Inhibition of sialidase activity and cellular invasion by the bacterial vaginosis pathogen Gardnerella vaginalis.

Bacterial vaginosis is a genital tract infection, thought to be caused by transformation of a lactobacillus-rich flora to a dysbiotic microbiota enriched in mixed anaerobes. The most prominent of these is Gardnerella vaginalis (GV), an anaerobic pathogen that produces sialidase enzyme to cleave terminal sialic acid residues from human glycans. Notably, high sialidase activity is associated with preterm birth and low birthweight. We explored the potential of the sialidase inhibitor Zanamavir against GV whole cell sialidase activity using methyl-umbelliferyl neuraminic acid (MU-NANA) cleavage assays, with Zanamavir causing a 30% reduction in whole cell GV sialidase activity (p < 0.05). Furthermore, cellular invasion assays using HeLa cervical epithelial cells, infected with GV, demonstrated that Zanamivir elicited a 50% reduction in cell association and invasion (p < 0.05). Our data thus highlight that pharmacological sialidase inhibitors are able to modify BV-associated sialidase activity and influence host-pathogen interactions and may represent novel therapeutic adjuncts.

Comparison of central venous and external jugular venous pressures during repair of proximal femoral fracture.

BACKGROUND: External jugular venous pressure (EJVP) is a close estimate of central venous pressure (CVP) in patients undergoing mechanical ventilation in the supine position, but the effects of spontaneous respiration and posture on this relationship are not known. In this study, we compared CVP with EJVP measurements in 36 patients undergoing repair of proximal femoral fracture breathing spontaneously in the supine or lateral positions. METHODS: A standard general anaesthetic was administered with patients breathing spontaneously via a laryngeal mask airway and i.v. fluids administered according to an algorithm guided by CVP measurements. CVP and EJVP catheters were placed on the right side of the neck where possible. RESULTS: In the supine position, 185 paired measurements of CVP and EJVP and 79 in the lateral position were recorded by a blinded observer during surgery. In the supine position, the mean difference between CVP and EJVP was -0.3 mm Hg (limits of agreement -2.6 to +1.9 mm Hg, 95% confidence intervals for both upper and lower limits of agreement, respectively, were -2.9 to -2.2 and +1.6 to +2.2 mm Hg). In the lateral position, the mean difference was -1.2 mm Hg (limits of agreement -5.8 to +3.8 mm Hg, 95% confidence intervals -6.8 to -4.5 and +2.7 to +4.9 mm Hg). CONCLUSIONS: These data suggest that EJVP is an acceptable estimate of CVP in the supine position. Agreement was poor in the lateral position but was stronger for estimates of trend rather than absolute values. This could be explained by the direct effects of posture.

Comparison of external jugular and central venous pressures in mechanically ventilated patient.

We compared central venous pressures, measured via a 150 mm triple lumen catheter in the internal jugular vein with simultaneous external jugular venous pressures, measured with a 5 mm cannula in the external jugular vein, in 24 patients undergoing major surgery. Patients were mechanically ventilated in the supine position. Six sets of paired measurements of mean central venous pressure and mean external jugular venous pressure were taken by a blinded observer, in random order and at end-expiration at 30-min intervals during surgery. Four patients were not studied because of a failure to cannulate the external jugular vein. The remaining 20 patients yielded 111 sets of paired measurements. The mean difference between external jugular venous pressure and central venous pressure was 0.3 mmHg over a range of central venous pressure of 0-22 mmHg. Limits of agreement were 3.6 to +3.0 mmHg (95% CI 4.1 to +3.5 mmHg). We conclude that external jugular venous pressure is an accurate estimate of central venous pressure in surgical patients undergoing mechanical ventilation.

Induction of anesthesia in the elderly ambulatory patient: a double-blinded comparison of propofol and sevoflurane.

IMPLICATIONS: Hypotension during induction of anesthesia is common and particularly undesirable in elderly patients. This study has shown that inhaled induction with sevoflurane is well tolerated by the elderly and is associated with higher mean arterial pressure than slow propofol induction.

Comparison of effects of remifentanil and alfentanil on cardiovascular response to tracheal intubation in hypertensive patients.

