Nitric oxide synthase inhibition with N(G)-monomethyl-l-arginine: Determining the window of effect in the human vasculature.

Nitric oxide synthase (NOS) inhibition with N(G)-monomethyl-l-arginine (L-NMMA) is often used to assess the role of NO in human cardiovascular function. However, the window of effect for L-NMMA on human vascular function is unknown, which is critical for designing and interpreting human-based studies. This study utilized the passive leg movement (PLM) assessment of vascular function, which is predominantly NO-mediated, in 7 young male subjects under control conditions, immediately following intra-arterial L-NMMA infusion (0.24 mg⋅dl-1⋅min-1), and at 45-60 and 90-105 min post L-NMMA infusion. The leg blood flow (LBF) and leg vascular conductance (LVC) responses to PLM, measured with Doppler ultrasound and expressed as the change from baseline to peak (ΔLBFpeak and ΔLVCpeak) and area under the curve (LBFAUC and LVCACU), were assessed. PLM-induced robust control ΔLBFpeak (1135 ± 324 ml⋅min-1) and ΔLVCpeak (10.7 ± 3.6 ml⋅min-1⋅mmHg-1) responses that were significantly attenuated (704 ± 196 ml⋅min-1 and 6.7 ± 2 ml⋅min-1⋅mmHg-1) immediately following L-NMMA infusion. Likewise, control condition PLM ΔLBFAUC (455 ± 202 ml) and ΔLVCAUC (4.0 ± 1.4 ml⋅mmHg-1) were significantly attenuated (141 ± 130 ml and 1.3 ± 1.2 ml⋅mmHg-1) immediately following L-NMMA infusion. However, by 45-60 min post L-NMMA infusion all PLM variables were not significantly different from control, and this was still the case at 90-105 min post L-NMMA infusion. These findings reveal that the potent reduction in NO bioavailability afforded by NOS inhibition with L-NMMA has a window of effect of less than 45-60 min in the human vasculature. These data are particularly important for the commonly employed approach of pharmacologically inhibiting NOS with L-NMMA in the human vasculature.

Evidence for the pathophysiological role of endogenous methylarginines in regulation of endothelial NO production and vascular function.

In endothelium, NO is derived from endothelial NO synthase (eNOS)-mediated L-arginine oxidation. Endogenous guanidinomethylated arginines (MAs), including asymmetric dimethylarginine (ADMA) and NG-methyl-L-arginine (L-NMMA), are released in cells upon protein degradation and are competitive inhibitors of eNOS. However, it is unknown whether intracellular MA concentrations reach levels sufficient to regulate endothelial NO production. Therefore, the dose-dependent effects of ADMA and L-NMMA on eNOS function were determined. Kinetic studies demonstrated that the Km for L-arginine is 3.14 microM with a Vmax of 0.14 micromol mg-1 min-1, whereas Ki values of 0.9 microM and 1.1 microM were determined for ADMA and L-NMMA, respectively. EPR studies of NO production from purified eNOS demonstrated that, with a physiological 100 microM level of L-arginine, MA levels of >10 microM were required for significant eNOS inhibition. Dose-dependent inhibition of NO formation in endothelial cells was observed with extracellular MA concentrations as low 5 microm. Similar effects were observed in isolated vessels where 5 microm ADMA inhibited vascular relaxation to acetylcholine. MA uptake studies demonstrated that ADMA and L-NMMA accumulate in endothelial cells with intracellular levels greatly exceeding extracellular concentrations. L-arginine/MA ratios were correlated with cellular NO production. Although normal physiological levels of MAs do not significantly inhibit NOS, a 3- to 9-fold increase, as reported under disease conditions, would exert prominent inhibition. Using a balloon model of vascular injury, approximately 4-fold increases in cellular MAs were observed, and these caused prominent impairment of vascular relaxation. Thus, MAs are critical mediators of vascular dysfunction following vascular injury.

Effects of nitric oxide synthase inhibitor on acid aspiration-induced lung injury in rats.

