Identification and functional distribution of intracellular ca channels in mouse lacrimal gland acinar cells

Abstract

We have determined the presence and cellular distribution of intracellular calcium channels, inositol 1, 4, 5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in adult and postnatal (P10) lacrimal gland acinar cells. Western blot analysis of both P10 cultures and adult tissue identified the presence of each IP(3)R and RyR isotypes. The immunocytochemistry analysis showed a differential cellular distribution of these calcium channels where the nuclear envelope, endoplasmic reticulum (ER) and Golgi apparatus membranes represent areas with highest levels of channel expression. This IP(3)R and RyR isotype distribution is confirmed by the immuno-EM results. The findings described in this study are in agreement with published pharmacological data that shows the participation of these channels in the secretion process of the lacrimal gland acinar cells. Furthermore, the differential subcellular distribution between the isoforms could indicate a potential role of these intracellular Ca(2+ )channels on the regulation of specific cellular functions.

Fig. (1)

Identification of IP₃Rs and RyRs by immunoblotting. Western blot detection of IP₃R1-3, RyR1-3 was performed in homogenates of adult lacrimal gland tissue (G) and primary cultures of P10 lacrimal acinar cells (P) yielding bands of approximately 250 and 550-600 kDa, respectively.

Fig. (2)

Immunohistochemical analysis of IP₃R and RyR distribution in adult mouse lacrimal gland. Representative images of (A) IP₃R1, IP₃R2, IP₃R3 and (B) RyR1, RyR2 and RyR3 immunoreactivity. (C) Negative control was performed by omission of the primary antibody. Scale bar in C for A-C, 25 µm. Red (Alexa Fluor® 594 labeled immunoreactivity) shows receptor signals and the nuclear counterstain (DAPI) is displayed in blue.

Fig. (3)

Immunocytochemical analysis of IP₃Rs and RyRs distribution in acutely isolated acinar cells of the adult mouse lacrimal gland. Representative images of (A) IP₃R1, IP₃R2, IP₃R3 and (B) RyR1, RyR2 and RyR3. (C) Omission of primary antibody yielded the negative control. Red (Alexa Fluor® 594 labeled immunoreactivity) shows receptor signals and the nuclear counterstain (DAPI) is displayed in blue. Scale bar in C for A-C, 10 µm.

Fig. (4)

Immunocytochemical analysis of IP₃Rs and RyRs distribution in cultured acinar cells of the P10 lacrimal gland after 10 DIV. Representative images of (A) IP₃R1, IP₃R2, IP₃R3 and (B) RyR1, RyR2 and RyR3 are shown. (C) Primary antibody was omitted for the negative control. Red (Alexa Fluor® 594 labeled immunoreactivity) shows receptor signals and the nuclear counterstain (DAPI) is displayed in blue. Scale bar in C for A-C, 10 µm.

Fig. (5)

EM analysis of IP₃R and RyR immunoreactivity in adult lacrimal gland sections. Representative images of IP₃R1, IP₃R2, IP₃R3, RyR1, RyR2, RyR3 staining. Arrows indicate 15 nm immunogold label in different subcellular compartments. Scale bar, 100nm (RyR1, RyR2, RyR3, IP₃R3, and 160nm (IP₃R1 and IP₃R2).

Fig. (6)

EM semi-quantitative analysis of ultrastructural immunoreactivity in adult mouse lacrimal gland tissue. Graph A shows immunoreactivity for IP₃R1, 2, and 3 and graph B; for RyR1, 2, and 3. Immunoreactivity in Mitochondria (M), Vesicles (V), Nucleus (N) ER-Golgi Apparatus (ER-G), and Plasma Membrane (PM) displayed along x axis. Percentage of total particles (minus controls) indicated along Y axis. The subcellular regions most highly associated with IP₃R1 and 2 immunoreactivity are the nucleus, ER and Golgi apparatus, while the nuclear structures are more highly associated with IP₃R3. Analysis shows RyR1 and 2 to be more highly expressed in nuclear structures and ER-Golgi apparatus, while RyR3 is expressed in ER-Golgi with less immunoreactivity in the nucleus.