In a randomized double-blind study, we compared the effect of remifentanil and alfentanil on the cardiovascular response to laryngoscopy and tracheal intubation in patients on long-term treatment for hypertension. Forty ASA II-III patients were allocated to receive (i) remifentanil 0.5 microg kg(-1) followed by an infusion of 0.1 microg kg min(-1) or (ii) alfentanil 10 microg kg(-1) followed by an infusion of saline; all patients received glycopyrrolate 200 microg before the study drug. Anaesthesia was induced with propofol and rocuronium and maintained with 1% isoflurane and 66% nitrous oxide in oxygen. Laryngoscopy and tracheal intubation were performed after establishment of neuromuscular block. Arterial pressure and heart rate (HR) were measured non-invasively at 1 min intervals from 3 min before induction until 5 min after intubation. Systolic (SAP), diastolic and mean arterial pressure decreased significantly after induction in both groups (P<0.05). Maximum increases in mean SAP after laryngoscopy and intubation were 35 and 41 mm Hg in the remifentanil and alfentanil groups, respectively. After intubation, arterial pressure did not increase above baseline values in either group. HR remained stable after induction of anaesthesia, but increased above baseline values after intubation. Mean maximum HR was 87 beats min(-1) for the remifentanil group (12 beats min(-1) above baseline; P=0.065) and 89 beats min(-1) for the alfentanil group (15 beats min(-1) above baseline; P<0.05). There were no significant differences between groups in HR or arterial pressure at any time. There were no incidences of bradycardia. Seven patients in the remifentanil group and four in the alfentanil group received ephedrine for hypotension (i.e. SAP<100 mm Hg).

Sarcoplasmic reticulum Ca(2+) uptake is impaired in coronary smooth muscle distal to coronary occlusion.

After chronic occlusion, collateral-dependent coronary arteries exhibit alterations in both vasomotor reactivity and associated myoplasmic free Ca(2+) levels that are prevented by chronic exercise training. We tested the hypotheses that coronary occlusion diminishes Ca(2+) uptake by the sarcoplasmic reticulum (SR) and that exercise training would prevent impaired SR Ca(2+) uptake. Ameroid constrictors were surgically placed around the proximal left circumflex (LCx) artery of female swine 8 wk before initiating 16-wk sedentary (pen confined) or exercise-training (treadmill run) protocols. Twenty-four weeks after Ameroid placement, smooth muscles cells were enzymatically dissociated from both the LCx and nonoccluded left anterior descending (LAD) arteries of sedentary and exercise-trained pigs, and myoplasmic free Ca(2+) was studied using fura 2 microfluorometry. After the SR Ca(2+) store was partially depleted with caffeine (5 mM), KCl-induced membrane depolarization produced a significant decrease in the time to half-maximal (t(1/2)) myoplasmic free Ca(2+) accumulation in LCx versus LAD cells of sedentary pigs. Furthermore, inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA; 10 microM cyclopiazonic acid) significantly reduced t(1/2) in cells isolated from the LAD but not from the LCx. Exercise training did not prevent the differences in t(1/2) myoplasmic free Ca(2+) accumulation observed between LCx and LAD cells. Occlusion or exercise training did not alter SERCA protein levels. These results support our hypothesis of impaired SR Ca(2+) uptake in coronary smooth muscle cells isolated distal to chronic occlusion. Impaired SR Ca(2+) uptake was independent of SERCA protein levels and was not prevented by exercise training.

Exercise training restores adenosine-induced relaxation in coronary arteries distal to chronic occlusion.

We previously reported that canine collateral-dependent coronary arteries exhibit impaired relaxation to adenosine but not sodium nitroprusside. In contrast, exercise training enhances adenosine sensitivity of normal porcine coronary arteries. These results stimulated the hypothesis that chronic coronary occlusion and exercise training produce differential effects on cAMP- versus cGMP-mediated relaxation. To test this hypothesis, Ameroid occluders were surgically placed around the proximal left circumflex coronary artery (LCx) of female Yucatan miniature swine 8 wk before initiating sedentary or exercise training (treadmill run, 16 wk) protocols. Relaxation to the cAMP-dependent vasodilators adenosine (10(-7) to 10(-3) M) and isoproterenol (3 x 10(-8) to 3 x 10(-5) M) were impaired in collateral-dependent LCx versus nonoccluded left anterior descending (LAD) arterial rings isolated from sedentary but not exercise-trained pigs. Furthermore, adenosine-mediated reductions in simultaneous tension and myoplasmic free Ca(2+) were impaired in LCx versus LAD arteries isolated from sedentary but not exercise-trained pigs. In contrast, relaxation in response to the cAMP-dependent vasodilator forskolin (10(-9) to 10(-5) M) and the cGMP-dependent vasodilator sodium nitroprusside (10(-9) to 10(-4) M) was not different in LCx versus LAD arteries of sedentary or exercise-trained animals. These data suggest that chronic occlusion impairs receptor-dependent, cAMP-mediated relaxation; receptor-independent cAMP- and cGMP-mediated relaxation were unimpaired. Importantly, exercise training restores cAMP-mediated relaxation of collateral-dependent coronary arteries.