The current study was designed to determine the effects of nitric oxide synthase (NOS) in the development of acid aspiration-induced lung injury in rats. Hydrochloric acid (HCl, 0.1 N; 2 ml/kg) or normal saline (NS, 2 ml/kg) was instilled into the lung of anesthetized, ventilated Sprague-Dawley rats. NG-monomethyl-L-arginine (L-NMMA, 20 mg kg(-1)) and a selective inducible nitric oxide synthase (iNOS) inhibitor, ONO-1714 (0.1 and 0.3 mg kg(-1)), were used to block NOS. Bronchoalveolar lavage fluid (BALF) and wet and dry measurements of lung (W/D) were obtained 5h after HCl or NS instillation. Unlike the control group, rats instilled with HCl showed significant increases in total nuclear cell counts (NCC), neutrophil counts, concentrations of albumin, tumor necrosis factor-alpha (TNF-alpha), interleukine-6 (IL-6) and nitrites/nitrates (NO(x)) in BALF. These parameters were associated with the significantly increased W/D in the HCl group compared with the NS group. ONO-1714 (0.1 mg kg(-1)) significantly prevented the increases in all these parameters. Its inhibitory effects were superior to those of L-NMMA and 0.3 mg kg(-1) of ONO-1714. NOS plays an important role in the pathogenesis of acid aspiration-induced lung injury. Furthermore, selective iNOS inhibition at the optimal dose was most effective in improving lung injury induced by acid aspiration in rats.

Pharmacodynamics of L-NMMA in type 1 diabetes patients and control subjects.

RATIONALE: L-NMMA is widely used in venous occlusion plethysmography studies to determine baseline NO production. Studies using L-NMMA indicate that endothelial dysfunction is present early in the course of diabetic microvascular complications. However, the optimal dose to maximally inhibit NO-production is unknown. OBJECTIVE: To determine the L-NMMA-dose that maximally reduces basal forearm blood flow (FBF). To investigate whether there are any differences in the response to L-NMMA between non-complicated type 1 diabetes patients and control subjects. METHODS: In eight non-complicated type 1 diabetes patients and nine healthy subjects FBF-responses to intra-arterial infusion of increasing doses of L-NMMA (0.01-1.6 mg/min/dL forearm volume [FAV]) were measured using the perfused forearm technique. RESULTS: Infusion of 0.8 mg/min/dL maximally reduced FBF. The dose of 1.6 mg/min/dL did not additionally reduce FBF. No differences existed between non-complicated type 1 diabetes patients and controls with regard to EC50 (0.017 +/- 0.02 resp. 0.22 +/- 0.02 mg L-NMMA/min/dL) or maximal vasoconstrictive response (Delta FBF: 1.13 +/- 0.4 resp. 0.97 +/- 0.4 mL/min/dL). Throughout the study blood pressure increased significantly in both groups, possibly reflecting a systemic vasoconstrictive effect of L-NMMA. CONCLUSIONS: The maximal vasoconstrictive dose was 0.8 mg/min/dL in type 1 diabetes patients as well as the control subjects. There were no significant differences between non-complicated type 1 diabetes subjects and controls with regard to the pharmacodynamics of L-NMMA. At high dosages of L-NMMA a systemic effect can not be ruled out.

Real-time direct measurement of nitric oxide in bovine perfused eye trabecular meshwork using a clark-type electrode.

