Subacute sepsis impairs vascular smooth muscle contractile machinery and alters vasoconstrictor and dilator mechanisms

INTRODUCTION: Sepsis results in hyporesponsiveness to alpha-adrenergic stimulation. This is thought to be mediated by the release of vasoactive compounds from the septic endothelium or by the direct effect of sepsis on vascular smooth muscle (VSM) contractile mechanics and machinery. Previous studies have used lethal models of sepsis or endotoxemia to examine this phenomenon. The present study utilizes a clinically relevant, nonlethal model of soft tissue infection to determine the effects of sepsis on alpha-adrenergic mechanisms. We hypothesize that subacute sepsis causes impaired alpha-adrenergic vascular responsiveness by a combination of effects on adrenergic constrictor mechanisms, endogenous dilator tone, and VSM contractile function. METHODS: Male Sprague-Dawley rats underwent implantation of a 2 x 2-cm2 gauze sponge into a subcutaneous pocket created at the base of the tail. Five days after implantation, sepsis (S) was induced by inoculation of the sponge with 10(9) CFU Escherichia coli and Bacteroides fragilis. Controls (C) were inoculated with saline. Thoracic aortic harvest was performed 24 and 48 h after sponge inoculation for organ bath ring studies. Receptor-mediated (phenylephrine) and nonreceptor-mediated (KCl) maximum force of contraction (Fmax) was measured. Vessel sensitivity (pD2) to phenylephrine, acetylcholine, and KCl was calculated from dose-response curves. RESULTS: At 24 h, sepsis resulted in a lower Fmax to phenylephrine (1.15 for C vs 0.5 for S, P < 0.05 by ANOVA), despite an increase in vessel sensitivity (pD2) to alpha-adrenergic stimulation (6.70 for C vs 6.88 for S, P < 0.05 by ANOVA). Fmax to KCl was lower in septic animals at 24 h (3. 50 for C vs 2.77 for S, P < 0.05 by ANOVA) and sensitivity to acetylcholine (pD2) was markedly increased (6.56 for C vs 7.23 for S, P < 0.05 by ANOVA). At 48 h, the impairment in Fmax to alpha-adrenergic stimulation (2.29 for C vs 1.72 for S, P < 0.05 by ANOVA) and KCl (3.5 for C vs 3.08 for S. P < 0.05 vs 24 h C by ANOVA) persisted without any change in sensitivity to phenylephrine or acetylcholine. CONCLUSIONS: Subacute sepsis results in an early suppression of maximum contractile force despite an increase in adrenergic receptor sensitivity (pD2). This may be secondary to an elevation in dilator sensitivity combined with a direct effect of sepsis on VSM contractile mechanisms. Later in the septic process, however, alpha-adrenergic hyporesponsiveness ( downward arrow Fmax) is primarily due to changes in VSM contractile machinery.     

Halothane enhances pulmonary artery endothelial eicosanoid release.

To determine whether anesthetics alter endothelial eicosanoid release, cultured bovine pulmonary artery endothelial cells were studied during constant flow and pressure perfusion at two oxygen tensions (hypoxia, 50 +/- 2 mm Hg; normoxia, 144 +/- 5 mm Hg; mean +/- SEM) with and without 1% halothane. Endothelialized microcarriers containing approximately 5 x 10(6) cells were loaded into cartridges and perfused (3 mL/min) with Krebs' solution (pH 7.4, at 37 degrees C) equilibrated with each gas mixture. Eicosanoids (6-keto prostaglandin F1 alpha, thromboxane B2, and total peptidoleukotrienes [C4, D4, E4, F4]) were measured by radioimmunoassay and quantified per gram of cellular protein per minute. Eicosanoid release did not vary over time. The 6-keto prostaglandin F1 alpha release increased during hypoxia (normoxia 291 +/- 27 vs hypoxia 395 +/- 35 ng.min-1 x g protein-1; P < 0.01). Halothane (H) increased release of each eicosanoid during both normoxia and hypoxia: 6-keto prostaglandin F1 alpha-normoxia 291 +/- 27 versus normoxia + H 356 +/- 32 ng.min-1 x g protein-1, hypoxia 395 +/- 35 versus hypoxia + H 464 +/- 40 ng.min-1 x g protein-1, P < 0.05; thromboxane B2-normoxia 19 +/- 2 versus normoxia + H26 +/- 2 ng.min-1 x g protein-1, hypoxia 20 +/- 2 versus hypoxia + H 38 +/- 5 ng.min-1 x g protein-1, P < 0.001; leukotriene-normoxia 363 +/- 35 versus normoxia + H 489 +/- 52 ng.min-1 x g protein-1, hypoxia 329 +/- 29 versus hypoxia + H 455 +/- 39 ng.min-1 x g protein-1, P = 0.001.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contraction coupled endothelial nitric oxide release: a new paradigm for local vascular control?

