S. R. Bailey

Rho Kinase Mediates Cold-Induced Constriction of Cutaneous Arteries Role of α2C-Adrenoceptor Translocation

Cold-induced vasoconstriction in cutaneous blood vessels is mediated in part by increased activity of vascular smooth muscle &agr;2-adrenoceptors (VSM &agr;2-ARs). In mouse cutaneous arteries, &agr;2C-ARs are normally silent at 37°C but mediate cold-induced augmentation of &agr;2-AR responsiveness. In transfected HEK293 cells, this functional rescue is mediated by cold-induced translocation of &agr;2C-ARs from the Golgi to the plasma membrane. Experiments were performed to determine the role of Rho/Rho kinase signaling in this process. Inhibition of Rho kinase (fasudil, Y27632 or H-1152) did not affect constriction of isolated mouse tail arteries to the &agr;2-AR agonist UK 14 304 at 37°C but dramatically reduced the augmented responses to the agonist at 28°C. After Rho kinase inhibition, cooling no longer increased constriction evoked by &agr;2-AR stimulation. Cooling (to 28°C) activated Rho in VSM cells and increased the calcium sensitivity of constriction in &agr; toxin-permeabilized arteries. Stimulation of &agr;2-ARs in VSM cells had no effect on Rho activity or calcium sensitivity at 37°C or 28°C. In HEK293 cells transfected with &agr;2C-ARs, cooling (to 28°C) stimulated the translocation of &agr;2C-ARs to the plasma membrane and this effect was prevented by inhibition of Rho kinase, using fasudil or RNA interference. Consistent with inhibition of the spatial rescue of &agr;2C-ARs, fasudil inhibited &agr;2-AR–mediated mobilization of calcium in tail arteries at 28°C but not 37°C. Therefore, cold-induced activation of Rho/Rho kinase can mediate cold-induced constriction in cutaneous arteries by enabling translocation of &agr;2C-ARs to the plasma membrane and by increasing the calcium sensitivity of the contractile process.

Hypertension and insulin resistance in a mixed-breed population of ponies predisposed to laminitis

OBJECTIVE To determine the metabolic phenotype of a group of laminitis-prone ponies when at pasture in summer, compared with when at pasture in winter. ANIMALS 40 ponies of various breeds predisposed to recurrent pasture-associated laminitis and 40 unaffected control ponies. PROCEDURES Body condition score and size of the crest of the neck were assessed, blood samples obtained, and blood pressure measured by use of an indirect oscillometric technique, while ponies were kept on winter pasture (last week of November or beginning of December) and again on summer pasture (June). Serum insulin concentration and plasma glucose, triglyceride, uric acid, and ACTH concentrations were measured. Insulin sensitivity was calculated with proxies derived from basal serum insulin and plasma glucose concentrations. RESULTS No significant differences were apparent between ponies predisposed to laminitis and control ponies during winter. However, in June, laminitis-prone ponies had increased serum insulin concentration and plasma triglyceride and uric acid concentrations, compared with control ponies. Also, laminitis-prone ponies were relatively insulin resistant, compared with control ponies. Mean blood pressure was significantly higher during summer in laminitis-prone ponies (median [interquartile range], 89.6 mm Hg [78.3 to 96.9 mm Hg]), compared with control ponies (76.8 mm Hg [69.4 to 85.2 mm Hg]). CONCLUSIONS AND CLINICAL RELEVANCE Summer pastures appear to induce metabolic responses in some ponies, leading to expression of the prelaminitic phenotype, which includes hypertension as well as insulin resistance. Signs of this metabolic syndrome may not be apparent in affected ponies during periods of grazing winter pasture. Understanding this syndrome may enable improved countermeasures to be devised to prevent laminitis.

Current research and theories on the pathogenesis of acute laminitis in the horse.

A large number of studies have been undertaken in recent years aimed at furthering our understanding of the complex mechanisms underlying the common and debilitating condition of acute laminitis in the horse. Many of these studies have either reinforced or cast doubt on previously held theories on the pathogenesis of this disease, while others have suggested new mechanisms which may play a key role in its development. This review seeks to put the current hypotheses into the context of this recent body of evidence. While a unifying theory may not yet seem to be achievable, this review demonstrates that most of the current theories are not mutually exclusive. Studies utilising in vitro and in vivo models of the disease, particularly addressing the areas of inflammation, haemodynamic disturbances and enzyme activation in the hoof, as well as the preceding events occurring in the hindgut, have helped to explain many clinical observations of the disease and may possibly lead to more effective therapies and means of prevention in the future.

Identification of equine cecal bacteria producing amines in an in vitro model of carbohydrate overload.

