Both serotonin and a nitric-oxide donor cause chloride secretion in rat colonocytes by stimulating cGMP

BACKGROUND: Previous studies have demonstrated that an antagonist of nitric oxide synthase inhibits neurally mediated chloride secretion in response to serotonin (5-HT). The purpose of this study was to demonstrate that chloride secretion in rat colonocytes that were caused by stimulation of neural 5-HT receptors is mediated by way of a nitrergic pathway that involves the activation of guanylate cyclase. METHODS: The nitric oxide (NO) donor, diethylenetriamine/NO (DNO), was added to an enriched suspension of rat colonocytes that were preloaded with (36)Cl(-). In parallel experiments, DNO (1 micromol/L) was added to cells that were pretreated with the specific inhibitor of soluble guanylate cyclase, NS2028 (2 micromol/L). In additional studies, the neural 5-HT(3) receptor agonist, 2-methyl-5-HT (10 micromol/L), was added to the serosal surface of muscle-stripped sheets of rat colonic mucosa that were mounted in Ussing chambers under voltage clamp conditions, both in the absence and presence of NS2028 (20 mircro). RESULTS: DNO induced 18.0% +/- 8.0% greater (36)Cl(-) efflux than controls (P <.05; n = 14 animals). This efflux was abolished by previous treatment with NS2028. In the chamber experiments, 2-methyl-5-HT induced electrogenic chloride secretion that was significantly inhibited by previous treatment with NS2028 (2.2 +/- 0.5 microA/cm(2) vs 13.1 +/- 2.1 microA/cm(2); P <.001; n = 9 animals). CONCLUSIONS: The predominant secretomotor neurotransmitter that mediates the chloride secretory effects of 5-HT in vitro is nitric oxide. Both the secretory effect initiated at the 5-HT(3) receptor on enteric neurons and at the NO(-) receptor on the rat colonocytes are mediated through the activation of intracellular guanylate cyclase and the production of cyclic guanosine monophosphate.     

Vasoactive intestinal peptide is a neuropeptide mediator of the secretory response to serotonin in rat

BACKGROUND: The chloride secretory response to serotonin (5-HT) has nonneural and neural mechanisms, the latter mediated through a 5-HT(3) receptor. We hypothesized that 5-HT(3)-induced C1(-) secretion is partially mediated by VIP as a neurosecretory transmitter. Therefore it should be inhibited by a VIP receptor antagonist, VIP 6-28. Furthermore, exogenous VIP should induce secretion in the presence of tetrodotoxin (TTX). METHODS: Unstripped sheets of rat colon (n = 6) were mounted in Ussing chambers. The 5-HT(3) receptor agonist 2-Me-5-HT (10 microM) was added in the absence and presence of VIP 6-28 (30 microM). In companion studies VIP (1 microM) was added to tissue with or without TTX. Changes in short-circuit current (DeltaI(SC)) were recorded and repeat-measure ANOVA was used to analyze data. RESULTS: Addition of 2-Me-5-HT induced a rise in DeltaI(SC) seen in controls at 1 to 5 min (3.2 +/- 1.5 to 12.3 +/- 3.7 microA/cm(2), P < 0.02). VIP 6-28 blunted DeltaI(SC) (1.2 +/- 0.4 to 3.7 +/- 1.3 microA/cm(2), P < 0.01). VIP caused DeltaI(SC) to increase above baseline in 15 min (4.7 +/- 2.6 to 10.4 +/- 3.0 microA/cm(2), P < 0.01). The addition of TTX prior to VIP did not alter DeltaI(SC). CONCLUSION: Activation of the neural 5-HT(3) receptor by 2-Me-5-HT induces a secretory response in rat colon that is inhibited by a VIP receptor antagonist. Exogenous VIP mimics this response and is unaffected by TTX. VIP is a likely nonadrenergic, noncholinergic neurotransmitter in this pathway.     

Inhibition of Endogenous Nitric Oxide Synthase Potentiates Nitrovasodilators in Experimental Pulmonary Hypertension

Background: The role of endogenous nitric oxide (NO) in the regulation of pulmonary vascular tone is complex. Inhibition of endogenous NO synthase, potentially through upregulation of guanylyl cyclase, results in an increase in potency of nitrovasodilators in the systemic circulation. This study considered whether inhibition of endogenous NO synthase would increase the potency of nitrovasodilators, but not of cyclic adenosine monophosphate-dependent vasodilators, in the pulmonary vasculature.

