Do endogenous opioids contribute to the bradycardia of rats with obstructive cholestasis?

Endogenous opioids have nitric oxide (NO)-dependent cardiovascular actions. In the light of biological evidence of accumulation of endogenous opioids in cholestasis and also existence of NO-dependent bradycardia in cholestatic subjects, this study was carried out to evaluate the role of endogenous opioids in the generation of bradycardia in a rat model of cholestasis. Male Sprague-Dawley rats were used to induce cholestasis by surgical ligation of the bile duct, with sham-operated animals serving as a control. The animals were divided into six groups which received naltrexone [20 mg/kg/day, subcutaneously (s.c.)], N(G)-L-nitro-arginine methyl ester (L-NAME, 3 mg/kg/day, s.c.), aminoguanidine (200 mg/kg/day, s.c.), L-arginine (200 mg/kg/day, s.c.), naltrexone + L-NAME (20 and 3 mg/kg/day, s.c) or saline. One week after the operation, a lead II electrocardiogram (ECG) was recorded and the spontaneously beating atria of the animals were then isolated and the chronotropic responses to epinephrine evaluated. The plasma L-nitro-tyrosine level and alanine amino transferase and alkaline phosphatase activities were also measured. The heart rate of cholestatic animals was significantly lower than that of control rats in vivo and this bradycardia was corrected with daily adminstration of naltrexone or L-NAME. The basal spontaneous beating rate of atria in cholestatic animals was not significantly different from that of sham-operated animals in vitro. Cholestasis induced a significant decrease in the chronotropic effect of epinephrine. This effect was corrected by daily injection of naltrexone or L-NAME, or concurrent administration of naltrexone + L-NAME, and was not corrected by aminoguanidine. L-arginine had an equivalent effect to L-NAME and increased the chronotropic effect of epinephrine in cholestatic rats but not in control animals. Bile duct ligation increased the plasma activity of liver enzymes as well as the level of L-nitro-tyrosine. L-arginine and naltrexone treatment significantly decreased the elevation of liver enzymes in bile duct-ligated rats. Pretreatment of cholestatic animals with naltrexone or L-NAME decreased the plasma L-nitro-tyrosine level. The results suggest that either prevention of NO overproduction or protection against liver damage is responsible for recovery of bradycardia after naltrexone administration.     

Enhancement of aspirin-induced gastric damage by cholestasis in rats

Summary— In this study the seventy of aspirin-induced gastric mucosal damage was investigated in rats with obstructive cholestasis. Cholestasis was induced by ligation and resection of the bile duct under general anesthesia. Two weeks after operation, the rats were fasted for 24 hours. Aspirin was administered orally in doses of 0, 128, 192, 266 and 335 mg/kg, and the animals were killed four hours after dosing. The dose of 266 mg/kg was chosen for a study of the time-dependency; other groups of animals were killed at time intervals of one, three, five, seven and nine hours after aspirin administration. The results showed that aspirin induces more severe gastric damage in bile duct resected rats compared with sham-operated and control animals. Salicylate levels of serums were also measured but there was no significant difference in serum salicylate levels between bile duct resected, sham-operated and control rats. It can be concluded that cholestasis can potentiate aspirin-induced gastric damage in rats.     

Interaction of cyclooxygenase isoenzymes, nitric oxide, and afferent neurons in gastric mucosal defense in rats

The cyclooxygenase (COX)-2 inhibitors 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl-2(5II)-furanone (DFU) (0.02-2 mg/kg) and N-[2-(cyclohexyloxy)-4-nitrofenyl]-methanesulfonamide (NS-398) (0.01-1 mg/kg), the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC-560) (0.05-5 mg/kg), and dexamethasone (1 mg/kg) were studied in rats challenged with intragastric acid (300 mM HCl). All compounds induced severe gastric damage when rats were treated concurrently with the inhibitor of constitutive and inducible nitric-oxide (NO) synthase N(G)-monomethyl-L-arginine methyl ester (L-NAME) (3 or 40 mg/kg). DFU and NS-398 caused significantly less damage in rats receiving the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl)acetamidine (1400W) (0.3 mg/kg). The COX-1 inhibitor SC-560 induced moderate damage in the acid-challenged stomach even without suppression of NO, but damage was aggravated by L-NAME. The COX-3 inhibitor phenacetin (400 mg/kg) did not injure the gastric mucosa despite suppression of NO. Furthermore, DFU, NS-398, SC-560, and dexamethasone caused severe injury in the acid-challenged stomach of rats pretreated with capsaicin to ablate afferent neurons. The mucosal damage induced by the COX-1 inhibitor, the COX-2 inhibitors, and dexamethasone in L-NAME- or capsaicin-treated rats was reversed by coadministration of 16,16-dimethyl-prostaglandin E2 (2 x 8 ng/kg). Gross mucosal damage was paralleled by histology. Our results support the concept that endogenous NO, prostaglandins, and afferent neurons act in concert in the regulation of gastric mucosal integrity. The prostaglandins necessary for mucosal defense in the face of NO suppression, and afferent nerve ablation can be derived either from COX-1 or COX-2. The data do not propose a protective role for a phenacetin-sensitive COX-3. Our findings suggest that not only COX-1 but also COX-2 has important functions in the maintenance of gastric integrity.     