INTRODUCTION: Nitric oxide (NO), a potent vasodilator, is presumed to be constitutively released in most mammalian blood vessels. In isolated rat thoracic aorta, however, hemoglobin (Hb), a nitric oxide scavenger, elicited contraction only when the vessels were precontracted with an alpha adrenergic agonist. Does vascular contraction induce endothelial NO release? METHODS: Thoracic aortic rings from male Sprague-Dawley rats were prepared with or without the endothelium. Vessel rings were contracted with several distinct types of contractile agonists and NO release was probed using a Hb contraction assay in the presence and absence of nitro-l-arginine methyl ester (NAME), a NO synthase inhibitor. RESULTS: In vessel rings precontracted with norepinephrine, potassium chloride, arginine vasopressin, prostaglandin F(2alpha), or serotonin, Hb elicited significant additional contractions. In contrast, Hb failed to elicit significant contractions in vessel rings without the functional endothelium or vessels pretreated with NAME. The Hb mediated additional contraction was not inhibited by calmidazolium, a calmodulin antagonist, and protein kinase inhibitors staurosporine and 2,5-dihydromethylcinnamate. Intercellular gap junction inhibitor 2,3-butanedione monoxime at a low dose (<2 mM) significantly attenuated the NE/Hb mediated contractions but at a high dose (>15 mM) completely prevented both contractions. The contraction coupled NO release may be mediated through a mechanism distinct from the Ca(2+)-calmodulin-dependent endothelial NOS pathway. CONCLUSIONS: In the isolated rat thoracic aorta, endothelial NO release may be coupled to contractile stimulus. This vascular property appears to render a unique local control mechanism independent of baroreflex and other central mechanisms.     

Lipopolysaccharide impairs endothelial nitric oxide synthesis in rat renal arteries

BACKGROUND: Impaired endothelium-dependent vasodilation may contribute to hypoperfusion and failure of abdominal organs, including the kidneys during endotoxin or septic shock. In this study, the short-term (2 h) effects of bacterial lipopolysaccharide (LPS) on endothelium-dependent vasodilation in rat renal and superior mesenteric arteries were documented. METHODS: Rat renal and mesenteric arteries were dissected and exposed in vitro to LPS for two hours. The effects of LPS on vascular reactivity were determined and compared with time-matched controls. Endothelial nitric oxide (NO) release was determined using an NO microsensor in adjacent vessel segments. RESULTS: LPS impaired maximal acetylcholine (ACh)-induced endothelium-dependent vasodilation in renal arteries (62.5 +/- 8.8% vs. 34.4 +/- 7.5% in controls and LPS-exposed arteries), but not in mesenteric arteries. LPS did not alter the sensitivity of renal arteries to exogenous NO. ACh-dependent vasodilation was abolished after blocking NO synthesis with 10-4 mol/L L-NA in control and LPS-incubated renal arteries. When compared with controls, NO release induced by ACh and the receptor-independent calcium ionophore A23187 was significantly decreased (P < 0.05) in LPS-exposed renal segments and was fully abolished in endothelium-denuded segments, indicating that LPS attenuated receptor-dependent as well as receptor-independent endothelial NO release. In contrast, ACh- and A23187-induced NO release was normal in LPS-exposed mesenteric arteries. CONCLUSIONS: These results indicate that LPS-induced selective impairment of ACh-induced endothelium-dependent relaxation in rat renal arteries is caused by decreased endothelial NO release. This may contribute to the propensity for acute renal failure during septic shock.     

The effects of sepsis and endotoxemia on gut glutamine metabolism.

