Polycystin-2 expression and function in adult mouse lacrimal acinar cells

Abstract

Purpose: Lacrimal glands regulate the production and secretion of tear fluid. Dysfunction of lacrimal gland acinar cells can ultimately result in ocular surface disorders, such as dry eye disease. Ca(2+) homeostasis is tightly regulated in the cellular environment, and secretion from the acinar cells of the lacrimal gland is regulated by both cholinergic and adrenergic stimuli, which both result in changes in the cytosolic Ca(2+) concentration. We have previously described the detailed intracellular distribution of inositol-1,4,5-trisphosphate receptors (IP(3)Rs), and ryanodine receptors (RyRs) in lacrimal acinar cells, however, little is known regarding the expression and distribution of the third major class of intracellular Ca(2+) release channels, transient receptor potential polycystin family (TRPP) channels.

Methods: Studies were performed in adult lacrimal gland tissue of Swiss-Webster mice. Expression, localization, and intracellular distribution of TRPP Ca(2+) channels were investigated using immunocytochemistry, immunohistochemistry, and electron microscopy. The biophysical properties of single polycystin-2 channels were investigated using a planar lipid bilayer electrophysiology system.

Results: All channel-forming isoforms of TRPP channels (polycystin-2, polycystin-L, and polycystin-2L2) were expressed in adult mouse lacrimal gland. Subcellular analysis of immunogold labeling revealed strongest polycystin-2 expression on the membranes of the endoplasmic reticulum, Golgi, and nucleus. Biophysical properties of lacrimal gland polycystin-2 channels were similar to those described for other tissues.

Conclusions: The expression of TRPP channels in lacrimal acinar cells suggests a functional role of the proteins in the regulation of lacrimal fluid secretion under physiological and disease conditions, and provides the basis for future studies focusing on physiology and pharmacology.

Figure 1.

Lacrimal gland ducts express polycystin-2 Ca²⁺ channels. Immunohistochemistry on sections of adult mouse lacrimal gland shows specific immunoreactivity for polycystin-2, PCL, and polycystin-2L2 in the ducts. Polycystins were detected using AlexaFluor 594-conjugated secondary antibody (Invitrogen), DAPI was used to label nuclei. Primary antibody was omitted in the control condition. Representative images are shown. Scale bar, 10 μm.

Figure 2.

Polycystins are expressed in freshly isolated acinar cell preparations of the lacrimal gland. Representative images of polycystin-2, PCL, and polycystin-2L2 specific immunoreactivity in freshly isolated acinar cell preparations of adult mouse lacrimal gland are shown. All three isoforms yielded strong cytosolic immunoreactivity, while PCL and polycystin-2L2 also showed strong plasma membrane labeling. Primary antibody was omitted in the control condition. Representative images are shown. Scale bar, 10 μm.

Figure 3.

Polycystins localize to the membranes of the ER/Golgi and the nucleus. (A) Representative images of the subcellular localization of polycystin-2, PCL, and polycystin-2L2. Arrows indicate 15 nm immunogold label in the different subcellular compartments. Scale bar, 100 nm. (B) Quantification of immunogold label expressed as percentage of total particles measured and corrected for control (omission of primary antibody). Polycystin expression is strongest in the membranes of the ER/Golgi and nucleus, in accordance with their function as intracellular Ca²⁺ release channels. Data are shown as mean ± SEM (n = 3). ER-G, ER-Golgi apparatus; M, mitochondria; N, nucleus; PM, plasma membrane; V, vesicles.

Figure 4.

Single channel electrophysiology of polycystin-2 isolated from mouse lacrimal gland. (A) Single-channel channel activity of polycystin-2 in ER microsomes isolated from mouse lacrimal gland was determined at pCa 7 as a function of the holding potential of the lipid bilayer membrane at varying concentrations of cytosolic Mg²⁺ and is shown as the absolute channel open probability. (B) The activity dependence of lacrimal gland polycystin-2 on the cytosolic Ca²⁺ concentration was measured and normalized to maximal activity at pCa 4. Increased activity was due to elevated mean open time and frequency of single-channel openings with lower activity at low and high physiological cytosolic Ca²⁺ concentrations. Data are shown as mean ± SEM (n = 3).