Evaluation of new instruments for screening and diagnosis of tics and tic disorders in a well-characterized sample of youth with tics and recruited controls.

Tics and tic disorders can significantly impact children, but limited screening tools and diagnostic challenges may delay access to care. The current study attempted to address these gaps by evaluating sensitivity and specificity of the Motor or Vocal Inventory of Tics (MOVeIT), a tic symptom screener, and the Description of Tic Symptoms (DoTS), a brief diagnostic assessment for tic disorders. Children (n=100, age 6-17 years old) with tic disorders attending a Tourette specialty clinic and a community-recruited sample without tics completed a gold-standard assessment by a tic expert; these evaluations were compared to child self-report and parent and teacher report versions of the MOVeIT, and child and parent versions of the DoTS. The parent and child MOVeIT met or exceeded pre-specified 85% sensitivity and specificity criteria for detecting the presence of tics when compared to a gold-standard tic expert diagnosis. The Teacher MOVeIT had lower sensitivity (71.4%) but good specificity (95.7%) for identifying any tic symptoms compared to gold standard. For determination of the presence or absence of any tic disorder, sensitivity of both parent and child DoTS was 100%; specificity of the parent DoTS was 92.7% and child DoTS specificity was 75.9%. More work may be needed to refine the teacher MOVeIT, but it is also recognized that tic expression may vary by setting. While the MOVeIT and DoTS parent and child questionnaires demonstrated adequate sensitivity and specificity for determining the presence of tics and tic disorders in this well-defined sample, additional testing in a general population is warranted.

Increasing precision in the measurement of change in pediatric neurodegenerative disease.

Due to the surge in new brain-directed treatments, metrics to detect the alteration in developmental trajectories in cognition and adaptive behavior have become increasingly important. We propose Growth Scale Values (GSVs) as a solution to monitoring children with severe neurologic/neurodegenerative conditions. This report stems from a panel of experts presenting at the Gorlin symposium (WORLD Symposium) and a subsequent open Webinar sponsored by the National MPS Society. Because norm-referenced scores (Standard Scores or Intelligence Quotient, i.e., IQ) do not yield information about gain, stability, or loss of skills, they are not suitable for natural history studies or clinical trials. Age-equivalent (AE) scores have been the standard metric used in natural history studies. While AEs are familiar and interpretable to clinicians and parents, they are imprecise due to lack of standard deviations, standard errors of measurement, and equal intervals between scores. Raw scores also have unequal intervals and are not comparable between ages or ability levels. The GSV, a nonlinear transformation of raw scores using item calibration to make an interval scale score, can be used for accurate measures of within-person change. GSVs have been identified as a useful metric for longitudinal measurement of other conditions involving neurodiversity. These growth scores circumvent inaccurate AEs in infants, are not limited by age and can be used for impaired patients who are chronologically above the normative age range. GSVs have interval properties (a given difference between GSV values represents the same difference in ability at all score levels) and each GSV value has a known standard error of measurement (SEM). GSVs are recommended to measure change in cognitive and adaptive behavior in natural history studies and in clinical trials for children with neurologic disease.

Design of a Multisite Study Assessing the Impact of Tic Disorders on Individuals, Families, and Communities.

BACKGROUND: Tic disorders, including Tourette syndrome, are complex, multisymptom diseases, yet the impact of these disorders on affected children, families, and communities is not well understood. METHODS: To improve the understanding of the impacts of Tourette syndrome, two research groups conducted independent cross-sectional studies using qualitative and quantitative measures. They focused on similar themes, but distinct scientific objectives, and the sites collaborated to align methods of independent research proposals with the aim of increasing the analyzable sample size. RESULTS: Site 1 (University of Rochester) was a Pediatric Neurology referral center. Site 2 (University of South Florida) was a Child Psychiatry referral center. A total of 205 children with tic disorders were enrolled from both studies. The University of Rochester also enrolled 100 control children in order to clearly isolate impacts of Tourette syndrome distinct from those occurring in the general population. The majority of children with tic disorders (n = 191, 93.1%) had Tourette syndrome, the primary population targeted for these studies. Children with Tourette syndrome were similar across sites in terms of tic severity and the occurrence of comorbid conditions. The occurrence of psychiatric comorbidities in the control group was comparable with that in the general pediatric population of the United States, making this a well-justified comparison group. CONCLUSIONS: Through collaboration, two sites conducting independent research developed convergent research methods to enable pooling of data, and by extension increased power, for future analyses. This method of collaboration is a novel model for future epidemiological research of tic disorders.