The effects of sepsis on gut glutamine (GLN) metabolism were studied to gain further insight into the regulation of the altered glutamine metabolism that characterizes critical illnesses. Studies were done in laboratory rats and in hospitalized patients. The human studies were done in seven healthy surgical patients (controls) and six septic patients who underwent laparotomy. Radial artery and portal vein samples were obtained during operation and were analyzed for GLN and oxygen content. Despite no reduction in arterial glutamine concentration in the septic patients, gut glutamine extraction was diminished by 75% (12.0% +/- 1.6% in controls vs. 2.8% +/- 0.8% in septic patients, p less than 0.01). Similarly gut oxygen extraction was diminished by nearly 50% in the septic patients (p less than 0.05). To further investigate these abnormalities, endotoxin (10 mg/kg intraperitoneally) or saline (controls) was administered to adult rats 12 hours before cannulation of the carotid artery and portal vein. The arterial GLN concentration was increased by 13% in the endotoxin-treated animals (p less than 0.05) but gut glutamine uptake was diminished by 46% (526 +/- 82 nmol/100 g BW/minute in controls vs. 282 +/- 45 in endotoxin, p less than 0.01). Simultaneously gut glutaminase activity was diminished by 30% (p less than 0.01) and intestinal glutamate release fell by two thirds. Blood cultures were negative in control animals (0 of 20), but were positive in 25% of endotoxemic animals (6 of 24) for gram-negative rods (p = 0.019). Sepsis and endotoxemia impair gut glutamine metabolism. This impairment may be etiologic in the breakdown of the gut mucosal barrier and in the development of bacterial translocation.     

Effects of human cathelicidin antimicrobial peptide LL-37 on lipopolysaccharide-induced nitric oxide release from rat aorta in vitro

BACKGROUND: Lipopolysaccharides (LPS), released by Gram-negative bacteria, cause vascular expression of inducible nitric oxide synthase (iNOS) leading to nitric oxide (NO) production and septic shock. Human cathelicidin antimicrobial peptide (LL-37) can bind and neutralize LPS. We wanted to study whether LL-37 affects LPS or interleukin-1beta (IL-1beta)-induced production, release and function of NO in intact rat aorta rings and cultured rat aorta smooth muscle cells. METHODS: Isolated segments of thoracic aorta and cultured cells were incubated in the presence of LPS, LL-37, LPS + IL-37, IL-1beta, IL-1beta + IL-37 or in medium alone. Smooth muscle contraction in response to phenylephrine and accumulation of the sdegradation products of NO, nitrate and nitrite, were measured on aorta segments. Levels of iNOS were assessed by Western blot and cytotoxic effects were detected by measurement of DNA fragmentation in cultured cells. Number of viable cells were determined after Trypan blue treatment. RESULTS: Both LPS and IL-1beta reduced contractility in response to phenylephrine and increased NO production as well as iNOS expression. LL-37 inhibited the LPS depression of vascular contractility induced only by LPS. LL-37 reduced both the LPS- and IL-1beta-induced NO production and iNOS expression. LL-37 at high concentrations induced DNA fragmentation and decreased the number of living cells. CONCLUSION: IL-37 reduces NO production induced by LPS and IL-1beta. The reduction does not seem to result only from neutralization of LPS but also from a cytotoxic effect, possibly via induction of apoptosis.     

Acute administration of L-arginine does not improve arterial endothelial function in chronic renal failure.

BACKGROUND: Reduced activity of the nitric oxide (NO) pathway has been implicated in the endothelial dysfunction that occurs in patients with renal failure. NO is generated from L-arginine by NO synthase, and certain uremic toxins including asymmetrical dimethyl-L-arginine (ADMA), inhibit NO synthase and might contribute to endothelial dysfunction. We hypothesized that exogenous L-arginine might improve endothelial function in patients with renal failure by overcoming the effects of uremic toxins. METHODS: Endothelial function of the forearm resistance vasculature was assessed using plethysmography to measure the dilator response to intra-arterial acetylcholine (25 to 100 nmol/min). Endothelial function of radial and brachial arteries was assessed using vascular ultrasound to measure the dilator response to flow during reactive hyperemia (flow-mediated dilation; FMD). Studies were performed before and after administration of L-arginine by intra-arterial infusion (50 micromol/min) in 8 pre-dialysis patients or by intravenous infusion (10 g) in 18 hemodialysis patients. RESULTS: Local L-arginine did not improve the dilator response of forearm resistance vessels (AUC 23.1 +/- 6.4 pre, 23.1 +/- 5.1 post; P = 0.9) or FMD of the radial artery (6.5 +/- 1.2% pre, 6.3 +/- 0.8% post; P = 0.8). Systemic L-arginine did not improve FMD of the brachial artery (4.1 +/- 1.1% pre, 3.0 +/- 1.1% post; P = 0.07). These data demonstrate that acute local or systemic administration of L-arginine did not improve endothelial function in resistance or conduit arteries of patients with chronic renal failure. CONCLUSION: The results suggest that competitive inhibition of nitric oxide synthase (NOS) by circulating inhibitors is not the principal explanation for impaired endothelial dilator function in chronic renal failure.     