Methods: We used the isolated buffer-perfused rabbit lung. Preparations were randomized to receive either pretreatment with NG -nitro-L-arginine methyl ester (or L-NAME, an inhibitor of endogenous NO synthase) or no pretreatment. Stable pulmonary hypertension was then produced by infusing the thromboxane A2 analog U46619. The dose-response characteristics of two nitrovasodilators, sodium nitroprusside and nitroglycerin, and two nonnitrovasodilators, prostaglandin E1 and 5'-N-ethylcarboxamidoadenosine, were studied.

Results: Inhibition of endogenous NO synthase caused no significant changes in baseline pulmonary artery pressure but did significantly reduce the U46619 infusion rate required to produce pulmonary hypertension. Pretreatment with L-NAME (vs. no L-NAME) resulted in significantly lower values of the log median effective dose with sodium nitroprusside and nitroglycerin. In contrast, pretreatment with L-NAME resulted in no changes in the dose-response characteristics of the cyclic adenosine monophosphate-mediated, NO-independent vasodilators prostaglandin E1 and 5'-N-ethylcarboxamidoadenosine.     

Effect of cyclooxygenase inhibition on serotonin-induced chloride secretion from rat distal colon

BACKGROUND: Serotonin is a well-known mediator of intestinal chloride secretion. The effects of exogenous 5-hydroxytryptamine (5-HT) can be demonstrated experimentally by a rise in short-circuit current, which is proportional to active electrogenic chloride secretion. Prostaglandin E2 is also an intestinal secretagogue and has been implicated in certain diarrheal illnesses. The aim of this study was to evaluate the effect of a cyclooxygenase inhibitor on 5-HT-stimulated ion transport. METHODS: Full- or partial-thickness sheets of rat colon were mounted in Ussing chambers and placed under short-circuit conditions. 5-HT or the specific 5-HT3 receptor agonist, 2-methyl-5-HT, was added in the absence and presence of the cyclooxygenase inhibitor, piroxicam. RESULTS: The overall drug effect was statistically significant at two concentrations (10(-5) and 5 x 10(-4) mol) of piroxicam when compared with 5-HT alone (P < or = .005; ANOVA, n = 8). The inhibitory effect of piroxicam (10(-5)) was statistically significant when compared to 2-methyl-5-HT alone (P < .01; ANOVA, n = 5). Piroxicam did not significantly affect baseline current. CONCLUSIONS: Exogenous 5-HT applied to an in vitro preparation of rat distal colon induces chloride secretion that is significantly inhibited by the cyclooxygenase inhibitor, piroxicam. It appears that the cyclooxygenase pathway plays a major role in the mediation of the secretory response to exogenous serotonin in vitro.     

A nonneuronal 5-hydroxytryptamine receptor 3 induces chloride secretion in the rat distal colonic mucosa

BACKGROUND: The 5-HT3 receptor is a serotonin receptor believed to reside on enteric neurons. However, several studies belie an exclusive neural localization. Our hypothesis is that the 5-HT3 receptor agonist, 2-methyl-5-HT (2Me5HT), induces chloride secretion despite neural blockade, which can be blocked by a 5-HT3 receptor antagonist. METHODS: Rat distal colon was stripped of its muscularis, mounted as mucosal sheets in Ussing chambers, and short-circuited. Adjacent sheets were pretreated with 1 micromol/L of the neurotoxin, tetrodotoxin, and incubated with 2Me5HT (50 micromol/L) alone or with a 5-HT3 (0.3 micromol/L ondansetron or 0.3 micromol/L tropisetron) or a 5-HT4 (0.3 micromol/L GR11808) receptor antagonist. Short-circuit current (I(sc)) was measured continuously. RESULTS: 2Me5HT caused an increase in I(sc), which was significantly (P <.01, repeated measures analysis of variance) inhibited by ondansetron (n = 8) and tropisetron (n = 5) but not by GR11808. CONCLUSIONS: A 5-HT3 receptor is present at the mucosal level that mediates chloride secretion by a nonneural pathway.     

Selective iNOS inhibition prevents hypotension in septic rats while preserving endothelium-dependent vasodilation