Virus-induced airway hyperresponsiveness in guinea pigs is related to a deficiency in nitric oxide.

Intratracheal inoculation of parainfluenza type 3 virus to guinea pigs induces a marked increase in airway responsiveness in vivo and in vitro. In spontaneously breathing anesthetized guinea pigs inhalation of an aerosol containing the nitric oxide (NO) precursor L-arginine (2.0 mM) completely prevented the virus-induced airway hyperresponsiveness to histamine. In addition, perfusion of L-arginine (200 microM) or the direct NO-donor S-nitroso-N-acetyl-penicillamine (SNAP, 1 microM) through the lumen of tracheal tubes from infected animals prevented the increase in airway responsiveness to histamine or the cholinoceptor agonist methacholine. The NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME, 120 microM) did not further increase the virus-induced airway hyperresponsiveness. In additional experiments, NO was measured with an Iso-NO nitric oxide meter and sensor. Stimulation of control tissues in vitro with histamine (10(-3) M) resulted in a contraction with a simultaneous release of NO (44.5 +/- 5.4 nM). The release of NO was markedly reduced by 75% (P < 0.01, 11.4 +/- 3.1 nM) in tracheas from virus-infected animals that demonstrated enhanced contractile responses. Preincubation of tissues from virus-treated guinea pigs with L-arginine (200 microM) completely prevented the enhanced contraction and simultaneously returned the NO production to control values (51.2 +/- 3.4 nM). An NO deficiency might be causally related to the development of airway hyperresponsiveness after a viral respiratory infection.     

Involvement of endogenous opioid peptides and nitric oxide in the blunted chronotropic and inotropic responses to beta-adrenergic stimulation in cirrhotic rats

It is well known that chronotropic and inotropic responses to beta-adrenergic stimulation are impaired in cirrhosis, but the exact reason is not clear. Considering the inhibitory effect of endogenous opioid peptides and nitric oxide (NO) on beta-adrenergic pathway, we examined their roles in hyporesponsiveness of isolated atria and papillary muscles to isoproterenol stimulation in cirrhotic rats. Cirrhosis was induced by chronic bile duct ligation. Four weeks after ligation or sham operation, the responses of the isolated atria and papillary muscles to isoproterenol stimulation were evaluated in the absence and presence of naltrexone HCl (10(-6) m), N(omega)-nitro-L-arginine methyl ester (L-NAME, 10(-4) m), and naltrexone plus L-NAME in the organ bath. Considering the role of inducible NOS (iNOS) in hemodynamic abnormalities of cirrhotic rats, the chronotropic and inotropic responses of cirrhotic rats to isoproterenol stimulation were also assessed in the presence of aminoguanidine (a selective inhibitor of iNOS, 3 x 10(-4) m). Sham operation had no significant effect on basal atrial beating rate, contractile force, and maximal time derivatives for the development and the dissipation of papillary muscle tension. The basal atrial beating rate of cirrhotic rats did not show any significant difference compared with the sham-operated ones; however, the basal contractile parameters were significantly decreased in cirrhosis. Although the maximum effects of isoproterenol on chronotropic and inotropic responses were significantly reduced in cirrhotic rats, there was no difference in half-maximal effective concentrations of isoproterenol in these concentration-response curves. The basal abnormalities and the attenuated chronotropic and inotropic responses to isoproterenol were completely corrected by the administration of naltrexone, L-NAME and aminoguanidine. Concurrent administration of naltrexone and L-NAME also restored to normal the basal abnormalities and the blunted responses to isoproterenol in cirrhotic rats, and did not show any antagonistic effect. Based on these findings, both the endogenous opioid peptides and NO may be involved in the attenuated chronotropic and inotropic responses to beta-adrenergic stimulation in cirrhosis. It seems that the iNOS activity results in NO-induced hyporesponsiveness to beta-adrenergic stimulation in cirrhosis.     