ABSTRACT Acute laminitis has been associated with the overgrowth of gram-positive bacteria within the equine hindgut, causing the release of factor(s) leading to ischemia-reperfusion of the digits. The products of fermentation which trigger acute laminitis are, as yet, unknown; however, vasoactive amines are possible candidates. The objectives of this study were to use an in vitro model of carbohydrate overload to study the change in populations of cecal streptococci and lactobacilli and to establish whether certain species of these bacteria were capable of producing vasoactive amines from amino acids. Cecal contents from 10 horses were divided into aliquots and incubated anaerobically with either corn starch or inulin (fructan; both at 1 g/100 ml). Samples were taken at 6-h intervals over a 24-h period for enumeration of streptococci, lactobacilli, and gram-negative anaerobes by a dilution method onto standard selective growth media. The effects of the antibiotic virginiamycin (1 mg/100 ml) and calcium hydrogen phosphate (CaHPO4; 0.3 g/100 ml) were also examined. Fermentation of excess carbohydrate was associated with increases in numbers of streptococci and lactobacilli (2- to 3.5-log unit increases; inhibited by virginiamycin) but numbers of gram-negative anaerobes were not significantly affected. A screening agar technique followed by 16S rRNA gene sequence analysis enabled the identification of 26 different bacterial strains capable of producing one or more vasoactive amines. These included members of the species Streptococcus bovis and five different Lactobacillus spp. These data suggest that certain bacteria, whose overgrowth is associated with carbohydrate fermentation, are capable of producing vasoactive amines which may play a role in the pathogenesis of acute laminitis.

Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis

The link between the fermentation of carbohydrate in the equine large intestine and the development of acute laminitis is poorly understood. Absorption of endotoxin (lipopolysaccharide; LPS) into the plasma has been observed in one experimental model of laminitis, but does not cause laminitis when administered alone. Thus, the potential role of endotoxin is unclear. Platelet activation has previously been demonstrated in the developmental stage of laminitis. Equine platelets are more sensitive than leukocytes to activation by endotoxin, and can be activated directly by LPS in the low pg/ml range, activating p38 MAP kinase and releasing serotonin (5-HT) and thromboxane. The objectives of this study were firstly to determine whether endotoxin and platelet activation could be measured in the plasma of horses in the developmental phase of laminitis induced with oligofructose. Secondly, the time course of events involving platelet activation and platelet-derived vasoactive mediator production was investigated. Laminitis was induced in six Standardbred horses by the administration of 10 g/kg bwt of oligofructose. Plasma samples were obtained every 4h, and platelet pellets were obtained by centrifugation. LPS was measured using a kinetic limulus amebocyte lysate assay, and platelet activation was assessed by Western blotting for the phosphorylated form of p38 MAP kinase. Plasma 5-HT was assayed by HPLC with electrochemical detection and thromboxane B(2) was measured by radioimmunoassay. Clinical signs of laminitis and histopathologic changes were observed in lamellar sections from five of the six horses. Onset of lameness was between 20 and 30 h after the administration of oligofructose. LPS increased above the limit of detection (0.6 pg/ml) to reach a peak of 2.4+/-1.0 pg/ml at 8 h. TNFalpha was also detectable in the plasma from 12 to 24 h. There was a time-dependent increase in platelet p38 MAPK phosphorylation, which peaked at approximately 12 h (3.8+/-1.3 fold increase); plasma 5-HT and thromboxane increased steadily after this time (2.9+/-0.6 and 11.3+/-5.0 fold increases, respectively). These data indicate that small quantities of endotoxin may move into the circulation from the large intestine after the sharp decrease in pH that occurs as a result of carbohydrate fermentation. Correlating these findings with in vitro studies suggests that LPS may primarily activate platelets, leading indirectly to the activation of leukocytes. Therefore, endotoxin may contribute in the initiation of the early inflammatory changes observed in experimental models of acute laminitis.

Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators

REASONS FOR PERFORMING STUDY Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined. OBJECTIVES To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations. METHODS Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E. coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made. Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured. RESULTS Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration. Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased. Systemic blood pressure remained unaltered. Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion. Plasma endothelin concentrations remained unchanged. CONCLUSIONS The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis. POTENTIAL RELEVANCE These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions.

Breed differences in insulin sensitivity and insulinemic responses to oral glucose in horses and ponies of moderate body condition score