Asymmetrical dimethylarginine plasma concentrations are related to basal nitric oxide release but not endothelium-dependent vasodilation of resistance arteries in peritoneal dialysis patients

Vascular dysfunction in chronic renal failure may be linked to reduced nitric oxide (NO) bioactivity and increased circulating concentrations of the endogenous NO synthase inhibitor asymmetrical dimethyl L-arginine (ADMA). The association between ADMA and basal endothelial NO release and endothelium-dependent vasodilation in resistance arteries of chronic renal failure patients is unknown. Forearm blood flow responses to the endothelium-dependent vasodilator acetylcholine, the endothelium-independent vasodilator nitroglycerine, and the endothelium-dependent vasoconstrictor N(G)-monomethyl-L-arginine (L-NMMA) were assessed in 37 peritoneal dialysis patients. L-arginine and ADMA plasma concentrations were measured by HPLC. ADMA (mean +/- SEM: 0.68 +/- 0.02 micromol/L) was associated with basal forearm blood flow (r = -0.33; P < 0.05) and L-NMMA induced vasoconstriction (r = -0.55; P < 0.0005), but not with dilator effects of acetylcholine or nitroglycerine. L-arginine (68 +/- 3 micromol/L) tended to correlate with acetylcholine-induced vasodilation (r = 0.32; P = 0.05) but was not associated with other parameters. ADMA is related to basal but not to acetylcholine-stimulated NO bioactivity in patients on peritoneal dialysis. Impaired endothelium-dependent vasodilation found in chronic renal failure is not explained by elevated circulating NO synthase inhibitors in renal failure.     

Differential expression of inducible nitric oxide synthase in septic shock

During cardiopulmonary bypass (CPB), the septic patient has markedly decreased peripheral vascular resistance as a consequence of endotoxin release from microorganisms. This decrease in peripheral vascular resistance is the result of endotoxin-induced nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). iNOS and eNOS are responsible for the synthesis of NO because of various stimuli, including the bacterial endotoxin, lipopolysaccharide (LPS). We tested the hypothesis that a differential expression of iNOS among human endothelial cells and murine macrophage is dependent upon exposure to endotoxin and various pro-inflammatory cytokines. Using a human endothelial cell line, ECV-304 and murine macrophage cell line, RAW 264.7, we quantified the expression of iNOS with specific FITC-conjugated antibodies using fluorescence activated cell sorter (FACS) and NO production with a Bioxytech nitric oxide spectrophotometric assay. This in vitro septic model utilized LPS supported with species-specific interferon-gamma, interleukin-1 beta, and tumor necrosis factor-alpha. The cell type were stimulated for 8 hours with combinations of the cytokines mentioned. The FACS data demonstrated a significant stimulus-dependent increase in iNOS expression among the macrophage groups; however, the stimulated endothelial cells showed no significant change in iNOS expression. The nitric oxide production data demonstrated significant increases in NO production among macrophage stimulated groups; whereas, endothelial stimulated groups exhibit no significant change. We conclude that NO secreted during septic shock is the result of activated macrophage, not the endothelium. The clinical relevance is that the more severe the infectious process, the lower the PVR may be during CPB because of increased NO production from activated macrophage.     