Lack of detrimental effects of nitric oxide inhibition in bile duct-ligated rats with hepatic encephalopathy

BACKGROUND: The pathogenetic mechanisms of hepatic encephalopathy (HE) are not fully understood. Vasodilatation induced by nitric oxide (NO) may be involved in the development of HE. There is no comprehensive data concerning the effects of NO inhibition on HE in chronic liver disease. METHODS: Male Sprague-Dawley rats weighing 240-270 g at the time of surgery were selected for experiments. Secondary biliary cirrhosis was induced by bile duct ligation (BDL). Counts of movements were compared between BDL rats and rats receiving a sham operation. In another series of experiments, BDL rats received either Nomega-nitro-L-arginine methyl ester (L-NAME, 25 mg kg-1 day-1 in tap water) or tap water (control) from the 36th to 42nd days after BDL. Besides motor activities, plasma levels of tumour necrosis factor (TNF)-alpha and nitrate/nitrite, liver biochemistry tests and haemodynamics were determined after treatment. RESULTS: Compared with the sham-operated rats, the total, ambulatory and vertical movements were significantly decreased in the BDL rats (P 相似文献   

Modulation of stress-induced gastric mucosal lesions by exogenous L-arginine

L-arginine, a nitric oxide (NO) precursor, can exert both ameriolative and deteriorative effects on gastric mucosal lesions. This study was designed to determine whether exogenous L-arginine modulates stress-induced gastric mucosal lesions through NO production by either constitutive NO synthase (cNOS) or inducible NO synthase (iNOS) in gastric mucosal tissues. In rats subjected to water immersion restraint stress over a 6-hour period, the concentration of gastric mucosal nitrite/nitrate, breakdown products of NO, increased with the development of gastric mucosal lesions and a decrease in cNOS activity and a drastic increase in iNOS activity in the gastric mucosal tissue. Preadministration of L-arginine (150 to 600 mg/kg intraperitoneally) attenuated the lesion development with prevention of increases in gastric mucosal nitrite/nitrate concentration and iNOS activity. In contrast, postadministration of L-arginine (150 to 600 mg/kg intraperitoneally) enhanced the lesion development with further increase in gastric mucosal nitrite/nitrate concentration. This deteriorative action of postadministration of L-arginine (300 mg/kg intraperitoneally) was prevented by pretreatment with aminoguanidine (100 mg/kg subcutaneously), a selective iNOS inhibitor, with inhibition of increases in gastric mucosal iNOS activity and nitrite/nitrate concentration. These results indicate that preadministered L-arginine protects against water immersion restraint stress-induced gastric mucosal lesions, possibly through restricted NO production by cNOS in gastric mucosal tissues, whereas postadministered L-arginine aggravates the stress-induced gastric mucosal lesions, possibly through excessive NO production by iNOS increasing in gastric mucosal tissues.     

埃索美拉唑对大鼠胃黏膜保护的作用

目的：探讨埃索美拉唑对大鼠胃黏膜保护作用。方法：在乙醇诱导大鼠胃黏膜损伤前,预先给予埃索美拉唑（20 mg/kg）灌胃,L-硝基-精氨酸甲酯（L-NAME,4 mg/kg）静脉注射。采用激光多普勒血流计（LDF）测定胃黏膜血流量（GMBF）,采用镉粒还原和比色法测定胃黏膜NO-2/NO3-含量,并观察了胃黏膜损伤指数（Ulcer index,UI）、溃疡坏死组织和中性粒细胞浸润严重程度的变化。结果：与模型损伤组比,埃索美拉唑组大鼠UI明显降低（P〈0.01）,溃疡坏死组织和中性粒细胞浸润程度明显减轻（P〈0.05）。预先用L-NAME处理后,埃索美拉唑保护胃黏膜损伤作用明显减弱。向胃内灌注埃索美拉唑,可增加GMBF、胃黏膜NO2-/NO-3,L-NAME可逆转这种作用,但对埃索美拉唑抑制酸分泌作用无明显影响。结论：埃索美拉唑对大鼠胃黏膜具有重要的保护作用,一氧化氮（Nitric oxide,NO）介导了这种作用。     