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) mediates hypotension and metabolic derangements in sepsis. We hypothesized that selective iNOS-inhibition would prevent hypotension in septic rats without inhibiting endothelium-dependent vasodilation caused by the physiologically important endothelial NOS. Rats were exposed to lipopolysaccharide (LPS) for 6 h and the selective iNOS-inhibitor L-N6-(1-iminoethyl)-lysine (L-NIL), the nonselective NOS-inhibitor N:(G)-nitro-L-arginine methyl ester (L-NAME), or control. Mean arterial pressure (MAP) and vasodilation to acetylcholine (ACh, endothelium-dependent), sodium nitroprusside (SNP, endothelium-independent), and isoproterenol (ISO, endothelium-independent beta agonist) were determined. Exhaled NO, nitrate/nitrite-(NOx) levels, metabolic data, and immunohistochemical staining for nitrotyrosine, a tracer of peroxynitrite-formation were also determined. In control rats, L-NAME increased MAP, decreased the response to ACh, and increased the response to SNP, whereas L-NIL did not alter these variables. LPS decreased MAP by 18% +/- 1%, decreased vasodilation (ACh, SNP, and ISO), increased exhaled NO, NOx, nitrotyrosine staining, and caused acidosis and hypoglycemia. L-NIL restored MAP and vasodilation (ACh, SNP, and ISO) to baseline and prevented the changes in exhaled NO, NOx, pH, and glucose levels. In contrast, L-NAME restored MAP and SNP vasodilation, but did not alter the decreased response to ACh and ISO or prevent the changes in exhaled NO and glucose levels. Finally, L-NIL but not L-NAME decreased nitrotyrosine staining in LPS rats. In conclusion, L-NIL prevents hypotension and metabolic derangements in septic rats without affecting endothelium-dependent vasodilation whereas L-NAME does not. IMPLICATIONS: Sepsis causes hypotension and metabolic derangements partly because of increased nitric oxide. Selective inhibition of nitric oxide produced by the inducible nitric oxide synthase enzyme prevents hypotension and attenuates metabolic derangements while preserving the important vascular function associated with endothelium-dependent vasodilation in septic rats.     

Effect of tacrolimus and cyclosporine on renal microcirculation and nitric oxide production

OBJECTIVES: Cyclosporine (CSA) and tacrolimus (TAC) frequently induce nephrotoxicity and similar pathologic changes. Acute CSA-induced nephrotoxicity has been reported to be mediated by activation of vasoconstrictors such as endothelin. The purpose of the present study was to investigate the acute effects of TAC and CSA on the renal microcirculation and upon a vasodilator such as nitric oxide (NO) production. METHODS: Renal blood flow (RBF) in the microcirculation was measured by a Laser Doppler flow meter in uninephrectomised rats. RBF, mean arterial pressure (MAP), and renal vascular resistance (RVR) were measured in the following groups: (a) TAC (0.1 to 2.0 mg/kg/h, n = 3 approximately 6); CSA (20 and 50 mg/kg/h, n = 5); (b) L-NAME (10 mg/kg), an NO synthase inhibitor, 8 minutes prior to TAC (0.5 and 1.5 mg/kg/h, n = 5), or CSA (20 and 50 mg/kg/h, n = 5). Stable NO end-products, serum NO(2) and NO(3), were measured by the Griess method (n = 5). RESULTS: None of the parameters were changed by TAC alone, whereas TAC with L-NAME significantly reduced RBF (-28 +/- 7%) and increased RVR (46 +/- 17%) in a dose-dependent manner. CSA alone significantly reduced RBF (-37 +/- 6%) and increased RVR (69 +/- 22%) without any changes in MAP. The effects of CSA were enhanced by L-NAME. Serum concentration of NO(2) + NO(3) was significantly reduced by both L-NAME alone and CSA (50 mg/kg) (P < .05), while there were no changes with TAC (1.5 mg/kg). CONCLUSIONS: Blockade of NO production enhance the vasoconstrictive effect of CSA, and unmasked such an effect of TAC. These results suggest that the nephrotoxicity of CSA and TAC may involve the NO system.     

Nitric oxide and thromboxane A2-mediated pulmonary microvascular dysfunction

OBJECTIVES: To examine whether the lung releases nitric oxide (NO) in response to thromboxane A2 and to examine the local release of NO as a protective compensatory mechanism by which the lung responds to the proinflammatory and vasoactive effects of thromboxane A2. DESIGN: The lungs of anesthetized Sprague-Dawley rats were perfused in vitro with Krebs-Henseleit buffer that contained an inhibitor of NO synthase (nitroglycerinenitro-L-arginine methyl ester [L-NAME]) (10(-4) mol/L), an NO donor (sodium nitroprusside) (10(-8) mol/L), or perfusate alone. Following equilibration, the thromboxane A2 receptor agonist 9,11-dideoxy-11alpha, 9alpha-epoxymethanoprostaglandin F2alpha(U-46619) (7.1 X 10(-8) mol/L) was added to the perfusate. Fifteen minutes later, the capillary filtration coefficient, pulmonary arterial pressure, and vascular resistance were measured. Pulmonary NO release was assessed by quantitating the release of cyclic guanosine monophosphate into the perfusate. RESULTS: The capillary filtration coefficient of lungs exposed to U-46619 was 3.5 times greater than that of lungs perfused with buffer alone (P<.05). The addition of sodium nitroprusside reduced the increase in capillary filtration coefficient associated with U-46619 by 50% (P<.05) whereas L-NAME had no effect. The addition of U-46619 to the perfused lung caused a 3.0+/-0.4 mm Hg increase in pulmonary artery pressure (P<.01) with a corresponding rise in total vascular resistance (P<.05). This effect was exacerbated by L-NAME (P<.05) and inhibited by sodium nitroprusside (P<.05). Exposure of the isolated lungs to U-46619 caused a 4-fold increase in cyclic guanosine monophosphate levels within the perfusate. CONCLUSION: These data are consistent with the hypothesis that NO release may be an important protective mechanism by which the lung responds to thromboxane A2.     