Exercise training improves endothelium-mediated vasorelaxation after chronic coronary occlusion.

The present study evaluated combined effects of chronic coronary occlusion and exercise training on endothelial function. Gradual occlusion was produced by placement of an ameroid constrictor around the proximal left circumflex (LCX) coronary artery of female swine. Two months after placement of the ameroid, animals were restricted to their pens or exercise trained for 16 wk. Epicardial arteries (>500 microm ID) were isolated from the collateral-dependent LCX coronary artery distal to the occlusion and the nonoccluded left anterior descending (LAD) coronary artery. Bradykinin- and ADP-mediated relaxation of LCX and LAD coronary arteries was enhanced after exercise training. Inhibition of nitric oxide synthase with N(G)-nitro-L-arginine methyl ester decreased bradykinin- and ADP-mediated relaxation in LCX and LAD myocardial regions. Importantly, combined inhibition of effects of endothelium-derived hyperpolarizing factor with increased extracellular K(+) (20-30 mM) and nitric oxide synthase completely abolished coronary LAD and LCX relaxation to bradykinin. Our data indicate that exercise training improves endothelium-mediated relaxation of arteries isolated after chronic coronary artery occlusion, likely as a result of enhanced production of nitric oxide and endothelium-derived hyperpolarizing factor.

Enhanced prostanoid-mediated vasorelaxation in pulmonary arteries isolated during experimental endotoxemia.

Endotoxemia secondary to gram-negative sepsis has been shown to inhibit endothelium-dependent vasomotion in numerous vascular beds, including guinea pig aortae and coronary arteries. We tested the hypothesis that in vivo endotoxin impairs endothelium-dependent nitric oxide-mediated relaxation responses of pulmonary arteries isolated from guinea pigs given intraperitoneal injections of Escherichia coli endotoxin lipopolysaccharide (LPS) or saline (control) 16 h before sacrifice. Pulmonary rings from the main artery and primary branches were isolated and studied in vitro using conventional isometric techniques. Interestingly, endotoxemia resulted in enhanced pulmonary artery relaxation in response to the endothelium-dependent receptor agonists acetylcholine (10(-10) -10(-5) M) and adenosine diphosphate (ADP; 10(-9) -10(-5) M), as compared with control responses (p < .05). Nitric oxide synthase inhibitors N-monomethyl-L-arginine (300 microM) and N-nitro-L-arginine methyl ester (100 microm) reduced acetylcholine- and adenosine diphosphate-mediated relaxation in both groups (p < .05); however, vasodilation responses in arteries from LPS animals remained enhanced relative to those of control arteries. In contrast to nitric oxide synthase inhibitors, the cyclooxygenase inhibitor indomethacin markedly inhibited acetylcholine- and adenosine diphosphate-mediated relaxation responses of pulmonary arteries isolated from LPS-treated animals (p < .05) but not control arteries; indomethacin effectively reversed LPS-induced enhanced vasodilation of pulmonary arteries. Relaxation responses to the receptor-independent calcium ionophore (A23187) and to the direct smooth muscle vasodilator sodium nitroprusside (+ N-nitro-L-arginine methyl ester) were not significantly altered by LPS treatment (p > .05). These data suggest that in pulmonary arteries, unlike aortae and coronary arteries isolated from the same model, in vivo LPS enhances agonist-mediated endothelium-dependent vasodilation responses to acetylcholine and adenosine diphosphate. Underlying mechanisms appear to involve increased dependency upon vasodilator prostanoids and decreased dependency on nitric oxide synthesis/release for LPS-induced alterations in pulmonary relaxation responses.

Reduction-oxidation (Redox) and vascular tissue level of homocyst(e)ine in human coronary atherosclerotic lesions and role in extracellular matrix remodeling and vascular tone.