NO was detected in bovine trabecular meshwork (TM). Bovine eyes were perfused (posterior ciliary artery). In some eyes (operated eyes) a NO electrode was inserted adjacent to the TM (scleral flap). Vascular perfusion/intraocular pressures (VPP/IOP) were recorded. In operated eyes, epinephrine (1 nM-100 microM) increased NO (maximally 979.9 +/- 117.6 nM, mean +/- SDM). Timolol (1 mM) shifted the epinephrine-NO concentration-response curve rightward (2.94 log units) without significantly changing the maximal response (903.0 +/- 67.7 nM, mean +/- SDM). The non-selective NO synthase (NOS) inhibitor L-NMMA (100 microM) virtually abolished the NO response to epinephrine. L-NMMA alone (1 microM-100 microM) significantly reduced tonic NO generation (maximally 109.5 +/- 24.9 nM, mean +/- SDM), whereas timolol alone (1 microM-1 mM) had no effect. In unoperated eyes, epinephrine (1 nM-100 microM) reduced IOP (maximally 2.56 +/- 0.64 mmHg, mean +/- SDM). Epinephrine (100 microM) mildly increased VPP (4.6 +/- 1.3 mmHg, mean +/- SDM). Baseline aqueous humor formation rate (11.5 +/- 3.2 microl/min, mean +/- SDM) was unaffected. Effluent perfusate (effusate) total NO(2)(-) was determined by enzymatically reducing all NO(3)(-) to NO(2)(-), then assessing resultant NO(2)(-) (Griess assay). Epinephrine (1 nM-1 microM) increased effusate NO(2)(-) (maximally 15.8 +/- 4.9 microM, mean +/- SDM). Timolol (1 mM) reduced, and L-NMMA (100 microM) virtually abolished effusate NO(2)(-) response to epinephrine. L-NMMA alone (1 microM-100 microM) reduced tonic effusate NO(2)(-) (maximally from 5.8 +/- 1.6 microM to 1.1 +/- 0.9 microM, mean +/- SDM), whereas timolol alone (1 microM-1 mM) had no effect. NO is generated tonically in bovine TM and increases in response to epinephrine.

The effect of inflammation on rat urinary bladder-dependent relaxation in coaxial bioassay system.

The effect of urinary bladder inflammation on the activity of a bladder-derived relaxant factor in the coaxial bioassay system was examined. Bladder inflammation was induced by intraperitoneal (i.p.) cyclophosphamide or intravesical lipopolysaccharide (LPS) injection to male rats. In precontracted rat anococcygeus muscle that was placed within rat bladder (coaxial bioassay system), acetylcholine induced a relaxation response, which was not altered by the denudation of urothelium or incubation with indomethacin and N(G)-methyl-L-arginine. Acetylcholine-induced relaxation was significantly attenuated, when bladders were removed from cyclophosphamide- and LPS-pretreated group of rats and were used with intact urothelium in the coaxial bioassay system. However, the impairment acetylcholine response in both pretreatment groups was not observed after denudation of the bladder urothelium. These results showed that bladder inflammation did not alter the synthesis and/or release of this bladder-derived relaxant factor, which is neither a cyclooxygenase product nor nitric oxide, but restricted its demonstration by coaxial bioassay assembly probably due to inflammation-induced mucosal oedema.

Reduced nitric oxide synthase in the brainstem contributes to enhanced sympathetic drive in rats with heart failure.

Recent studies have suggested that central nervous mechanisms are involved in the enhanced sympathetic drive observed in heart failure (HF). Nitric oxide (NO) in the brainstem has been shown to reduce sympathetic nerve activity. The aim of this study was to determine whether the expression of neuronal nitric oxide synthase (nNOS) in the brainstem is reduced in rats with HF. Heart failure was produced by myocardial infarction in Wistar-Kyoto rats (HF group). Hemodynamic and echocardiographic examinations were performed. Western blot analysis for nNOS in the nucleus tractus solitarii (NTS) and the rostral ventrolateral medulla (RVLM) in the brainstem were performed to determine the expression of the nNOS gene in the HF group or sham-operated (control) group. We also performed in situ hybridization for nNOS mRNA and distribution in the brainstem. The expression of nNOS protein in the NTS and the RVLM were reduced in the HF group compared to the control group. The expression of nNOS mRNA in the brainstem was also reduced in the HF group, particularly in the NTS, compared to the control group. Intracisternal injection of NG-monomethyl-L-arginine elicited a smaller pressor response in the HF group than in the control group. These results suggest that reduced nNOS expression in the NTS and the RVLM, and the resulting reduced NO production of these sites, contribute to the enhanced sympathetic drive in HF.

