Submitted on November 6, 2006
Accepted on January 21, 2007
Multiple P2X receptors are involved in the modulation of apoptosis
in human mesangial cells: evidence for a role of P2X4
Anna Solini1*, Eleonora Santini1, Daniele Chimenti1, Paola Chiozzi2, Federico Pratesi3, Sabina Cuccato3, Simonetta Falzoni2, Roberto Lupi4, Ele Ferrannini3, Giuseppe Pugliese5, and Francesco Di Virgilio6
1 Department of Internal Medicine, University of Pisa, Pisa, Italy
2 Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy
3 Department of Internal Medicine, University of Pisa, Italy
4 Section of Diabetes and Metabolic Diseases, University of Pisa, Italy
5 Department of Clinical Sciences, "La Sapienza" University, Italy
6 Department of Experimental and Diagnostic Medicine, University of Ferrara, Italy
* To whom correspondence should be addressed. E-mail: a.solini{at}med.unipi.it.
Apoptosis, a normal event in renal tissue homeostasis, has been considered as a major mechanism for either resolution of glomerular hypercellularity in glomerulonephritis or loss of cellularity and progression to glomerulosclerosis in chronic renal disease. This study was aimed at investigating the role of extracellular ATP (eATP) in mediating apoptosis in human mesangial cells (HMC) and identifying the subtype(s) of purinergic receptors involved. eATP, but not uridin-5'-triphosphate (UTP), caused dose-dependent modifications of cellular morphology, as assessed by contrast-phase microscopy, and late apoptosis, as measured by Annexin V/propidium iodide based flow-cytometry and caspase-3 activation. Both phenomena were prevented by the P2X antagonist oxidized-ATP. 2', 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) was less effective than ATP, whereas 1[N,O-bis (5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl] -4-phenylpiperazine (KN62), a selective inhibitor of human P2X7, prevented morphological changes but potentiated apoptosis induced by BzATP. P2X7 was barely expressed in HMC, and showed a relatively scarce functional activity, as assessed by monitoring nucleotide-induced intracellular calcium surge and plasma membrane depolarization by Fura-2/AM and bis[1,3-diethylthiobarbiturate]trimethineoxonal uptake, respectively. These data indicated a negligible role of P2X7 in eATP-mediated apoptosis and pointed to the involvement of other P2X receptor(s). Molecular and inhibitor studies suggested a main role for P2X4 receptor in nucleotide-induced apoptosis in HMC, indicating a relevant role for purinergic signalling in regulating death rate in these cells.
