UP-REGULATION OF P2Y(2) NUCLEOTIDE RECEPTORS IN RAT SALIVARY-GLAND CELLS DURING SHORT-TERM CULTURE

In contrast to the widespread expression of G protein-coupled P2Y(2) r eceptors for extracellular nucleotides in permanent cell Lines of sali vary gland origin, there is less evidence for robust P2Y(2) receptor a ctivity in normal rat salivary gland cells assayed immediately after i solation. We examined the effect of shortterm culture (3 h to 6 days) of normal rat submandibular gland (SMG) cells on P2Y(2) receptor activ ity and mRNA expression. Results indicate that increases in the intrac ellular free Ca2+ concentration in SMG cells in response to the P2Y(2) receptor agonist UTP (100 mu M) were detectable after 3 h in culture and that after 3 days in culture the magnitude of the response to UTP was similar to that obtained with maximal muscarinic cholinoceptor act ivation. The Ca2+ mobilization response exhibited the pharmacological profile (UTP = ATP > 2-methylthioadenosine 5'-triphosphate) typical of the P2Y(2) receptor subtype and was accompanied by enhanced productio n of inositol phosphates, reflecting the activation of phospholipase C ubiquitously associated with P2Y(2) receptors. The time-dependent inc rease in P2Y(2) receptor activity was accompanied by an increase in th e steady-state level of P2Y(2) receptor mRNA, as assessed by reverse t ranscription-polymerase chain reaction. Other studies revealed that th e increased P2Y(2) receptor activity was independent of cell prolifera tion, was similar in serum-containing and defined culture media, and w as blocked by inhibitors of transcription and translation. Upregulatio n of the P2Y(2) receptor was observed in both acinar cell-and ductal c ell-enriched cultures of the SMG and in cells isolated from rat paroti d and sublingual glands but not in cells isolated from the pancreas. T hese in vitro results were complemented by in vivo studies in which P2 Y(2) receptor activity and mRNA levels were increased in SMG after lig ation of the main excretory duct but were not increased in the contral ateral, nonligated gland. These Endings suggest that changes in the ex pression and activity of the P2Y(2) receptor in salivary gland cells m ay he related to pathological challenges to the gland in vivo.