J Biol Chem. 2002 Sep 13;277(37):33987-94. Epub 2002 Jun 27. Calmodulin activates electron transfer through neuronal nitric-oxide synthase reductase domain by releasing an NADPH-dependent conformational lock.
Craig DH, Chapman SK, Daff S.
Department of Chemistry, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh EH9 3JJ, Scotland, United Kingdom.
Neuronal nitric-oxide synthase (nNOS) is activated by the Ca(2+)-dependent binding of calmodulin (CaM) to a characteristic polypeptide linker connecting the oxygenase and reductase domains. Calmodulin binding also activates the reductase domain of the enzyme, increasing the rate of reduction of external electron acceptors such as cytochrome c. Several unusual structural features appear to control this activation mechanism, including an autoinhibitory loop, a C-terminal extension, and kinase-dependent phosphorylation sites. Pre-steady state reduction and oxidation time courses for the nNOS reductase domain indicate that CaM binding triggers NADP(+) release, which may exert control over steady-state turnover. In addition, the second order rate constant for cytochrome c reduction in the absence of CaM was found to be highly dependent on the presence of NADPH. It appears that NADPH induces a conformational change in the nNOS reductase domain, restricting access to the FMN by external electron acceptors. CaM binding reverses this effect, causing a 30-fold increase in the second order rate constant. The results show a startling interplay between the two ligands, which both exert control over the conformation of the domain to influence its electron transfer properties. In the full-length enzyme, NADPH binding will probably close the conformational lock in vivo, preventing electron transfer to the oxygenase domain and the resultant stimulation of nitric oxide synthesis.
PMID:_12089147
J Biol Chem. 2002 Sep 13;277(37):33995-4002. Epub 2002 Jun 28. Endogenous methylarginines regulate neuronal nitric-oxide synthase and prevent excitotoxic injury.
Cardounel AJ, Zweier JL.
Molecular and Cellular Biophysics Laboratories, Department of Medicine, Division of Cardiology, The Johns Hopkins Medical Institutions, Baltimore, MD 21224, USA.
Nitric oxide (NO) has a critical role in neuronal function; however, high levels lead to cellular injury. While guanidino-methylated arginines (MA) including asymmetric dimethylarginine (ADMA) and N(G)-methyl-l-arginine (NMA) are potent competitive inhibitors of nitric oxide synthase (NOS) and are released upon protein degradation, it is unknown whether their intracellular concentrations are sufficient to critically regulate neuronal NO production and secondary cellular function or injury. Therefore, we determine the intrinsic neuronal MA concentrations and their effects on neuronal NOS function and excitotoxic injury. Kinetic studies demonstrated that the K(m) for l-arginine is 2.38 microm with a V(max) of 0.229 micromol mg(-1) min(-1), while K(i) values of 0.67 microm and 0.50 microm were determined for ADMA and NMA, respectively. Normal neuronal concentrations of all NOS-inhibiting MA were determined to be approximately 15 microm, while l-arginine concentration is approximately 90 microm. These MA levels result in >50% inhibition of NO generation from neuronal NOS. Down-modulation or up-modulation of these neuronal MA levels, respectively, dramatically enhanced or suppressed NO-mediated excitotoxic injury. Thus, neuronal MA profoundly modulate NOS function and suppress NO mediated injury. Pharmacological modulation of the levels of these intrinsic NOS inhibitors offers a novel approach to modulate neuronal function and injury.
PMID:_12091378
J Biol Chem. 2002 Sep 6;277(36):33518-28. Epub 2002 Jul 01. Inhibition of cytokine-induced matrix metalloproteinase 9 expression by peroxisome proliferator-activated receptor alpha agonists is indirect and due to a NO-mediated reduction of mRNA stability.
Eberhardt W, Akool el-S, Rebhan J, Frank S, Beck KF, Franzen R, Hamada FM, Pfeilschifter J.
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitat, Frankfurt am Main D-60590, Germany. w.eberhardm.uni-frankfurt.de
Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
PMID:_12093797
J Pharmacol Exp Ther. 2003 Apr;305(1):123-30. Renal function in a rat model of analgesic nephropathy: effect of chloroquine.
Ahmed MH, Ashton N, Balment RJ.
School of Biological Sciences, University of Manchester, Manchester, United Kingdom.
The antimalaria drug chloroquine is often taken against a background of analgesic nephropathy caused by nonsteroidal anti-inflammatory drugs such as paracetamol (acetaminophen). Chloroquine has marked effects on the normal kidney and stimulates an increase in plasma vasopressin via nitric oxide. The aim of this study was to determine the renal action of chloroquine in a model of analgesic nephropathy. Sprague-Dawley rats (n = 6-8/group) were treated with paracetamol (500 mg kg(-1) day(-1)) for 30 days in drinking water to induce analgesic nephropathy; control rats received normal tap water. Under intraval anesthesia (100 mg kg(-1)) rats were infused with 2.5% dextrose for 3 h to equilibrate and after a control hour they received either vehicle, chloroquine (0.04 mg h(-1)), N(omega)-nitro-L-arginine methyl ester (L-NAME, nitric-oxide synthase inhibitor, 60 micro g kg(-1) h(-1)) or combined chloroquine and L-NAME over the next hour. Plasma was collected from a parallel group of animals for vasopressin radioimmunoassay. Long-term paracetamol treatment resulted in a decrease in glomerular filtration rate (p < 0.05), sodium excretion (p < 0.001), and urine osmolality (p < 0.001), but no change in urine flow rate compared with untreated animals. Chloroquine administration in paracetamol treated rats induced a significant reduction (p < 0.05) in urine flow rate and a significant increase in plasma vasopressin (p < 0.001). These effects were blocked by coadministration of L-NAME and thus seem to be mediated by a pathway involving nitric oxide. However, these responses contrast with the chloroquine-induced diuresis previously observed in untreated rats, possibly reflecting paracetamol inhibition of renal prostaglandin synthesis and consequent moderation of vasopressin's action.
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