Role of the L-arginine/nitric oxide pathway in ischaemic/reoxygenation injury of the human myocardium

The role of the L-arginine/nitric oxide (NO) pathway in myocardial ischaemic/reperfusion injury remains controversial in experimental animal models. The aim of the present studies was to investigate the role of this pathway in the human myocardium. Myocardial specimens from right atrial appendages of patients undergoing elective coronary bypass graft surgery were incubated in crystalloid buffer at 37 degrees C and subjected to 120 min of simulated ischaemia followed by 120 min of reoxygenation. Tested drugs were added 15 min before ischaemia, and maintained during ischaemia and throughout reoxygenation. Ischaemia resulted in severe myocardial damage, as assessed by the leakage of lactate dehydrogenase (LDH) into the incubation medium and by the capacity of the tissue to reduce 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan product. L-Arginine (10 mM), a precursor of NO, significantly decreased LDH leakage (from 9.0+/-0.6 to 5.3+/-0.3 units/g wet wt; P<0.05), but had no effect on MTT reduction or oxygen consumption. D-Arginine (10 mM), N(G)-nitro-L-arginine methyl ester (L-NAME; 0.5 mM), an NO synthase inhibitor, and S-nitroso-N-acetylpenicillamine (at 1, 100, 500 and 1000 microM), an NO donor, had no significant effects on the measured indices, and L-NAME did not reverse the protection afforded by L-arginine against LDH leakage. In addition, the formation of nitrotyrosine was not influenced by ischaemia/reoxygenation alone or by the agents investigated. In conclusion, these data suggest that L-arginine affords modest protection against ischaemic/reoxygenation injury of the human myocardium, an action that is NO-independent, and that NO metabolism does not play a significant role in this model.     

Endogenously synthesized nitric oxide prevents endotoxin-induced glomerular thrombosis.

Escherichia coli endotoxin (LPS) can induce the clinical syndrome of septic shock and renal cortical necrosis and can stimulate nitric oxide (NO) production from macrophages, vascular smooth muscle, and glomerular mesangial cells in vitro. NO is an endogenous vasodilator, which also inhibits platelet aggregation and adhesion. We therefore sought to determine whether LPS would stimulate NO production in vivo and, if so, whether this NO would modulate endotoxin-induced glomerular thrombosis. The stable NO end-products, NO2 and NO3, were measured in serum and urine collections from rats during baseline and after injection of LPS, with or without substances that modulate NO synthesis. The urinary excretion of NO2/NO3 was 1,964 +/- 311 nm/8 h during the baseline and increased to 6,833 +/- 776 nm/8 h after a single intraperitoneal injection of 0.1 mg/kg LPS (P < 0.05). The serum concentration of NO2/NO3 also significantly increased after LPS injection. Both the urine and serum stimulation was significantly prevented by the NO synthesis inhibitor, Nw-nitro-L-arginine methyl ester (L-NAME). L-Arginine, given with LPS+L-NAME significantly restored the NO2/NO3 levels in the urine. Ex vivo incubation of tissues from rats treated with LPS demonstrated NO production by the aorta, whole kidney, and glomeruli, but not cortical tubules. Histological examination of kidneys from rats given either LPS or L-NAME alone revealed that 2 and 4.5% of the glomeruli contained capillary thrombosis, respectively. In contrast, rats given LPS+L-NAME developed thrombosis in 55% of glomeruli (P < 0.001), which was significantly prevented when L-arginine was given concomitantly. We conclude that LPS stimulates endogenous production of NO in vivo and that this NO is critical in preventing LPS-induced renal thrombosis.     

Nitric oxide production modulates cyclosporin A-induced distal renal tubular acidosis in the rat