Preserved glucoregulation but attenuation of the vascular actions of insulin in mice heterozygous for knockout of the insulin receptor

Type 2 diabetes is preceded by years of insulin resistance and is characterized by reduced bioavailability of the antiatherosclerotic signaling molecule nitric oxide (NO) and premature atherosclerosis. The relationship between resistance to the glucoregulatory actions of insulin and its effects on the vasculature (in particular NO-dependent responses) is poorly characterized. We studied this relationship in mice heterozygous for knockout of the insulin receptor (IRKO), which have a mild perturbation of insulin signaling. Male heterozygous IRKO mice aged 8-12 weeks were compared with age- and sex-matched littermates. IRKO mice had fasting blood glucose, insulin, free fatty acid, and triglyceride levels similar to those of wild-type mice. Intraperitoneal glucose and insulin tolerance tests were also similar in the two groups. Insulin levels in response to a glucose load were approximately twofold higher in IRKO compared with wild-type mice (1.08 +/- 0.11 vs. 0.62 +/- 0.13 ng/ml; P = 0.004). Despite this mild metabolic phenotype, IRKO mice had increased systolic blood pressure (124 +/- 4 vs. 110 +/- 3 mmHg; P = 0.01). Basal NO bioactivity, assessed from the increase in tension of phenylephrine preconstricted aortic rings in response to the NO synthase inhibitor N(G)-monomethyl-l-arginine, was reduced in IRKO (61 +/- 14 vs. 152 +/- 30%; P = 0.005). Insulin-mediated NO release in aorta, assessed as the reduction in phenylephrine constrictor response after insulin preincubation, was lost in IRKO mice (5 +/- 8% change vs. 66 +/- 9% reduction in wild-type; P = 0.03). Insulin-stimulated aortic endothelial NO synthase phosphorylation was also significantly blunted in IRKO mice (P < 0.05). These data demonstrate that insulin-stimulated NO responses in the vasculature are exquisitely sensitive to changes in insulin-signaling pathways in contrast to the glucoregulatory actions of insulin. These findings underscore the importance of early intervention in insulin-resistant states, where glucose homeostasis may be normal but substantial abnormalities of the vascular effects of insulin may already be present.     

Sepsis increases NOS-2 activity and decreases non-NOS-mediated acetylcholine-induced dilation in rat aorta

INTRODUCTION: Acetylcholine (Ach) is frequently used to assess endothelium-dependent vasodilation during sepsis. However, the effects of sepsis on constitutive nitric oxide synthase activity (NOS-1 and -3) and other non-NOS effects of Ach are unclear. METHODS: Sepsis was induced in rats by inoculation of an implanted sponge with Escherichia coli and Bacteroides fragilis (10(9) CFU each). Thoracic aortic rings (2 mm) were harvested at 24 h from septic (N = 9) and control (N = 9) rats and were suspended in physiological salt solution (PSS), PSS + l-N(6)-(1-iminoethyl)lysine (l-NIL: NOS-2 inhibitor, 10 microM), or PSS + l-N(G)-monomethylarginine (l-NMMA: NOS-1, -2, and -3 inhibitor, 60 microM). Rings were set at 1-g preload and precontracted with phenlyephrine (10(-8) M). Relaxation dose-response curves were generated with six doses of Ach (3 x 10(-8) to 10(-5) M). RESULTS: Sepsis increased the maximal relaxation to Ach under basal conditions. NOS 2 inhibition with l-NIL decreased Ach-induced relaxation in controls (66% vs 84%, P < 0.05, two-way ANOVA) and more so in septic rats (44% vs 93%, P < 0.05). Total NOS inhibition with l-NMMA decreased Ach-induced relaxation to 45% (P < 0.05) in controls and to 30% (P < 0.05) in septic animals. CONCLUSIONS: Inhibition of NOS-1, -2, and -3 failed to abolish Ach-induced relaxation, suggesting the presence of other Ach-induced vasodilator mechanisms. NOS-2 inhibition reduced Ach-induced relaxation by 20-25% in the normal thoracic aorta, but by 50% in septic animals. The remaining Ach-induced non-NOS vasodilation (after inhibition of NOS-1 + NOS-2 + NOS-3) was reduced from 45% in normals to 30% in septic animals. Vascular dysregulation in sepsis is a complex event involving increased NOS-2, decreased NOS-1 + NOS-3, and decreased Ach-induced non-NOS vasodilator mechanisms.     

Aprotinin decreases release of 6-keto-prostaglandin F1 alpha and increases release of thromboxane B2 in cultured human umbilical vein endothelial cells.

