Objective—To compare responses of equine digital arteries (EDAs) and veins (EDVs) to human-acalcitonin gene-related peptide (hαCGRP), evaluate effect of the endothelium, and characterize receptors and sources of endogenous CGRP.
Sample—Palmar digital vessels (5 to 9/experiment) from healthy adult horses killed at an abattoir.
Procedures—Vessel rings were mounted under tension in organ baths containing Krebs-Henseleit solution at 30°C, with relaxation responses examined in vessels preconstricted with a thromboxane-mimetic (3 × 10−8M). Responses of endothelium-intact (+e) and -denuded (−e) EDAs and EDVs to hαCGRP C10−10 to 3 × 10−7M) were compared. Following incubation with an hαCGRP receptor antagonist (hαCGRP8–37; 1μM), responses of EDA(−e) and EDV(−e) to hαCGRP (10−7M) were obtained. Responses of endothelium-intact and -denuded arteries and veins to hαCGRP (3 × 10−7M) or capsaicin (10−5M) were evaluated as well as responses of endothelium-intact and -denuded EDA and EDV to hαCGRP (10−10 to 10−6M) after incubation with endothelin-1 (ET-1; 10−12M).
Results—hαCGRP resulted in nonendothelium, concentration-dependent relaxation in EDAs and EDVs, with greater responses in EDAs. Treatment with hαCGRP8–37 had minimal effect on responses to hαCGRP in either vessel type. Capsaicin induced relaxation in both vessel types. There were no differences between responses to hαCGRP for vessels pretreated with ET-1 or vehicle.
Conclusions and Clinical Relevance—Both hαCGRP and capsaicin induced digital vasodilation unaffected by a functional endothelium. This suggested that endogenous CGRP likely emanates from sensory-motor nerves and may contribute to digital vasodilation.
Objective—To determine in vitro vasoactive potency
of monoamines formed in the cecum and found in the
systemic circulation of horses.
Sample Population—Segments of digital blood vessels
obtained from 6 healthy mixed-breed horses and
ponies euthanatized at an abattoir and platelets isolated
from 4 healthy ponies.
Procedure—Paired rings of digital artery and vein
from the same horse were examined, and isometric
tension was recorded. Concentration-response
curves for tryptamine (TRP), tyramine (TYR),
phenylethylamine (PEA), isoamylamine (IAA), and
isobutylamine (IBA) were obtained. Vasoconstrictor
mechanisms were investigated for TRP and TYR by
the use of antagonists. Washed platelets loaded with
[3H]-5-hydroxytryptamine (5-HT) were incubated with
monoamines; the amount of radioactivity displaced
after 30 minutes was estimated.
Results—TRP, TYR, and PEA were potent constrictors
of arteries and veins, with TRP and TYR being more
potent in veins than arteries. Constrictions induced by
TYR were inhibited by benextramine (α-antagonist)
and nisoxetine (neuronal-uptake blocker), whereas
TRP responses were inhibited by ketanserin (5-HT
receptor antagonist). All 5 amines displaced 5-HT
from platelets with the order of potency being TYR >
TRP > PEA > IAA > IBA.
Conclusions and Clinical Relevance—Amines from
the equine cecum cause digital vasoconstriction. The
most potent (TRP and TYR) cause selective venoconstriction.
Tyrosine activates predominantly α-adrenoceptors
through the release of neuronal norepinephrine,
whereas TRP activates 5-HT receptors. All
amines tested released 5-HT from platelets. Amines
formed in the cecum and released into the systemic
circulation warrant additional investigation as trigger
factors for digital ischemia and subsequent laminitis.
(Am J Vet Res 2003;64:1124–1131)
Objective—To compare the responses of equine digital
arteries (EDAs) and equine digital veins (EDVs) to
endothelin-1 (ET-1) and determine the role of the
endothelium and type of receptors involved in the
modulation and mediation of those responses,
Sample Population—5 to 9 palmar digital
vessels/experiment from 28 healthy horses.
Procedure—Rings of dissected vessels were mounted
under tension between force transducer wires in
organ baths containing Krebs-Henseleit solution at
30oC. Responses of EDAs and EDVs (with intact [+e]
or denuded [–e] endothelium) to cumulative concentrations
of ET-1 (10–10 to 3 × 10–7 M) were compared.
For (+e)EDAs and (+e)EDVs precontracted with a
thromboxane-mimetic (U44069; 10–8 M) and (–e)EDAs
and (–e)EDVs, responses to an ETB receptor agonist
(S6c; 10–10 to 3 × 10–7 M) were evaluated. Responses to
ET-1 (10–7 M) in (–e)EDAs and (–e)EDVs were evaluated
after incubation with an ETA receptor antagonist (BQ-
123; 3 × 10–7 M), an ETB receptor antagonist (BQ-788;
3 × 10–7 M), or vehicle solution.
