Domestic dog health worsens with socio-economic deprivation of their home communities.

Dogs play an important role in infectious disease transmission as reservoir hosts of many zoonotic and wildlife pathogens. Nevertheless, unlike wildlife species involved in the life cycle of pathogens, whose health status might be a direct reflection of their fitness and competitive abilities, dog health condition could be sensitive to socio-economic factors impacting the well-being of their owners. Here, we compare several dog health indicators in three rural communities of Panama with different degrees of socio-economic deprivation. From a total of 78 individuals, we collected blood and fecal samples, and assessed their body condition. With the blood samples, we performed routine hematologic evaluation (complete blood counts) and measured cytokine levels (Interferon-γ and Interleukin-10) through enzyme-linked immunosorbent assays. With the fecal samples we diagnosed helminthiases. Dogs were also serologically tested for exposure to Trypanosoma cruzi and canine distemper virus, and molecular tests were done to assess T. cruzi infection status. We found significant differences between dog health measurements, pathogen prevalence, parasite richness, and economic status of the human communities where the dogs lived. We found dogs that were less healthy, more likely to be infected with zoonotic pathogens, and more likely to be seropositive to canine distemper virus in the communities with lower economic status. This study concludes that isolated communities of lower economic status in Panama may have less healthy dogs that could become major reservoirs in the transmission of diseases to humans and sympatric wildlife.

Environmental variabilities and the distribution of octocorals and black corals in Hong Kong.

A recent comprehensive survey covering 125 sites in Hong Kong waters recorded 29 soft coral species in 14 genera, 38 species of gorgonians in 19 genera and six species of black corals in two genera. Environmental variabilities based on water quality data collected by Hong Kong Environmental Protection Department were analyzed using multivariate statistics to find variables that are significantly correlated with coral distribution patterns. Eleven water quality zones with similar environmental variabilities were recognized, which could further be classified into five groups, namely Inner Bay, Outer Bay, Eastern, Western and Southern waters. LINKTREE analysis provided an overall trend indicating the importance of salinity, sediment and nutrient loadings in affecting octocoral and black coral distribution from west to east of Hong Kong waters, and from inner to outer bays. Furthermore, water turbidity and wave exposure could also affect the coral distribution patterns from north, northeast to southern waters.

Systemic biochemical effects of inhaled NO in rats: increased expressions of NOS III, nitrotyrosine-, and phosphotyrosine-immunoreactive proteins in liver and kidney tissues.

Inhaled nitric oxide (iNO) has been shown to reduce pulmonary hypertension associated with several disease states. The effects of iNO are thought to be restricted to the pulmonary vasculature because of its rapid inactivation by hemoglobin. Recent data have suggested, however, that iNO can form nitrosothiols, which can be carried throughout the circulation, thus increasing the half life and bioactivity on NO. Other studies have shown that iNO can affect intestinal ischemia and renal hemodynamics. In this study, rats were exposed to 49 +/- 4 ppm or 107 +/- 13 ppm NO for 4 h and the lung, spleen, liver, and kidney tissues were removed and measured for NOS II and NOS III protein, nitrotyrosine (NT), and phosphotyrosine (PT) immunoreactivity. Following 107 ppm iNO, increases in NOS III protein expression, NT, and PT were observed in the liver and kidney, but not in the lung or spleen. No such increases were noted after the lower dose of iNO. These results paralleled those shown for isobutyl nitrite that we reported earlier and indicated that iNO can cause changes in protein chemistry in organs and tissues beyond the lungs. Since iNO produced little systemic hemodynamic effects, it is unlikely that the observed biochemical alterations were derived secondarily from physiological changes.

Nitroglycerin-induced relaxation of anorectal smooth muscle: evidence for apparent lack of tolerance development in the anaesthetized rat.

