title: ATP priming of macrophage-derived chemokine responses in CHO cells expressing the CCR4 receptor.
abstract: The mechanism by which ATP primes for subsequent macrophage-derived chemokine (MDC) mediated intracellular calcium (Ca2+(i)) responses at the human CCR4 receptor stably expressed in Chinese hamster ovary (CHO) cells was investigated. MDC alone was unable to elicit a Ca2+(i) response, but pre-stimulation of cells with ATP enabled a subsequent MDC-mediated Ca2+(i) response with a pEC50 of 8.66+/-0.16. The maximal response elicited by MDC was dependent upon the concentration of ATP used to prime, but the pEC50 was stable at all ATP concentrations tested. Pertussis toxin pre-treatment did not effect the ATP response, but abolished that to MDC, demonstrating that priming with ATP did not alter G protein-coupling specificity of the CCR4 receptor. Ionomycin and thapsigargin both increased Ca2+(i) concentrations (pEC50s of 7.59+/-0.57 and 6.81+/-0.31 respectively), but were unable to prime for MDC responses, suggesting the priming mechanism was not dependent upon increases in Ca2+(i) concentrations. Priming of the MDC response was still observed when experiments were performed with low Ca2+(e) (70 microM), indicating that Ca2+ influx was not required for ATP to prime the CCR4 receptor. Neither Ro31-8220 nor wortmannin affected priming, suggesting that protein kinase C and phosphoinositol 3-kinase were not involved. In conclusion, pre-stimulation of endogenous P2Y receptors with ATP facilitates Ca2+ signalling at the recombinant CCR4 receptor in CHO cells, although the mechanism by which this occurs remains to be defined.
authors.affiliation: Novartis Horsham Research Centre, Wimblehurst Road, RH12 5AB, UK.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Rosethorne
authors.forename: Juliet R
authors.initials: JR
authors.lastname: Leighton-Davies
authors.forename: David
authors.initials: D
authors.lastname: Beer
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Charlton
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D000730:Androstadienes / Q000008:administration & dosage; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D054414:Chemokine CCL17; D054422:Chemokine CCL22; D019742:Chemokines, CC / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007211:Indoles / Q000008:administration & dosage; D007457:Iodine Radioisotopes; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D037342:Pertussis Toxin / Q000494:pharmacology; D011869:Radioligand Assay / Q000379:methods; D054398:Receptors, CCR4; D019707:Receptors, Chemokine / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D014530:Uridine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; C106230:CCL17 protein, human; C515482:CCL22 protein, human; C515354:CCR4 protein, human; D054414:Chemokine CCL17; D054422:Chemokine CCL22; D019742:Chemokines, CC; D007211:Indoles; D007457:Iodine Radioisotopes; D054398:Receptors, CCR4; D019707:Receptors, Chemokine; D018048:Receptors, Purinergic P2; D015759:Ionomycin; D014530:Uridine Diphosphate; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D002118:Calcium; C064758:Ro 31-8220; D000077191:Wortmannin