Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells

Apical ATP, ATP, UTP and UDP evoked transient increases in short circuit current (ISC, a direct measure of transepithelial ion transport) in confluent Caco-2 cells grown on permeable supports. These responses were mediated by a population of at least three pharmacologically distinct receptors. Experiments using cells grown on glass coverslips showed that ATP and UTP consistently increased intracellular free calcium ([Ca2+]i) whilst sensitivity to UDP was variable. Cross desensitization experiments suggested that the responses to UTP and ATP were mediated by a common receptor population. Messenger RNA transcripts corresponding to the P2Y2, P2Y4 and P2Y6 receptors genes were detected in cells grown on Transwell membranes by the reverse transcriptase–polymerase chain reaction. Identical results were obtained for cells grown on glass. Experiments in which ISC and [Ca2+]i were monitored simultaneously in cells on Transwell membranes, confirmed that apical ATP and UTP increased both parameters and showed that the UDP-evoked increase in ISC was accompanied by a [Ca2+]i-signal. Ionomycin consistently increased [Ca2+]i in such polarized cells but caused no discernible change in ISC. However, subsequent application of apical ATP or UTP evoked a small rise in ISC but no rise in [Ca2+]i. UDP evoked no such response. As well as evoking increases in [Ca2+]i, the ATP/UTP-sensitive receptors present in Caco-2 cells thus allow direct control over ion channels in the apical membrane. The UDP-sensitive receptors, however, appear to simply evoke a rise in [Ca2+]i. Keywords: Purinoceptor, pyrimidinoceptor, apical membrane, epithelial cells, anion secretion, Caco-2 cells Introduction Nucleotides in the luminal fluid exert control over epithelial ion transport processes in many polarized epithelia, and these responses are mediated by P2Y receptors in the apical plasma membrane (see e.g. Devor & Pilewski, 1999; Inglis et al., 1999; Kerstan et al., 1998; Wilson et al., 1996; Wong, 1988). Whilst this apical control system's physiological role is unknown, a body of evidence suggests that the apical receptors may be activated by ATP originating from within the epithelial cells themselves, and that they thus form part of an autocrine control system (Schwiebert, 1999; Taylor et al., 1998). Whatever their physiological significance, these receptors have attracted recent interest as they may allow pharmacological correction of the ion transport defects seen in cystic fibrosis, a potentially lethal genetic disease. Epithelial cells' sensitivity to apical nucleotides is often attributed to receptors belonging to the P2Y2 subclass (Knowles et al., 1996; Parr et al., 1994) that are equally sensitive to UTP and ATP but insensitive to nucleotide diphosphates (Nicholas et al., 1996). However, it has recently become clear, that at least two other P2Y receptor subtypes, P2Y4 and P2Y6, allow cells to respond to pyrimidine nucleotides such as UTP (Communi & Boeymans, 1997; Nicholas et al., 1996). Interestingly, these receptors are essentially insensitive to ATP (Communi & Boeymans, 1997; Nicholas et al., 1996) and our earlier work (Ko et al., 1997) suggested that such ‘pyrimidinoceptors' may also allow apical nucleotides to control epithelial transport processes (see also Inoue et al., 1997; Lazarowski et al., 1997). In the present study we have therefore explored the extent to which such receptors allow apical nucleotides to control ion transport processes in a human epithelial cell line cell line. Some of the data have been presented to the Physiological Society (McAlroy et al., 1999).