1. Recent studies indicate that nitroglycerin (NTG) can produce beneficial clinical effects in healing anal fissures through the relaxation of the internal anal sphincter. The in vivo relaxation effects of NTG on the anorectal smooth muscle have not been studied and it is not known whether this tissue may also exhibit pharmacological tolerance toward NTG. 2. We have developed an in vivo procedure in the anaesthetized rat that permits continual monitoring of anorectal pressure after intravenous (i.v.) and intra-rectal application of NTG. The relaxant effects of NTG were quantified via the area-under-the-contraction-waveforms vs time curve (AUEC). 3. AUEC decreased significantly after intra-rectal bolus doses of NTG (5 - 25 microg), in a dose- and time-dependent manner. Sustained relaxation effects on anorectal pressure were also observed after continuous intra-rectal infusions of NTG. 4. Two-hours of i.v. NTG infusion led to a significant reduction in the mean arterial blood pressure (MAP) response toward a i.v. NTG (30 microg) bolus challenge. In contrast, relaxation of the anorectal pressure toward the challenge dose was not altered after NTG infusion. 5. In isolated tissues, cyclic GMP accumulation was significantly decreased after NTG pre-incubation in the rat aorta but not in the rat anorectal smooth muscle and anal sphincter. 6. These results indicate that the relaxation response toward NTG was not diminished in the anorectum under conditions that produced vascular tolerance. Thus, NTG causes significant and sustained in vivo relaxation of anorectal smooth muscle in the anaesthetized rat without evidence of tolerance development.

Intestinal and hemodynamic impairment following mesenteric ischemia/reperfusion.

BACKGROUND: Clinical intestinal ischemia/reperfusion (I/R) injury results in local and systemic dysfunction. A rat model of transient mesenteric occlusion has been used to study this phenomenon. However, a systematic analysis of the rat model with respect to intestinal permeability and hemodynamics has not been carried out. MATERIALS AND METHODS: In anesthetized rats, the superior mesenteric artery was occluded for 60 min, followed by reperfusion for 4 h. Intestinal impairment was evaluated via histological examination and by measuring ex vivo apparent permeability coefficients (Papp) of mannitol (0.18 kDa), inulin (5 kDa), and dextran (70 kDa). Hemodynamic effects of intestinal I/R were determined by monitoring mean arterial pressure (MAP) and heart rate (HR) via a catheter placed in the femoral artery. RESULTS: The animal model was associated with increased ex vivo Papp for mannitol and inulin. Although I/R injury was accompanied by significant histological disruption, there was no observable alteration in dextran permeability, suggesting that the loss in normal barrier function was limited to low-molecular-weight compounds. Hemodynamic measurements indicated that reperfusion induced a precipitous and sustained fall in MAP. HR values fell sharply following reperfusion but gradually increased and eventually "overshot" to values greater than baseline. CONCLUSIONS: Our findings demonstrate the selective loss of barrier function of the small bowel following intestinal I/R. Furthermore, these results also illustrate the importance of selecting appropriate permeability markers for the evaluation of intestinal damage. In light of the significant hemodynamic disruption accompanying the animal model, our investigation also points toward the need for developing therapeutic strategies that mitigate the local and systemic effects of intestinal I/R injury.

Impact of mechanism-based enzyme inactivation on inhibitor potency: implications for rational drug discovery.

Mechanism-based enzyme inactivators (MBEIs) have unique kinetic actions that make predictions of potency, selectivity, and potential for metabolic drug interactions more complex than for competitive antagonists. We have derived a mathematical relationship that links the influence of substrate concentration and binding constant ([S] and K(m), respectively), inhibitor concentration and binding constant ([I] and K(I), respectively), and inactivation rate constant (k(inact)) to enzyme activity (v) and maximal activity (V(max)) at any time (t). The kinetic behavior of this relationship was validated in murine-macrophage cell cultures using MBEIs of nitric oxide synthase (NOS). This initial equation was also used in the derivation of a new relationship that directly links the kinetic parameters of mechanism-based inactivation to inhibitory potency at a particular time (IC((t))(50)). Using this direct relationship, we observed that the predicted rank inhibitory potency of a series of MBEIs was improved over that predicted by the K(I) parameter alone. These relationships offer a fundamental understanding of the kinetics of MBEI action and may be useful in the evaluation of these compounds during the discovery process.

Evaluation of nitric oxide synthase activity and inhibition kinetics by chemiluminescence.

The binding affinity (K(I)) and inactivation rate (k(inact)) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.

