Cytoprotective properties of novel nonpeptide calpain inhibitors in renal cells.
Calpains are cytosolic, Ca(2+)-activated, neutral cysteine proteases. Rabbit renal proximal tubule (RPT) cells express both mu- and m-calpain. Although multiple calpain inhibitors protect against RPT cell death, most calpain inhibitors lack specificity, membrane permeability, and/or potency. A group of novel catalytic site-directed calpain inhibitors, including chloroacetic acid N'-[6,7-dichloro-4-(4-methoxy-phenyl)-3-oxo-3,4-dihydroquinoxalin-2-yl]hydrazide (SJA7019) and chloroacetic acid N'-(6,7-dichloro-4-phenyl-3-oxo-3,4-dihydroquinoxalin-2-yl) hydrazide (SJA7029), were identified to be potent calpain inhibitors in vitro. The goals of this study were to determine the action of these two compounds on 1) RPT calpain activity using fluorescein isothiocyanate-casein zymography, 2) antimycin A-induced RPT extracellular (45)Ca(2+) influx and cell death, and 3) hypoxia/reoxygenation-induced RPT cellular dysfunction and death. The results showed that the SJA compounds inhibited RPT mu- and m-calpain with equal potency (approximate IC(50), 30 microM) and efficacy, and blocked antimycin A-induced extracellular Ca(2+) influx and cell death. In addition, SJA7029 blocked cell death and allowed the recovery of mitochondrial function and active Na(+) transport in RPTs subjected to hypoxia/reoxygenation. In summary, the SJA compounds 1) were more potent inhibitors of calpains than catalytic site-directed peptide inhibitors in this model, 2) prevented extracellular Ca(2+) influx during the late phase of cell death, and 3) are true cytoprotectants and allow recovery of RPT cellular functions after injury.
Liu
Xiuli
X
Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
Harriman
Jay F
JF
Schnellmann
Rick G
RG
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors*; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D000860:Hypoxia / Q000473:pathology; D007527:Isoenzymes / Q000037:antagonists & inhibitors; D007668:Kidney / Q000166:cytology* / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects; D010101:Oxygen Consumption / Q000187:drug effects; D011817:Rabbits
D015853:Cysteine Proteinase Inhibitors; D005456:Fluorescent Dyes; D007527:Isoenzymes; D000968:Antimycin A; D002154:Calpain; D016650:Fluorescein-5-isothiocyanate; D002118:Calcium