Alpha 1-acid glycoprotein is upregulated in severe COVID-19 patients and decreases neutrophil NETs in SARS-CoV-2 infection.

Orosomucoid, or alpha-1 acid glycoprotein (AGP), is a major acute-phase protein expressed in response to systemic injury and inflammation. AGP has been described as an inhibitor of neutrophil migration on sepsis, particularly its immunomodulation effects. AGP's biological functions in coronavirus disease 2019 (COVID-19) are not understood. We sought to investigate the role of AGP in severe COVID-19 infection patients and neutrophils infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Epidemiological data, AGP levels, and other laboratory parameters were measured in blood samples from 56 subjects hospitalized in the ICU with SARS-CoV-2 infection. To evaluate the role of AGP in NETosis in neutrophils, blood samples from health patients were collected, and neutrophils were separated and infected with SARS-CoV-2. Those neutrophils were treated with AGP or vehicle, and NETosis was analyzed by flow cytometry. AGP was upregulated in severe COVID-19 patients (p<0.05). AGP level was positively correlated with IL-6 and C-reactive protein (respectively, p=0.005, p=0.002) and negatively correlated with lactate (p=0.004). AGP treatment downregulated early and late NETosis (respectively, 35.7% and 43.5%) in neutrophils infected with SARS-CoV-2 and up-regulated IL-6 supernatant culture expression (p<0.0001). Our data showed increased AGP in COVID-19 infection and contributed to NETosis regulation and increased IL-6 production, possibly related to the Cytokine storm in COVID-19.

Effect of methylene blue on hemodynamic response in the early phase of septic shock: A case series.

RATIONALE: Methylene blue (MB) has been used to increase blood pressure in septic shock, acting on the activity of guanylate cyclase and nitric oxide synthase. PATIENCE CONCERNS: The aim of this study is to demonstrate the benefit of MB in early phase of septic shock.Diagnoses: We report 6 cases of patients with septic shock with up to 72 hours of evolution. INTERVENTIONS: We used MB after fluid replacement, use of norepinephrine and vasopressin. Patients received a loading dose of MB and maintenance for 48 hours. OUTCOMES: All patients presented a reduction in the dose of vasopressors and lactate levels soon after the administration of the loading dose of MB, an effect that was maintained with the maintenance dose for 48 hours. Interleukin 6 and interleukin 8 were elevated at the beginning of the septic condition, with a progressive and marked reduction after the beginning of MB infusion, demonstrating a role of MB in reducing the inflammatory activity. LESSONS: This case series suggests that MB used early in the treatment of septic shock may be useful in reducing vasopressor dose and lactate levels. Further studies are still required to further validate these findings.

Imbalanced matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 activities in patients with thromboangiitis obliterans.

The pathogenic mechanisms of thromboangiitis obliterans (TAO) are not entirely known and the imbalance of matrix metalloproteinases (MMPs) plays a role in vascular diseases. We evaluated the MMP-2 and MMP-9 circulating levels and their endogenous tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) in TAO patients with clinical manifestations. The study included 20 TAO patients (n = 10 female, n = 10 male) aged 38-59 years under clinical follow-up. The patients were classified into two groups: (1) TAO former smokers (n = 11) and (2) TAO active smokers (n = 9); the control group included normal volunteer non-smokers (n = 10) and active smokers without peripheral artery disease (n = 10). Patient plasma samples were used to analyze MMP-2 and MMP-9 levels using zymography, and TIMP-1 and TIMP-2 concentrations were determined by enzyme-linked immunosorbent assays. The analysis of MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios (which were used as indices of net MMP-2 and MMP-9 activity, respectively) showed significantly higher MMP-9/TIMP-1 ratios in TAO patients (p < 0.05). We found no significant differences in MMP-2/TIMP-2 ratios (p > 0.05). We found higher MMP-9 levels and decreased levels of TIMP-1 in the TAO groups (active smokers and former smokers), especially in active smokers compared with the other groups (all p < 0.05). MMP-2 and TIMP-2 were not significantly different in patients with TAO as compared to the control group (p > 0.05). In conclusion, our results showed increased MMP-9 and reduced TIMP-1 activity in TAO patients, especially in active smokers compared with non-TAO patients. These data suggest that smoke compounds could activate MMP-9 production or inhibit TIMP-1 activity.

Methylene Blue not Contraindicated in Treating Hemodynamic Instability in Pediatric and Neonate Patients.

