Effect of N-butylscopolammonium bromide on equine ileal smooth muscle activity in an ex vivo model.

REASONS FOR PERFORMING STUDY: N-butylscopolammonium bromide (NBB) is an anticholinergic agent used to treat spasmodic colic in horses. Intestinal smooth muscle spasm also occurs in horses with intraluminal intestinal obstructions, such as ileal impactions. The antispasmodic effects of NBB may be useful in managing ileal impactions, but the effects of NBB on equine ileal smooth muscle are unknown. OBJECTIVES: To investigate the effects of NBB on spontaneous and induced contraction of the equine ileum in an ex vivo model. STUDY DESIGN: Ex vivo biomechanical study assessing contractile properties in isolate equine ileal smooth muscle with and without exposure to NBB. METHODS: Ileal tissue was collected from 6 healthy horses after euthanasia, and isolated circular and longitudinal smooth muscle strips were connected to isometric force transducers in organ baths. After equilibration, the effect of NBB (1 nmol/l to 100 µmol/l) on spontaneous and carbachol-induced contraction was determined and compared with responses in control tissue. RESULTS: At ≥30 µmol/l, NBB inhibited spontaneous contractions in all muscle strips that exhibited spontaneous activity. N-butylscopolammonium bromide pre-treatment inhibited carbachol-induced contraction in circular (NBB-treated half maximal effective concentration [EC50] 0.530 × 10(-8) mol/l vs. control EC50 41.57 × 10(-8) mol/l) and longitudinal muscle strips (NBB-treated EC50 0.243 × 10(-8) mol/l vs. control EC50 90.84 × 10(-8) mol/l). Abolition of carbachol-induced contraction with NBB was observed at lower concentrations in circular than longitudinal muscle strips. Pretreatment with NBB significantly inhibited carbachol-induced contractions; NBB-treated tissue required greater carbachol concentrations to produce sustained contractions than control muscle strips. Histamine-evoked contraction was not affected by NBB. CONCLUSIONS: N-butylscopolammonium bromide inhibits spontaneous and cholinergically mediated contraction in equine ileal smooth muscle strips ex vivo. Thus, NBB might reduce intestinal spasm in equine ileal impactions and could be useful for medical management of these cases, although further study is needed to confirm these effects in vivo.

Disparate effects of LPS infusion and carbohydrate overload on inflammatory gene expression in equine laminae.

Although clinical evidence of endotoxemia has been associated with the development of acute laminitis in hospitalized horses with gastrointestinal diseases and endotoxins have been detected in the circulation of horses with experimentally-induced laminitis, it is unclear what role, if any, endotoxins have play the pathogenesis of the disease. Therefore, in the present study we compared the effects of endotoxin infusion to that of intra-gastric administration of mixed carbohydrate (CHO) on clinical signs of laminitis, plasma concentrations of TNF-α and IL-10, and laminar tissue expression of 20 genes associated with inflammation. Horses were divided into 4 groups: Control (water placebo, n=7), endotoxin infusion (LPS, n=6), CHO/Developmental (30% decrease in central venous pressure, n=6) and CHO/Lame (Obel grade I laminitis, n=7). Horses in the LPS group developed clinical signs consistent with systemic inflammation, had rapid increases in plasma concentrations of both TNF-α and IL-10, and leukopenia, but did not have any changes in laminar tissue expression of the genes associated with inflammation. In contrast, horses administered CHO developed clinical signs consistent with systemic inflammation, had more delayed increases in TNF-α, IL-10 and total leukocyte counts, and had marked increases in laminar tissue expression of the genes associated with inflammation. Only the horses administered CHO developed clinical signs of laminitis, providing additional credence to the concept that factors other than endotoxin are responsible for the changes in laminar tissue gene expression that occur during the development of acute equine laminitis.

Pharmacokinetic assessment of ketanserin in the horse.