Nitrite inhalation in rats elevates tissue NOS III expression and alters tyrosine nitration and phosphorylation.

Organic nitrites are nitric oxide (NO) donors that are used predominantly as inhalant drugs of abuse and have been shown to have immunomodulating effects. NO donors can modulate NOS activity and expression, thus altering the level of endogenous NO production. NO can react with superoxide (O(*)(2)(-)) to form peroxynitrite (ONOO(-)), which can nitrate tyrosine residues in proteins and alter tyrosine phosphorylation. We investigated the effects of inhaled isobutyl nitrite (ISBN) on NOS expression, tyrosine nitration, and tyrosine phosphorylation in selected organs of rats. Following exposures of 109 and 1517 ppm ISBN for 4 h, the lung, spleen, liver, and kidney were removed and assayed by SDS-PAGE for NOS III (eNOS), NOS II (iNOS), nitrotyrosine (NT)- and phosphotyrosine (PT)-immunoreactive proteins using specific antibodies. ISBN at 1517 ppm, but not 109 ppm, caused an increase in NOS III expression in the liver and kidney, but not in the lung and spleen. No apparent effect on NOS II expression was observed in these organs. The expressions of NT and PT protein bands (30-200 kDa) were increased in the liver and kidney, but not in the lung and spleen. This increase in NT persisted for 24 h post-exposure. Increased NOS III expression in the liver and kidney may promote peroxynitrite formation and contribute to the increase in NT and PT immunoreactivity. ISBN inhalation may thus cause changes in cellular signaling involving tyrosine phosphorylation. These findings may suggest a mechanistic basis for the apparent immunotoxicity associated with nitrite abuse.

Examination of N-hydroxylation as a prerequisite mechanism of nitric oxide synthase inactivation.

L-N5-(1-Hydroxyiminoethyl)-ornithine (L-NHIO) and L-N6-(1-hydroxyiminoethyl)-lysine (L-NHIL) were synthesized and tested as potential intermediates in the mechanism-based inactivation of nitric oxide synthase (NOS) by L-N5-iminoethylornithine (L-NIO) and L-N6-iminoethyllysine (L-NIL). Although these compounds were determined to be competitive inhibitors, mechanism-based inactivation was not observed.

Attenuated nitric oxide synthase activity and protein expression accompany intestinal ischemia/reperfusion injury in rats.

Intestinal ischemia/reperfusion (I/R) leads to bowel impairment via the release of reactive oxygen species (ROS) and neutrophil infiltration. In addition to modulating intestinal integrity, nitric oxide (NO(*)) inhibits neutrophil activation and scavenges ROS. Attenuated endogenous NO(*) formation may result in the accrual of these deleterious stimuli. Therefore, we determined nitric oxide synthase (NOS) activity in anesthetized rats subjected to 1 h of superior mesenteric ischemia or ischemia followed by reflow. NOS activity was measured in intestinal tissue homogenates as the conversion rate of (3)H-L-arginine to (3)H-L-citrulline. Our results demonstrate that intestinal ischemia leads to a decrease in NOS activity indicating lower NO(*) formation in the animal model. The attenuation in NOS activity was not reversed following 4 h of reperfusion. Western blot analysis revealed that the decline in enzyme activity was accompanied by reduced intestinal NOS III (endothelial constitutive NOS) expression. These findings provide biochemical evidence for impaired NO(*) formation machinery in intestinal I/R injury.

Regulation of the RON receptor tyrosine kinase expression in macrophages: blocking the RON gene transcription by endotoxin-induced nitric oxide.