Hyperhomocyst(e)inemia in patients with coronary and peripheral arterial occlusion has been demonstrated by others. Redox-state of homocyst(e)ine causes dysfunction of endothelial cells and promote growth of vascular smooth muscle cells. The role of tissue, protein bound and unbound, oxidative mixed disulfides in the development of fibrous plaque in atherosclerotic lesion is not known. Redox-state around the fibroblasts and vascular smooth muscle cells modulates the expression of extracellular matrix (ECM) components (Tyagi et al. 1996, J Cell Biochem, 61: 139-151). To determine the role of tissue homocystine in fibrotic atherosclerotic plaque development, coronary arteries were isolated from ischemic explanted hearts (n = 10). Apparently normal vascular tissue was obtained from idiopathic cardiomyopathic explanted hearts (n = 10). Tissue extract were prepared from atherosclerotic lesions and from normal arteries devoid of adventitia. Interaction of homocystine with Ellman's reagent (5, 5'-dithio-bis-2-nitro benzoic acid) catalyzed by limiting amount of reducing agent (catalyst) generated change in optical density (OD) at 412 nm in dose dependent fashion. We have generated a standard curve between change at 412 nm and amount of homocystine. The change in OD at 412 nm with increasing amount (0-25 microg) of homocystine demonstrated linearity. The protein-bound oxidized disulfides were precipitated by trichloroacetic acid (TCA) and free-oxidative disulfides in the supernatant were collected. The pathophysiological amount of protein-bound disulfide in atherosclerotic tissue (1.0 +/- 0.2 microg/mg total protein) was 10 times that in normal tissue (0.1 +/- 0.01 microg/mg, p < 0.001). The amount of free oxidative disulfide in atherosclerotic tissue (1.5 +/- 0.3 microg/mg) was 15 times that in normal tissue (0.12 +/- 0.02 microg/mg, p < 0.001). To determine the role of homocystine in ECM expression, ECM collagenase activity in the presence and absence of homocystine was measured by zymography. The effect of homocysteine on collagenase activity was biphasic, increased at < [0.01 mM] and inhibited at > [0.1 mM]. To determine whether homocystine regulates vascular tone, isometric measurements were carried out using normal coronary rings. Results suggested that homocystine induced endothelial-modulated vasoconstriction in coronary vessels. Tissue oxidative disulfides and the homocystine may contribute to the development of fibrotic atherosclerotic lesions and vascular dysfunction.

Altered reactivity of coronary arteries located distal to a chronic coronary occlusion.

The coronary vasculature located distal to a chronic occlusion (collateral-dependent) has been shown to exhibit altered reactivity to vasoactive agonists. Thus we evaluated effects of chronic coronary artery occlusion on vasomotor responsiveness of collateral-dependent arteries isolated from a canine model of Ameroid occlusion of the left circumflex (LCX) coronary artery. We compared in vitro responses of large (approximately 1.3- to 1.4-mm-ID) and small (approximately 0.6-mm-ID) LCX arteries located distal to an occlusion with responses of similar-sized segments of the unoccluded left anterior descending (LAD) coronary artery. Alpha-adrenergic receptor-mediated contractile responses to norepinephrine (10(-9)-10(-4) M) and phenylephine (10(-9)-10(-4) M) in the presence of propranolol were markedly enhanced in large LCX arteries compared with LAD arteries (P < 0.001). Prazosin (1 microM), an alpha1-adrenergic receptor antagonist, abolished contractile responses of LCX and LAD arteries to norepinephrine. Inhibition of nitric oxide synthesis with N(omega)-nitro-L-arginine methyl ester (100 microM) enhanced norepinephrine-induced contractions of LAD arteries to a greater extent than contractions of LCX arteries. We simultaneously measured myoplasmic free Ca2+ (fura 2 fluorescence ratio) and contractile responses in LCX and LAD arteries denuded of endothelium; norepinephrine-induced increases in myoplasmic free Ca2+ and contractile tension were significantly enhanced in LCX arteries compared with LAD arteries. In addition, large and small LCX arteries exhibited impaired relaxation in response to adenosine (10(-8)-10(-3) M) compared with LAD arteries (P < 0.05). In contrast, relaxation in response to the beta-adrenergic agonist isoproterenol (10(-9)-10(-4) M) and sodium nitroprusside (10(-10)-10(-4) M) was not significantly different in LCX and LAD arteries. Thus collateral-dependent coronary arteries exhibit enhanced alpha-adrenergic vasoconstriction and impaired vasorelaxation in response to adenosine. The enhanced alpha-adrenergic contractile responsiveness involves at least two mechanisms: 1) enhanced alpha1-adrenergic reactivity of smooth muscle and 2) decreased alpha-adrenergic-induced synthesis of nitric oxide by the endothelium.