Cyclosporine A (CsA) causes distal renal tubular acidosis (dRTA) in humans and rodents. Because mice deficient in nitric-oxide (NO) synthase develop acidosis, we examined how NO production modulated H+ excretion during acid loading and CsA treatment in a rat model. Rats received CsA, L-arginine (L-Arg), or N omega-nitro-L-arginine methyl ester (L-NAME), or combinations of CsA and L-NAME or L-Arg, followed by NH4Cl (acute acid load). In vehicle-treated rats, NH4Cl loading reduced serum and urine (HCO3-) and urine pH, which was associated with increases in serum [K+] and [Cl-] and urine NH3 excretion. Similar to CsA (7.5 mg/kg), L-NAME impaired H+ excretion of NH4Cl-loaded animals. The combination CsA and L-NAME reduced H+ excretion to a larger extent than either drug alone. In contrast, administration of L-Arg ameliorated the effect of CsA on H+ excretion. Urine pH after NH4Cl was 5.80 +/- 0.09, 6.11 +/- 0.13*, 6.37 +/- 0.16*, and 5.77 +/- 0.09 in the vehicle, CsA, CsA + L-NAME and CsA + L-Arg groups, respectively (*P < 0.05). The effect of CsA and alteration of NO synthesis were mediated at least in part by changes in bicarbonate absorption in perfused cortical collecting ducts. CsA or L-NAME reduced net HCO3- absorption, and, when combined, completely inhibited it. CsA + L-Arg restored HCO3- absorption to near control levels. Administration of CsA along with L-NAME reduced NO production to below levels observed with either drug alone. These results suggest that CsA causes dRTA by inhibiting H+ pumps in the distal nephron. Inhibition of NO synthesis may be one of the mechanisms underlying the CsA effect.     

Carbenoxolone gastroprotective mechanism: participation of nitric oxide/(c) GMP/K(ATP) pathway in ethanol-induced gastric injury in the rat

Carbenoxolone, a semi-synthetic triterpenoid, exhibits gastroprotective activity related to the participation of nitric oxide (NO); however, the complete NO/(c) GMP/K(ATP) channels pathway for carbenoxolone is unknown. Therefore the aim of this study was to examine the NO/(c) GMP/K(ATP) channels pathway as the gastroprotective mechanism of carbenoxolone in the ethanol-induced gastric injury model in the rat. Oral administration of carbenoxolone (30 mg/kg, p.o.) exhibited gastroprotective effect against ethanol-induced gastric injury in rats. Pretreatment with N(G) -nitro-l-arginine methyl ester (L-NAME, 70 mg/kg, i.p.); 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, guanylate cyclase inhibitor, 10 mg/kg, i.p.); or glibenclamide (K(ATP) channels inhibitor, 1 mg/kg, i.p.) reversed the gastroprotective effect of carbenoxolone for ethanol-induced gastric injury. Furthermore, gastric prostaglandins and NO levels increased after carbenoxolone administration in ethanol-induced gastric injury in rats. In conclusion, our results suggest that the increase of NO levels in gastric tissue after pretreatment with carbenoxolone activates the NO/(c)GMP/K(ATP) channels pathway, the principal gastroprotective mechanism of carbenoxolone.     

雷贝拉唑对胃黏膜损伤保护的作用

目的 探讨雷贝拉唑对大鼠胃黏膜保护作用.方法 在乙醇诱导大鼠胃黏膜损伤前,预先给予雷贝拉唑(20mg/kg)灌胃,L-硝基-精氨酸甲酯(L-NAME.4mg/kg)静脉注射.采用激光多普勒血流计(LDF)测定胃黏膜血流量(GMBF),采用镉粒还原和比色法测定胃黏膜和血浆NO2-/NO3-含量,并观察胃黏膜损伤指数(Ulcer index,UI)、溃疡坏死组织和中性粒细胞浸润严重程度的变化.结果 与模型损伤组比,雷贝拉唑组大鼠UI明显降低(P＜0.01),溃疡坏死组织和中性粒细胞浸润程度明显减轻(P＜0.05).预先用L-NAME处理后,雷贝拉唑保护胃黏膜损伤作用明显减弱.向胃内灌注雷贝拉唑,可增加GMBF、胃黏膜和血浆NO2/NO3,L-NAME可逆转这种作用,但对雷贝拉唑抑制酸分泌作用无明显影响.结论 雷贝拉唑对大鼠胃黏膜具有重要的保护作用,一氧化氨(Nitric oxide,NO)介导了这种作用.     