Increased T-regulatory cells within lymphocyte follicles in moderate COPD.

Lymphoid follicles in the lung parenchyma are a characteristic feature of chronic obstructive pulmonary disease (COPD). There are reports of altered CD4 T-regulatory cell numbers in COPD lungs, but the location of these cells within COPD lung tissue specific follicles has not been investigated. The presence of CD4(+)FOXP3(+) T-regulatory cells was assessed in surgically resected lung tissue from 12 COPD patients, 11 smokers with normal lung function and seven nonsmokers by combined immunofluorescence and immunohistochemistry. Organised lymphoid follicles were observed in all three groups of patients, as well as lymphoid clusters lacking organisation. The percentage of CD4 cells that were T-regulatory cells were significantly increased (p = 0.02) within COPD (16%) follicles compared with smokers (10%) and nonsmokers (8%). In contrast, there was no change (p>0.05) in the percentage of T-regulatory cells in clusters or the subepithelium between groups. Lymphoid follicles in COPD patients have increased T-regulatory cells. Therefore, T-regulatory activity may be altered within COPD lymphoid follicles.

Endotoxin impairs agonist-stimulated intracellular free calcium (Ca(i)) responses in freshly dispersed aortic endothelial cells.

Impairment in endothelial cell intracellular free calcium (Ca(i)) mobilization mechanisms may contribute to decreased nitric oxide (NO) biosynthesis and impaired vasorelaxation responses of endotoxemic guinea pigs to endothelium-dependent vasodilators. We tested this hypothesis using fura-2 microfluorometry to compare agonist-stimulated Ca(i) responses of aortic endothelial cells freshly dispersed from guinea pigs 16 h after intraperitoneal injection of Escherichia coli endotoxin (lipopolysaccharide, LPS; 4 mg/kg) or saline (CON). In the presence of normal extracellular Ca2+ (2 mmol/L), basal (non-stimulated) endothelial Ca(i) (340/380 nm fluorescence ratio, R) was not different between CON and LPS cells (1.1 +/- 0.03 and 1.1 +/- 0.03, respectively). However, exposure to ADP (10 micromol/L) produced a biphasic increase in Ca(i) that was markedly decreased in cells from LPS-treated animals (P < 0.0001). Peak ADP-stimulated Ca(i) responses averaged 2.2 +/- 0.21 in CON cells and 1.5 +/- 0.11 (P < 0.01) in cells dispersed from LPS-treated animals. Exposure to acetylcholine (ACh; 10 micromol/L) produced sustained increases in Ca(i) (R = 1.4 +/- 0.13) in CON cells; however, LPS abolished Ca(i) responses to ACh. Exposure of endothelial cells to substance P (100 nmol/L) produced a biphasic increase in Ca(i) that was not different between groups. In the absence of extracellular Ca2+ (plus 10 micromol/L EGTA), exposure to ADP (10 micromol/L) produced transient increases in Ca(i) (Ca2+ release) that were decreased in cells from LPS-treated versus CON animals. Exposure to ACh in zero Ca2+ (10 micromol/L) produced smaller increases in Ca(i) (peak R = 1.3 +/- 0.12) in CON cells (when compared to ADP); however, Ca(i) responses to ACh remained absent in cells from LPS-treated animals. Re-exposure to Ca2+ produced sustained ACh-induced Ca(i) responses (Ca2+ influx) in cells from CON, but not LPS-treated animals; LPS markedly impaired (P< 0.05) ADP-induced sustained Ca(i) responses. Our data demonstrate that in vivo LPS exposure elicits decreased agonist-stimulated endothelial Ca(i) responses primarily involving impaired Ca2+ influx mechanisms. Known dependence of endothelial agonist-stimulated NO synthesis on Ca(i) suggests that defects in cell Ca2+ mobilization may contribute to LPS-induced impaired NO biosynthesis and decreased endothelium-dependent relaxation.