Endothelin-induced increased nitric oxide mediates augmented distal nephron acidification as a result of dietary protein

Tested was the hypothesis that enhanced nitric oxide (NO) production that is stimulated by increased renal endothelin activity mediates decreased distal nephron HCO(3) secretion that is induced by dietary protein. Munich-Wistar rats that ate minimum electrolyte diets with 50% casein-provided protein (HiPro) compared with controls that ate 20% protein for 3 wk had higher urine excretion of endothelin-1 (80 +/- 15.7 versus 29 +/- 3.9 fmol/kg body wt per d; P < 0.02) and of the NO metabolites NO(2)/NO(3) (21.2 +/- 1.9 versus 14.9 +/- 0.8 mumol/kg body wt per d; P < 0.03). Bosentan, an endothelin A/B receptor antagonist, reduced HiPro rats' urine excretion of net acid (5859 +/- 654 versus 8017 +/- 1103 micromol/d; P < 0.03, paired t test) and NO(2)/NO(3) (18.1 +/- 1.1 versus 22.9 +/- 2.0 micromol/kg body wt per d; P < 0.05, paired t test). N-nitro-l-arginine methyl ester (L-NAME), an NO synthase inhibitor, also decreased urine net acid excretion (6621 +/- 717 versus 8449 +/- 1086 micromol/d; P < 0.05, paired t test) but was not additive to bosentan. L-NAME increased in situ late distal nephron HCO(3) delivery in HiPro rats (18.8 +/- 1.7 versus 9.6 +/- 1.4 pmol/mm per min; P < 0.001) that was mediated by increased distal nephron HCO(3) secretion (-7.2 +/- 0.7 versus -3.5 +/- 0.4 pmol/mm per min; P < 0.001) without changes in distal nephron transtubule HCO(3) permeability or H(+) secretion. Bosentan decreased H(+) secretion and increased HCO(3) secretion in the distal nephron of HiPro rats, but L-NAME had no additive effect on either component. The data support that dietary protein augments distal nephron acidification through decreased HCO(3) secretion that is mediated through endothelin-stimulated NO.     

Interaction of angiotensin II and nitric oxide in isolated perfused afferent arterioles of mice

The present study was performed to evaluate angiotensin II (Ang II)-nitric oxide (NO) interaction in afferent arterioles (Af) of wild-type mice and mice that are homozygous (-/-) for disruption of the endothelial NO synthase (eNOS) gene. Af were microperfused, and the dose responses were assessed for the NO precursor L-arginine (n = 4), NO inhibitor NG-nitro-L-arginine methyl ester (L-NAME, n = 5), L-NAME after pretreatment with L-arginine (n = 5), Ang II (n = 8), and Ang II after pretreatment with L-NAME (n = 7). Acute administration of L-arginine and L-NAME (both in doses from 10(-6) to 10(-3) mol/L) did not change arteriolar diameter. Moreover, pretreatment with L-arginine did not change the response to L-NAME. However, Ang II, applied in doses of 10(-12), 10(-10), 10(-8), and 10(-6) mol/L, significantly reduced the lumen to 66.5 +/- 7.0% and 62.2 +/- 8.0% at 10(-8) and 10(-6) mol/L Ang II, respectively. The contraction was augmented after L-NAME pretreatment (19.5 +/- 13.6% and 25.5 +/- 10.2% at 10(-8) and 10(-6) mol/L Ang II, respectively). In eNOS (-/-) mice (n = 8), the response to Ang II also was enhanced (9.1 +/- 6.0% and 11.2 +/- 8.2% at 10(-8) and 10(-6) mol/L Ang II, respectively). Female mice did not differ from male mice in their reactivity to Ang II (n = 9) and Ang II + L-NAME pretreatment (n = 11). The study shows that (1) it is feasible to microperfuse mouse Af, (2) the basal production of endothelial NO is very low and not inducible by L-arginine in Af of mice, and (3) a counteracting effect of NO is initiated by Ang II. High Ang II sensitivity in eNOS (-/-) mice underscores the considerable role of endothelial-derived NO to balance Ang II vasoconstriction in Af.     

