Analytic subcellular fractionation of acini from rat lacrimal gland

A recent hypothesis for the cellular mechanism of fluid secretion by lacrimal acini has been based, in part, on the results of subcellular fractionation analyses of lacrimal gland fragments which had been incubated for a brief period in vitro. An important assumption in those studies was that the ion transporters and neurotransmitter receptors measured in isolated subcellular fractions were associated with membranes derived from the acinar cells, since these comprise the bulk of the lacrimal gland mass. This study was undertaken to validate this assumption. Acinar complexes were isolated from rat exorbital lacrimal glands by digestion with collagenase, hyaluronidase, and DNase. Although terminal intralobular duct segments and myoepithelial cells were occasionally noted, the preparations appeared to be free of larger ducts, blood cells, blood vessels, and interstitial cells. Acinar cells were then disrupted, and the homogenates underwent the fractionation procedure used previously for lacrimal gland fragment preparations. This procedure involved a sequence of analyses by differential sedimentation, isopycnic centrifugation on sorbitol gradients, and partitioning in dextran-polyethyleneglycol two-phase systems. Calculated initial specific activities for sodium/potassium adenosinetriphosphatase (Na⁺/K⁺-ATPase), alkaline phosphatase, acid phosphatase, and succinate dehydrogenase were identical to those obtained from fragment preparations. Major membrane populations resolved by the sequential analyses, including one believed to represent endoplasmic reticulum membranes, two believed to be derived from the acinar cell basal-lateral membrane, and two believed to be derived from the Golgi complex, corresponded closely to populations resolved from lacrimal fragment preparations. In addition to validating the previous use of lacrimal gland fragment preparations in studies of acinar cell function, these results suggest that preparations of isolated lacrimal acini will be useful for future work on neurotransmitter-receptor regulation and basal-lateral plasma membrane dynamics in the lacrimal gland.