The present review was carried out to describe publications on the use of methylene blue (MB) in pediatrics and neonatology, discussing dose, infusion rate, action characteristics, and possible benefits for a pediatric patient group. The research was performed on the data sources PubMed, BioMed Central, and Embase (updated on Aug 31, 2020) by two independent investigators. The selected articles included human studies that evaluated MB use in pediatric or neonatal patients with vasoplegia due to any cause, regardless of the applied methodology. The MB use and 0 to 18-years-old patients with vasodilatory shock were the adopted criteria. Exclusion criteria were the use of MB in patients without vasoplegia and patients ≥ 18-years-old. The primary endpoint was the increase in mean arterial pressure (MAP). Side effects and dose were also evaluated. Eleven studies were found, of which 10 were case reports, and 1 was a randomized clinical study. Only two of these studies were with neonatal patients (less than 28 days-old), reporting a small number of cases (1 and 6). All studies described the positive action of MB on MAP, allowing the decrease of vasoactive amines in several of them. No severe side effects or death related to the use of the medication were reported. The maximum dose used was 2 mg/kg, but there was no consensus on the infusion rate and drug administration timing. Finally, no theoretical or experimental basis sustains the decision to avoid MB in children claiming it can cause pulmonary hypertension. The same goes for the concern of a possible deleterious effect on inflammatory distress syndrome.

Effect of methylene blue on hemodynamic and metabolic response in septic shock patients.

INTRODUCTION: Septic shock is a lethal disease responsible for a large proportion of deaths in the Intensive Care Unit (ICU), even with therapy centered on fluid resuscitation, use of vasopressors and empirical antibiotic therapy applied within the first hour of diagnosis. Considering the multifactorial pathophysiology of septic shock and the mechanism of action of vasopressors, some patients may not respond adequately, which can lead to the maintenance of vasodilatation, hypotension and increased morbidity, and mortality. This protocol aims to verify whether the use of methylene blue in septic patients with an early diagnosis can contribute to an earlier resolution of a shock compared to standard treatment. METHODS AND ANALYSIS: This is a study protocol for a single-center randomized clinical trial design in an ICU of a tertiary university hospital. In this study, we intend to include 64 patients aged between 18 and 80 years with a diagnosis of septic shock, of any etiology, with up to 72 hours of evolution after volume restoration, using norepinephrine at a dose ≥0.2 µg/kg/min and vasopressin at a dose of 0.04 IU/min. After the initial approach, we will randomize patients into two groups, standard care, and standard care plus methylene blue. The sample size was calculated in order to show 30% differences in septic shock resolution between groups. The Research Ethics Committee approved the study, and all patients included will sign an informed consent form (Clinical registration: RBR-96584w4).

Inflammatory markers and restenosis in peripheral percutaneous angioplasty with intravascular stenting: current concepts.

In this article, we review the current status of inflammation linked to percutaneous transluminal angioplasty with stent implantation, especially as it relates to restenosis and its clinical implications. Common to multiple vascular diseases, including atherosclerosis, interventional restenosis is a localized inflammatory reaction. Activated smooth muscle cells respond to local inflammation and migrate from the media into the lumen of the vessel, where they proliferate and synthesize cytokines which they respond to in an autocrine manner, sustaining the progression of the lesion. The deleterious effects of proinflammatory cytokines, particularly immunomodulatory interleukins, on vascular pathophysiology and development of these maladaptive processes have been the subject of intense study. Matrix metalloproteinases and tissue inhibitors of metalloproteinases are important in many physiologic and pathologic processes and their expression is related with the classic cardiovascular risk factors as well as with inflammation. They seem to play a central role in atherosclerosis and restenosis. The primary use of drug-eluting stents has become routine clinical practice for coronary artery disease, but the use in peripheral arteries remains to be further studied, like that demonstrated in sirolimus-coated Cordis trials. New studies to understand this complex process in peripheral arteries are warranted.

Chronic alcoholism associated with diabetes impairs erectile function in rats.