Effects of castration on nitric oxide-mediated relaxations in male rat corpus cavernosum smooth muscle

BACKGROUND: The effects of castration on nitric oxide- mediated relaxations and nitric oxide synthase activity in male rat corpus cavernosum smooth muscles. METHODS: Eight-week-old male rats were assigned to two groups: control (sham operated) and castrated animals. After 8 weeks, corpus cavernosum smooth muscle strips were mounted in an organ bath for isometric tension recordings. Electrical field stimulation (EFS) was applied to the strips precontracted with 30 microM phenylephrine. The microdialysis probe was inserted into the strip, and Krebs-Henseleit solution was perfused into the probe. The dialysate during EFS and cholinergic stimulation was collected, and the amount of NO(-)(2)/NO(-)(3) (NOx) released in the dialysate was measured by the Greiss method. Sodium nitroprusside and carbachol were cumulatively added to the strips precontracted with 30 microM phenylephrine. RESULTS: EFS caused frequency-dependent relaxations and NOx releases in the strips. Pretreatment with N(omega)-nitro-L-arginine (100 microM) and tetrodotoxin (1 microM) completely inhibited relaxations and NOx releases. The maximum relaxation in the castration group was significantly greater than that in the control group. The release of NOx was significantly greater in the castration group than in the control group. Sodium nitroprusside relaxed the tissues in both groups similarly. Carbachol failed either to relax the tissue or to increase the amount of NOx production in the tissue. CONCLUSION: The present data suggest that castration enhances nitric oxide synthase activity and nitric oxide-mediated relaxations in the male rat corpus cavernosum.     

Effect of nitric oxide in ischemia/reperfusion of the pancreas

BACKGROUND: Ischemia/reperfusion injury, and thus graft pancreatitis, remains a major problem in pancreas transplantation. Contradictory results about the role of nitric oxide (NO) in pancreatic ischemia/reperfusion have been reported; however, in none of the reports has a detailed comparison between inhibition of NO synthase and NO supplementation been carried out. METHODS: Vascular isolation of the pancreatic tail was performed in landrace pigs. After splenectomy catheters placed in the distal part of the splenic vessels allowed collection of the venous effluent and perfusion of the pancreatic tail. Three hours of complete warm ischemia was followed by 6 h of reperfusion. The effect of the NO donor sodium nitroprusside (SNP) and L-arginine was compared to a control group and NO synthase inhibition with L-NAME. RESULTS: Lipase in the venous effluent of the pancreas was significantly decreased in the SNP and the L-arginine groups. Vascular resistance was markedly elevated in the L-NAME group and reduced in the NO donor groups. Tissue pO2 after reperfusion was only significantly elevated in the SNP group. Granulocyte infiltration and also overall histological tissue injury were most severe in the control group followed by the L-NAME group, the SNP group, and the L-ARG group. CONCLUSION: The data show that supplementation of nitric oxide is clearly protective in pancreatic ischemia/reperfusion. However, inhibition of NO synthesis does not lead to an equally clear aggravation of tissue injury.     

Serotonin release is mediated by muscarinic receptors on duodenal mucosal cells

Serotonin (5-HT), found in abundance in intestinal enterochromaffin cells, has been shown to be released in response to neural, humoral, and intraluminal stimuli but the mechanisms controlling release are poorly understood. The purpose of this study was to determine whether 5-HT release induced by the muscarinic agonist, methacholine, is mediated by enteric nerves or is a direct action at the mucosal cell level. We mounted rabbit mucosal sheets containing intact submucosal neural elements, but stripped of muscularis propria and myenteric plexus, in modified Ussing chambers and measured the release of 5-HT in response to 5 X 10(-5) M methacholine added to both mucosal and serosal surface bathing solutions, in the presence and absence of selective neural blockade with 1 X 10(-6) M tetrodotoxin. 5-HT release in control tissues (no drugs) as measured by radioimmunoassay was 25.7 +/- 6 ng/cm2/30 min (mean +/- SEM). In the presence of mucosal and serosal methacholine, total 5-HT release increased significantly (P less than 0.05) to 66.7 +/- 9 ng/cm2/30 min. When tetrodotoxin alone was applied, 5-HT release significantly increased to 55.2 +/- 9 ng/cm2/30 min compared to matched control. In the presence of tetrodotoxin, methacholine increased 5-HT release to 79.3 +/- 9 ng/cm2/30 min, which was significantly greater than with tetrodotoxin alone. These results provide evidence of an inhibitory neural regulation of basal 5-HT release since release increased in the presence of neural blockade. They also suggest that a muscarinic receptor at or near the enterochromaffin cells mediates mucosal 5-HT release since 5-HT is further increased by methacholine even in the presence of neural blockade.     