Results—Endothelin-1 induced a concentrationdependent
contraction of endothelium-intact and
-denuded EDAs and EDVs; EDVs were more sensitive.
Neither vessel type relaxed in response to S6c,
although 2 of the (–e)EDAs contracted mildly.
Whereas BQ-123 inhibited the (–e)EDA and (–e)EDV
responses to ET-1, BQ-788 had no effect.
Conclusions and Clinical Relevance—Endothelin-1
induced digital vasoconstriction (marked constriction
in veins). This action was unaffected by endothelium
and mediated predominantly by ETA receptors. These
findings suggest ET-1 can induce selective digital
venoconstriction. (Am J Vet Res 2003;64:1438–1443
Objective—To compare flow-mediated vasodilation (FMD) measurements in brachial and femoral arteries of healthy dogs habituated to the assessment method, evaluate repeatability of these measurements, and investigate effects of blood pressure cuff inflation time on femoral artery FMD measurements.
Animals—11 healthy adult Miniature Schnauzers.
Procedures—Arterial luminal diameter and blood flow velocity integral (FVI) were measured before and after cuff inflation of 5 minutes' (brachial and femoral arteries) or 3 minutes' duration (femoral artery) in separate experiments. A blood pressure cuff was inflated to > 200 mm Hg distal to each imaging site to increase local blood flow to induce reactive hyperemia. Changes in FVI after cuff deflation, FMD, and between-dog and within-dog coefficients of variation (CVs) were determined.
Results—After cuff inflation of 5 minutes' duration, greater changes were detected in median change in FVI and FMD of brachial arteries (174.0% and 8.0%, respectively), compared with values determined for femoral arteries (32.0% and 2.1%, respectively). Between-dog CV for brachial artery FMD was 34.0%, compared with 89.6% for femoral arteries, and within-dog CV was 32.5% for brachial arteries versus 51.6% for femoral arteries after cuff inflation of 5 minutes' duration.
Conclusions and Clinical Relevance—In healthy Miniature Schnauzers, FMD was greater and more repeatable in brachial arteries than in femoral arteries. Reactive hyperemia was inconsistently induced in femoral arteries following 3- or 5-minute cuff inflation times. Brachial, but not femoral, artery FMD measurement is a potentially useful research technique for measurement of endothelial function in dogs.
Objective—To validate a nonautomated technique for the measurement of urinary N-acetyl-β-D-glucosaminidase (NAG) activity in cats and assess the correlation between NAG index, plasma creatinine concentration, and proteinuria.
Animals—197 client-owned cats (≥ 9 years old; 119 neutered males and 78 neutered females) of which 103 had previously been determined to have chronic kidney disease (CKD).
Procedures—Preliminary assay validation was performed for a nonautomated colorimetric technique for quantification of NAG activity. The effect of storage of samples was examined. A cross-sectional study was performed to assess urinary NAG index in cats with variable plasma creatinine concentrations and with proteinuria, as quantified by use of the urine protein-to-creatinine ratio (UP:C).
Results—Interassay coefficients of variance (CVs) in cats with low (mean, 0.64 U/L), medium (mean, 4.38.U/L), and high (mean, 8.48 U/L) urine NAG activity were 25.9%, 14.4%, and 25.1%, respectively, but intra-assay CVs were < 20%. Urine NAG activity was stable for 4 freeze-thaw cycles and for storage at −20°C. There was no significant difference in log NAG index when cats (n = 197) were grouped according to plasma creatinine concentration, but a moderate positive correlation was found between log NAG index and log UP:C (r2 = 0.259).
Conclusions and Clinical Relevance—N-acetyl-β-D-glucosaminidase activity can be quantified in feline urine by use of a nonautomated colorimetric technique. However, data should be interpreted cautiously because of high interassay CVs. The NAG index in cats with CKD may be indicative of ongoing lysosomal activity rather than active proximal tubular cell damage.
Objective—To compare concentrations of urinary iodide (UI) in euthyroid and untreated hyperthyroid cats.
Animals—118 euthyroid and 88 hyperthyroid client-owned cats from 2 nonreferral veterinary practices.
Procedures—Iodide concentration was measured in 5 urine samples collected every 3 to 12 months from selected cats, and variability of results between euthyroid cats and hyperthyroid cats prior to the diagnosis of hyperthyroidism was evaluated via 1-way ANOVA, after logarithmic transformation of UI concentrations (logUIs). The UI concentration in hyperthyroid cats was measured at diagnosis and 2 to 6 weeks and 3 to 6 months after treatment for hyperthyroidism. The pretreatment logUI in hyperthyroid cats was compared with that in euthyroid cats, taking into account the effects of renal function on UI concentration. Iodine intake was estimated in euthyroid cats following calculation of the volume of daily urine output, with a fixed value for iodine concentration in feces.