OBJECTIVE: To investigate the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)-mediated relaxation of cavernosal smooth muscle (CSM). MATERIAL AND METHODS: Male Wistar rats were divided into four groups: control, isocaloric, diabetic and ethanol-diabetic. The CSMs were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (ACh; 0.01-1000 micromol/L), sodium nitroprusside (SNP, 0.01-1000 micromol/L) or EFS (1-32 Hz) in strips pre-contracted with phenylephrine (10 micromol/L). Immunoexpression of endothelial NO synthase (eNOS) and inducible NOS (iNOS) was also accessed. RESULTS: The endothelium-dependent relaxation induced by ACh was decreased in CSM from ethanol-diabetic rats when compared with the controls, with a mean (sem) of 21 (4) vs 37 (2)%. Similarly, the potency and maximal responses induced by SNP were reduced in the ethanol-diabetic [3.97 (0.38) and 85 (1)%, respectively] and diabetic groups [3.78 (0.56) and 81 (2)%, respectively] when compared with the controls [5.3 (0.22) and 90 (3)%, respectively] and isocaloric [5.3 (0.19) and 92 (1)%, respectively] groups. Noradrenergic nerve-mediated contractions of CSM in response to EFS were increased in rats from ethanol-diabetic and diabetic groups when compared with the control and isocaloric groups. Conversely, there were no differences in EFS-induced relaxation among the groups. The immunostaining assays showed overexpression of eNOS and iNOS in the CSM from diabetic and ethanol-diabetic rats when compared with the control and isocaloric rats. CONCLUSION: There was an impairment of relaxation of CSM from ethanol-diabetic and diabetic rats that involved a decrease in the NO-cyclic guanosine monophosphate signalling pathway by endothelium-dependent mechanisms accompanied by a change in the CSM contractile sensitivity.

Comparisons of the release of vasodilator substances from left and right cardiac chambers of the isolated perfused rabbit heart: implications for intraventricular thrombus formation.

Prostacyclin (PgI(2)) and endothelium-derived nitric oxide (EDNO) are produced by the arterial and venous endothelium. In addition to their vasodilator action on vascular smooth muscle, both act together to inhibit platelet aggregation and promote platelet disaggregation. EDNO also inhibits platelet adhesion to the endothelium. EDNO and PgI(2) have been shown to be released from the cultured endocardial cells. In this study, we examined the release of vasoactive substances from the intact endocardium by using isolated rabbit hearts perfused with physiological salt solution (95% O(2)/5% CO(2), T=37 degrees C). The right and left cardiac chambers were perfused through separate constant-flow perfusion loops (physiological salt solution, 8 ml min(-1)). Effluent from left and right cardiac, separately, was bioassayed on canine coronary artery smooth muscle, which had been contracted with prostaglandin F(2alpha_)(2 x 10(-6)M) and no change in tension was exhibit. However, addition of calcium ionophore A23187 (10(-6)M) to the cardiac chambers' perfusion line induced vasodilation of the bioassay coronary ring, 61.4+/-7.4% versus 70.49+/-6.1% of initial prostaglandin F(2alpha) contraction for the left and right cardiac chambers perfusate, respectively (mean+/-SEM, n=10, p>0.05). Production of vasodilator was blocked totally in the left heart but, only partially blocked in the right heart by adding indomethacin (10(-5)M) to the perfusate, respectively, 95.2+/-2.2% versus 41.5+/-4.8% (mean+/-SEM, n=10, p<0.05). 6-Keto prostaglandin F(1alpha), measured in the endocardial superfusion effluent was also higher for the left cardiac chambers than for the right at the time of stimulation with the A23187, respectively, 25385.88+/-5495 pg/ml (n=8) versus 13,132.45+/-1839.82 pg/ml (n=8), (p<0.05). These results showed that cyclooxygenase pathway plays major role in generating vasoactive substances for the left cardiac chamber endocardium; while it is not the main pathway for the right ventricular endocardium at which EDNO and PgI(2) could act together and potentiate their antithrombogenic activities in isolated perfused rabbit heart. This may be an explanation for the intraventricular thrombus mostly seen in left ventricle rather than in right ventricle as a complication of myocardial infarction.

Blood cardioplegia with N-acetylcysteine may reduce coronary endothelial activation and myocardial oxidative stress.