Impact of mechanism-based enzyme inactivation on inhibitor potency: implications for rational drug discovery.

Mechanism-based enzyme inactivators (MBEIs) have unique kinetic actions that make predictions of potency, selectivity, and potential for metabolic drug interactions more complex than for competitive antagonists. We have derived a mathematical relationship that links the influence of substrate concentration and binding constant ([S] and K(m), respectively), inhibitor concentration and binding constant ([I] and K(I), respectively), and inactivation rate constant (k(inact)) to enzyme activity (v) and maximal activity (V(max)) at any time (t). The kinetic behavior of this relationship was validated in murine-macrophage cell cultures using MBEIs of nitric oxide synthase (NOS). This initial equation was also used in the derivation of a new relationship that directly links the kinetic parameters of mechanism-based inactivation to inhibitory potency at a particular time (IC((t))(50)). Using this direct relationship, we observed that the predicted rank inhibitory potency of a series of MBEIs was improved over that predicted by the K(I) parameter alone. These relationships offer a fundamental understanding of the kinetics of MBEI action and may be useful in the evaluation of these compounds during the discovery process.

Treatment of psoriasis with topical NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis.

A double blind left, right comparative study was carried out in 17 psoriatic subjects to examine the influence of a topically applied inhibitor of nitric oxide (NO) synthesis on the pathogenic events of psoriasis. The inhibitor NG-monomethyl-L-arginine (L-NMMA) in aqueous cream BP was applied to one plaque while aqueous cream BP alone served as control. Compared with the control, the L-NMMA-treated side showed significant (77%) inhibition of NO production and a reduction in blood flow, confirming its bioavailability. L-NMMA significantly reduced staining for endothelial cells and intercellular adhesion molecule 1, while CD1a-positive Langerhans cells and CD8-positive suppressor cytotoxic T cells increased. CD4-positive lymphocytes and epidermal proliferation, as indicated by Ki-67 staining, were unaffected by this degree of inhibition of NO synthesis, and correspondingly significant clinical improvement was not found.

Strategies to reduce side effects of interleukin-2: evaluation of the antihypotensive agent NG-monomethyl-L-arginine.

PURPOSE: The clinical utility of high-dose intravenous recombinant interleukin (IL)-2 therapy is limited by severe toxicity including hypotension, fever, chills, pulmonary edema, and oliguria Hypotension has been previously shown to result from excessive vascular relaxation due to overproduction of the endogenous vasodilator nitric oxide. Nitric oxide production can be decreased by administration of the competitive enzyme inhibitor NG-monomethyl-L-arginine (NMA). A clinical trial to investigate the dose-dependent effects of NMA on blood pressure was undertaken in patients with metastatic renal cell carcinoma. PATIENTS AND METHODS: Patients with metastatic renal cell carcinoma receiving a 5-day continuous infusion of IL-2 (18 million IU/m2/d) who developed hypotension were treated with increasing doses of NMA, ranging from 3 to 36 mg/kg. RESULTS: Twenty-three patients received a total of 61 courses of IL-2; 18 of these patients developed hypotension and received NMA. Antihypotensive activity was observed at all dose levels, and the duration of the effect varied directly with the dose of NMA. At the higher dose levels tested (12 to 36 mg/kg), increased pulmonary vascular resistance and decreased cardiac output were observed. Patients experiencing a significant decrease in cardiac output received dobutamine (2.5 to 10 microg/kg/min). Pulmonary capillary wedge pressure was unaffected by administration of NMA. One patient treated at 24 mg/kg (bolus) experienced a major motor seizure, but no neurologic disorders were observed in other patients treated with NMA doses of 24 to 36 mg/kg. No other adverse events involving hepatic, renal, or hematologic systems were attributed to NMA. Three patients received NMA by an initial bolus followed by a continuous infusion. Similar antihypotensive effects were noted, and these patients were able to complete a full 5-day course of IL-2. CONCLUSION: The antihypotensive effects of NMA appear to be optimal at a dose of 24 mg/kg, with maintenance doses of 8 mg/kg every 4 to 6 hours. At this dose level, blood pressure was restored, and IL-2-associated vasodilatation was fully reversed. Coincident with the reversal of hypotension, the state of high cardiac output was also reversed by NMA administration. These results suggest that NMA may be effective for alleviating the hypotensive effects of high-dose IL-2 therapy in cancer patients.