Contractile responsiveness of coronary arteries from exercise-trained rats.

The purpose of this study was to determine whether exercise training alters vasomotor reactivity of rat coronary arteries. In vitro isometric microvessel techniques were used to evaluate vasomotor properties of proximal left anterior artery rings (1 ring per animal) from exercise-trained rats (ET; n = 10) subjected to a 12-wk treadmill training protocol (32 m/min, 15% incline, 1 h/day, 5 days/wk) and control rats (C; n = 6) restricted to cage activity. No differences in passive length-tension characteristics or internal diameter (158 +/- 9 and 166 +/- 9 micron) were observed between vessels of C and ET rats. Concentration-response curves to K+ (5-100 mM), prostaglandin F2alpha (10(-8)-10(-4) M), and norepinephrine (10(-8)-10(-4)) were unaltered (P > 0.05) in coronary rings from ET rats compared with C rats; however, lower values of the concentration producing 50% of the maximal contractile response in rings from ET rats (P = 0.05) suggest that contractile sensitivity to norepinephrine was enhanced. Vasorelaxation responses to sodium nitroprusside (10(-9)-10(-4) M) and adenosine (10(-9)-10(-4) M) were not different (P > 0.05) between vessels of C and ET rats. However, relaxation responses to the endothelium-dependent vasodilator acetylcholine (ACh; 10(-10)-10(-4) M) were significantly blunted (P < 0.001) in coronary rings from ET animals; maximal ACh relaxation averaged 90 +/- 5 and 46 +/- 12%, respectively, in vessels of C and ET groups. In additional experiments, two coronary rings (proximal and distal) were isolated from each C (n = 7) and ET (n = 7) animal. Proximal coronary artery rings from ET animals demonstrated decreased relaxation responses to ACh; however, ACh-mediated relaxation of distal coronary rings was not different between C and ET groups. NG-monomethyl-L-arginine (inhibitor of nitric oxide synthase) blocked ACh relaxation of all rings. L-Arginine (substrate for nitric oxide synthase) did not improve the blunted ACh relaxation in proximal coronary artery rings from ET rats. These studies suggest that exercise-training selectively decreases endothelium-dependent (ACh) but not endothelium-independent (sodium nitroprusside) relaxation responses of rat proximal coronary arteries; endothelium-dependent relaxation of distal coronary arteries is unaltered by training.

Chronic endotoxemia and endothelium-dependent vasodilation in coronary arteries.

Endotoxin acutely decreases the production of nitric oxide, leading to abnormal regulation of coronary vascular tone: however, the effects of chronic endotoxemia on vasomotion are unknown. We therefore tested the hypothesis that chronic, low-level endotoxemia inhibits endothelium-dependent vasodilation. Rabbits were continuously infused with a subclinical dose of Escherichia coli endotoxin (.6 microgram/24 h, intraperitoneal) or saline for 5 wk. Endotoxin at this concentration elicited no significant sustained pyretic or hemodynamic responses. Both endothelium-dependent and independent vasomotor responses were determined in coronary arteries (250-500 mu). Vasorelaxation in response to acetylcholine, but no adenosine diphosphate (ADP), was significantly enhanced in endotoxin-challenged animals (EC50 = 62.6 +/- 11.1 nM, control vs. 33.97 +/- 5.7 nM, endotoxin-challenged; p < .05). Vasoconstriction to PGF2 alpha, but not KCl, was significantly decreased in endotoxin-challenged animals. These results indicate that endothelium-dependent and independent vasomotor responses are altered during chronic endotoxemia and are due, in part, to alterations in signal-transduction mechanisms specific for certain types of receptors.

Effects of exercise training on responses of peripheral and visceral arteries in swine.