PET evaluation of [(18)F]FCWAY, an analog of the 5-HT(1A) receptor antagonist, WAY-100635.

We synthesized [(18)F]FCWAY, an analog of [carbonyl-(11)C]WAY-100635 ¿[(11)C]N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl)ethyl))-N-(2-(pyridi nyl))cyclohexanecarboxamide¿, by replacing the cyclohexanecarbonyl group acid with a trans-4-fluorocyclohexanecarbonyl group (FC). Control and preblocking studies were performed in anesthetized monkeys. Plasma radioactive metabolite analysis showed the presence of [(18)F]FC and [(18)F]fluoride. Tissue time-radioactivity curves were corrected for metabolite contamination based on separate positron-emission tomography studies of these two labeled metabolites. Analysis using a two-tissue compartment model gave distribution volume (V) estimates (mL/mL) ranging from 33 in frontal cortex to 4 in cerebellum. Preblocking data showed uniform V of 2-3 mL/mL. These studies demonstrate that [(18)F]FCWAY has very similar kinetic characteristics to [(11)C]WAY-100635.

Kinetic analysis of the 5-HT2A ligand [11C]MDL 100,907.

The goal of this study was to develop a suitable kinetic analysis method for quantification of 5-HT2A receptor parameters with [11C]MDL 100,907. Twelve control studies and four preblocking studies (400 nmol/kg unlabeled MDL 100,907) were performed in isoflurane-anesthetized rhesus monkeys. The plasma input function was determined from arterial blood samples with metabolite measurements by extraction in ethyl acetate. The preblocking studies showed that a two-tissue compartment model was necessary to fit the time activity curves of all brain regions including the cerebellum--in other words, the need for two compartments is not proof of specific binding. Therefore, a three-tissue compartment model was used to analyze the control studies, with three parameters fixed based on the preblocking data. Reliable fits of control data could be obtained only if no more than three parameters were allowed to vary. For routine use of [11C]MDL 100,907, several simplified methods were evaluated. A two-tissue (2T') compartment with one fixed parameter was the most reliable compartmental approach; a one-compartment model failed to fit the data adequately. The Logan graphical approach was also tested and produced comparable results to the 2T' model. However, a simulation study showed that Logan analysis produced a larger bias at higher noise levels. Thus, the 2T' model is the best choice for analysis of [11C]MDL 100,907 studies.

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.

Development of fluorine-18-labeled 5-HT1A antagonists.

