Epidermal and hepatic glucocorticoid receptors in cats and dogs

Low capacity, high affinity [³H] dexamethasone binding receptors were identified in cytosolic preparations of liver (mean number 45±10·1 fmol mg⁻¹ protein, apparent dissociation constant 0·4±0·1 nM) and skin (mean number 46·4±23·8 fmol mg⁻¹ protein, apparent dissociation constant 1 ± 0·2 nM) of clinically normal dogs. For clinically normal cats, approximately half these numbers of receptors with a lower affinity, were detected in liver (mean number 23·1±10·4 fmol mg⁻¹ protein, apparent dissociation constant 3·2±0·9 nM) and skin (mean number 23·90±10·9 fmol mg⁻¹ protein, apparent dissociation constant 2·2±1·5 nM). This difference between dogs and cats in [³H] dexamethasone binding receptors may contribute to the relative glucocorticoid resistance observed in cats.     

Pharmacologic identification of putative D1 dopamine receptors in feline kidneys

The presence of dopamine (DA) receptors in feline kidneys is a matter of contention. Radioligand binding and Western blotting studies were employed to determine whether DA receptors are present in feline kidneys. The pharmacologic profile of the selective D1-receptor antagonist [3H]-SCH 23390 was studied in renal cortical membrane preparations from cats by conducting saturation binding isotherm and competitive binding experiments. [3H]-SCH 23390 bound to feline renal cortical membranes in a manner consistent with labeling of a D1-like receptor. The binding profile revealed a single site D1-like or D1 receptor in the feline renal cortex with a Kd = 7.8 +/- 1.0 nmol/L and Bmax = 76.5 +/- 19.5 fmol/mg. Competitive binding studies for [3H]-SCH 23390 against unlabeled agonists yielded the following Ki values and rank order of competition: SKF38393 (Ki = 0.47 +/- 0.26 micro m) > fenoldopam (Ki = 3.12 +/- 1.1 micro m) > DA (Ki = 933.1 +/- 1.6 micro m). Competitive binding studies for [3H]-SCH-23390 against unlabeled antagonists yielded the following rank order of competition: SCH 23390 (Ki = 1.97 +/- 0.81 micro m) > spiperone (Ki = 3.79 +/- 0.79) > metoclopramide (Ki = 4.26 +/- 2.4 micro m). Western blot analysis with anti-DA D1 receptor antibodies detected a single band with Mr of 74 kDa corresponding to a D1 DA receptor. These results suggest that a putative D1-like or D1 receptor exists in feline kidneys different from those previously identified in rat, dog or human kidneys.     

Specific binding of naloxone to ovine brain tissue: comparison of brain regions and endocrine states

Binding of [3H]naloxone ([3H]NAL) to brain membranes was quantified by Scatchard analysis using two methods of separating bound from free [3H]NAL. In the centrifugation method, membranes that were soluble at 1,000 x g, but sedimented at 20,000 x g, were incubated with [3H]NAL. For filtration, all membranes that sedimented at 20,000 x g were incubated and filtered through glass filter fibers. Nonspecific binding was estimated using greater than 500-fold excess of unlabeled naloxone (10(-6) M). Specific binding of [3H]NAL was used to generate linear multiple-point Scatchard plots, which indicated a single class of high-affinity sites. In Exp. 1, 10 ovariectomized (OVX) ewes were injected with estradiol-17 beta alone or in combination with progesterone. Compared with OVX controls, these hormonal treatments did not affect binding of [3H]NAL (centrifugation method) to combined hypothalamus (HYP) + preoptic (POA) tissues. In cyclic ewes (Exp. 2, filtration method), affinity constants (2.4 +/- .2 x 10(8) M-1) did not differ among HYP, POA and basal forebrain (BF) tissues, but BF had more sites (39 +/- 3 fmol/mg) than either HYP (14 +/- 1) or POA (17 +/- 1). Binding affinity and concentration of sites within each brain area (HYP, POA, BF) did not differ between d 8 and d 16 (preovulatory but after luteolysis) in normally cycling ewes. Overall, neural tissue dissected from BF had a greater concentration of binding sites than HYP or POA. Exogenous and endogenous fluctuations in ovarian steroids did not affect binding of [3H]NAL to these tissues.     