Use of the proteinase inhibitor aprotinin significantly improves hemostasis and reduces bleeding after operations in which extracorporeal circulation is used. The mechanism of action, however, has been only partially clarified. In this work we investigated whether aprotinin influenced the production and release of the eicosanoids prostacyclin, measured as the stable metabolite 6-keto-prostaglandin F1 alpha, and thromboxane A2, measured as the stable metabolite thromboxane B2, from endothelial cells. Human umbilical vein endothelial cells were incubated with different concentrations of aprotinin (5.5, 20, 55, and 100 mumol/L). The levels of 6-keto-prostaglandin F1 alpha and thromboxane B2 were measured at baseline and after thrombin stimulation. A concentration-dependent effect of aprotinin on 6-keto-prostaglandin F1 alpha synthesis was demonstrated. After incubation with 100 mumol/L of aprotinin, a 90% reduction in 6-keto-prostaglandin F1 alpha production was seen (31.69 versus 307.44 picograms per million cells; p less than 0.001). Conversely, thromboxane B2 production showed a 345% increase after incubation with aprotinin (287.80 versus 83.82 picograms per million cells; p less than 0.0001). Since 6-keto-prostaglandin F1 alpha inhibits and thromboxane B2 strongly enhances platelet aggregation, it appears that one mechanism of the clinically observed effectiveness of aprotinin lies in the altered ratio of 6-keto-prostaglandin F1 alpha: thromboxane B2 in endothelial cells, which leads to enhanced platelet aggregation and improved vessel sealing.     

Muscle prostaglandin production in the rat. Effect of abdominal sepsis and different amino acid formulations

Recent in vitro studies attribute regulatory functions to prostaglandins (PGs) in muscle protein metabolism, particularly enhancing proteolysis. In the present study, the amount of muscle PG production from endogenous precursors was determined in control and septic animals (cecal ligation and puncture) that were infused with 5% dextrose or dextrose with three amino acid formulations differing in their branched-chain amino acid (BCAA) content. We could not detect any differences in prostaglandin E, 6-keto-prostaglandin F1 alpha, and thromboxane B2 production between control and septic animals. Furthermore, the infusion of BCAAs, which have previously been shown to be nitrogen sparing following injury, did not influence the production of any of the PGs studied in either control or septic muscle. It is likely that the effects of the BCAAs on muscle synthesis and degradation are independent of the PGs.     

Induced nitric oxide impairs relaxation but not contraction in endotoxin-exposed rat pulmonary arteries

BACKGROUND: Many patients with severe acute lung injury do not respond to nitric oxide (NO) inhalational therapy with alleviation of pulmonary arterial hypertension and hypoxemia, so this treatment remains controversial. MATERIALS AND METHODS.: We investigated in endotoxin-exposed Wistar rat pulmonary arteries whether endogenous NO alters contractile and relaxing responses, by electrochemical NO and isometric force measurements. RESULTS: Receptor-independent contraction was similar in control and endotoxin-exposed arteries, while thromboxane analogue (TxA)-dependent contraction was less in the latter. Neither non-selective NO synthase (NOS) inhibition by N(G)-nitro-l-arginine (l-NA) or selective inducible-NOS2 inhibition by aminoguanidine (AG) improved TxA-induced contraction in endotoxin-exposed arteries. Acetylcholine-induced relaxation was impaired in endotoxin-exposed pulmonary arteries, despite a comparable acetylcholine-induced NO release in control arteries. Additionally, NO solution-induced relaxation of endotoxin-exposed arteries was impaired, but could be improved by l-NA or AG. Application of a phosphodiesterase-insensitive cyclic guanosine monophosphate analogue induced similar relaxation in both control and endotoxin-exposed arteries. CONCLUSIONS: Endotoxin-associated NOS2-derived NO is thus associated with impaired NO-mediated relaxation, but does not underlie reduced receptor-mediated pulmonary contractile responses. An increased phosphodiesterase activity may underlie the former, so this route can be explored to replace or improve the effect of inhalational NO therapy in severe sepsis-induced acute lung injury in patients.     

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.     