Blood flow to skeletal muscle during exercise is greater in the trained state. We hypothesized that intrinsic vasomotor reactivity of arteries to active muscle during training bouts would be altered to favor a relative vasodilation after training. To test this hypothesis, miniature swine were pen confined (Sed; n = 30) or treadmill trained for 5 days/wk over 16-20 wk (Trn; n = 32). Efficacy of training was indicated by myocardial hypertrophy (4.84 +/- 0.11 and 5.81 +/- 0.12 g/kg body wt for Sed and Trn, respectively, P < 0.0005), training bradycardia at several submaximal running speeds of a maximal exercise test, increased running time to exhaustion (26 +/- 1 and 35 +/- 1 min for Sed and Trn, respectively, P < 0.0005), and increased oxidative capacities of several locomotory skeletal muscles. Segments of femoral, brachial, mesenteric, renal, and hepatic arteries were isolated from Sed and Trn swine. Isometric contractile and relaxation properties of vascular rings cut from these segments were determined in vitro. Contractile responses to KCl and norepinephrine (NE) were determined, as were relaxation responses to sodium nitroprusside and adenosine, agents acting directly on vascular smooth muscle, and the endothelium-dependent agents bradykinin and the calcium ionophore A-23187. Responses to vasocontractile and vasorelaxation agents were not different between Sed and Trn swine for vessels serving active muscles (i.e., femoral, brachial). On the other hand, renal arterial rings from Trn swine exhibited lesser contractile responses than those from Sed swine across a range of NE concentrations (P < 0.05) and approximately 25% less maximal contractile response to NE (32.7 +/- 2.6 and 24.2 +/- 2.1 g for Sed and Trn, respectively, P < 0.01). Responses of other vessels serving viscera (i.e., mesenteric, hepatic) were unchanged with training. These data indicate that vasomotor reactivity of porcine conduit-type arteries generally does not change with exercise training. An exception is the lesser contractile response to NE in renal artery, which could permit better preservation of renal blood flow during acute exercise in trained animals.

Endothelium-dependent vasodilation of proximal coronary arteries from exercise-trained pigs.

We recently reported that alpha-adrenergic vasoconstriction is blunted and adenosine-induced vasodilation is enhanced in proximal coronary arteries of exercise-trained miniature swine [C. L. Oltman, J. L. Parker, H. R. Adams, and M. H. Laughlin. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H372-H382, 1992]. The purpose of the present study was to determine whether this model of exercise training also alters endothelium-dependent vasodilator responses of proximal coronary arteries. Female Yucatan miniature swine were exercise trained (ET) on a motor-driven treadmill or were cage confined (Sed) for 13-20 wk. Exercise tolerance, heart weight-to-body weight ratios, and skeletal muscle oxidative capacity were all significantly greater in ET than in Sed animals. Vasodilator responses were evaluated in vitro by determining concentration-response curves by using vascular rings (3.5-4 mm in axial length) isolated from right and left coronary arteries. Vasorelaxation responses were determined, after tone had been produced with either 30 microM prostaglandin F2 alpha, 30 mM KCl, or 30 nM endothelin. Concentration-response curves were obtained to endothelium-dependent vasodilators including bradykinin (10(-9)-10(-6) M), substance P (10(-12)-10(-6) M), clonidine (10(-9)-10(-6) M), serotonin (10(-10)-10(-5) M), and the Ca2+ ionophore A-23187 (10(-10)-10(-6) M). Endothelium-independent vasodilator responses to sodium nitroprusside (10(-9)-10(-4) M) were not different between arteries from Sed and ET. Bradykinin, substance P, and A-23187 were potent vasodilators in arteries from both groups, whereas serotonin and clonidine did not consistently produce vasodilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of EDRF and NO in ex vivo perfused aorta: inhibition by in vivo E. coli endotoxemia.