Detrimental effects of nitric oxide inhibition on hepatic encephalopathy in rats with thioacetamide-induced fulminant hepatic failure

Hepatic encephalopathy is a complex neuropsychiatric syndrome seen secondary to acute liver failure, chronic parenchymal liver disease, or portal-systemic anastomosis. Vasodilatation induced by nitric oxide (NO) may be involved in the development of hepatic coma. However, there are no comprehensive data concerning the effects of NO inhibition on the severity of hepatic encephalopathy. Male Sprague-Dawley rats weighing 300-350 g were used. Fulminant hepatic failure was induced by intraperitoneal injection of thioacetamide (TAA, 350 mg kg-1 day-1) for 3 days. Rats were divided into two groups to receive either NG-nitro-L-arginine methyl ester (L-NAME, 20 mg kg-1 day-1 via intragastric gavage) or normal saline (N/S) from 2 days prior to TAA administration for 5 days. Severity of encephalopathy was assessed by counts of motor activity and neurobehaviour test scores. Plasma levels of endotoxin, tumour necrosis factor-alpha and nitrate/nitrite were determined by the chromogenic Limulus assay, enzyme-linked immunosorbent assay and colorimetric assay, respectively. Compared with N/S-treated rats, the mortality rate was significantly higher in rats receiving L-NAME (59% vs. 18%, P < 0.01). Inhibition of NO had detrimental effects on the counts of motor activities (P < 0.05) and neurobehaviour score (P < 0.01). Rats treated with L-NAME had significantly higher plasma levels of endotoxin (26.7 +/- 3.8 pg mL-1) and tumour necrosis factor-alpha (29.4 +/- 6.5 pg mL-1) compared with rats treated with N/S (13.2 +/- 2.7 pg mL-1 and 11.2 +/- 2.6 pg mL-1, respectively, P < 0.01). Plasma levels of endotoxin and tumour necrosis factor-alpha, but not of nitrate/nitrite, were significantly correlated with the severity of hepatic encephalopathy (P < 0.05). Chronic L-NAME administration had detrimental effects on the severity of encephalopathy in TAA-treated rats, suggesting a protective role of NO in the development of fulminant hepatic failure.     

Nitric oxide synthase inhibition increases venular leukocyte rolling and adhesion in septic rats

OBJECTIVE: Excess production of nitric oxide (NO) has been implicated in hypotension and blood flow abnormalities in sepsis, but NO is also an important inhibitor of leukocyte rolling and adhesion. Leukocyte adhesion is increased in sepsis despite elevated NO production. We hypothesized that inhibition of NO synthase (NOS) could increase leukocyte adhesion in sepsis. DESIGN: Prospective animal study. SETTING: Experimental animal laboratory. SUBJECTS: Twenty-five male rats, anesthetized with ketamine and acepromazine. INTERVENTIONS: Topical superfusion of the nonselective NOS inhibitor N(G)-monomethyl-L-arginine (NMA) on skeletal muscle postcapillary venules. MEASUREMENTS AND MAIN RESULTS: Rats made septic by cecal ligation and puncture were compared with controls that underwent sham ligation. Leukocyte rolling and adhesion were measured in cremasteric postcapillary venules of septic and control rats using in vivo videomicroscopy. The effects of NOS inhibition on leukocyte rolling and adhesion were also measured. After a stable baseline was reached, 1 microM of the nonselective NOS inhibitor NMA was suffused topically followed by physiologic buffer. The effects of L-arginine on leukocyte rolling and adhesion were also measured, both before and after suffusion of NMA. Leukocyte rolling and adhesion was increased in septic rats as compared with controls (control 5.5+/-0.9 rolling cells/min, 1.0+/-0.3 adherent cells/min; septic 13.7+/-2.0 rolling cells/min, 3.1+/-0.6 adherent cells/min; p < .001), and NOS inhibition further increased leukocyte rolling and adhesion in both septic and control rats (control 14.0+/-1.7 rolling cells/min, 2.8+/-0.5 adherent cells/min; septic 25+/-2.1 rolling cells/min, 5.4+/-0.5 adherent cells/min; both p < .001 vs. baseline). Prior suffusion of excess L-arginine prevented the increase in leukocyte adhesion with NMA in septic rats (2.6+/-0.4 adherent cells/min vs. 3.0+/-0.6 adherent cells/min; n = 3; p > .05). When administered after NMA, excess L-arginine partially reversed leukocyte adhesion in septic rats (5.4+/-0.7 adherent cells/min, with NMA vs. 4.3+/-0.7 adherent cells/min, after L-arginine; n = 5; p < .05). Venular shear did not differ between septic and control rats (600+/-109 (sec(-1)) vs. 620+/-37 (sec(-1)); p > .05). CONCLUSIONS: Although NOS inhibition may ameliorate hypotension in sepsis, such therapy may be deleterious by increasing leukocyte adhesion.     