The purpose of this study was to determine the pharmacokinetics (PK) of the 5-HT(2A) receptor antagonist ketanserin in healthy adult horses, and to develop a computational model that could be used to optimize dosing. Plasma concentrations of ketanserin were determined using liquid chromatography with mass spectrometry after single and multiple intravenous administration in the horse. A two-compartment linear pharmacokinetic model described the plasma concentration-time profile of ketanserin after single and multiple doses in healthy horses; the terminal half-life was 11.5 h; steady-state volume of distribution was 10.5 L/kg; AUC was 115 ng · h/mL; and clearance was 0.87 L/h/kg. Model simulations followed by the examination in three healthy horses suggest 0.3 mg/kg q.8 h exhibited linear PK and produced consistent systemic blood concentrations of ketanserin above 3 ng/mL.

5-HT activates vagal afferent cell bodies in vivo: role of 5-HT2 and 5-HT3 receptors.

Occipital artery (OA) injections of 5-HT elicit pronounced reductions in heart rate and mean arterial blood pressure (MAP) in urethane-anesthetized rats by activation of vagal afferent cell bodies in the ipsilateral nodose ganglion. In contrast, internal carotid artery (ICA) and i.v. injections elicit similar cardiovascular responses by activation of peripheral vagal afferent terminals. The aim of this study was to examine the roles of 5-HT3 and 5-HT2 receptors in the 5-HT-induced activation of vagal afferent cell bodies and peripheral afferent terminals in urethane-anesthetized rats. OA, ICA and i.v. injections of 5-HT elicited dose-dependent reductions in heart rate and MAP that were virtually abolished after i.v. administration of the 5-HT3 receptor antagonists, MDL 7222 or ICS 205-930. The responses elicited by the OA injections of 5-HT were markedly diminished after i.v. injection of the 5-HT2 receptor antagonists, xylamidine or ketanserin, whereas the responses elicited by i.v. or ICA injections of 5-HT were not affected. The present findings suggest that (1) 5-HT3 and 5-HT2 receptor antagonists gain ready access to nodose ganglion cells upon i.v. administration, and (2) functional 5-HT3 and 5-HT2 receptors exist on the cell bodies of vagal afferent neurons mediating the cardiovascular responses elicited by OA injections of 5-HT. These findings also support a wealth of evidence that 5-HT3 receptors exist on the peripheral terminals of vagal afferents, and although they do not discount the possibility that 5-HT2 receptors exist on peripheral vagal afferent terminals, it appears that activation of these receptors does not have pronounced effects on 5-HT3 receptor activity on terminals that mediate the hemodynamic responses to 5-HT.

Occipital artery injections of 5-HT may directly activate the cell bodies of vagal and glossopharyngeal afferent cell bodies in the rat.

The primary objective of this study was to determine whether circulating factors gain direct access to and affect the activity of vagal afferent cell bodies in the nodose ganglia and glossopharyngeal afferents cell bodies in the petrosal ganglia, of the rat. We found that the occipital and internal carotid arteries provided the sole blood supply to the nodose ganglia, and that i.v. injections of the tracer, Basic Blue 9, elicited strong cytoplasmic staining in vagal and glossopharyngeal afferent cell bodies that was prevented by prior ligation of the occipital but not the internal carotid arteries. We also found that occipital artery injections of 5-HT elicited pronounced dose-dependent reductions in heart rate and diastolic arterial blood pressure that were (1) virtually abolished after application of the local anesthetic, procaine, to the ipsilateral nodose and petrosal ganglia, (2) markedly attenuated after transection of the ipsilateral vagus between the nodose ganglion and brain and virtually abolished after subsequent transection of the ipsilateral glossopharyngeal nerve between the petrosal ganglion and the brain, (3) augmented after ipsilateral transection of the aortic depressor and carotid sinus nerves, and (4) augmented after transection of all ipsilateral glossopharyngeal and vagal afferent nerves except for vagal cardiopulmonary afferents. These findings suggest that blood-borne 5-HT in the occipital artery gains direct access to and activates the cell bodies of vagal cardiopulmonary afferents of the rat and glossopharyngeal afferents of undetermined modalities.