Previous studies have yielded contradictory results about interrelations between endotoxin and endothelium-derived relaxing factor (EDRF). We tested the hypothesis that in vivo endotoxemia inhibits basal and/or agonist-mediated release of EDRF and nitric oxide (NO). EDRF bioactivity, NO production, and NO synthase (NOS) activity were measured in aorta from guinea pigs following 16 h of Escherichia coli endotoxemia (4 mg/kg endotoxin i.p.). Endothelium-dependent relaxation of aortic rings was studied under standard isometric conditions. Endotoxemia resulted in an 89% reduction in basal EDRF bioactivity and a 62% reduction in basal NO production in perfused aorta. EDRF bioactivity and NO production in response to the receptor-dependent agonists acetylcholine and ADP were significantly reduced in perfused aorta from endotoxemic animals. In contrast, endotoxin did not significantly inhibit EDRF bioactivity and NO production by the receptor-independent agonist A-23187. Aortic rings from endotoxemic animals likewise showed decreased vasodilator responses to acetylcholine and ADP but not to A-23187. Inducible (Ca2+ independent) NOS activity was not significantly different in control and endotoxin-treated animals. These findings indicate that prolonged endotoxemia resulted in diminution of release of EDRF, consistent with the interpretation that endotoxemia decreases basal and agonist-stimulated EDRF bioactivity and NO production with loss of endothelium-dependent vasodilator reserves during gram-negative sepsis.

Inhibition of endothelium-dependent vasodilation by Escherichia coli endotoxemia.

To test the hypothesis that release of endothelium-derived relaxing factor/nitric oxide is inhibited by Gram-negative lipopolysaccharide (LPS; endotoxin), we examined endothelium-independent and endothelium-dependent vasodilator agents in aortic vascular smooth muscle isolated from guinea pigs 4 h after injection of saline (controls) or induction of Escherichia coli endotoxemia. LPS significantly inhibited vasodilator responses to the endothelium-dependent agonists acetylcholine (ACh; 10(-10)-10(-5) M) and ADP (10(-8)-10(-5) M). However, LPS did not affect vasodilator responses to the endothelium-independent agonist nitroprusside (10(-10)-10(-4) M). The nitric oxide synthase (NOS) inhibitor N gamma-nitro-L-arginine methyl ester (L-NAME) inhibited the vasodilator response to ACh; whereas, the cyclooxygenase inhibitor indomethacin (INDO) did not reduce vasodilator effects of ACh. Neither L-NAME nor INDO affected the vasodilator effects of nitroprusside in LPS or control vessels. In contrast, L-NAME converted the vasodilator action of ADP to a vasoconstrictor response that was blocked individually by INDO and the thromboxane synthase inhibitor dazoxiben, suggesting that ADP releases NO and also the vasoconstrictor and platelet aggregating eicosanoid thromboxane A2. These findings suggest that acute (4 h) endotoxemia inhibits function of the constitutive isoform of NOS in vascular endothelial cells. Since L-NAME unmasked a vasoconstrictor action of the endogenous purinoceptor agonist ADP, pharmacologic agents that inhibit NOS may exacerbate LPS-induced inhibition of endothelial NOS; this series of events could lead to diminution of vasodilator reserves and perhaps to augmentation of platelet aggregation during Gram-negative sepsis.

Effects of angiotensin II on canine and porcine coronary epicardial and resistance arteries.

Coronary resistance arteriolar diameter importantly regulates myocardial blood flow, and is influenced by circulating neurohumoral agents. Angiotensin II (A-II) is a circulating polypeptide that is chronically elevated in heart failure and serves as a potent peripheral vasoconstrictor agent. However, its effects on isolated coronary resistance arterioles is relatively unknown. We compared the vasomotor effects of A-II on coronary epicardial and resistance arterioles in vitro from both the canine and porcine heart in order to determine the effects of A-II in different vascular beds and species. Epicardial rings were studied under isometric recording conditions, while resistance arterioles (50-150 microns) were studied in vitro using a video imaging system to record diameter. A-II, whether applied to passively distended or preconstricted porcine resistance arterioles, did not cause vasoconstriction when applied as a bolus or as cumulative doses. In preconstricted canine resistance arterioles, A-II elicited dose-dependent vasodilation (EC50 = 0.2 nM). In passively distended canine arterioles, high concentrations of A-II (0.1 microM) applied as a bolus elicited transient vasoconstriction in 28% of the vessels studied. In large epicardial rings, A-II was a weak vasoconstrictor, with greater potency in canine arteries compared to porcine arteries. In canine arteries, vasoconstriction to A-II was augmented after incubation with indomethacin. In contrast to the findings in canine arteries, the A-II vasoconstrictor response in porcine coronary arteries was decreased after incubation with indomethacin or removal of the endothelium. Thus, A-II elicits the release of a vasodilator prostanoid in epicardial canine coronary arteries and a vasoconstrictor prostanoid in porcine vessels which modulate the vasomotor action of A-II.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of exercise training on regulation of tone in coronary arteries and arterioles.