Previous studies have shown that activation of the RON receptor tyrosine kinase inhibits inducible NO production in murine peritoneal macrophages. The purpose of this study is to determine whether inflammatory mediators such as LPS, IFN-gamma, and TNF-alpha regulate RON expression. Western blot analysis showed that RON expression is reduced in peritoneal macrophages collected from mice injected with a low dose of LPS. The inhibition was seen as early as 8 h after LPS challenge. Experiments in vitro also demonstrated that the levels of the RON mRNA and protein are diminished in cultured peritoneal macrophages following LPS stimulation. TNF-alpha plus IFN-gamma abrogated macrophage RON expression, although individual cytokines had no significant effect. Because LPS and TNF-alpha plus IFN-gamma induce NO production, we reasoned that NO might be involved in the RON inhibition. Two NO donors, S-nitroglutathione (GSNO) and (+/-)-S-nitroso-N-acetylpenicillamine (SNAP), directly inhibited macrophage RON expression when added to the cell cultures. Blocking NO production by NO inhibitors like TGF-beta prevented the LPS-mediated inhibitory effect. In Raw264.7 cells transiently transfected with a report vector, GSNO or SNAP inhibited the luciferase activities driven by the RON gene promoter. Moreover, GSNO or SNAP inhibited the macrophage-stimulating protein-induced RON phosphorylation and macrophage migration. We concluded from these data that RON expression in macrophages is regulated during inflammation. LPS and TNF-alpha plus IFN-gamma are capable of down-regulating RON expression through induction of NO production. The inhibitory effect of NO is mediated by suppression of the RON gene promoter activities.

Pharmacokinetics of a model organic nitrite inhalant and its alcohol metabolite in rats.

Volatile organic nitrites were originally used to relieve the chest pain that is associated with angina pectoris. Today, these inhalants are predominantly used as drugs of abuse. Little is known regarding the bioavailability and disposition of volatile nitrites. In this study, the pharmacokinetics of a major organic nitrite inhalant, isobutyl nitrite (ISBN), and its primary metabolite, isobutyl alcohol (ISBA), were investigated after inhalation and i.v. administration. ISBN blood concentrations in the rat declined mono-exponentially with a half-life of 1.4 min and a blood clearance of 2.9 l/min/kg that vastly exceeded cardiac output (0.3 l/min/kg). Approximately 98% of ISBN was metabolized to ISBA, which declined monoexponentially with a half-life of 5.3 min when the infusion of ISBN was terminated. The bioavailability of inhaled ISBN, over a range of 300 to 900 ppm, was estimated to be 43%. After inhaled ISBN, the half-life of ISBA decreased approximately 4-fold (t(1/2) inh = 1.5 min versus t(1/2) i.v. = 5.3 min; P <.001), whereas no pharmacokinetic difference was observed for ISBN. Inhalation of another nitrite, isoamyl nitrite, accelerated the apparent clearance of ISBA, suggesting that nitrite inhalation could change the disposition of another compound. A pharmacokinetic model was developed to describe the concentration-time profile of ISBA and ISBN after inhalation and i.v. administration.

Differential sensitivity among nitric oxide donors toward ODQ-mediated inhibition of vascular relaxation.

Nitric oxide (NO) donors are believed to exert their vasodilatory action through the activation of soluble guanylate cyclase (sGC), the heme site of which can be specifically inhibited by 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). We examined the vascular relaxation of the rat aorta mediated by eight different NO donors in the presence of ODQ (0.1, 1, or 10 microM), and demonstrated that these NO donors displayed different sensitivities toward ODQ inhibition (ANOVA, P <.05). Among the NO donors studied, S-nitrosothiols such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione exhibited partial resistance toward ODQ inhibition at 0.1 microM ODQ, whereas nitroglycerin (NTG) showed nearly complete inhibition at this concentration of ODQ. Three NO donors representing increasing sensitivity toward ODQ inhibition, SNAP < sodium nitroprusside (SNP) < NTG, were chosen for additional mechanistic studies. ODQ (1 microM) inhibition of vascular relaxation by SNAP and SNP, but not that by NTG, was partially reversed by a sulfhydryl donor, N-acetylpenicillamine (100 microM), and by a phosphodiesterase inhibitor, zaprinast (10 microM), specific for cGMP. Our results strongly indicate that the vascular relaxation mechanism(s) of NO donors is not identical for each. In the rat aorta, NTG appeared to exhibit its vasodilatory effect exclusively through activation of the heme site of sGC. On the other hand, in the intact vascular tissue, SNAP and SNP could bring about vasodilation through a secondary pathway. These results are consistent with the view that SNAP and SNP, but not NTG, can induce vascular relaxation additionally through the activation of the sulfhydryl site of sGC.

