Purinoceptor-mediated calcium mobilization and proliferation in HaCaT keratinocytes

Abstract

To investigate the effect of nucleotides on cytosolic free calcium mobilization and proliferation activity in HaCaT keratinocytes, nucleotides-induced intracellular free calcium concentration ([Ca 2+] i) and cell proliferation observed. [Ca 2+] i to the extracellular nucleotides was determined using Ca 2+ 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 [Ca 2+] 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 [Ca 2+] i increase in a concentration-dependent manner. However, adenosine induced a slight increase of [Ca 2+] i only at 10 −3 M. α,-methylene-ATP did not evoke any rise in [Ca 2+] i. The maximal response observed occurred with ATP and UTP at a concentration of 10 −4 M. The ATP-induced transient [Ca 2+] i increase was attenuated by the pretreatment with phospholipase C (PLC) inhibitor, U-73122 (10 μM) for 30 min. ATP-induced [Ca 2+] 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 [Ca 2+] i mobilization