Characterization of ryanodine receptors in rat colonic epithelium

Abstract

Functional evidence suggests the presence of two types of intracellular Ca(2+) channels responsible for the release of Ca(2+) from Ca(2+)-stores, i.e. inositol-1,4,5-trisphosphate (IP(3)R) and ryanodine receptors (RyR), in rat colonic epithelium. Generally, three ryanodine receptor isoforms (RyR1-RyR3) are known; however, the type of RyR at this epithelium is unknown and was the focus of the present study. RyRs were characterized by molecular biological and immunohistochemical methods in the rat colon. A transcript of RyR1 was found in mRNA from colonic crypts. In contrast, RyR2 and RyR3 were found in their corresponding reference tissues, but not in the cDNA from colonic crypts suggesting a predominant expression of the RyR1 isoform in this epithelium. In order to characterize the subcellular localization of RyR1, immunohistochemical experiments were performed. They showed that RyR1 is present in the lamina epithelialis mucosae and smooth muscle cells and is distributed equally along the whole crypt axis with no difference between surface and crypt cells. A double staining with IP(3)R3, the dominant cytoplasmic isoform of IP3Rs in this epithelium, revealed that there is only little colocalization of the two receptor subtypes within the epithelial cells. Furthermore, the epithelium is equipped with the enzyme CD38 responsible for the production of cyclic adenosine diphosphate ribose, the physiological agonist of RyR. RyRs are known to be activated by changes in the redox state. The oxidant, monochloramine evoked a ruthenium red-sensitive Ca(2+) release all over the crypt axis. This release was unaffected by prior stimulation of IP(3) receptors with ATP (and vice versa). The present data suggest a functional separation of IP(3)- and ryanodine receptor-carrying Ca(2+) stores in the colonic epithelium.