Pharmacokinetic-pharmacodynamic profile of systemic nitric oxide-synthase inhibition with L-NMMA in humans.

AIMS: It has been demonstrated that inhibition of endothelium derived nitric oxide with NG-monomethyl-L-arginine (L-NMMA) results in a different cardiac and peripheral vascular response. The purpose of this study was to investigate the pharmacokinetic-pharmacodynamic profile of L-NMMA and pharmacokinetic interactions with L-arginine in healthy subjects. METHODS: Plasma pharmacokinetics were analysed from two different studies: In study 1, 3 mg kg-1 L-NMMA was administered i.v. over 5 min and systemic haemodynamics, cardiac output (CO), fundus pulsation amplitude (FPA), and NO-exhalation (exhNO) were measured at baseline and 15, 65, 95, 155, and 305 min after start of drug administration (n=7). In study 2, 17 mg kg-1 min-1 of the physiologic substrate for nitric oxide synthase, L-arginine, was coinfused i.v. over 30 min with a primed constant infusion of 50 microg kg-1 min-1 L-NMMA (n=8). RESULTS: Bolus infusion of L-NMMA resulted in a maximum plasma concentration of 12. 9+/-3.4 microg ml-1 (mean+/-s.d.) with elimination half-life of 63. 5+/-14.5 min and clearance of 12.2+/-3.5 ml min-1 kg-1 and caused a small hypertensive response, decreased CO by 13%, FPA by 26%, exhNO by 46% and increased systemic vascular resistance by 16% (P<0.05 each) 15 min after start of drug administration. Although only limited data points were available in the L-NMMA plasma concentration range between 0 and 4 microg ml-1, drug effects over time were in good agreement with an Emax model (r2>0.98 each), which also suggested that concentrations producing half-maximum effects were higher for FPA than for CO and exhNO. The coinfusion with L-arginine caused a nearly two-fold increase in plasma L-NMMA levels, indicating a pharmacokinetic interaction. CONCLUSIONS: In the absence of a systemic hypertensive response, L-NMMA significantly decreased CO, exhNO, and FPA. The concentration calculated to produce a half maximal effect was equivalent for exhNO and CO, but markedly higher for FPA. Furthermore, measurement of FPA is susceptible to changes in L-NMMA levels at small plasma concentrations.

Pharmacokinetics of the nitric oxide synthase inhibitor L-NG-methylarginine hydrochloride in patients with septic shock. Glaxo Wellcome International Septic Shock Study Group.