Results—The variability of UI concentrations did not differ significantly between hyperthyroid (n = 10) and euthyroid (8) cats. The logUI increased 2 to 6 weeks after initiation of treatment in hyperthyroid cats (n = 80) and was lower in azotemic versus nonazotemic cats. Hyperthyroid cats had a lower logUI than euthyroid cats, and there was no evidence of deficient iodine intake in euthyroid cats.
Conclusions and Clinical Relevance—The logUI was lower in cats with azotemia and with untreated hyperthyroidism, compared with that in euthyroid cats from the same population. Additional studies are needed to determine whether iodine intake plays a role in the development of hyperthyroidism in cats.
Objective—To evaluate the between- and within-dog repeatability of a flow-mediated vasodilation (FMD) measurement technique in healthy dogs.
Animals—43 male and female dogs of various breeds (weight range, 6.9 to 31.7 kg; age range, 11 months to 11 years).
Procedures—5 dogs were used to refine the technique; other dogs were classified as large (> 15 kg) or small (≥ 15 kg) before use in the main study. In each dog, a brachial artery was occluded for 5 minutes by inflating a blood pressure cuff (applied pressure was more than 50 mm Hg greater than that required to occlude flow). Two-dimensional ultrasonographic images of the artery were recorded during a 30-second period prior to cuff inflation (baseline) and during a 3-minute period after cuff deflation by each of 2 sonographers. Relative percentage increases in luminal size from baseline (ie, FMD) were calculated. Independent contributing factors to FMD (eg, body weight, age, and room temperature) were assessed.
Results—Median FMD was significantly greater in small dogs (77%; range, 0% to 19.3%) than it was in large dogs (2.2%; range, −2.2% to 10.6%); values were significantly greater in dogs < 6 years old, compared with dogs > 6 years old. Weight was the only independent contributing factor for FMD. Coefficients of variation for between- and within-dog repeatability were 99.7% and 62.8%, respectively.
Conclusions and Clinical Relevance—Application of the FMD measurement technique used in humans appears to be feasible in dogs and may provide a means of assessing canine endothelial function, although between and within-dog variations were large. (Am J Vet Res 2010;71:1154–1161)
Objective—To evaluate proteomic delineation of feline urine by mass spectrometry as a method for identifying biomarkers in cats at risk of developing azotemia.
Samples—Urine samples from geriatric cats (> 9 years old) with chronic kidney disease and nonazotemic cats that either remained nonazotemic (n = 10) or developed azotemia (10) within 1 year.
Procedures—Optimization studies with pooled urine were performed to facilitate the use of surface enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for analysis of the urinary proteome of cats. Urine samples from nonazotemic cats at entry to the study were analyzed via SELDI-TOF-MS with weak cation exchange and strong anion exchange arrays. Spectral data were compared to identify biomarkers for development of azotemia.
Results—Low protein concentration in feline urine precluded direct application to array surfaces, and a buffer exchange and concentration step was required prior to SELDI-TOF-MS analysis. Three preparation conditions by use of weak cation and strong anion exchange arrays were selected on the basis of optimization studies for detection of biomarkers. Eight potential biomarkers with an m/z of 2,822, 9,886, 10,033, 10,151, 10,234, 11,653, 4,421, and 9,505 were delineated.
Conclusions and Clinical Relevance—SELDI-TOF-MS can be used to detect urinary low-molecular weight peptides and proteins that may represent biomarkers for early detection of renal damage. Further study is required to purify and identify potential biomarkers before their use in a clinical setting.
Objective—To evaluate urine cauxin immunoreactivity in geriatric cats with variable plasma creatinine concentrations and proteinuria and to assess urinary cauxin-to-creatinine concentration ratio (UC/C) as a predictor of developing azotemia.
Animals—188 client-owned geriatric (≥ 9 years of age) cats.
Procedures—A direct immunoassay was developed and validated for the quantification of urinary cauxin relative to a standard curve generated from a urine sample with high cauxin immunoreactivity. Relationships among UC/C, plasma creatinine concentration, and proteinuria were assessed. Nonazotemic cats were recruited and followed for 12 months. Urinary cauxin-to-creatinine concentration ratio was evaluated as a predictor of development of azotemia in these cats.
Results—No relationship was evident between UC/C and plasma creatinine concentration. A weak positive correlation was identified between UC/C and urine protein-to-creatinine concentration ratio (r = 0.212). At entry to the longitudinal study, those cats that later developed azotemia had a UC/C that was significantly higher than in those remaining nonazotemic after 12 months.
Conclusions and Clinical Relevance—The UC/C did not vary with severity of azotemia but appeared contributory to the feline urinary proteome. High UC/C values were predictive of the geriatric cats in our study developing azotemia. However, it seems unlikely that UC/C will provide additional information about the measurement of urine protein-to-creatinine concentration ratio as a biomarker for the development of azotemia in cats.
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.