A large number of studies now support the concept that exercise training alters functional control of the coronary circulation. Recent work has approached this area using ex vivo coronary arterial preparations (proximal coronary arteries, near-resistance arteries, resistance arterioles) isolated from exercise-trained animals and contracting independently of confounding in vivo influences. The combined results of these studies indicate that training-induced alterations in vascular control mechanisms do not occur uniformly throughout the coronary vascular tree. Proximal epicardial coronary arteries (approximately 2.0 mm diameter) isolated from exercise-trained pigs exhibited significantly reduced contractile responsiveness to the alpha-adrenergic receptor agonist, norepinephrine, but unaltered contractile responsiveness to K+, acetylcholine, and endothelin. Also, proximal arteries from exercise-trained animals demonstrated enhanced sensitivity to the vasodilator effects of adenosine. At the other end of the vascular spectrum, in resistance arterioles (< 150 microns diameter) the relaxation responses to adenosine were unaffected by exercise training, but bradykinin-induced vasodilation (endothelium-dependent) was significantly enhanced. In near-resistance arteries (150-240 microns diameter) responses to both bradykinin and adenosine were enhanced by exercise training. Thus, exercise training is associated with intrinsic vessel size-dependent alterations in coronary smooth muscle and endothelium-mediated regulatory mechanisms.

Escherichia coli endotoxin inhibits agonist-mediated cytosolic Ca2+ mobilization and nitric oxide biosynthesis in cultured endothelial cells.

Altered release of endothelium-derived relaxing factor/nitric oxide (EDRF/NO) has been proposed as a final common pathway underlying the abnormal vasodilator responses to gram-negative lipopolysaccharide (endotoxin). However, mechanisms responsible for lipopolysaccharide-induced changes in EDRF/NO release from endothelial cells have not been clarified. We evaluated direct effects of Escherichia coli endotoxin on agonist-stimulated cytosolic Ca2+ mobilization and NO biosynthesis in cultured bovine and porcine aortic endothelial cells (ECs). Two methods were used to assay for NO: (1) analysis of NO-induced endothelial levels of cGMP as a biological indicator of NO generation and (2) direct quantitative measurement of NO release (chemiluminescence method). Cytosolic free Ca2+ ([Ca2+]i) was evaluated using fura 2 fluorescence methodology (340/380-nm ratio excitation and 500-nm emission). Incubation of ECs with endotoxin (0.5 microgram/mL, 1 hour plus 1-hour wash) significantly inhibited bradykinin (100 nmol/L)- and ADP (10 mumol/L)-mediated increases in endothelial cell cGMP to 37% and 22% of control responses, respectively. In contrast, endotoxin failed to inhibit the increase in cGMP produced by the non-receptor-dependent Ca2+ ionophore A23187 (1 mumol/L) or sodium nitroprusside (1 mmol/L). Similarly, incubation with endotoxin inhibited ADP-stimulated increases in NO release and EDRF bioactivity to 55% and 56% of control values, respectively, but did not affect A23187-stimulated increases in NO release or EDRF bioactivity. Endotoxin produced significant decreases in both transient and sustained [Ca2+]i responses of ECs to bradykinin and ADP. For example, the initial rapid increase in bovine EC [Ca2+]i in response to bradykinin was reduced to 31% of the initial increases in control cells, and the secondary plateau phase was reduced to only 3% of respective control responses. Concentration-response relation to endotoxin (10(-3)) to 10(0) micrograms/mL) indicated high correlation and similar IC50 values (0.025 and 0.021 micrograms/mL, respectively) for inhibitory effects on cGMP and [Ca2+]i. Endotoxin had no effect on inositol trisphosphate formation ([3H]myo-inositol incorporation) and intracellular Ca2+ release ([Ca2+]i responses in Ca(2+)-free medium) induced by bradykinin. However, agonist-stimulated Mn2+ quenching (index of Ca2+ influx) was significantly attenuated by endotoxin treatment. These studies demonstrate that endotoxin directly decreases agonist (bradykinin and ADP)-mediated biosynthesis and release of EDRF/NO from ECs. These effects can be explained by altered [Ca2+]i mobilization mechanisms, which in turn produce subsequent decreases in activity of the Ca(2+)-calmodulin-dependent constitutive isoform of NO synthase and, ultimately, impairment of agonist-mediated NO release and endothelium-dependent vasodilation.