Effect Of New Inducible Nitric Oxide Synthase Inhibitor On Cytosolic Free Calcium Concentration In Cultured Neonatal Rat Cardiac Myocytes

Background: Three nitric oxide synthase(NOS) enzymes are described in mammalian systems, all of which oxidize the terminal guanidino nitrogen of L-arginine to form nitric oxide (NO) and the amino acid L-citrulline. These isoforms-neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial NOS (eNOS or NOS3)-play modulatory roles in essentially all organ systems; including (but not limited to) the nervous, immune, respiratory, urologic, gastrointestinal, and cardiovascular systems. All of them are present in the heart: NOS 1 has been detected in cardiac conduction tissue and intracardiac neurons; NOS2 can be expressed by virtually all cells in the heart, often in conjunction with the expression of inflammatory cytokines; and finally, NOS3 is expressed in coronary endothelium, endocardium, and cardiac myocytes. It has been established that NO also has important effects on myocardial function which include the modulation of contractile function, the tone of vascular smooth muscle cells, energetics, substrate metabolism, cell growth and survival. There are at least two distinct downstream signaling modes for NO: 3',5'-cyclic guanosine monophosphate ( cGMP) production and protein thiol nitrosylation (S-nitrosylation). NO activates soluble guanylyl cyclase (S-GC) by binding to its heme moiety, forming an Fe-nitrosyl complex, leading to the production of cGMP, which in turn activates protein kinase G (PKG) and a cascade of biological signaling events.Subcellular localization of NOS with effector molecules is an important regulatory mechanism for NO signalling.NOS3 localizes to caveolae, where compartmentalization with P-adrenergic receptors and L-type Ca²⁺ channels allows NO to inhibitβ-adrenergic-induced inotropy. NOS1,however, is targeted to cardiac SR. NO stimulation of SR Ca²⁺ release via the ryanodine receptor (RyR) in vitro suggests that NOS1 has an opposite, facilitative effect on contractility. NOS2 is not normally present in myocardium, inflammation and/or cytokine activation cause induction of NOS2 and producing NO. It has been reported that NOS2 induction suppresses myocyte Ca²⁺ transients and contributes to depressed myocardial contractility andβ-adrenergic hyporesponsiveness. There is accumulating evidence that NO participates in all aspects of EC coupling, including receptor signal transduction, L-type Ca²⁺-channel activity, SR calcium release through the RYR, and mitochondrial respiration.L-Arginine, the sole substrate for the NOS enzyme in producing NO, is also a substrate for arginase. Thus arginase has a potential role in limiting substrate available for NO production. Arginase, often perceived solely as the last of the now six enzymes of the urea cycle, exists in 2 isoforms, cytosolic form arginase I and mitochondrial form arginaseâ…¡. Arginaseâ… is located in the cytosol and thought to be primarily involved in ureagenesis., whereas arginaseâ…¡is located in the mitochondrial matrix and has been thought to be more widely expressed and to be involved in the biosynthesis of polyamines, the amino acids ornithine, proline, and glutamate and in the inflammatory process, among others. Both arginaseâ… andâ…¡were detected in crude myocardial homogenates, but only arginaseâ…¡was present in isolated rat myocytes. Arginase inhibition with S-(2-boronoethyl)-L-cysteine (BEC) augmented Ca²⁺-dependent NOS activity and NO production in myocytes.Arginine concentration also governs NOS activity. The intracellular concentration of L-arginine in endothelial cells exceeds its K_m for the NOS enzyme by 2- to 3-fold, indicating that L-arginine availability should not limit NOS activity or NO production. Also, exogenous L-arginine administration should not influence NOS activity and NO production. However, in certain conditions, the addition of extracellular L-arginine enhances NO-dependent relaxation, giving rise to the "arginine paradox". The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of NOS2 protein expression.Objective: Solusion phase synthesis of tripeptide contained L-Arginine and L-Lysine. Primary cultured Human Umbilical Vein Endothelial