Multiple P2X receptors are involved in the modulation of apoptosis in human mesangial cells: evidence for a role of P2X4.
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 P2X(7), prevented morphological changes but potentiated apoptosis induced by BzATP. P2X(7) 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 P2X(7) in eATP-mediated apoptosis and pointed to the involvement of other P2X receptor(s). Molecular and inhibitor studies suggested a main role for P2X(4) receptor in nucleotide-induced apoptosis in HMC, indicating a relevant role for purinergic signaling in regulating death rate in these cells.
Solini
Anna
A
Department of Internal Medicine, University of Pisa, Pisa, Italy. a.solini@med.unipi.it
Santini
Eleonora
E
Chimenti
Daniele
D
Chiozzi
Paola
P
Pratesi
Federico
F
Cuccato
Sabina
S
Falzoni
Simonetta
S
Lupi
Roberto
R
Ferrannini
Ele
E
Pugliese
Giuseppe
G
Di Virgilio
Francesco
F
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000008:administration & dosage / Q000031:analogs & derivatives / Q000494:pharmacology / Q000502:physiology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D004594:Electrophysiology; D004789:Enzyme Activation; D045604:Extracellular Fluid / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D050527:Mesangial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008858:Microscopy, Phase-Contrast; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; D013997:Time Factors
C548226:P2RX4 protein, human; C546468:P2RX7 protein, human; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058848:Receptors, Purinergic P2X4; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; C045674:2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D053148:Caspase 3; D002118:Calcium