Immunolocalization of CRHSP28 in exocrine digestive glands and gastrointestinal tissues of the rat

The 28-kDa (on SDS-PAGE) Ca2+-regulated heat stable protein (CRHSP28) was r ecently purified as novel phosphoprotein in exocrine pancreas, since it und ergoes an immediate increase in serine phosphorylation when acini are stimu lated with Ca2+-mobilizing agonists. Examination of CRHSP28 protein express ion in rat revealed that most was highly expressed in pancreas and other mo rphologically related exocrine tissues, including the parotid, lacrimal, an d submandibular glands. Immunofluorescence staining in pancreas indicated t hat CRHSP28 was specifically concentrated in zymogen granule-rich areas in the apical cytoplasm of acinar cells. Lack of colocalization with pancreati c lipase in dual immunofluorescence studies confirmed localization of CRHSP 28 to the area immediately surrounding the granules. Western analysis of pa ncreatic zymogen granule membrane proteins indicated CRHSP28 was not associ ated with the granules following their purification. A similar pattern of a pical cytoplasmic secretory granule staining was noted in lacrimal and subm andibular glands. CRHSP28 protein was also expressed at relatively high lev els in mucosal epithelial cells of the stomach and small intestine. CRHSP28 was found in the supranuclear apical cytoplasm of cells lining the small i ntestinal crypts, including Paneth cells, and was abundant in the cytoplasm of goblet cells. In the stomach, strong CRHSP28 staining was seen in mucus -secreting cells in the upper portion of the gastric glands and in the apic al, granule-rich cytoplasm of chief cells located in the lower portions of the glands. Dual labeling with anti-H+-K+-ATPase demonstrated a comparative ly lower expression of CRHSP28 in parietal cells. Collectively, the high re lative expression of CRHSP28 in various secretory cell types within the dig estive system, together with its intracellular localization surrounding the acinar cell secretory granules, strongly supports a role for CRHSP28 in Ca 2+-mediated exocrine secretion.