Black walnut extract-induced laminitis in horses is associated with heterogeneous dysfunction of the laminar microvasculature.

REASONS FOR PERFORMING STUDY: Equine laminitis purportedly involves haemodynamic dysfunction at the level of the laminar vasculature. However, to date, no studies have been performed characterising the function of laminar arteries and veins during the prodromal stages of equine laminitis. HYPOTHESIS: That the prodromal stages of laminitis are associated with contractile dysfunction of the equine laminar vasculature. OBJECTIVE: To assess contractile function of laminar arteries and veins to phenylephrine (PE) and 5-hydroxytryptamine (5-HT). METHODS: Horses were administered black walnut heartwood extract (BWHE) or water (control horses) via nasogastric intubation. After euthanasia, laminar vessels (100-800 microm internal diameter) were isolated and mounted on small vessel myographs to assess contractile function. RESULTS: Contractile responses to PE or 5-HT were identical in laminar arteries isolated from either control horses or those administered BWHE. In contrast, responses to PE or 5-HT were significantly reduced in laminar veins isolated from BWHE-administered horses when compared with laminar veins isolated from control horses. CONCLUSIONS AND POTENTIAL RELEVANCE: These results are consistent with the prodromal stages of laminitis being associated with selective dysfunction of laminar veins. Further studies are required to discern the precise nature of this dysfunction and its potential relevance to the pathogenesis of acute laminitis in the horse and possible therapeutic targets for treatment.

Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor.

1. The mechanisms responsible for sensing hypoxia and initiating hypoxic pulmonary vasoconstriction (HPV) are unclear. We therefore examined the roles of the mitochondrial electron transport chain (ETC) and glycolysis in HPV of rat small intrapulmonary arteries (IPAs). 2. HPV demonstrated a transient constriction (phase 1) superimposed on a sustained constriction (phase 2). Inhibition of complex I of the ETC with rotenone (100 nM) or complex III with myxothiazol (100 nM) did not cause vasoconstriction in normoxia, but abolished both phases of HPV. Rotenone inhibited the hypoxia-induced rise in intracellular Ca(2+) ([Ca(2+)](i)). Succinate (5 mM), a substrate for complex II, reversed the effects of rotenone but not myxothiazol on HPV, but did not affect the rise in NAD(P)H fluorescence induced by hypoxia or rotenone. Inhibition of cytochrome oxidase with cyanide (100 microM) potentiated phase 2 constriction. 3. Phase 2 of HPV, but not phase 1, was highly correlated with glucose concentration, being potentiated by 15 mM but abolished in its absence, or following inhibition of glycolysis by iodoacetate or 2-deoxyglucose. Glucose concentration did not affect the rise in [Ca(2+)](i) during HPV. 4. Depolarisation-induced constriction was unaffected by hypoxia except in the absence of glucose, when it was depressed by approximately 50 %. Depolarisation-induced constriction was depressed by rotenone during hypoxia by 23 +/- 4 %; cyanide was without effect. 5. Hypoxia increased 2-deoxy-[(3)H]glucose uptake in endothelium-denuded IPAs by 235 +/- 32 %, and in mesenteric arteries by 218 +/- 38 %. 6. We conclude that complex III of the mitochondrial ETC acts as the hypoxic sensor in HPV, and initiates the rise in smooth muscle [Ca(2+)](i) by a mechanism unrelated to changes in cytosolic redox state per se, but more probably by increased production of superoxide. Additionally, glucose and glycolysis are essential for development of the sustained phase 2 of HPV, and support an endothelium-dependent Ca(2+)-sensitisation pathway rather than the rise in [Ca(2+)](i).

Hypoxia induces the release of a pulmonary-selective, Ca(2+)-sensitising, vasoconstrictor from the perfused rat lung.