Cells (HUVEC), cardiac myocytes, macrophages with different treatment and examined the inhibitory effect of tripeptide on NOS activety. Expression of NOS2 was induced by lipopolysaccharide (LPS) and interleukin-6 (IL-6) for 24 hours before NOS2 protein was assayed by western blotting and cardiac myocyes (CMs) were incubated by new arginine analog for 12h before changes of the fluorescence signal of free calcium caused by isoproterenol (ISO), aβ-AR agonist, were measured under laser scanning confocal microscope (LSCM). The roles of ryanodine receptor (RyRs), inositol 1,4,5,-triphosphate rceptor (IP3R) and L-type calcium channel involved tripeptide effects was examined.Methods: 1, Macrophages treated with LPS for 24 h to express N0S2. We examined the inhibitory effect of tripeptine on NO production in HUVEC, CMs and Macrophages.2, CMs were treated with LPS and IL-6 for 6. 12, 24 h. NOS2 expression measured by western blotting.3, CMs expressed NOS2 and treated with different concentration tripeptide or L-NNA for 12 h, to measure NO production.4, CMs expressed NOS2 and treated with BEC and/or L-VNIO, then to test the NO levels.5, CMs expressed NOS2 and treated with different NOS inhibitor for 12 h. LSCM measured the changes of fluorescence singal of free calcium caused by ISO.6, CMs treated with verapamil(inhibitor of L-type channel), procaine(inhibitor of RyRs) and heparin(inhibitor of IP3R). LSCM measured the changes of fluorescence singal. To study the role of different calcium channel inhibitors in tripeptine effects on free calcium concentration..7, Normal CMs treated with ISO, ISO+BEC, ISO+L-VNIO, ISO+BEC+L-VNIO, respectively. LSCM measured the changes of fluorescence singal caused by ISO.8, CMs expressed NOS2 and treated with tripeptide, L-VNIO, BEC, tripeptide+L-VNIO. LSCM measured the changes of fluorescence singal caused by ISO.Statistical analysisAll data are expressed as means±SD, and differences among groups were analyzed by one-way ANOVA followed by LSD test or Tambane test when equal variance was not assumed. Differences between two groups were analyzed by Independent - Samples T Test. A value of p<0.05 was considered statistically significant.Results: 1,1, Effects of tripeptide on NO productionIn macrophages expressing NOS2, after treated with tripeptide, L-NNA, the NO levels was 208.2±19.3, 279.5±20.7 and 308.9±17.1, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=73.553, P=0.000). The data analyzed by LSD shows that compared with control group, tripeptide significantly inhibited NO production in macrophages (P=0.000) and the effect of tripeptide is better than that of L-NNA (P=0.000).HUVEC expresses NOS3. The levels of NO (umol/L) in tripeptide group, L-NNA and control group is 179.5±9.4, 157.8±13.9 and 188.2±11.6, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F= 17.660, P=0.000). Compared with control group, L-NNA could significantly inhibit NO production (P=0.000) and tripeptide had no the effect (P=0.110). Tripeptide has little influence on NO production in HUVEC.CMs expressed NOS1 and NOS3. The levels of NO (umol/L) in tripeptide, L-NNA and control group is 55.5±7.5, 30.2±5.9 and 61.4±9.9, respectively. The data was analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=41.061, P=0.000). Compared with control group, L-NNA could significantly inhibit NO production (P=0.000) and tripeptide had no the effect (P=0.101).2, NOS2 protein expression in response to different treatmentCardiac myocytes treated with IL-6 and LPS for 6, 12 and 24 h, NOS2 protein expressions were measured by western blotting. The result showed that NOS2 protein began to express at 6h. The data was analyzed by one-way ANOVA. Significant difference was found in 6h, 12h and 24h group (F=187.101, P=0.000). NOS2 protein expression at 24h was significantly high compared with 6h (P=0.000) and 12h (P=0.003).3, Effect of NOS inhibtor on NO production in CMs expressed NOS2In CMs expressed NOS2, The NO values were analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=29.230, P=0.000). The data analyzed by LSD shows that compared with control group, tripeptide (P=0.000) and L-NNA (P=0.001) significantly inhibit NO production and arginine increased NO production (P=0.003). The