麻醉药对血管收缩反应性的影响

全身麻醉药在临床上常引起剂量依赖性血压下降，直接扩血管作用是其诱发低血压的重要原因之一。血管平滑肌（VSM）收缩反应由细胞内钙离子浓度（[Ca^2＋]i）升高激发，也受钙离子敏感化机制包括蛋白激酶C（PKC）、Rho激酶、p44／42丝裂素激活蛋白激酶（p44／42MAPK）等传导通路的调节。血管张力也受内膜衍生的松弛因子（EDRFs）如一氧化氮（NO）、前列腺素I2（PGI2）、内膜衍生的超极化因子（EDHF）以及ATP敏感性钾（KATP）通道调节。吸入性麻醉药如氟烷、异氟醚、七氟醚等被证明通过抑制平滑肌[Ca^2＋]i升高和PKC、Rho激酶、p44／42MAPK的活性、同时也通过抑制EDRFs的释放和增加KATP通道的开放来扩张血管。所以，全身麻醉药的扩血管作用是其同时抑制VSM的收缩作用和血管内膜介导的舒血管作用的综合结果。     

Vascular smooth muscle contraction is an independent regulator of endothelial nitric oxide production

This investigation was conducted to determine whether endothelial nitric oxide (NO) production is regulated by vascular smooth muscle contraction. Unperfused ring segments of rat aorta and mesenteric artery were studied using isometric tension recording (n = 6-8 in all experiments). Following a reference contraction to K+ 80 mM (100%), arteries were left either unstimulated or stimulated by different concentrations of K+ or prostaglandin F2alpha (PGF2alpha) to induce different levels of vascular precontraction. N(G)-nitro-L-arginine methyl ester (L-NAME 0.1-300 microM) or NS 2028 (0.03-3 microM), which is a new specific inhibitor of the NO-sensitive guanylate cyclase, was then added at increasing concentrations to evaluate endothelial NO production. L-NAME and NS 2028 produced a concentration-dependent vasoconstrictor response which was progressively enhanced with increasing levels of precontraction. For L-NAME, this amounted in aorta to (% of reference contraction): 35+/-1% and 105 +/- 4% (precontraction by K(+) 20 and 30 mM) and 22+/-1%, 89+/-1%, 138+/-1% and 146+/-2% (precontraction by PGF2alpha 0.5, 1, 2 and 3 microM). A similar coupling was found in the mesenteric artery. A precontraction as little as 2% was enough to trigger a vasoconstrictor response to L-NAME. In contrast, L-NAME and NS 2028 had no effect in non-contracted arteries, not even when passive mechanical stretch was increased by 100%. The results suggest (i) that endothelial NO formation is progressively increased with increasing vascular tone, and (ii) that vascular isometric contraction per se stimulates endothelial NO formation. It is concluded, that active vascular smooth muscle contraction is an independent regulator of endothelial NO production.     

Traction injury during minimally invasive harvesting of the saphenous vein is associated with impaired endothelial function

OBJECTIVE: Many methods of minimally invasive surgical harvesting of the great saphenous vein have been developed because of the morbidity related to the long skin incision after traditional (open) great saphenous vein harvesting. One such method involves the use of multiple small incisions separated by 10- to 15-cm skin bridges through which the saphenous vein is harvested. We hypothesized that this method of saphenous vein harvesting might subject the saphenous vein to considerable traction forces, resulting in impaired endothelial cell function. METHODS: Four-millimeter great saphenous vein segments were obtained from patients undergoing elective coronary artery bypass graft surgery. Group A (minimally invasive surgery) consisted of 23 rings from 20 patients (age, 65.8 +/- 11.1 years, mean +/- SD). Group B (open harvesting) consisted of 33 rings from 8 patients (age, 69.8 +/- 8.6 years). All great saphenous vein segments were undistended and were used within 24 hours of harvesting. Isometric tension experiments were performed on each ring of the great saphenous vein by using a force-displacement transducer to measure the force of contraction in grams. Measurements included developed force after exposure to high-potassium depolarizing solution and 50 micromol/L phenylephrine and decrease in force of contraction (relaxation) after exposure to 1 and 10 micromol/L acetylcholine. RESULTS: There were no differences between the minimally invasive surgery and open harvesting groups in their responses to high-potassium depolarizing solution or phenylephrine: high-potassium depolarizing solution, contractions of 4.26 +/- 0.72 g (mean +/- SEM) and 3.95 +/- 0.38 g, respectively (P =.70); phenylephrine, contractions of 3.49 +/- 0.63 g and 2.73 +/- 0.39 g, respectively (P =.41). There was no net relaxation in segments from the minimally invasive surgery group after exposure to 1.0 or 10 micromol/L acetylcholine. In contrast, rings from the open harvesting group demonstrated relaxation of -0.41 +/- 0.07 g and -0.32 +/- 0.09 g after exposure to 1.0 and 10 micromol/L acetylcholine, respectively. CONCLUSIONS: In undistended saphenous vein segments isolated from patients undergoing minimally invasive surgical and open techniques of harvesting, there was no acetylcholine-mediated endothelium-dependent relaxation in the minimally invasive surgery group. Therefore harvesting of the great saphenous vein through multiple small incisions might result in endothelial dysfunction, possibly caused by traction injury.     