OBJECTIVES: The aim of this prospective study was to compare the efficacy of intermittent antegrade blood cardioplegia with or without n-acetylcysteine (NAC) in reducing myocardial oxidative stress and coronary endothelial activation. METHODS: Twenty patients undergoing elective isolated coronary artery bypass graft surgery were randomly assigned to receive intermittent antegrade blood cardioplegia (32 degrees C-34 degrees C) with (NAC group) or without (control group) 300 mg of NAC. For these 2 groups we compared clinical outcome, hemodynamic evolution, systemic plasmatic levels of troponin I, and plasma concentrations of malondialdehyde (MDA) and soluble vascular adhesion molecule 1 (sVCAM-1) from coronary sinus blood samples. RESULTS: Patient demographic characteristics and operative and postoperative data findings in both groups were similar. There was no hospital mortality. Comparing the plasma levels of MDA 10 min after the aortic cross-clamping and of sVCAM-1 30 min after the aortic cross-clamping period with the levels obtained before the aortic clamping period, we observed increases of both markers, but the increase was significant only in the control group (P= .039 and P= .064 for MDA; P= .004 and P= .064 for sVCAM-1). In both groups there was a significant increase of the systemic serum levels of troponin I compared with the levels observed before cardiopulmonary bypass (P< .001), but the differences between the groups were not significant (P= .570). CONCLUSIONS: Our investigation showed that NAC as an additive to blood cardioplegia in patients undergoing on-pump coronary artery bypass graft surgery may reduce oxidative stress and the resultant coronary endothelial activation.

Why Methylene Blue Have to Be Always Present in the Stocking of Emergency Antidotes.

Evidence-based review of the existing literature ultimately recommends stocking of Methylene Blue (MB) as an emergency antidote in the United States. The same is reported around the world in Japan, Greece, Italy and Canada. The observation that MB is always present as the main antidote required in emergency and critical care units calls for a revisit on its effects on the NO/cGMP system to reemphasize its multisystem actions. Therefore, the present review aimed to display the role of MB in emergency units, concerning: 1) Polytrauma and circulatory shock; 2) Neuroprotection, 3) Anaphylaxis and, 4) Overdose and poisoning.

Methylene blue reduces progression of burn and increases skin survival in an experimental rat model.

Following burn, increased nitric oxide (NO) combine with superoxide anion forming peroxynitrite. Methylene blue (MB) has NO blocking and antioxidant effects. Male Wistar rats (250g) were burned bilaterally in dorsum with a comb metal plate heated inside boiling water and applied during 30s, creating four rectangular 10×20mm full-thickness burned areas separated by three 5×20mm unburned interspaces (stasis zone). 30 rats were randomized into three groups (n=10): treated groups received one dose of intraperitoneal (IP) MB injections (2mg/kg), one or six hours after injury, and control group received saline. Seven days after injury, wounds were visually analyzed for interspaces necrosis; full-thickness sections were evaluated with Masson staining; tissue fragments were processed for nitrite/nitrate (NOx) and malondialdehyde (MDA) dosages. Photographic analysis: interspaces progression to necrosis were higher in control (64.8%) than in one (44.7%) and six (13.3%) hours MB groups (P=0.0060). Histopathology showed lower necrosis percentage in one (34.85%) and six (41.62%) hours MB groups than control (77.03%) (P=0.0034) and higher normal skin percentage in one (25.33%) and six (26.85%) hours MB groups than control (8.32%) (P=0.0037). Re-epithelialization skin areas were higher in both MB groups (39.94% for one and 31.89% for six hours) than control (14.63%) (P=0.0210). Interspace's NOx increased in both MB groups (P=0.0130) with no difference in burned areas. No MDA difference was observed. IP MB injection one or six hours after injury reduced necrosis progression in stasis area in the rat comb burn model suggesting an antioxidant effect reducing oxidative stress.

Bradykinin Impairs and HOE 140 does not Protect Rat Hindlimb Skeletal Muscle Against Tourniquet-induced Reperfusion Injury.

BACKGROUND: Bradykinin (BK) is used in different tissues. Dose-dependent studies have demonstrated that low doses protect against ischemia/reperfusion (I/R) injury while higher doses lead to adverse effects. Although the beneficial effects of BK infusion were observed in myocardium, its role on the I/R impact in skeletal muscle (SM) has not been fully clarified. OBJECTIVE: This study was carried out to evaluate the effects of BK, administered in the hindlimbs of rats subjected to I/R. METHODS: The study design included three experimental groups: Group 1 control (saline), Group 2 (bradykinin), and Group 3 (HOE 140, a BK2 receptor blocker). In all three groups, rats were subjected to hindlimb ischemia for a total of 2 h followed by continuous 4 h of reperfusion with pharmacological interventions. The methods include analysis of enzymes (lactate dehydrogenase-LDH and creatinine phosphokinase-CPK), cell membrane marker of injury (malondialdeyde-MDA), recruitment of neutrophils (myeloperoxidase-MPO), and apoptosis index (immunohistochemistry TUNEL in situ peroxidase dead end). RESULTS: Except for the apoptotic index, all parameters studied were shown to be elevated in the reperfusion group intervened with BK. The blocking of BK2 receptors by HOE 140 did not affect the I/R injury. CONCLUSION: After 2 h of total ischemia, infusion of bradykinin during 4 h of reperfusion, worsened the I/R injury in the hindlimb skeletal muscle.