OBJECTIVE: Sustained hypoxic pulmonary vasoconstriction is dependent upon the presence of an intact endothelium, strongly suggesting that an endothelium-derived constrictor factor is involved in this response. In the present study we have attempted to determine whether hypoxia induces the release of a vasoconstrictor(s) from the lung, and whether this vasoconstrictor shares mechanistic features with the hypoxic constrictor response. METHODS: The salt-perfused rat lung, coupled with a simple solid-phase extraction process, and a rat intrapulmonary artery functional bioassay were utilised in this study. RESULTS: Hypoxic, but not normoxic, perfusion of the isolated lung of the rat induced the release of a vasoconstrictor(s) which appeared to be selective for pulmonary over mesenteric arteries of the rat. The vasoconstriction observed was unaffected by inhibition of voltage-gated Ca(2+) channels, and was not associated with a rise in intracellular [Ca(2+)], suggesting Ca(2+)-sensitisation of the contractile apparatus. The vasoconstriction was also unaffected by the protein kinase C (PKC) inhibitor Ro-31-8220, or the endothelin-1 antagonists BQ123/BQ788 but was markedly potentiated in the presence of prostaglandin F(2alpha). CONCLUSION: We conclude that hypoxic perfusion of the rat lung results in the release of a vasoconstrictor(s) which shares some of the facets of the sustained hypoxic constriction of isolated intrapulmonary arteries of the rat, since it involves PKC-independent Ca(2+) sensitisation, is independent of voltage-gated Ca(2+) entry, and is potentiated by the presence of preconstriction.

Inhibition of sustained hypoxic vasoconstriction by Y-27632 in isolated intrapulmonary arteries and perfused lung of the rat.

We have examined the effects of Y-27632, a specific inhibitor of Rho-activated kinases (ROCK I and ROCK II) upon sustained hypoxic pulmonary vasoconstriction (HPV) in both rat isolated small intrapulmonary arteries (IPA) and perfused rat lungs in situ. Y-27632 (100 nM - 3 microM) was found to cause a concentration-dependent inhibition of acute sustained HPV in rat IPA. Application of Y-27632 (10-600 nM) in perfused rat lungs caused no change in basal perfusion pressure, but was found to inhibit HPV in a concentration-dependent manner, resulting in complete ablation of the pressor response to hypoxia at a concentration of 600 nM. Furthermore, addition of Y-27632 at any point during hypoxia caused a reversal of HPV in perfused rat lungs. These results suggest that activation of Rho-associated kinase may be a pivotal step in the generation of sustained HPV.

Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat.

It has been proposed that hypoxic pulmonary vasoconstriction (HPV) is mediated via K+ channel inhibition and Ca2+ influx through voltage-gated channels. HPV depends strongly on the degree of preconstriction, and we therefore examined the effect of Ca2+ channel blockade on tension and intracellular [Ca2+] ([Ca2+]i) during HPV in rat intrapulmonary arteries (IPAs), whilst maintaining preconstriction constant. We also investigated the role of intracellular Ca2+ stores. HPV demonstrated a transient constriction (phase I) superimposed on a sustained constriction (phase II). Nifedipine (1 microM) partially inhibited phase I, but did not affect phase II. In arteries exposed to 80 mM K+ and nifedipine or diltiazem the rises in tension and [Ca2+]i were blunted during phase I, but were unaffected during phase II. At low concentrations (< 3 microM), La3+ almost abolished the phase I constriction and rise in [Ca2+]i, but had no effect on phase II, or constriction in response to 80 mM K+. Phase II was inhibited by higher concentrations of La3+ (IC50 approximately 50 microM). IPA treated with thapsigargin (1 microM) in Ca2+-free solution to deplete Ca2+ stores showed sustained constriction upon re-exposure to Ca2+ and an increase in the rate of Mn2+ influx, suggesting capacitative Ca2+ entry. The concentration dependency of the block of constriction by La3+ was similar to that for phase I of HPV. Pretreatment of IPA with 30 microM CPA reduced phase I by > 80 %, but had no significant effect on phase II. We conclude that depolarization-mediated Ca2+ influx plays at best a minor role in the transient phase I constriction of HPV, and is not involved in the sustained phase II constriction. Instead, phase I appears to be mainly dependent on capacitative Ca2+ entry related to release of thapsigargin-sensitive Ca2+ stores, whereas phase II is supported by Ca2+ entry via a separate voltage-independent pathway.