Luminal thrombus disrupts nitric oxide-dependent endothelial physiology

BACKGROUND: The goals of this study were: (1) to develop a large animal model to study endothelial function, and (2) to determine if arterial thrombosis induces endothelial dysfunction in vivo. METHODS: Surgical exposure of the porcine iliac and femoral arteries was performed. Normal porcine arteries were compared with arteries subjected to 90 min of arterial thrombosis. External iliac artery (EIA) luminal diameters were measured using M- and B-mode duplex ultrasound. Endothelium-dependent relaxation (EDR) and endothelium-independent relaxation (EIR) were measured using acetylcholine (ACh) and sodium nitroprusside (NTP), respectively. Endothelial integrity was determined by factor VIII immunohistochemistry (F8) and scanning electron microscopy (SEM). Nitric oxide levels were determined using a chemiluminescence assay of nitrite/nitrate metabolites (NO(x)). Continuous variables were analyzed using the two-tailed Student t test. RESULTS: Control artery EDR was 80 +/- 7.1% (+/- SE), while arteries exposed to luminal thrombus for 90 min had an EDR of 55.2 +/- 5.7% (ACh = 15 microg/min, n = 11, P = 0.0231). EIR was preserved in normal and thrombosis groups with uniform response to NTP (4.92 +/- 0.1 cm vs 5.07 +/- 0.42 cm, P = 0.76). F8 staining identified endothelium in all groups. SEM analysis revealed an intact monolayer of endothelium after thrombosis. Local NO(x) levels were 17.3% lower after 90 min of thrombosis (49.3 microM vs 40.8 microM, n = 16, P < 0.001). CONCLUSIONS: Luminal thrombus induces arterial dysfunction acutely without causing endothelial cell loss. EIR remains unaffected, indicating normal smooth muscle cell function. NO(x) levels suggest that nitric oxide levels are decreased acutely after thrombosis. The development of this porcine large animal model allows the in vivo study of vasospasm and alternative thrombolytic regimens.     

Prostaglandin biochemistry of seeded endothelial cells on Dacron prostheses

The beneficial effect of seeding endothelial cells on synthetic vascular conduits has been well established. The biochemical production and interaction of the prostaglandins, prostacyclin (PGI2) and thromboxane (TxA2), were studied on Dacron vascular grafts that were seeded with autogenous venous endothelial cells. Seventy-three seeded and nonseeded grafts were implanted into the carotid arteries of dogs. Animals were medicated with either cyclooxygenase inhibitors (aspirin and dipyridamole, or ibuprofen, or U-53,059), or dipyridamole alone, or a thromboxane synthase inhibitor, U-63557A. All animals were killed at 5 weeks and analyzed for patency, thrombus-free surface (TFS), and PGI2 and TxA2 production from mid-graft punch biopsies. PGI2 and TxA2 identifications were made by radioimmunoassay determination of 6-keto PGF1 alpha and TxB2, respectively. Results of the study demonstrated in nonmedicated animals a slightly increased patency rate in seeded vs. nonseeded grafts (50% vs. 40%) and a more significant difference in TFS (49% vs. 24%). The addition of cyclooxygenase inhibitors or TxA2 synthase inhibitors significantly improved both patency (90% vs. 47%) and TFS (87% vs. 9%) in seeded vs. nonseeded grafts. PGI2 production was decreased in seeded grafts with the use of cyclooxygenase inhibitors in all cases. It is concluded that seeded endothelial cells on Dacron velour grafts can synthesize PGI2; these PGI2 levels are far less than PGI2 levels produced by endothelial cells from the adjacent carotid artery; and TxA2 synthase inhibitors best improve thromboresistance of seeded grafts without significant reduction in PGI2 production.