We have synthesized five fluorinated derivatives of WAY 100635, N-{2-[4-(2-methoxyphenyl)piperazino]ethyl}-N-(2-pyridyl)cyclohe xaneca rboxamide (4a), using various acids in place of the cyclohexanecarboxylic acid (CHCA, 2a) in the reaction scheme. The five acids are 4-fluorobenzoic acid (FB, 2b), 4-fluoro-3-methylbenzoic acid (MeFB, 2c), trans-4-fluorocyclohexanecarboxylic acid (FC, 2d), 4-(fluoromethyl)benzoic acid (FMeB, 2e), and 3-nitro-4-(fluoromethyl)benzoic acid (NFMeB, 2f) (see Scheme 1). These compounds were radiolabeled with fluorine-18, and their biological properties were evaluated in rats and compared with those of [11C]carbonyl WAY 100635 ([carbonyl-11C]4a). [Carbonyl-11C]4a cleared the brain with a biological half-life averaging 41 min. The metabolite-corrected blood radioactivity had a half-life of 29 min. [18F]FCWAY ([18F]4d) gave half-lives and intercepts comparable to [carbonyl-11C]4a in the brain, but the blood clearance was faster. [18F]FBWAY ([18F]4b) showed an early rapid net efflux from the whole brain, clearing with a biological half-life of 35 min. The metabolite-corrected blood half-life was 41 min. The comparable whole brain and blood half-lives for Me[18F]FBWAY ([18F]4c) were 16 and 18 min, respectively. For each compound, the corresponding carboxylic acid was identified as a major metabolite in blood. Fluoride was also found after injection of [18F]4d. However, for all compounds there was a good correlation (R > 0.97) between the differential uptake ratio (DUR, (%ID/g) x body weight (g)/100) in individual rat brain regions at 30 min after injection and the concentration of receptors as determined by in vitro quantitative autoradiography in rat. Specific binding ratios [region of interest (ROI)/cerebellum-1] in control studies for cortex (Ctx) and hippocampus (H) were higher for [carbonyl-11C]4a and [18F]4d compared to [18F]4b and [18F]4c. [18F]4d has similar pharmacokinetic properties and comparable specific binding ratios to [carbonyl-11C]4a. Fifty nanomoles of 4a blocked only 30% of the specific binding of [18F]4d, while complete blockade was obtained from co-injection of 200 nmol of 4a (H/Cb-1 from 17.2 to 0.6). [18F]4b and [18F]4c showed lower specific binding ratios than [carbonyl-11C]4a and [18F]4d. [18F]4c was superior to [18F]4b since its specific binding was more readily blocked by 4a. These studies suggest that [18F]4c should be a useful compound to assess dynamic changes in serotonin levels while [18F]4d, with its high contrast and F-18 label, should provide better statistics and quantification for static measurement of 5-HT1A receptor distribution.

Contractile function and myoplasmic free Ca2+ (Cam) in coronary and mesenteric arteries of endotoxemic guinea pigs.

Endotoxin-induced vascular hyporesponsiveness could potentially involve alterations of vascular smooth muscle (VSM) myoplasmic free calcium (Ca(m)) mobilization mechanisms. Contractile function and Ca(m)(fura-2 microfluorometry) regulation were evaluated in vitro using coronary (COR) and mesenteric (MES) artery preparations (100-250 microm inner diameter) isolated from guinea pigs 16 h after intraperitoneal (i.p.) injection of either saline (control; CON) or Escherichia coli endotoxin lipopolysaccharide (LPS; 4 mg/kg). Concentration-response relationships to K+ (5-100 mM) were significantly enhanced in both COR and MES arteries isolated from LPS-treated animals. In contrast, contractile responses to prostaglandin F2alpha (PGF2alpha; 1-100 microM) were markedly impaired in COR and MES arteries from LPS-treated animals, while endothelin-1 (ET; 1-100 nM)-mediated contractile responses of these arteries were enhanced at the maximal dose (100 nM). In COR arteries, PGF2alpha (1-100 microM) and ET (1-100 nM) produced biphasic increases in Ca(m) in both CON and LPS groups. No significant differences were observed in either the initial transient peak or secondary sustained Ca(m) responses between groups, suggesting a lack of effect of LPS upon intracellular Ca2+ release or Ca2+ influx mechanisms in COR arteries. Exposure of MES arteries to PGF2alpha and ET produced concentration-dependent increases in Ca(m) in both groups. However, Ca(m) responses of MES arteries lacked initial peak responses, suggesting potential differences in Ca(m) mobilization between COR and MES arteries. Ca(m) responses to K+ (80 mM) and PGF2alpha (1-100 microM) were similar in MES arteries from both groups; however, ET-mediated increases in Ca(m) were significantly blunted in LPS compared with CON MES arteries. Thus, endotoxemia produced differential effects upon depolarization (K4) and receptor (PGF2alpha, ET)-mediated contractile responses in both COR and MES arteries. Reductions in VSM Ca(m) mobilization appear unlikely as a mechanism for LPS-induced impairment of contractile function of COR and MES arteries; other mechanisms (i.e., decreased Ca2+ sensitivity of contractile proteins) may be involved in effects of LPS upon VSM function of COR and MES arteries.

Endotoxemia-induced myocardial dysfunction is not associated with changes in myofilament Ca2+ responsiveness.