Membrane potential-dependent and -independent vasodilation in small pulmonary arteries from chronically hypoxic rats.

Chronic hypoxia is associated with altered pulmonary vasoreactivity, and it has been suggested that an increased response to voltage-dependent vasodilators may relate to enhanced Ca++ entry via voltage-dependent channels, secondary to depolarization. Few studies have been performed on small pulmonary arteries, and it is unknown whether they are depolarized after chronic hypoxia. We examined the resting membrane potential, and the actions of voltage-dependent (verapamil, levcromakalim) and -independent (isoproterenol, forskolin, papaverine) vasodilators in small ( approximately 300 microm internal diameter) pulmonary arteries from chronically hypoxic rats. The resting membrane potential was more positive in arteries after chronic hypoxia (control: -60 +/- 0.5 mV; hypoxic: -54.4 +/- 1.1 mV; P < .01), and this was reflected by a shift to the left of the response curves for K+ and 4-aminopyridine. In arteries constricted with prostaglandin F2alpha the response to verapamil and levcromakalim was increased after chronic hypoxia, although maximum prostaglandin F2alpha-induced tension was unchanged, which implies a reduction in voltage-independent constrictor mechanisms. Although vasorelaxation to isoproterenol was depressed in arteries from hypoxic rats, forskolin-induced relaxation was enhanced substantially, and because the response to the phosphodiesterase inhibitor papaverine was unchanged, we suggest that this reflects an up-regulation of adenylate cyclase. In conclusion, chronic hypoxia resulted in a significant depolarization in small pulmonary arteries, but this may explain only partly the increased efficacy of voltage-dependent vasodilators. Whether the reduction in voltage-independent constrictor mechanisms is related to the apparent up-regulation of adenylate cyclase remains to be elucidated.

The role of the endothelium in hypoxic pulmonary vasoconstriction.

The precise mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are still elusive. The recent discovery of K+ channels that are depressed by hypoxia in pulmonary vascular smooth muscle has provided a potential signal transduction mechanism for linking a reduction in Po2 to Ca2+ entry, but there are many reports suggesting that sustained HPV depends on the presence of the endothelium. Many endothelium-derived vasoactive factors have been investigated as possible mediators of HPV, including endothelium-derived relaxing factor (EDRF-NO), leukotrienes, prostanoids and endothelin, yet none have been found to be indispensable for HPV. They do, however, act as powerful modulators of the response to hypoxia. HPV is probably multifactorial in origin, as exemplified by the biphasic response to hypoxia seen in isolated pulmonary arteries over 40 min. The first phase is of rapid onset but transient, endothelium independent and partly related to Ca2+ release from intracellular stores. The second phase is slowly developing but sustained, dependent on the endothelium and associated with a stable elevation of cytosolic Ca2+. Since tension continues to rise while intracellular [Ca2+] remains constant, this implies Ca2+ sensitization of the contractile apparatus. This is independent of protein kinase C or pH. It is proposed that HPV depends upon both smooth muscle and endothelium resident mechanisms. Inhibition of K+ channels causes an elevation of cytosolic Ca2+, which may not be sufficient to generate substantive contraction on its own. However, release from the endothelium of an as yet unidentified mediator increases Ca2+ sensitivity of the contractile apparatus, and sustained contraction ensues.

Hypoxic vasoconstriction and intracellular Ca2+ in pulmonary arteries: evidence for PKC-independent Ca2+ sensitization.