The correlation between nitric oxide and vascular endothelial growth factor in spinal cord injury

STUDY DESIGN: Prospective, randomized, placebo-controlled, experimental study. OBJECTIVES: The issue of whether nitric oxide (NO) production is beneficial or deleterious on ischemic injuries of the central nervous system still remains doubtful. Vascular endothelial growth factor (VEGF) is known to induce the release of NO from endothelial cells. However, the effect of NO on VEGF synthesis is not clear. We aimed to determine the effects of L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) on VEGF synthesis and free radicals in a rat model of spinal cord ischemia-reperfusion (IR) injury. SETTING: Surgical Research Laboratory of a Medical School. MATERIAL AND METHODS: Twenty-eight Wistar rats were divided into four groups as follows (n=7): Sham, IR injury, L-arginine, and L-NAME. Infrarenal abdominal aorta was occluded to induce spinal cord ischemia. L-Arginine (100 mg/kg) and L-NAME (10 mg/kg) were given before aortic occlusion. Biochemical assays of malondialdehyde (MDA), NO and VEGF were carried out in spinal cord specimens. RESULTS: L-Arginine treatment significantly increased MDA and NO, but decreased VEGF levels in spinal cord. However, nonselective inhibition of NOS with L-NAME significantly decreased MDA and NO, but increased VEGF levels. Besides, the positive linear correlation between MDA and NO, and negative linear correlations between MDA, NO and VEGF levels have also been demonstrated. CONCLUSION: Nonselective inhibition of NO synthase activity with L-NAME attenuated free radical formation and increased VEGF level when compared with NO precursor L-arginine in a rat model of spinal cord ischemia. We suggest that inhibition of NO synthase, as well as induction of VEGF, may be a therapeutic option in spinal cord IR injury.     

Basal release of nitric oxide and its interaction with endothelin-1 on single vessel hydraulic permeability

BACKGROUND: Both endothelin-1 (ET-1) and nitric oxide (NO) are released by the endothelium and are implicated in modulating the permeability of the endothelial barrier. The present study was designed to examine the interaction between ET-1 and NO and its influence on microvascular permeability as well as the role of NO in maintaining microvascular permeability. To isolate the direct effect of ET-1 and NO, experiments were conducted under conditions where hydraulic and oncotic pressures were controlled. METHODS: Postcapillary venules in the rat mesentery were perfused in situ and paired measurements of hydraulic permeability (Lp) obtained using the modified Landis micro-occlusion method. The effect of basal endogenous NO was tested by measuring the effects of perfusion with the NO synthase inhibitor Nw-nitro-L-arginine-methyl-ester (L-NAME) (100 micromol/L) on Lp (n = 6). In addition, Lp measured after a 15-minute perfusion with L-NAME (100 micromol/L) was compared with measures of Lp obtained after perfusion with a combined mixture of L-NAME (100 micromol/L) and ET-1 (80 pmol/L) (n = 6). RESULTS: Units for Lp are mean +/- SE x 10(-8) cm x sec(-1) x cm H2O(-1). Under basal conditions, in the absence of exogenous ET-1, NO synthase inhibition led to a significant increase in Lp from 5.7 +/- 0.5 to 9.8 +/- 1.4 (p = 0.02). Compared with L-NAME alone, ET-1 + L-NAME significantly decreased Lp from 10.3 +/- 0.8 to 5.7 +/- 0.6 (p = 0.006). CONCLUSION: Constitutive release of NO from the microvascular endothelium plays a role in maintaining a basal level of microvascular permeability. Decreases in microvascular permeability seen with the administration of ET-1 are not mediated via the release of NO. These findings suggest important roles for ET-1 and NO in maintaining and modulating microvascular permeability.     

Stimulation of P2y purinoceptors induces, via nitric oxide production, endothelium-dependent relaxation of human isolated corpus cavernosum

