Abstract
In polarized epithelial cells [Ca2+]i waves are initiated in discrete regions and propagate through the cytosol. The structural basis for these compartmentalized and coordinated events are not well understood. In the present study we used a combination of [Ca2+]i imaging at high temporal resolution, recording of Ca2+-activated Cl- current, and immunolocalization by confocal microscopy to study the correlation between initiation and propagation of [Ca2+]i waves and localization of Ca2+ release channels in pancreatic acini and submandibular acinar and duct cells. In all cells Ca2+ waves are initiated in the luminal pole and propagate through the cell periphery to the basal pole. All three cell types express the three known inositol 1,4,5-trisphosphate receptors (IP3Rs). Expression of IP3Rs was confined to the area just underneath the luminal and lateral membranes, with no detectable receptors in the basal pole or other regions of the cells. In pancreatic acini and SMG ducts IP3R3 was also found in the nuclear envelope. Expression of ryanodine receptor was detected in submandibular salivary gland cells but not pancreatic acini. Accordingly, cyclic ADP ribose was very effective in mobilizing Ca2+ from internal stores of submandibular salivary gland but not pancreatic acinar cells. Measurement of [Ca2+]i and localization of IP3Rs in the same cells suggests that only a small part of IP3Rs participate in the initiation of the Ca2+ wave, whereas most receptors in the cell periphery probably facilitate the propagation of the Ca2+ wave. The combined results together with our previous studies on this subject lead us to conclude that the internal Ca2+ pool is highly compartmentalized and that compartmentalization is achieved in part by polarized expression of Ca2+ channels.
Highlights
In polarized epithelial cells [Ca21]i waves are initiated in discrete regions and propagate through the cytosol
Properties of [Ca21]i Waves in SMG Cells—In a previous study we showed that in maximally stimulated pancreatic acini, Ca21 waves tend to initiate in the apical pole and spread through the cell periphery to the basal pole [10]
It is important to note that acinar cells are about 2.5 times bigger than duct cells, which indicates that the wave travels about 2–3 times faster in SMG acinar compared with duct cells
Summary
Materials—cADPR was purchased from Calbiochem or Sigma. Preparation and characterization of the pAb raised against IP3R1–3 are described in Ref. 12. To maximize the temporal resolution, fluorescence was measured at a single excitation wavelength of 380 nm, averaging four consecutive images for each time point Under these conditions using a frame size of 256 3 240 pixels allowed recording the fluorescence of 4 –5 acinar cells at a resolution of up to 7.7 Hz. During perfusion with the control solution and just before the first stimulation, the image of the resting cells was acquired. The cells were washed with PBS and incubated in 0.5 ml of PBS containing 50 mM glycine for 10 min at room temperature This buffer was aspirated and the nonspecific sites were blocked by a 1 h incubation at room temperature with 0.25 ml of a PBS solution con-. Significant differences (p , 0.05) were measured in time to spread and time to peak in duct cells when stimulation with epinephrine is compared to that of other agonists
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