Comparison of opioid and alpha-2 adrenergic receptor binding in horse and dog brain using radioligand autoradiography

Objective To test the hypothesis that the distribution, density, and subtype of opioid and alpha (α)‐2 adrenergic receptors within the central nervous system (CNS) are significantly different between horse and dog. Study design Prospective experimental study. Animals Three dogs (3 years of age) and three horses (2–5 years of age). Animals were opioid‐ and α‐2 agonist‐free at the time of euthanasia. Methods Brain tissue was obtained at 126 days post‐surgery from dogs and 72 days post‐surgery from horses. The brains were removed, sectioned coronally into 1‐cm slabs, frozen in methylbutane, which was cooled by liquid nitrogen, and stored at ?70 °C. Receptor autoradiography was performed using established techniques. [³H]DAMGO, [³H]U‐69593, and [³H]RX821002 were used for mu (µ)‐opioid, kappa (κ)‐opioid, and α‐2 adrenergic‐binding assays, respectively. Species differences were analyzed separately for each major brain region by repeated measures anova for subregions followed by Fisher's protected Latin square design (LSD). p < 0.05 was considered significant. Results There was higher binding of µ‐opioid receptors in the frontal cortex, left somatosensory cortex, colliculus (mid‐brain), and granule cell layer of the cerebellum of horses than that of dogs. There was higher binding to κ‐opioid receptors in the frontal cortex of dogs compared to horses, whereas binding to κ‐opioid receptors in the cerebellum was higher in horses. Binding to α‐2 adrenergic receptors in the mid‐brain was significantly higher in dogs than in horses. There was higher binding of α‐2 adrenergic receptors in the dorsomedial and dorsolateral periaqueductal grey of dogs as compared to that of horses. Conclusion The results of this study show that the distribution of these receptors is different between horses and dogs. Further work is needed to understand the relevance of these differences to clinical responses to opioids and α‐2 adrenergic agonists in these species.     

Measurement of estrogen receptors in normal and neoplastic lymph node tissue from dogs.

Estrogen receptors were measured in normal canine lymph nodes and neoplastic tissue from dogs with lymphoma, using a commercially available [3H]estradiol dextran-coated charcoal assay. Using the same assay, estrogen receptors were detected in the positive-control tissues--dog uterus, rat uterus, and lyophilized bovine uterus. Specific binding of [3H]estradiol was not detected in rat skeletal muscle or in any of the canine lymphoid tissues, indicating that the specimens did not contain estrogen receptors.     

Regulation of equine lymphocyte beta-adrenoceptors under the influence of clenbuterol and dexamethasone

In 12 healthy horses, the effects of the beta2-agonist clenbuterol and the glucocorticoid dexamethasone on the lymphocyte beta2-adrenoceptor density and affinity (determined by (-)-[125I]-iodocyanopindolol binding) as well as its responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 micromol/l (-)-isoprenaline) were studied. Clenbuterol treatment, 2 x 0.8 microg/kg/day i.v. for 12 days, decreased significantly ICYP binding sites by approximately 30-40%; concomitantly, lymphocyte cAMP response to (-)-isoprenaline was reduced. After withdrawal of clenbuterol, beta2-adrenoceptor density and responsiveness gradually increased, reaching predrug levels after 4 days. The effects of dexamethasone on clenbuterol-induced desensitisation were further investigated. Administration of dexamethasone (1 x 0.1 mg/kg/day, i.v. for 5 days) immediately after clenbuterol withdrawal accelerated beta2-adrenoceptor recovery: only 24 h after administration dexamethasone restored the number of binding sites and cAMP response to (-)-isoprenaline to levels statistically indistinguishable from values before clenbuterol treatment. Three days after dexamethasone administration, lymphocyte beta2-adrenoceptors were further increased about 2-fold the pretreatment values, and this increase declined gradually after dexamethasone withdrawal, reaching baseline values after 4 days. Furthermore, in groups exposed simultaneously to both drugs, dexamethasone completely prevented clenbuterol-induced decrease in lymphocyte beta2-adrenergic receptor density and responsiveness. No significant change was observed in the dissociation constant for ICYP in any of the situations. We conclude that dexamethasone (glucocorticoids) can reverse and prevent Clenbuterol-induced desensitisation (down-regulation) of the lymphocyte beta2-adrenoceptors and therefore, a combined therapy with clenbuterol and dexamethasone may be potentially beneficial in horses suffering from chronic obstructive pulmonary disease (COPD).     

Binding characteristics of swine erythrocyte insulin receptors

Crossbred gilts (controls; n = 7) had 8.8 +/- 1.1% (mean +/- SEM) maximum binding of [125I]insulin to insulin receptors on erythrocytes. The number of insulin-binding sites per cell was 137 +/- 19, with a binding affinity ranging from 7.4 X 10(7)M-1 to 11.2 X 10(7)M-1 and mean of 8.8 X 10(7)M-1. Pregnant sows (n = 5) had a significant increase (P less than 0.01) in maximum binding due to an increase in number of receptor sites per cell. Lactating sows fed a high-fiber diet (n = 3) and a low-fiber diet (n = 4) did not develop a significant difference in maximum binding of insulin. Sows fed the low-fiber diet had a significantly higher number of binding sites and a significantly lower binding affinity than did sows fed a high-fiber diet. Receptor-binding affinity was lower in the low-fiber diet group than in cycling gilts, whereas data from sows fed the high-fiber diet did not differ from data for cycling gilts. Data from this study indicated that insulin receptors of swine erythrocytes have binding characteristics similar to those in other species. Pregnancy and diet will alter insulin receptor binding in swine.     

