Synthesis Of A New Inducible Nitric Oxide Synthase Inhibitor And Its Effects On NOS

ObjectivePersistent hypotension and hyporeactivity to vasopressor agents of septic shock are always difficult problems to deal with. The mortality rate from septic shock has remained high( 35-50%) over the last three decades. Now it is realized that excess production of NO by iNOS has play a major role in septic shock induced hypotension and vascular hyporeactivity to other vasoconstrictors. Some studies had proved that inhibition of the iNOS/NO pathway has a beneficial effect to survival rate from septic shock by improving the altered hemodynamics, alleviating tissue damages and augmenting myocardial contractile responses to β -adrenergic stimulation. Specifically selective iNOS inhibitors have shown promising therapeutic potential. We have synthesized the new highly selective iNOS inhibitor, N~G-nitro-Arginine-Lysine tripeptide. In the present study, we examined the inhibitory effect of tripeptide on nitrogen monoxide (NO) synthesis, nitricoxide synthase (NOS ) activity and the beneficial effects on hypotension in a canine septic shock model. Methods 1. Solusion phase synthesis of tripeptide contained L-Arginine and L-Lysine.2. THP-1 cells were induced to differentiate into macrophages by PMA treatment. LPS plus tripeptide and LPS plus L-NNA were add to the medium, and the NO level, iNOS activity, iNOS protein expression were determined. Subsequently macrophages incubated with varying concentrations of tripeptide to investigate the inhibitory effect on NO synthesis.3. Primary cultured Human Umbilical Vein Endothelial Cells (HUVEC) treated with insulin, which increases the expression of eNOS, and tripeptide. Production of NO and eNOS activity were studied.4. Canine septic shock model were founded by infusion E. coli. Tripeptide was infused beginning 5 hrs after onset of infection. Mean arterial pressure and concentration of NO in serum were measured.Results1. New tripeptide [HCrArg(NO2)]-Lys(OCH3)-Arg(NO2) HC1 (MW=635 ) ] was synthesized and its character was stable in water.2. LPS increased NO synthesis in macrophages, and this effect was reversed by tripeptide (P<0.01) and L-NNA (P<0.05) ; The tripeptide group was significantly greater than L-NNA group (P<0.05) .3. At the concentration of 1 X lO^mol/L to 7.5 X 10'4mol/L, the NO synthesis in macrophages was markedly inhibited in a dose-dependent manner through treatment with tripeptide (P<0.01) and L-NNA (P<0.05) .respectively. Under the same conditions, the tripeptide group was significantly greater than the L-NNA group (P<0.05) .4. The addition of tripeptide (P<0.01)and L-NNA (P<0.05) inhibited iNOS activity in macrophages espectively, and tripeptide had more potency than L-NNA (P<0.05) . L-NNA could significant inhibit eNOS activity compared with control group (P<0.05) , the tripeptide had no significanteffects on cNOS activity.5. Both tripeptide and L-NNA had no effect on iNOS protein expression..6. L-NNA could significantly inhibit NO synthesis and eNOS activity in HUVEC (P<0.05) . Tripeptide had little effect on eNOS in HUVEC compared with control group.7. Infusion E. coli successfully produced septic shock animal model. 5 hours after injecting E. coli, the MAP of dogs significantly decreased and serum NO concentration increased significantly. Infusion of tripeptide significantly increased MAP compared with both the controK P<0.01) and L-NNA (P<0.05) alone treatment group and decreased serum NO concentration.Conclusion1. Tripeptide could notably inhibit NO synthesis.2. The mechanism of action of tripeptide is to inhibit iNOS activity and its action is concentration dependence.3. Tripeptide has little effect on cNOS activity.4. Tripeptide is a useful agent to reserve hypotension and decrease plasma NO level in a canine septic shock model.