PURPOSE: Endothelial P2y purinoceptor stimulation is known to induce vasodilatation mediated by NO release from endothelial cells. We examined the effect of a potent P2y agonist, adenosine-5'-O-(2-thiodiphosphate) (ADPbetaS), on human corporal cavernosal strips and its dependence on a functional endothelial lining. MATERIALS AND METHODS: The preparations mounted in isometric conditions were precontracted by noradrenaline (NA) at a concentration of 0.1 microM. Increasing concentrations of ADPbetaS from 1 microM to 100 microM were added in the presence and absence of a functional endothelium or in the presence and absence of an NO synthase inhibitor and a selective P2y antagonist. Acetylcholine (Ach)-induced relaxation was used in each experiment for control. RESULTS: In human precontracted corporal cavernosal strips with a functional endothelium (relaxed by acetylcholine) ADPbetaS induces a dose-dependent relaxation with maximal relaxation of 45.5+/-5.0% and an EC50 of 11.7 microM. The relaxant effect of ADPbetaS was reduced by 77.1+/-7.0% by reactive blue 2 (20 microM)(a P2y antagonist). L-NAME (L-Nitro Arginin Methyl Ester), an NO synthase inhibitor (100 microM), reduced Ach- and ADPbetaS- induced relaxations by 86.59+/-3.24% and 86.83+/-0.5% respectively. Ach- and ADPbetaS- induced relaxations were significantly inhibited after dislodging of the endothelial lining of the corporal cavernosal strips, 90.11+/-6.2% and 87.1+/-5% respectively. CONCLUSIONS: Human corporal cavernosal strips can be relaxed by stimulation of P2y purinoceptors via NO release. This relaxation is an endothelium-dependent mechanism. Purines may be implicated in physiological erection in man.     

Luminal hypertonic solutions stimulate concentration-dependent duodenal serotonin release

The serotonin (5-HT) response to varying concentrations and types of luminal hyperosmolar solutions was examined in rabbit duodenum to define the mechanisms and direction (luminal or vascular) of release. Segments of duodenum were studied in either the Ussing chamber or an isolated, vascularly perfused model. Adjacent mucosal sheets devoid of muscularis were studied in parallel Ussing chambers with exposure of the mucosal surface to 0 to 50 gm/dl dextrose or 0 to 20 gm/dl mannitol. The 5-HT response to hyperosmolar dextrose was measured in the presence of autonomic receptor blockers and tetrodotoxin. In the isolated, perfused duodenum, venous and luminal drainage were sampled during basal, dextrose-infusion, and recovery periods. 5-HT content was measured by high-performance liquid chromatography. In chambered mucosal sheets the percentage of change in mucosal 5-HT release was a linear function of luminal dextrose (r = 0.96; p less than 0.05) or mannitol (r = 0.98; p less than 0.02) concentrations. A significant 65.8 +/- 19% inhibition of 5-HT release was observed for tetrodotoxin but not for atropine, hexamethonium, phentolamine, or propranolol. In the isolated, perfused duodenum, 50 gm/dl dextrose caused an integrated 5-HT venous response of 3.0 +/- 0.7 micrograms/100 gm (p less than 0.05 vs luminal or control venous release). It was therefore concluded that duodenal 5-HT release is concentration dependent for intraluminal glucose and mannitol, is partially neurally mediated by a nonadrenergic, noncholinergic pathway, and is predominantly into the venous drainage.     

Inhibiting the NO pathway with intracoronary L-NAME infusion increases endothelial dysfunction and intimal hyperplasia after heart transplantation.

BACKGROUND: The endothelium protects the vascular wall through the nitric oxide (NO) release. Coronary endothelial dysfunction occurs early after heart transplantation and predicts the development of intimal thickening typical of graft coronary vasculopathy. OBJECTIVE: We designed this study to examine the effect of endothelial NO synthase (eNOS) inhibition on the endothelial dysfunction caused by rejection and on the development of accelerated atherosclerosis after heart transplantation. METHODS: To study the effect on these 2 end-points of inhibiting eNOS with intracoronary L-nitro arginine methyl ester (L-NAME; 1 mg/kg/day), infused with an osmotic pump for 30 days, we used a porcine model of heterotopic heart transplantation with pre-operative immunologic typing, to permit slow rejection without the need for immunosuppression. The endothelium-dependent relaxations of allografted coronary arteries, allografted arteries infused with L-NAME, allografted arteries mounted with the pump, and vehicle and native coronary arteries were compared 30 days after graft implantation using standard organ chamber experiments. We evaluated intimal thickening using a semi-quantitative scale (0-4+ grading). RESULTS: A significant decrease in relaxations to serotonin (5-HT) occurred in allografted arteries infused directly with L-NAME compared with allografted arteries from swine receiving 5-HT, and relaxations in the latter were decreased compared with those of swine receiving the vehicle and native coronary arteries (p < 0.05). We found no significant differences in endothelium-dependent relaxations to bradykinin among coronary rings from all groups. We observed a significant increase in the prevalence and severity of intimal thickening in allografted coronary arteries infused with L-NAME compared with allografts not infused (p < 0.05), which had significantly more intimal thickening compared with native coronary arteries (p < 0.05). CONCLUSION: These results demonstrate that inhibiting the NO pathway worsens the endothelial dysfunction caused by rejection after heart transplantation and accelerates the intimal thickening process, leading to graft coronary vasculopathy. Strategies designed to preserve endothelial integrity and function of the endothelial NO pathway should be used to prevent graft coronary vasculopathy.     