OBJECTIVES: To characterize the pharmacokinetics of L-NG-methylarginine in patients with septic shock. METHODS: This was an international, uncontrolled, open-label study of L-NG-methylarginine (546C88) therapy given to 32 patients with septic shock. It was conducted in hospital-based intensive care units that admit general surgical and medical patients. Patient cohorts received an infusion of L-NG-methylarginine at fixed dose rates of 1, 2.5, 5, 10, and 20 mg/kg/h for up to 8 hours. The 5 dosing regimens were administered sequentially to separate groups of patients. RESULTS: Of the 32 patients studied, 23 received complete 8-hour infusions. In the other 9 patients, the infusion was terminated prematurely within the first 1/2 to 4 hours. Median clearance of L-NG-methylarginine averaged 485 mL/h/kg for the 1 and 2.5 mg/kg/h dosing cohorts combined but decreased to 283, 181, and 98 mL/h/kg for the 5, 10, and 20 mg/kg/h dosing cohorts, respectively. Median renal clearance was similar at 9 to 26 mL/h for the 1, 2.5, and 5 mg/kg/h dosing cohorts but increased to 156 and 284 mL/h for the 10 and 20 mg/kg/h dosing cohorts, respectively. Median steady-state volume of distribution was similar in all 5 dosing cohorts, averaging 0.66 to 0.82 L/kg. CONCLUSIONS: The 80% decrease in clearance from 485 to 98 mL/h/kg with the increase in dose suggests that a predominant metabolic pathway(s) of L-NG-methylarginine, accounting for at least 80% of clearance, is becoming progressively saturable in association with L-NG-methylarginine infusion rates > or = 5 mg/kg/h. Therefore the use of L-NG-methylarginine infusion rates > or = 5 mg/kg/h are typically expected to result in progressive inhibition of nitric oxide synthase activity. Consequently, patient hemodynamics should be monitored closely to avoid an excessive increase in vasomotor tone, which would be manifest by either an increase in mean arterial pressure or a decrease in cardiac output. The infusion rates of conventional vasopressor(s) (eg, norepinephrine [BAN, noradrenaline]) or L-NG-methylarginine or both may need to be reduced accordingly.

Nonlinear pharmacokinetics of L-N(G)-methyl-arginine in rats: characterization by an improved HPLC assay.

L-N(G)-methyl-arginine (L-NMMA) is an inhibitor of nitric oxide synthase (NOS) enzymes. We have characterized the pharmacokinetics of L-NMMA in rats using HPLC. The HPLC assay requires pre-column derivatization, gradient elution and ultraviolet detection. The limit of sensitivity in plasma was 3.0 microM (0.75 microg mL(-1)). Using this assay, the pharmacokinetics of L-NMMA were characterized following iv bolus doses of 25, 50 and 100 mg kg(-1). Compartmental and noncompartmental data analysis suggest that L-NMMA pharmacokinetics are nonlinear at these doses. From the nonlinear compartmental analysis, we estimated the K(m) and V(max) parameters of L-NMMA elimination to be 70.2 microM and 4.59 microM min(-1), respectively. This estimated K(m) value of L-NMMA elimination is consistent with its nonlinear elimination characteristics in humans and its saturable metabolism by the N(G), N(G)-dimethylarginine dimethylamino-hydrolase enzyme in isolated rat tissue.

Interaction of L-arginine analogs with L-arginine uptake in rat renal brush border membrane vesicles.

The dibasic amino acid, L-arginine, is a substrate for both nitric oxide synthase (NOS) and arginase and therefore, plays an important role in cell signaling and cell growth. We examined the effects of various NOS inhibitors on L-arginine transport into rat renal brush border membrane (BBM) vesicles. L-Arginine uptake was stimulated in the presence of an inwardly directed Na+ gradient and an imposed inside negative potential in BBM but not basolateral membrane vesicles. In BBM vesicles, the L-arginine analogs, N-iminoethyl-L-orinithine and Nw-monomethyl-L-arginine (L-NMMA) were potent inhibitors of L-arginine uptake (IC50 of 0.48 and 0.82 mM, respectively), while Nw-nitro-L-arginine was less active (IC50 = 10 mM) and Nw-nitro-L-arginine methyl ester (L-NAME) was inactive. The inhibition of L-arginine transport by L-NMMA was competitive in nature. L-NIO, L-NMMA as well as L-arginine and L-lysine but not Nw-nitro-L-arginine methyl ester, trans-stimulated L-arginine uptake when preloaded into BBM vesicles. The L-arginine analogs had no effect on the transport of the neutral amino acid, L-leucine, in the same preparations. The data suggest that in addition to inhibiting NOS, the L-arginine analogs, N-iminoethyl-L-orinithine, L-NMMA and to a lesser extent L-NA, also inhibit L-arginine transport across the BBM of proximal tubules.