATPase in resting and stimulated fragments of rat exorbital lacrimal gland. After a two-dimensional separation procedure based on differential sedimentation and density gradient centrifugation, we defined sixdensity windows, which differ from one another in their contents of biochemical markers. The membranes equilibrating inwindow I could be identified as a sample of basal-lateral membranes; in resting preparations these membranes contained Na,K-ATPase enriched 16.6-fold with respect to the initial homogenates.Windows II throughVI contained various cytoplasmic membrane populations; these accounted for roughly 80% of the total recovered Na,K-ATPase activity. Thirty-minute stimulation with 10 μm carbachol caused a 1.4-fold increase (P<0.05) in the total Na,K-ATPase content ofwindow I; this increase could be largely accounted for by a 1.7-fold decrease in the total Na,K-ATPase content ofdensity window V. Acid phosphatase activity also redistributed following stimulation, but it was recruited from a different source, and it was inserted into membranes equilibrating inwindows II andIII as well as into the membranes ofwindow I.

Subcellular Organization of Ion Transporters in Lacrimal Acinar Cells: Secretagogue-Induced Dynamics

The electrolyte-driven secretion of water is one of the major functions of the lacrimal gland, and impairment of this function is an obvious cause of dry eyes. As reviewed elsewhere (Mircheff, 1986, 1989), Alexander et al. showed in 1972 that the lacrimal gland produces fluid in two stages, secretion of a NaCl-rich solution in the acini, and secretion of a KCl-rich solution in the ducts. Studies of ductal transport have begun only recently (Saito, this volume). In contrast, modern concepts of acinar secretory mechanisms began to emerge more than ten years ago, when Dartt and coworkers (1981) presented the first evidence that the lacrimal glands conform to the principles of epithelial electrolyte secretion formulated by Silva and coworkers (1977),

Use of Phase Partitioning in Multidimensional Subcellular Fractionation

Conventional subcellular fractionation procedures incorporate two separation dimensions, one based on membrane sedimentation coefficient, the other on equilibrium density. Questions about the homogeneity of samples obtained with these procedures have been addressed with additional separations based on manipulation of membrane density or on partitioning in dextran-poly(ethylene glycol) two-phase systems. In some cases it has proven useful to employ a total of 4 separation dimensions, i.e. sedimentation, equilibrium density in 2 differently designed density gradients, and phase partitioning. In other cases, parallel third dimensions analyses in two-phase systems with different pH values have been used to delineate the multiplicity of populations present in conventional subcellular samples.