Comparison of methods for analyzing kinetic data from mechanism-based enzyme inactivation: application to nitric oxide synthase.

The goals of this study were (1) to investigate the performance of 2 classical methods of kinetic analysis when applied to data from enzyme systems in which mechanism-based inactivation and enzyme degradation are present, and (2) to develop and validate a nonlinear method of kinetic data analysis that may perform better under these situations. A composite equation was derived to link various parameters that govern the kinetics of mechanism-based inactivation, viz., enzyme activity, inhibitor-binding affinity (K(I)), inactivation rate (k(inact)), and enzyme degradation (k(deg)). The relative accuracy and precision of parameter estimation by the Dixon and Kitz-Wilson methods and a new nonlinear method were evaluated by computer simulation. The behavior of these methods of analysis were validated experimentally, using the nitric oxide synthase enzyme, both in purified form and as expressed in murine macrophage cell cultures. We showed that the Dixon method, as expected, could not provide accurate estimates of K(I) in the presence of either enzyme inactivation or instability. The Kitz-Wilson method could provide accurate estimates of these parameters; however, the precisions of these estimates were poorer than those obtained using the nonlinear method of analysis. We conclude that the nonlinear approach is superior to classical methods of data analysis for enzyme inhibitor kinetics, based on better efficiency, accuracy, and precision.

Relationship between pharmacokinetics and hemodynamic effects of inhaled isobutyl nitrite in conscious rats.

Our objective was to examine the pharmacokinetic/hemodynamic properties of inhaled isobutyl nitrite (ISBN) in rats. ISBN is one of the volatile organic nitrites that has been used primarily as a drug of abuse. Recent studies indicate, however, that these compounds may be superior to organic nitrates for cardiovascular use because they do not produce vascular tolerance. Rats inhaled ISBN over an exposure range of 20 to 1200 ppm for 1 hour. The effects of ISBN on blood pressure and heart rate were determined and blood concentrations of ISBN were analyzed with use of gas chromatography. Apparent steady-state blood levels of ISBN were achieved during inhalation and were linear with exposure concentration (blood concentration: 0.05 to 3.5 microM; exposure concentration: 23 to 1177 ppm; r2= 0.92). Inhaled ISBN caused rapid, dose-dependent, and parallel reductions in systolic and diastolic pressure, while heart rate increased maximally to 22%. A sigmoid Emax model could describe the mean arterial pressure effect of inhaled ISBN (Emax= 55%; EC50= 0.51 microM). After inhalation, blood pressure and heart rate quickly returned to baseline, without any withdrawal rebound effect. Inhaled ISBN produced a rapid onset of action on heart rate and blood pressure, and these effects were sustained over 60 minutes of exposure. Abrupt drug withdrawal did not lead to hemodynamic rebound. The blood pressure effects were related to ISBN blood concentration by the sigmoid Emax model. These results provide new information on the pharmacokinetic/pharmacodynamic relationship of a representative nitrite inhalant.

Analysis of isobutyl nitrite inhalant in rat and human blood: application for pharmacokinetic investigations.

Organic nitrites have been used therapeutically for the treatment of angina pectoris and as diagnostic agents for the evaluation of cardiac heart murmurs. In addition, these highly volatile vasodilators are being used as inhalant drugs of abuse. We developed a gas chromatographic assay using electron capture detection for the analysis of a representative nitrite inhalant, isobutyl nitrite (ISBN), in rat and human whole blood. Unconventional sampling and processing techniques were required because of the high volatility and chemical instability of nitrites in biological fluids. Our method produced a mean recovery of ISBN from rat blood of about 86% over a concentration range of 1.0 to 400 ng/ml. The inter-day coefficient of variation was below 15% at the lowest quantifiable concentration of 1 ng/ml ISBN in rat blood. In this report, we applied the analytical method to obtain new pharmacokinetic information about ISBN. Results show that rats inhaling 900 ppm ISBN for 45 min produced steady-state blood concentrations of about 290 ng/ml, and a rapid elimination half-life of 1.4 min.

Differential interactions of nitric oxide donors with rat oxyhemoglobin.

