Purinoceptor-mediated calcium mobilization and proliferation in HaCaT keratinocytes

To investigate the effect of nucleotides on cytosolic free calcium mobilization and proliferation activity in HaCaT keratinocytes, nucleotides-induced intracellular free calcium concentration ([Ca2+](i)) and cell proliferation observed. [Ca2+](i) to the extracellular nucleotides was determined using Ca2+ sensitive indicator, Fura-2/AM with digital video fluorescence imaging microscopy, and cell proliferation was evaluated by counting of cell number. An adenosine 5'-triphosphate (ATP)-induced [Ca2+](i) increase was observed from the concentration of 10(-8) M and was more conspicuous at higher concentrations in a concentration-dependent manner. Additionally, other nucleotides such as ADP, UTP, and 2-me-S-ATP also induced a [Ca2+](i) increase in a concentration-dependent manner. However, adenosine induced a slight increase of [Ca2+](i) only at 10(-3) M. alpha,-methylene-ATP did not evoke any rise in [Ca2+](i). The maximal response observed occurred with ATP and UTP at a concentration of 10(-4) M. The ATP-induced transient [Ca2+](i) increase was attenuated by the pretreatment with phospholipase C (PLC) inhibitor, U-73122 (10 muM) for 30 min. ATP-induced [Ca2+](i) increase and cell proliferation were inhibited by putative P2Y receptor antagonist, suramin (10(-4) M). When the HaCaT cells were stimulated with nucleotides on a concentration of 10(-4) M and cultured for 5 days, the order of effect on cell proliferation was observed to be ATP > UTP > ADP > 2-me-S-ATP. Based on these results, we suggest that extracellular ATP stimulate HaCaT keratinocytes proliferation via purinoceptor-mediated [Ca2+](i) mobilization (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.