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 ([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. alpha,-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 microM) 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
Lee
W K
WK
Department of Pharmacology, The Catholic University of Korea, 505 Banpo-dong, Seocho-gu, Seoul 137-040, South Korea.
Choi
S W
SW
Lee
H R
HR
Lee
E J
EJ
Lee
K H
KH
Kim
H O
HO
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000008:administration & dosage / Q000378:metabolism*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D009711:Nucleotides / Q000494:pharmacology; D009994:Osmolar Concentration; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D058914:Purinergic Antagonists; D011760:Pyrrolidinones / Q000494:pharmacology; D011983:Receptors, Purinergic / Q000502:physiology*; D013498:Suramin / Q000494:pharmacology
D003470:Culture Media; D004791:Enzyme Inhibitors; D004963:Estrenes; D009711:Nucleotides; D010726:Phosphodiesterase Inhibitors; D058914:Purinergic Antagonists; D011760:Pyrrolidinones; D011983:Receptors, Purinergic; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D013498:Suramin; D000255:Adenosine Triphosphate; D019833:Genistein; D002118:Calcium