Neuronal nitric oxide synthase (NOS) overexpression in polymorphonuclear cells from Parkinson’s disease patients

GATTO EM; CARRERAS MC; RIOBÓ N; CHEÑAVSKY A; RUBIO A; SATZ L; FERNÁNDEZ MM; PODEROSO JJ

Los Angeles, EEUU

Congreso; Third International Conference Biochemistry and Molecular Biology of Nitric Oxide.; 1998

NO Society

Nitric oxide (NO) and peroxynitrite have been implicated in the physiopathology of Parkinson’s disease (PD). In accord, inhibition of neuronal NOS (nNOS) in primates or knock out of nNOS gene in mice could almost completely prevent MPTP-induced parkinsonism; while overexpression of Cu-Zn-SOD partially ameliorated the síndrome. Recently, we have reported a 50% increased NO release during PMA-stimulated respiratory Bursa in polymorphonuclear cells (PMN) from Esther de novo or chronically treated PD patients, suggesting a role for NO in idiophatic PD. This study was conducted to compare expresión levels of nNOS in peripheral PMN from patients with PD and healthy controls. To that purpose, blood simples were obtained from 4 PD patients meeting CAPIT criteria for idiopathic PD and 4 healthy control donors. Total RNA was obtained from 2x106 purified PMN cells, together with human skeletal muscle simples during surgical procedures as positive control for nNOS. The b-actin and nNOS specific cDNA were synthesized from RNA of 1-3x104 and 50-140x103 cells, respectively. Fifhteen PCR ampllification cycles were performed for b-actin and thirty PCR were performed for nNOS and hybridization temperatura of 64ºC. The seto f primers used for nNOS amplification were 5’-TCC TGC CCA TCA TCG TAG GC-3’ and 5’-CCG GAG ACA TCA TTC TTG CGG-3’. PCR products were electrophoresed and analyzed by Southern blot and hybridization at 42ºC with apecific probe for nNOS (5’P32- CAC GTG GTC CTC ATT CTG-3’ and afterwards washed at 62 ºC; densitometric data were normalizad to b-actin content. Neuronal NOS was detected in all 4 PD and 3 out of 4 controls. When comparing the expresión level, ¾ PD showed increased nNOS, one of them reaching a 10-fold increase respect to the corresponding paired control; in one patient, nNOS expresión was similar to the paired control. These findings suggest that the increased NOS activity detected in PMN from PD patients is related to an overexpression of nNOS, supporting a role of NO in the physiopathology of PD. Whether nNOS overexpression in PD is genetically determined or secondary to changes of gene regulation remains to be elucidated.