Myocardial contractile function is depressed after onset of endotoxemia and is intrinsic to the ventricular myocyte. We tested the hypothesis that decreased Ca2+ responsiveness of the contractile myofilaments underlies this inotropic depression. Specifically, we evaluated the relationship between Ca2+ and unloaded cell shortening and isometric tension development of skinned guinea pig ventricular myocytes. Myocytes were isolated 4 h after intraperitoneal injection of 4 mg/kg Escherichia coli lipopolysaccharide (LPS) or saline (control; Ctl). Myofilament Ca2+ responsiveness assessed by image analysis of shortening of skinned myocytes at pH 7.0 was not different between Ctl[pCa value that resulted in half-maximal shortening (pCa50): 5.78 +/- 0.04] and LPS (pCa50: 5.72 +/- 0.02). Similarly, myofilament Ca2+ responsiveness measured by isometric tension of skinned myocytes was not different between Ctl (pCa50: 5.73 +/- 0.02) and LPS (pCa50: 5.76 +/- 0.02). Maximal tension generated by LPS myocytes (2.89 +/- 0.23 g/mm2) was significantly less (P < 0.05) than Ctl (3.75 +/- 0.34 g/mm2). However, when myocytes were isolated and skinned in the presence of protease inhibitors, maximal tension generated by LPS myocytes (3.53 +/- 0.98 g/mm2) was similar to Ctl (3.01 +/- 0.80 g/mm2). We conclude that in vivo administration of LPS resulting in endotoxemia without shock does not alter myofilament Ca2+ responsiveness of ventricular myocytes. Rather, reduced contractility is more likely a result of decreased Ca2+ availability because systolic Ca2+ transients of fura 2-loaded LPS myocytes were significantly decreased (P < 0.05) compared with Ctl myocytes.

Reduced L-type calcium current in ventricular myocytes from endotoxemic guinea pigs.

The circulatory response to gram-negative sepsis and its experimental counterpart, endotoxemia, includes a profound dysfunction in myocardial contractility that is resident to the myocyte and associated with reduced systolic free intracellular Ca2+ concentration ([Ca2+]i). We explored the possibility that decreased systolic [Ca2+]i in endotoxemic myocytes is correlated with reduced L-type Ca2+ current (ICa,L). Ventricular myocytes were isolated from guinea pigs 4 h after an intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS; 4 mg/kg). Membrane potentials and Ca2+ currents were measured using whole cell patch-clamp methods. The action potential duration of endotoxemic myocytes was significantly shorter than control values (time to 50% repolarization: LPS, 314 +/- 23 ms; control, 519 +/- 36 ms, P < 0.05). Correspondingly, endotoxemic myocytes demonstrated significantly reduced peak ICa,L density (3.5 +/- 0.2 pA/pF) and Ba2+ current (IBa) density (7.3 +/- 0.5 pA/pF) compared with respective values of control myocytes (ICa,L) density 6.1 +/- 0.3 pA/pF, IBa density 11.3 +/- 0.8 pA/pF; P < 0.05). Endotoxemia-induced reduction in peak ICa,L could not be attributed to alterations in current-voltage relationships, steady-state activation and inactivation, or recovery from inactivation. The beta-adrenoceptor agonist isoproterenol, but not the Ca2+ channel activator BAY K 8644, reversed the LPS-induced reduction in peak ICa,L, cell contraction, and systolic [Ca2+]i. These data demonstrate that part of the host response to endotoxemia involves diminished sarcolemmal ICa,L of ventricular myocytes.

Cytosolic Ca2+ concentration and contraction-relaxation properties of ventricular myocytes from Escherichia coli endotoxemic guinea pigs: effect of fluid resuscitation.