Gene transfer of endothelial nitric oxide isoform decreases rat hindlimb vascular resistance in vivo

The objective of this study was to design a methodology of gene transfer into a resistance vascular bed and to show if such a method can be used to examine the physiological function of a given gene product in vivo. We developed such a method and validated it by defining the role in vivo of endothelial nitric oxide synthase (eNOS). In a constant flow perfused rat hindlimb, gene transfer to the vascular endothelium was accomplished by incubating a "first-generation" serotype 5, replication-deficient, adenoviral vector (1.2 X 10(9) plaque-forming units/ml) containing cDNA encoding either the eNOS or the beta-galactosidase (beta-Gal) gene in the hindlimb vasculature for 30 min. Five days after infection, immunohistochemical staining for eNOS localized recombinant gene expression to vascular endothelial cells and eNOS protein levels were increased fourfold (11.9 +/- 6.6 vs. 2.9 +/- 1.3 intensity units/microg protein, n = 4, p < 0.05). Perfusion pressures were measured at different flow rates (10-50 ml/min). In addition, basal and acetylcholine (ACh)-stimulated vascular resistance (VR) in phenylephrine (PE)-precontracted (100 microM) hindlimb was measured at constant flow. There were flow-dependent increases (p < 0.05) in perfusion pressure. Overexpression of eNOS shifted the pressure-flow curve downward and administration of N(G)-nitro-L-arginine methyl ester (L-NAME) shifted the curve upward. Compared with beta-Gal-transfected rats, PE-induced VR decreased (p < 0.05) in eNOS-transfected rats (100 +/- 27 vs. 164 +/- 49 mmHg, n = 5). Addition of 100 microM L-NAME increased (p < 0.05) PE-induced VR in both eNOS-transfected and control rats (145 +/- 50 and 232 +/- 38 mmHg, n = 5, p < 0.05), respectively, which was partially abolished by L-arginine pretreatment. ACh-induced vasorelaxation was increased 45% (p < 0.05) in eNOS-transfected hindlimbs. L-NAME decreased (p < 0.05) ACh-induced vasorelaxation by 58% in eNOS-transfected hindlimbs versus 25% in beta-Gal-transfected hindlimbs (p < 0.05). We used this gene transfer method to examine the physiological function of a gene product in vivo and showed that (1) the flow-pressure relationship in the hindlimb vascular bed is NO dependent and (2) the eNOS enzyme modulates NO-mediated vasorelaxation in the rat hindlimb resistance arteries in vivo.     

Prolactin secretion in hypothyroid endotoxemic rats: involvement of L-arginine and nitric oxide synthase

The identification of nitric oxide (NO) within the hypothalamus and pituitary gland has suggested its role as modulator of the activity on hypothalamic-pituitary axis. Hypothalamic NO synthase (NOS) is known to be regulated by thyroid hormones. We investigated the effects of previous injection of N-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, and L-arginine (L-Arg), the substrate for NO synthesis, on prolactin (PRL) secretion, through the lipopolysaccharide (LPS)-induced inflammatory response in thyroidectomized (TX) rats. TX or sham-operated (N) rats were intraperitoneally (i.p.) injected with L-NAME (10 mg kg) or L-Arg (200 mg kg) or the same volume of vehicle (saline solution) 30 min before endotoxemia-induction with LPS at 250 mug (100 g body weight), i.p.. In N rats, NO increased PRL release in response to endotoxemia, whereas in hypothyroid rats, NO appeared to have the opposite effect. Our data support the hypothesis that NO exerts a modulatory influence on PRL secretion after LPS-induced inflammatory response.     

Comparison between selective and nonselective nitric oxide synthase inhibition and phenylephrine in normal and endotoxic swine