Breed-related differences may occur in the innate insulin sensitivity (SI) of horses and ponies, an important factor believed to be associated with the risk of laminitis. The aim of this study was to measure the glucose and insulin responses of different breeds of horses and ponies in moderate body condition to a glucose-containing meal and to compare these responses with the indices of SI as determined by a frequently sampled intravenous glucose tolerance test (FSIGT). Eight Standardbred horses, 8 mixed-breed ponies, and 7 Andalusian-cross horses with a mean ± SEM BCS 5.0 ± 0.3 of 9 were used in this study. Each animal underwent an oral glucose tolerance test (OGTT) in which they were fed a fiber-based ration (2.0 g/kg BW) containing 1.5 g/kg BW added glucose, as well as a standard FSIGT with minimal model analysis. The glucose response variables from the OGTT were similar between groups; however, the peak insulin concentration was higher in ponies (94.1 ± 29.1 μIU/mL; P = 0.003) and Andalusians (85.3 ± 18.6; P = 0.004) than in Standardbreds (21.2 ± 3.5). The insulin area under the curve was also higher in ponies (13.5 ± 3.6 IU · min · L(-1); P = 0.009) and Andalusians (15.0 ± 2.7; P = 0.004) than in Standardbreds (3.1 ± 0.6). Insulin sensitivity, as determined by the FSIGT, was lower in Andalusians (0.99 ± 0.18 × 10(-4)/[mIU · min]) than in Standardbreds (5.43 ± 0.94; P < 0.001) and in ponies (2.12 ± 0.44; P = 0.003) than in Standardbreds. Peak insulin concentrations from the OGTT were negatively correlated with SI (P < 0.001; rs = -0.75). These results indicate that there are clear breed-related differences in the insulin responses of horses and ponies to oral and intravenous glucose. All animals were in moderate body condition, indicating that breed-related differences in insulin dynamics occurred independent of obesity.

A review of recent advances and current hypotheses on the pathogenesis of acute laminitis

With the increasing number of studies being published on the different experimental models used to induce and study acute laminitis, the pathophysiological events associated with these various models (i.e. starch overload, oligofructose overload, black walnut extract and hyperinsulinaemia) can be compared more realistically. Within this review, the mechanisms for metabolic vs. inflammatory laminitis are discussed, and the question of how pasture laminitis may fit into any of the proposed mechanisms is addressed.

The effects of vasoactive amines found in the equine hindgut on digital blood flow in the normal horse

REASONS FOR PERFORMING STUDY Disturbances of digital blood flow are thought to be fundamental to the pathophysiology of acute laminitis. However, factors linking the initiating events in the equine hindgut with these disturbances in the foot remain to be determined. HYPOTHESIS Amine compounds, formed by bacteria in the equine hindgut, have digital vasoconstrictor effects in vivo. METHODS Tryptamine (1.6 microg/kg/min) and phenylethylamine (2.13 microg/kg/min) were infused i.v. into standing nonsedated horses. Digital blood flow was measured by Doppler ultrasound and foot surface temperature was monitored. Plasma 5-hydroxytryptamine (5-HT) concentrations were measured by HPLC. RESULTS Tryptamine and phenylethylamine infusions had no effect on systemic arterial blood pressure or heart rate, but caused significant decreases in digital arterial blood flow (mean +/- s.e. 29.2 +/- 8.5 and 18.4 +/- 6.8%, respectively). Both amines also caused decreases in dorsal hoof wall temperature (0.6 +/- 0.1 and 0.5 +/- 0.1 degrees C for tryptamine and phenylethylamine, respectively) and concomitant increases in plasma 5-HT concentration. CONCLUSIONS Tryptamine and phenylethylamine caused reduction of digital blood flow, effects which may have been mediated, in part, via displacement of 5-HT from platelets. POTENTIAL RELEVANCE Amine compounds occurring in the equine hindgut, if released into the circulation following carbohydrate overload, could contribute to selective digital vasoconstriction. Further work in ponies and horses, with naturally occurring laminitis, is necessary to determine whether amines represent a therapeutic target in this disease.

Gastrointestinal Derived Factors Are Potential Triggers for the Development of Acute Equine Laminitis

Equine laminitis is the painful and debilitating condition resulting from cellular damage and inflammation of the tissues comprising the bonds supporting the pedal bone within the hoof capsule. One of the reasons why this condition is so complicated and enigmatic is its association with gastrointestinal disturbances, particularly a diet of lush grass at certain times of the year. Determining the link between disturbances to the hindgut flora and pathophysiology in the foot is one of the keys to preventing this condition. Therefore, one of the biggest challenges in equine laminitis research is to determine those events that precede the onset of the classical clinical signs of lameness, bounding digital pulses, and warm feet. A main theory for the pathogenesis of acute laminitis involves the development of vasoconstriction within the foot, which leads to ischemic and/or inflammatory tissue damage. Serotonin is an important constrictor mediator controlling digital blood flow. Certain equine hindgut bacteria produce amino acid decarboxylase enzymes that convert free amino acids into monoamines. Therefore, amines formed and released from the gastrointestinal tract are hypothesized to act as the link between the ingestion of lush grass and the digital ischemia thought to precede laminitis. Equine cecal contents contain a range of amines that are present in micromolar concentrations. Tryptamine is the most potent cecum-derived amine, causing vasoconstriction in vitro and in vivo through direct activation of serotonin receptors and displacing serotonin from platelets. Monoamines found in the cecum of the horse could potentially induce hemodynamic disturbances in the digit that result in laminar ischaemia, and so trigger laminitis.