The effect of hypoxia on intracellular Ca2+ ([Ca2+]i) and tension in small intrapulmonary arteries (IPA) of the rat was examined using the Ca2+ fluorophore fura 2. Induction of hypoxia in IPA preconstricted with 3 microM prostaglandin F2 alpha (PGF2 alpha) resulted in a biphasic contractile response, the first phase of which was associated with a transient rise in [Ca2+]i. No additional rise in [Ca2+]i was observed during the more slowly developing second phase constriction. Upon reoxygenation [Ca2+]i and tension returned to prehypoxic levels. Ro-31-8220 [a specific protein kinase C (PKC) inhibitor] reduced the first phase in IPA preconstricted with PGF2 alpha or 20 mM KCl, but had no effect on the second phase constriction in either of these groups. These results demonstrate that the first phase of the hypoxic constriction is associated with a transient rise in [Ca2+]i via either Ca2+ influx and/or release, and may have a PKC-dependent component, whereas the second phase involves a PKC-independent sensitization of the contractile machinery to Ca2+.

Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries.

Hypoxic vasoconstriction was investigated in isolated pulmonary and mesenteric arteries of the rat. Experiments were performed on large (approximately 2 mm pulmonary, approximately 0.8 mm mesenteric) and small (100-350 microns) arteries. Hypoxia [oxygen partial pressure (PO2) approximately 33 mmHg] elicited a biphasic response in arteries precontracted with prostaglandin F2 alpha (10 microM). A transient contraction reaching a peak within 2-3 min was observed in both large and small pulmonary and mesenteric arteries (phase 1). In pulmonary arteries, this was followed by a slowly developing contraction over 45 min (phase 2). In mesenteric arteries, there was no phase 2 but instead a profound relaxation. Mechanical disruption of the endothelium had no significant effect on phase 1 in preconstricted large pulmonary arteries but reduced phase 1 in small arteries by 40%. Phase 2 was abolished in both large and small arteries. Inhibition of endothelium-derived relaxing factor synthesis or cyclooxygenase pathways had no effect on either phase. Verapamil substantially reduced phase 1 but abolished phase 2. In conclusion, we have found a clear biphasic response to hypoxia in pulmonary arteries of the rat, but, in contrast to some previous reports, phase 1 was only partially dependent on the endothelium, whereas phase 2 was entirely dependent on the endothelium. Small and large arteries had qualitatively similar responses. These results are consistent with the involvement of at least two mechanisms for hypoxic vasoconstriction, one of which may involve release of an as yet unidentified endothelium-derived constrictor factor.

Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells.

Pulmonary hypertension due to long-term hypoxia occurs as a result of both chronic obstructive pulmonary disease and habitation at high altitudes. Studies in animal models of chronic hypoxia have demonstrated the development of a persistent depolarization of pulmonary artery (PA) smooth muscle cells (SMCs). In seeking to explain this effect, we compared under normoxic conditions the K+ currents in SMCs isolated from small PA of chronically hypoxic and normoxic rats. Chronic hypoxia was associated with a marked (40-50%) reduction in amplitude of a K+ current, which had the pharmacological and kinetic characteristics of a delayed rectifier. The resting potential of the isolated PA cells from chronically hypoxic animals was significantly more positive (-43.5 +/- 2 mV) than that of cells from normoxic animals (-54.3 +/- 2 mV), and this depolarization could be approximately mimicked in the cells from normoxic animals by application of 1 mM 4-aminopyridine, a blocker of the delayed rectifier K+ current. Glibenclamide (1 microM), a blocker of ATP-sensitive K+ (KATP) channels, also caused a substantial (14.5 +/- 2.2 mV) depolarization of the membrane. These results suggest that both delayed rectifier and ATP-dependent K+ currents contribute to setting the membrane potential in these cells and are consistent with the possibility that downregulation of the delayed rectifier K+ current contributes to the depolarization and altered responsiveness to vasoactive agents of PAs that occurs during long-term hypoxia.