Hearts isolated from a guinea pig model of Escherichia coli endotoxemia exhibit decreased systolic contractile function and reduced diastolic compliance of the left ventricle within 4 h after injection of endotoxin. Fluid resuscitation prevented the endotoxin-induced decrease in diastolic compliance without affecting systolic contractile depression. Because intrinsic myocardial dysfunction after endotoxemia may result from alterations in intracellular Ca2+ handling, we tested the hypothesis that in vivo fluid resuscitation improved diastolic function by altering Ca2+ handling of the myocardium. We tested this hypothesis by measuring cell shortening and intracellular Ca2+ of ventricular myocytes isolated from endotoxemic guinea pigs. E. coli endotoxin (LPS, 1 mg/kg)-injected guinea pigs were divided into resuscitated and nonresuscitated groups. Fluid resuscitated animals received a Ringer's infusion (8 mL.kg-1.h-1) intravenously (i.v.) beginning immediately after endotoxin injection. Four hours later, ventricular myocytes were isolated enzymatically and loaded with fura-2/AM. When myocytes were field stimulated at .8 Hz, peak systolic Ca2+ transients of LPS-resuscitated (619 +/- 75 nM) and LPS-nonresuscitated (599 +/- 60 nM) myocytes were not significantly different from each other, but both were significantly less than values from control myocytes (1187 +/- 118 nM, p < .05). The percentage of cell shortening of LPS-resuscitated (6.2 +/- .9%) and LPS-nonresuscitated (6.2 +/- .3%) myocytes were also less than control (11.8 +/- .5%, p < .05). In contrast to improved diastolic compliance of fluid-resuscitated hearts, diastolic [Ca2+]i of myocytes (at .8 Hz) from LPS-resuscitated animals (138 +/- 47 nM) was not statistically different from LPS-nonresuscitated animals (129 +/- 19 nM). Diastolic values of both LPS groups were consistently lower than control value (251 +/- 38 nM, p < .05). These data suggest that improved diastolic compliance of LPS hearts following fluid resuscitation is not associated with improved myocyte contractility or myoplasmic Ca2+ handling.

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.

Evidence for endothelium-derived relaxing factor/nitric oxide in equine digital arteries.

OBJECTIVE: To test the hypothesis that endothelium-derived nitric oxide modulates vasomotor reactivity in equine digital arteries. DESIGN: Digital arteries were isolated from adult horses, and their vasodilator properties were examined in an in vitro controlled environment. ANIMALS: Five adult horses (1 gelding, 4 mares) without evidence of hoof or vascular disease were studied. PROCEDURE: Arterial rings with or without endothelium were exposed to endothelium-dependent vasodilator drugs in the presence or absence of a pharmacologic inhibitor of the enzyme nitric oxide synthase. RESULTS: Vasodilator effects of 3 endothelium-dependent vasorelaxant agents were significantly greater in endothelium-intact vessels than in endothelium-denuded vessels. Moreover, a nitric oxide synthase inhibitor reduced vasodilator responses to endothelium-dependent vasodilators in endothelium-intact arteries, but had no discernable effects in endothelium-denuded arteries. CONCLUSIONS: These findings indicate the presence of endothelium-derived relaxing factor/nitric oxide in blood vessels of horses, and identify vascular endothelium as an endogenous modulator of vasomotor tone in the digital arteries of this species.

Endotoxin-induced myocardial dysfunction: is there a role for nitric oxide?

Nitric oxide has been implicated in the regulation of cardiac contractile function as well as the depressed myocardial contractility associated with sepsis and endotoxemia. We examined the effects of nitric oxide synthase (NOS) inhibitors and a nitric oxide generator on contractile responses of left atrial preparations and ventricular myocytes isolated from endotoxemic guinea pigs, which exhibit depressed myocardial contractile function. The NOS inhibitor L-NAME had no effect on contractile tension developed by control atria or atria isolated from guinea pigs 4 or 16 h after an intraperitoneal injection of endotoxin. Similarly, contraction of ventricular myocytes isolated from control or endotoxemic guinea pigs (4 h after endotoxin injection) was unchanged by exposure to several NOS inhibitors. In addition, neither Ca(2+)-dependent nor Ca(2+)-independent ventricular NOS activity was affected by endotoxemia. These data suggest that nitric oxide alone is not responsible for the cardiac contractile dysfunction of endotoxemic guinea pigs.

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.