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.