OBJECTIVE: To compare the cardiopulmonary and peripheral circulatory effects of the nonselective nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) to the more selective inducible NOS inhibitor S-methylisothiourea (SMT) and to phenylephrine (PE) in endotoxic and normal swine. DESIGN: Prospective, randomized, unblinded study. SETTING: Research laboratory of academic medical center. SUBJECTS: Nonanesthetized, sedated, mechanically ventilated, minimally invasive swine model. INTERVENTIONS: Animals received either lipopolysaccharide (LPS, LPS groups) or equivalent volume of saline (normal groups). LPS animals were further randomized into four groups when mean arterial pressure (MAP) had dropped to <60 mm Hg: the LPS/saline group received saline only; the other groups received either L-NAME, SMT, or PE. These were titrated to elevate MAP by 20-25 mm Hg, and animals were followed for another 3 hrs. Pulmonary artery occlusion pressure was maintained at one to two times baseline with the infusion of saline. Normal groups received the same agents 1 hr after baseline measurements, and drugs were titrated to achieve similar increases in MAP. We measured gastric-arterial PCO2 gradient by tonometry as an index of gastric mucosal perfusion. Left ventricular volumes were determined echocardiographically; right ventricular volumes were determined by a pulmonary arterial catheter equipped with a rapid thermistor. Plasma nitrite/nitrate (NOx) concentrations were measured hourly. MEASUREMENTS AND MAIN RESULTS: In the LPS groups, all agents elevated MAP and systemic vascular resistance similarly. By hr 4, cardiac output had decreased in all groups, but the decrease with L-NAME (35% +/- 16%) occurred earlier (at hr 3) and was larger than the decrease with SMT at hrs 3 and 5 and larger than the decrease with saline at hrs 3 to 5. L-NAME resulted in a larger increase in mean pulmonary arterial pressure (MPAP) when compared with saline (130% +/- 44% vs. 61% +/- 25%; p < .001) and SMT groups (130% vs. 97% +/- 80%; p < .007). Only L-NAME had detrimental effects on right ventricular function as indicated by an increase in right ventricular end-systolic volume (54 +/- 10 to 87 +/-6 mL; p < .05) and right ventricular end-diastolic volume (90 +/-11 to 128 +/- 18 mL; p < .05). SMT decreased both left ventricular end-systolic volume (10.4 +/- 2 to 7.7 +/- 4 mL; p < .05) and left ventricular end-diastolic volume (18.5 +/- 3 to 14.2 +/- 5 mL; p < .05), indicating improved left ventricular function, whereas L-NAME did not affect left ventricular volumes. Both SMT and PE corrected LPS-induced gastric mucosal acidosis, but L-NAME did not. We did not detect changes in plasma NOx concentrations in any of LPS groups. In the normal groups, all agents increased MAP without changes in plasma NOx concentrations. L-NAME caused a larger decrease in cardiac output, but the increase in MPAP was higher with SMT. Both NOS inhibitors led to left ventricular dilation, but PE did not. Only L-NAME caused right ventricular dilation. There were no changes in gastric-arterial PCO2 gradient. CONCLUSIONS: In LPS animals, we failed to detect changes in plasma NOx concentrations. Furthermore, for similar increases in MAP, SMT improved gastric mucosal acidosis, had less adverse effects on right ventricular function and MPAP, and may have improved left ventricular function. However, apart from its bene-ficial effects on left ventricular function, SMT was not superior to PE. The results from normal animals indicate that both NOS inhibitors have adverse effects on cardiac function beyond those attributed to increased MAP.     

Investigating the role of endogenous opioids and KATP channels in glycerol-induced acute renal failure

The present study was designed to investigate the possible role of endogenous opioids and K(ATP) channels in glycerol-induced acute renal failure (ARF) in rats. The rats were subjected to rhabdomyolytic ARF by single intramuscular injection of hypertonic glycerol (50% v/v; 8 mL/kg), and the animals were sacrificed after 24 h of glycerol injection. The plasma creatinine, blood urea nitrogen, creatinine clearance, and histopathological studies were performed to assess the degree of renal injury. Naltrexone (2.5, 5.0 and 10.0 mg/kg s.c.), glibenclamide (5.0 and 10.0 mg/kg i.p.), and minoxidil (25 and 50 mg/kg) were employed to explore the role of endogenous opioids and K(ATP) channels in rhabdomyolysis-induced ARF. Pretreatment with naltrexone and glibenclamide attenuated hypertonic glycerol-induced renal dysfunction in a dose-dependent manner, suggesting the role of endogenous opioids and K(ATP) channels in the pathogenesis of myoglobuniric renal failure. However, the simultaneous pretreatment with naltrexone (10 mg/kg s.c.) and glibenclamide (10 mg/kg i.p.) did not enhance the reno-protective effects of individual drugs, suggesting that release of endogenous opioids and opening of K(ATP) channels constitute a single pathway in acute renal injury triggered by hypertonic glycerol-induced rhabdomyolysis. Furthermore, administration of minoxidil abolished the attenuating effects of naltrexone in glycerol-induced renal failure, suggesting that opening of K(ATP) channels is downstream to opioid receptor activation. It is concluded that hypertonic glycerol-induced rhabdomyolysis may involve release of endogenous opioids that in turn modulate K(ATP) channels to contribute in the pathogenesis of ARF.