Nitrict Oxide Synthase Inhibitors, 7-Nitro Indazole and Nitro sup G -L-Arginine Methyl Ester, Dose Dependently Reduce the Threshold for Isoflurane Anesthesia

Background: Nitric oxide (NO), a recognized cell messenger for activating soluble guanylate cyclase, is produced by the enzyme NO synthase in a wide variety of tissues, including vascular endothelium and the central nervous system. The authors previously reported the possible involvement of the NO pathway in the anesthetic state by showing that a specific NO synthase inhibitor, nitroG -L-arginine methyl ester (L-NAME), dose dependently and reversibly decreases the minimum alveolar concentration (MAC) for halothane anesthesia. The availability of a structurally distinct inhibitor selective for the neuronal isoform of NO synthase, 7-nitro indazole (7-NI), allowed for the possibility of dissociating the central nervous system effects of neuronal NO synthase inhibition from the cardiovascular effects of endothelial NO synthase inhibition.

Methods: The effect of two structurally distinct inhibitors of NO synthase, L-NAME and 7-NI, on the MAC of isoflurane was investigated in Sprague-Dawley rats while concurrently monitoring the animals' arterial blood pressure and heart rate. L-NAME (1 to 30 mg/kg given intravenously, dissolved in 0.9% saline) and 7-NI (20 to 1,000 mg/kg given intraperitoneally, dissolved in arachis oil) were administered after determining control MAC and 30 min before determining MAC in the presence of NO synthase inhibitor.

Results: L-NAME and 7-NI caused a dose-dependent decrease from isoflurane control MAC (maximal effect: 35.5 +/- 2.5% and 43.0 +/- 1.7%, respectively) with a ceiling effect observed for both NO synthase inhibitors (above 10 mg/kg and 120 mg/kg, respectively). L-NAME administration significantly increased systolic and diastolic blood pressures (maximal effect: 39.9 +/- 2.2% and 64.3 +/- 4.0%, respectively), which were not accompanied by any changes in heart rate. 7-NI administration resulted in no changes in blood pressure and a small but clinically insignificant decrease in heart rate.     

Roles of nitric oxide and oxidative stress in the regulation of blood pressure and renal function in prehypertensive Ren-2 transgenic rats

AIMS: The present study was performed to evaluate the role of nitric oxide (NO) and its interaction with superoxide anion (O2-) in the regulation of blood pressure (BP) and renal function during the developmental phase of hypertension in Ren-2 transgenic rats (TGR). The first aim was to compare BP and renal functional responses to acute NO synthase (NOS) inhibition achieved by intravenous (i.v.) infusion of Nomega-nitro-L-arginine-methyl ester (L-NAME) in prehypertensive heterozygous TGR and in transgene-negative Hannover Sprague-Dawley (HanSD) rats. The second aim was to evaluate whether scavenging of O2- by infusion of the superoxide dismutase mimetic tempol increases NO bioavailability which therefore should augment BP and renal functional responses to L-NAME. Methods: Rats were anesthetized, prepared for clearance experiments and BP and renal functional responses were evaluated in response to i.v. L-NAME administration (20 microg.100 g(-1).min(-1)) without or with tempol pretreatment (i.v., 300 microg.100 g(-1).min(-1)). In renal cortical tissue, nitrotyrosine protein expression was assessed by immunoblotting as marker of O2- production and urinary 8-epi-PGF(2alpha) excretion as marker of intrarenal oxidative stress was assessed by enzyme immunoassay. Results: BP, glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion were similar in TGR and HanSD. L-NAME infusion induced greater increases in BP in TGR than in HanSD (+42 +/- 4 vs. +25 +/- 3 mmHg, p < 0.05). In the absence of a significant change in GFR, L-NAME caused similar decreases in RPF (-32 +/- 6 and -25 +/- 4%, p < 0.05) in TGR and HanSD. Despite significantly higher renocortical expression of nitrotyrosine and urinary 8-epi-PGF2alpha excretion in TGR than in HanSD, pretreatment with tempol did not augment the rise in BP and the decrease in RPF induced by L-NAME. CONCLUSIONS: The greater BP response to L-NAME in TGR suggests that prehypertensive TGR exhibit an enhanced NO activity in the systemic vasculature as compared with HanSD. Despite increased intrarenal oxidative stress in TGR, the dependency of the intrarenal vascular tone on NO appears to be similar in TGR and HanSD. The lack of a compensatory increase in renal NO activity may partially account for the enhanced renal vascular response to ANG II present in TGR.