effect of tripeptide on inhibiting NO production is better than that of L-NNA (P=0.000).CMs treated with different concentration tripeptide. The levels of NO were analyzed by one-way ANOVA. Significant difference was found in control group, tripeptide group and L-NNA group (F=20.290, P=0.000). Compared with control group, tripeptide (0.2mmol/L) could inhibit NO production significantly (P=0.004). On decrease NO production, there was no significant difference between tripeptide (0.4mmol/L) and L-NNA(lmmol/L) group (P=0.490).4, Effect of BEC and L-VNIO on NO production in CMsIn normal CMs treated with L-VINO, a NOS1 inhibitor, and BEC, a arginase inhibitor, the levels of NO were measured. The data was analyzed by factorial analyze. BEC increased NO production significantly (F=10.620, P=0.002) and L-VNIO decreased NO production significantly (F=48.909, P=0.000). BEC and L-VNIO had no interaction (F=0.838, P=0.366).5, Effect of NOS inhibitor on free calcium concentration caused by ISO in myocytes expressed NOS2Increased NOS2 activity contributes to decreased ISO-induced free calcium concentration. Free calcium concentration induced by ISO in normal myocytes is 172.0±8.0 and that in myocytes expressed NOS2 is 125.6±14.0. The data was analyzed by independent-samples t test. The result showed there is significant difference between two groups (t=9.099, P=0.000).The free calcium concentrations in every group were analyzed by one-way ANOVA. Significant difference was found in every group (F=40.726, P=0.000). Compared with control group, specific inhibition of NOS2 by Tripeptide (1mmol/L) dramatically increased free calcium concentration induced by ISO (P=0.000) and arginine significantly decreased free calcium concentration induced by ISO (P=0.007). Tripeptide (0.4mmol/L) is better than L-NNA (lmmol/L) on increasing free calcium concentration (P0.003).6, Effects of some drugs inhibiting Ca²⁺ channelsCMs treated with verapamil-a inhibitor of L-type calcium channel, heparin-a inhibitor of IP3R, and procaine-a inhibitor of RyR, the free calcium concentrations were 152.0±13.3 in control group, 137.1±10.9 in verapamil grouop, 150.0±11.0 in heparin group and 129.8±8.3 in procaine group. The data was analyzed by one-way ANOVA. Significant difference was found in every group (F=9.273, P=0.000). Verapamil (P=0.009) and procaine (P=0.000) could significantly inhibit tripeptide induced increasing in ISO-induced free calcium concentration in myocytes. Heparin has no the effect(P=0.957).7, Effect of arginase inhibitor on normal myocyte free calcium concentration caused by ISOCMs treated with BEC, L-VNIO and BEC+L-VNIO, the data of free calcium concentrations were analyzed by factorial analyze. Specific inhibition of NOS1 by L-VNIO decreased the ISO-induced free calcium concentration (F=147.008, P=0.000) and arginase inhibitor increased that in CMs (F=11.460, P=0.002). There was significant interaction between BEC and L-VNIO (F=5.356, P=0.026), L-VNIO could inhibit the effect of BEC on free calcium concentration in CMs. 8, Effect of arginase inhibitor on free Ca²⁺ concentraton caused by ISO in myocyte expressed NOS2.CMs expressed NOS2, the value of free calcium concentration was analyzed by one-way ANOVA. Significant difference was found in groups (F=17.633, P=0.000). Compared with control group, L-VNIO could significantly decreased free calcium concentration induced by ISO (P=0.017), L-VNIO+tripeptide could significantly increased free calcium concentration induced by ISO (P=0.045) and BEC had no significant influence on free calcium concentration induced by ISO (P=0.427).Conclusions:1, macrophages treated with LPS and CMs treated with LPS and IL-6 could express NOS2 protein.2, Tripeptide has highly selective and inhibitive effect on NOS2 activety.3, Arginase strongly limited ISO-induced increases in free Ca²⁺ concentration in a NOS1-dependent manner.4, Increased NOS2 activity contributed to limit ISO-induced myocardial free Ca²⁺ concentration. Tripeptide restored myocardial responsiveness toβ-adrenergic agonist via improving the functions of several calcium channels.5, Arginine strongly limited ISO-induced increases in free Ca²⁺ concentration via increasing NO production and expression of NOS2.