The use of methylene blue in the treatment of anaphylactic shock induced by compound 48/80: experimental studies in rabbits.

In this study, the isolated use of methylene blue (MB) in the treatment of anaphylactic shock induced by Compound 48/80 (C48/80), a potent histamine releaser, was examined, and the study of the effects of MB on the function of the aorta artery endothelium was accomplished in vitro. MB was used in a single 3.0 mg/kg dose, and C48/80 was used in a single 4.5 mg/kg dose. The study protocol included the following experimental groups, containing six animals each: group I (control), animals in the absence of any drug action; group II (MB), MB infusion; Group III (C48/80), anaphylactic shock induced by using C48/80; group IV (C48/80 + MB), anaphylactic shock treated with MB infusion at the moment of major hypotension; and group V (MB + C48/80), prevention of anaphylactic shock with MB by means of MB infusion minutes before the 4.5 mg/kg C48/80 infusion. Nitric oxide plasma levels were measured in each of the experimental groups. After the in vivo studies were performed, an in vitro study was conducted using segments of the abdominal aortas of the rabbits to determine the effect of MB on the arterial endothelium. The results obtained in the present investigation have shown that MB intravenous infusion does not change the mean arterial pressure when compared with the control group (n = 6 in each group, P < 0.05); that C48/80 is effective in producing experimental anaphylactic shock (n = 6, P < 0.05); that the attempt to prevent anaphylactic shock with MB results in a mean prolongation of animal survival ranging from 17 to 34 min (n = 6 in each group, P < 0.05); that MB is effective in reversing anaphylactic shock in all the studied rabbits (n = 6, P < 0.05); that absolute and percentage plasma nitrate values obtained with the experimental groups do not differ (n = 6, each group, P < 0.05); and that the in vitro study of segments of abdominal aorta has shown that there has not been endothelial dysfunction in any of the groups (n = 6 in each group, P < 0.05). The good results obtained in this study open a research path that may offer data to define new paradigms for treating anaphylaxis.

Acetylcholine-induced aortic relaxation studied in salbutamol treated rats.

It has been proposed that the acetylcholine (ACh)-induced relaxation of the rat aorta is entirely mediated by endothelium derived-nitric oxide (NO). However, some authors have reported that indomethacin pretreatment attenuates ACh-induced relaxation of rat aortic ring preparations. Moreover, it has also been suggested that cAMP accumulation may regulate either nitric oxide synthase (NOS) or cyclooxygenase (COX) expression in different tissues. Thus, in this in vitro study we have investigated the endothelial mechanisms involved in the ACh-induced relaxation of ring preparations of the rat thoracic aorta, as well as the influence chronic treatment with the selective beta(2)-agonist salbutamol had upon such mechanisms. Results of functional experiments show that N(G)-monomethyl-L-arginine (L-NMMA, 3 x 10(-4) M) considerably inhibited the ACh-induced relaxation of rat aortic ring preparations. However, indomethacin (10(-5) M) was also found to partially attenuate this ACh response, suggesting that although NO is the most important mediator of the ACh-induced relaxation of the rat aortic ring preparations, vasorelaxation may also involve prostanoids. Moreover, the results suggest that treatment with salbutamol failed to produce any change in the ACh-induced relaxation of rat aortic ring preparations.

Chronic fluoxetine treatment increases NO bioavailability and calcium-sensitive potassium channels activation in rat mesenteric resistance arteries.