To estimate the reaction of two primary redox-related species of nitric oxide (i.e. NO+ vs NO*) from a variety of NO donors, we employed the differential interactions of these NO forms with oxyhemoglobin (oxyHb) as a chemical assay. NO+ formation was estimated by the S-nitrosation reaction with oxyHb, and NO* formation via its reaction with the oxygen-heme complex of oxyHb. Under the conditions employed, all NO donors caused concentration-dependent formation of methemoglobin, indicative of NO* liberation. However, the extent of S-nitrosation was substantially different among the NO donors studied. A representative S-nitrosothiol, S-nitroso-N-acetyl-penicillamine, caused significantly more S-nitrosation than nitroglycerin, isobutyl nitrite, sodium nitroprusside, and 3-morpholino-sydnonimine (ANOVA, P < 0.05). These results indicated that NO donors can differ in their interactions with oxyHb, and possibly with other target proteins, in part because they liberate or transfer different ratios of NO redox forms. This difference may contribute, in part, to the diversity of pharmacological effects elicited by NO donors.

In vivo disposition of 3-nitro-L-tyrosine in rats: implications on tracking systemic peroxynitrite exposure.

In many pathological conditions such as inflammatory and neurodegenerative diseases, the in vivo toxicity of nitric oxide has been attributed to the toxic oxidant peroxynitrite. Interaction of peroxynitrite with biological molecules can modify tyrosine residues on the proteins at the ortho position resulting in the formation of the stable end-product, 3-nitro-L-tyrosine (3-NT). Recent investigations indicate that changes in the circulating concentrations of 3-NT in pathological conditions may reflect the extent of nitric oxide-dependent oxidative damage and peroxynitrite toxicity. In the present study, we examined the in vivo disposition characteristics of 3-NT in rats after either a single i.v. bolus dose (10 mg/kg) or a loading and maintenance infusion at 10 or 30 mg/kg. Plasma concentrations of 3-NT were analyzed by a reversed-phase HPLC method. After a single bolus dose of 3-NT at 10 mg/kg, the average half-life of the elimination phase for the drug was 68.5+/-18.4 min (n = 5). Infusions of 3-NT at two different doses (10 and 30 mg/kg) indicated that the pharmacokinetic properties of 3-NT below plasma concentrations of 100 microM were both linear and stationary. Urinary excretion of unchanged 3-NT was minimal, but two distinct metabolites of 3-NT were identified in the urine collected throughout the study. These findings may be useful in the interpretation of the plasma and urine 3-NT concentrations as possible indices of systemic peroxynitrite exposure.

Diethyldithiocarbamate prolongs survival of mice in a lipopolysaccharide-induced endotoxic shock model: evidence for multiple mechanisms.

It is now known that overproduction of nitric oxide (NO) by nitric oxide synthase (NOS) is an important contributing factor for the development of cardiovascular collapse and subsequent death in endotoxic shock. Diethyldithiocarbamate (DETC) is a molecular scavenger of NO and can inhibit overexpression of a number of cytokines during shock through inactivation of transcription factors such as nuclear factor (NF)-kappaB. Thus, DETC may be a useful adjunct in the therapy of endotoxic shock. In our study, we examined the effect of DETC on survival time in a murine model of severe endotoxic shock. Our results indicated that selected in vivo dosage regimens of DETC (intraperitoneal: at -2, -1, 3, 6, and 10 h or at -2, -1, 3, 6, 9, 12, 15, and 18 h relative to lipopolysaccharide administration, 180 mg/kg, at t = 0) in endotoxic mice were effective in increasing survival time when compared with untreated animals and DETC pretreatment was more effective than methylprednisolone (p<.05). DETC was shown to exert multiple beneficial mechanisms, including 1) a decrease in circulating NO, as determined by plasma nitrite/nitrate levels, 2) a reduction in plasma tumor necrosis factor-alpha after lipopolysaccharide induction, and 3) decreased expressions of metalloproteinases such as gelatinase A and B which may be responsible for cellular release of cytokines. These results indicate that DETC and its analogs may be useful in the treatment of endotoxic shock.