P-2U PURINOCEPTOR MODULATION OF INTRACELLULAR CA2-LINE - DOWN-REGULATION OF CA2+ INFLUX BY PROTEIN-KINASE-C( IN A HUMAN LUNG ADENOCARCINOMACELL)

The human lung small cell adenocarcinoma cell line, A549, demonstrates a concentration-dependent rise in [Ca2+](i) in response to extracellu lar nucleotides. The cells show Ca2+ mobilization on addition of vario us nucleotides, with an order of agonist potency: UTP greater than or equal to ATP > ADP > ADP beta S > AMP; adenosine is ineffective. The E (50) values for UTP and ATP are 12.5 +/- 0.4 mu M and 18.9 +/- 0.5 mu M, respectively. Together, these results are strongly indicative of th e P-2U subclass being the major nucleotide receptor expressed in these cells. The Ca2+ response was typically biphasic consisting of an init ial spike, representing release of Ca2+ from internal stores, and a su bsequent plateau representing Ca2+ influx. The majority of cells showe d an agonist-induced Ca2+ increase that was unaffected by pretreatment with the Ca2+-ATPase inhibitors 2,5-di(tert-butyl)1,4-benzohydroquino ne or thapsigargin. Caffeine did not raise [Ca2+](i) above basal level s and applied in conjunction with nucleotide did not attenuate the ago nist-mediated response. The Ca2+ influx was sensitive to protein kinas e C, and agonist addition in the presence of a protein kinase C inhibi tor, D-erythro-sphingosine, produced a significantly potentiated Ca2influx. Furthermore, agonist-mediated Ca2+ influx was abolished in the presence of a protein kinase C activator, phorbol 12,13-dibutyrate. I t is concluded that these cells posses a functional P-2U receptor that , upon activation, causes Ca2+ mobilization from TBQ and thapsigargin insensitive stores followed by protein kinase C regulated Ca2+ influx.