Fluoxetine, a selective serotonin reuptake inhibitor (SSRI), has effects beyond its antidepressant properties, altering, e.g., mechanisms involved in blood pressure and vasomotor tone control. Although many studies have addressed the acute impact of fluoxetine on the cardiovascular system, there is a paucity of information on the chronic vascular effects of this SSRI. We tested the hypothesis that chronic fluoxetine treatment enhances the vascular reactivity to vasodilator stimuli by increasing nitric oxide (NO) signaling and activation of potassium (K+) channels. Wistar rats were divided into two groups: (I) vehicle (water for 21 days) or (II) chronic fluoxetine (10 mg/kg/day in the drinking water for 21 days). Fluoxetine treatment increased endothelium-dependent and independent vasorelaxation (analyzed by mesenteric resistance arteries reactivity) as well as constitutive NO synthase (NOS) activity, phosphorylation of eNOS at Serine1177 and NO production, determined by western blot and fluorescence. On the other hand, fluoxetine treatment did not alter vascular expression of neuronal and inducible NOS or guanylyl cyclase (GC). Arteries from fluoxetine-treated rats exhibited increased relaxation to pinacidil. Increased acetylcholine vasorelaxation was abolished by a calcium-activated K+ channel (KCa) blocker, but not by an inhibitor of KATP channels. On the other hand, vascular responses to Bay 41-2272 and 8-bromo-cGMP were similar between the groups. In conclusion, chronic fluoxetine treatment increases endothelium-dependent and independent relaxation of mesenteric resistance arteries by mechanisms that involve increased eNOS activity, NO generation, and KCa channels activation. These effects may contribute to the cardiovascular effects associated with chronic fluoxetine treatment.

Endothelium-dependent relaxation to poly-L-arginine in canine coronary arteries.

The endothelium-dependent relaxation elicited by poly-L-arginine was investigated in canine coronary arteries. Poly-L-arginine (10(-11) to 10(-7) M) induced concentration-dependent endothelium-dependent relaxation in segments of canine coronary arteries incubated with indomethacin. In the presence of indomethacin, arteries contracted with prostaglandin F2alpha and exposed also to ouabain, oxyhemoglobin, or N(omega)-nitro-L-arginine, as well as those contracted with 20 mM of KCl, presented reduced endothelium-dependent relaxation to poly-L-arginine. The combination of N(omega)-nitro-L-arginine with indomethacin produced further reduction of endothelium-dependent relaxation in arteries contracted with prostaglandin F2alpha as well as in arteries contracted with 20 mM of KCl. However, only quinacrine or calmidazolium, both in presence of N(omega)-nitro-L-arginine plus indomethacin, abolished the endothelium-dependent relaxation elicited by poly-L-arginine. The relaxation to poly-L-arginine was not significantly affected by glybenclamide. The present study demonstrates that in addition to the prostacyclin and nitric oxide-dependent mechanisms, poly-L-arginine also elicits endothelium-dependent relaxation in canine coronary arteries, stimulating the release of endothelial relaxing factor(s) produced by the phospholipase A2 pathway, but independent of the cyclooxygenase pathway, which may cause hyperpolarization of the vascular smooth muscle.

Dorsal minithoracotomy for ductus arteriosus clip closure in premature neonates.

We present a new surgical technique for patent ductus arteriosus (PDA) occlusion in premature neonates (PN). Through a dorsal minithoracotomy the PDA is dissected extrapleurally with q-tips and clipped. The short surgical time, avoidance of pleural drainage, and prevention of late breast deformity are the operation highlights.

Endothelium-dependent relaxation in response to poly-L-arginine in canine coronary arteries: implications about hyperpolarization as a mechanism of vasodilatation.

OBJECTIVE: To study the mechanism by which poly-L-arginine mediates endothelium-dependent relaxation. METHODS: Vascular segments with and without endothelium were suspended in organ chambers filled with control solution maintained at 37 C and bubbled with 95% O2 / 5% CO2. Used drugs: indomethacin, acetycholine, EGTA, glybenclamide, ouabain, poly-L-arginine, methylene blue, N G-nitro-L-arginine, and verapamil and N G-monomethyl-L-arginine. Prostaglandin F2 and potassium chloride were used to contract the vascular rings. RESULTS: Poly-L-arginine (10-11 to 10-7 M) induced concentration-dependent relaxation in coronary artery segments with endothelium. The relaxation to poly-L-arginine was attenuated by ouabain, but was unaffected by glybenclamide. L-NOARG and oxyhemoglobin caused attenuation, but did not abolish this relaxation. Also, the relaxations was unaffected by methylene blue, verapamil, or the presence of a calcium-free bathing medium. The endothelium-dependent to poly-L-arginine relaxation was abolished only in vessels contracted with potassium chloride (40 mM) in the presence of L-NOARG and indomethacin. CONCLUSION: These experiments indicate that poly-L-arginine induces relaxation independent of nitric oxide.