P2u purinoceptor modulation of intracellular Ca2+ in a human lung adenocarcinoma cell line: down-regulation of Ca2+ influx by protein kinase C.
The human lung small cell adenocarcinoma cell line, A549, demonstrates a concentration-dependent rise in [Ca2+]i in response to extracellular nucleotides. The cells show Ca2+ mobilization on addition of various nucleotides, with an order of agonist potency: UTP > or = ATP > ADP > ADP beta S > AMP; adenosine is ineffective. The EC50 values for UTP and ATP are 12.5 +/- 0.4 microM and 18.9 +/- 0.5 microM, respectively. Together, these results are strongly indicative of the P2U subclass being the major nucleotide receptor expressed in these cells. The Ca2+ response was typically biphasic consisting of an initial spike, representing release of Ca2+ from internal stores, and a subsequent plateau representing Ca2+ influx. The majority of cells showed an agonist-induced Ca2+ increase that was unaffected by pretreatment with the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)1,4-benzohydroquinone or thapsigargin. Caffeine did not raise [Ca2+]i above basal levels and applied in conjunction with nucleotide did not attenuate the agonist-mediated response. The Ca2+ influx was sensitive to protein kinase C, and agonist addition in the presence of a protein kinase C inhibitor, D-erythrosphingosine, produced a significantly potentiated Ca2+ influx. Furthermore, agonist-mediated Ca2+ influx was abolished in the presence of a protein kinase C activator, phorbol 12,13-dibutyrate. It is concluded that these cells posses a functional P2U receptor that, upon activation, causes Ca2+ mobilization from TBQ and thapsigargin insensitive stores followed by protein kinase C regulated Ca2+ influx.
Clunes
M T
MT
Department of Child Health, University of Dundee, Ninewells Hospital, UK.
Kemp
P J
PJ
D000230:Adenocarcinoma; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D016227:Benzoquinones / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000201:enzymology; D014544:Uridine Triphosphate / Q000494:pharmacology
D016227:Benzoquinones; D004791:Enzyme Inhibitors; D010726:Phosphodiesterase Inhibitors; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D013873:Thionucleotides; C067344:2,5-di-tert-butylbenzoquinone; C030812:adenosine 5'-O-(2-thiodiphosphate); D002110:Caffeine; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D002118:Calcium; D014544:Uridine Triphosphate