Effect of fasting and dexamethasone on binding characteristics of canine erythrocyte insulin receptors

Insulin binding characteristics of canine erythrocyte insulin receptors were studied before and after a 72-hour fast, and one and three days following glucocorticoid (dexamethasone) administration. The 72-hour fast tended to increase maximum insulin binding, but no significant differences were found. The administration of dexamethasone resulted in an increased maximum binding of insulin to its receptors which, at day 1, was due to an increase in receptor concentration, and at day 3, to an increased insulin binding affinity of the receptor. These data suggest that the erythrocyte insulin receptor may be useful in clinical and experimental studies in the dog.     

Erythrocyte insulin receptors in dogs with spontaneous hyperadrenocorticism

Erythrocyte insulin receptor binding measurements were evaluated in 8 dogs with spontaneous hyperadrenocorticism. These dogs had normal serum glucose concentration, with normal to high serum insulin concentration (range, 45 to 1,400 pmol/L; normal, 40 to 170 pmol/L). Dogs with hyperadrenocorticism had significant (P less than 0.01) decrease in mean +/- SEM percentage of maximal binding for erythrocyte insulin receptors (2.25 +/- 0.21%), compared with results in 11 clinically normal pet dogs (4.29 +/- 0.42%). The decrease in erythrocyte receptor binding was attributed to significant (P less than 0.01) decrease in high-affinity receptor sites in dogs with hyperadrenocorticism (14.5 +/- 2.8), compared with clinically normal dogs (31.2 +/- 4.3). Significant differences in receptor affinity were not apparent between the 2 groups. Percentage of maximal binding for erythrocyte insulin receptors for dogs with hyperadrenocorticism was inversely correlated with serum insulin concentration (r = -0.85, P less than 0.01). Results indicate that the observed decrease in erythrocyte insulin receptor binding could contribute to insulin resistance and hyperinsulinemia associated with hyperadrenocorticism. Alternatively, decreased binding of insulin receptors in animals with hyperadrenocorticism may result from down-regulation secondary to hyperinsulinemia itself caused by insulin resistance at a postreceptor site (decreased responsiveness).     

Ontogeny of hepatic bovine growth hormone receptors in cattle

A series of studies examined the binding characteristics and ontogeny of hepatic growth hormone binding sites in dairy bulls on d 2, 30, 180, and 365 of age. Binding of iodinated recombinant bovine growth hormone ([125I]rbGH) to liver membrane receptors was membrane protein-dependent. Receptors were considered growth hormone-specific, because physiological concentrations of bovine prolactin (bPRL) failed to displace [125I]rbGH from bovine hepatocyte membranes. Only 50% of [125I]rbGH was bound reversibly to hepatic microsomes. Addition of dithiothreitol (DTT) to the receptor-assay buffer increased the binding of [125I]rbGH to hepatic membranes in a time-dependent manner. Moderate concentrations of Ca++ and Mg++ in the receptor-assay buffer had no detectable effects on binding of [125I]rbGH to hepatic microsomes. In growing dairy bulls, specific binding of [125I]rbGH per milligram of membrane protein increased from 1.9 +/- 1.8% at d 2 to 14.1 +/- 1.8% at d 180 and then declined to 5.2 +/- 1.6% at d 365. Likewise, concentration of insulin-like growth factor (IGF)-I in serum was low during the 1st mo of age (d 2, 13.3 +/- 8.8 ng/ml; d 30, 9.7 +/- 8.8 ng/ml), but it became maximal at d 180 (151.0 +/- 8.8 ng/ml). Circulating concentrations of IGF-II increased linearly during the 1st yr of growth. Serum concentrations of GH, triiodothyronine, and thyroxine declined from 39.9 +/- 6.5, 2.7 +/- .2, and 75.4 +/- 4.6 ng/ml at d 2 to 16.5 +/- 6.5, 1.3 +/- .2, and 53.4 +/- 4.6 ng/ml at d 30, respectively, and remained low through 1 yr of age. Insulin concentration in serum did not change significantly with development. Results indicated that increasing concentrations of specific bGH receptors in the bovine liver may play a key role in regulating postnatal growth in cattle.     

Cell proliferation of epidermis, hair follicles, and sebaceous glands of beagles and cocker spaniels with healthy skin

Cell proliferation kinetic values were established for the epidermis, hair follicle epithelium, and sebaceous glands of 10 Beagles and 4 Cocker Spaniels with healthy skin and coats. Values were established by intradermal pulse-labeling injections of [3H]thymidine, examination of cutaneous biopsied tissues, and autoradiography. The epidermal basal cell-labeling index was 1.41 +/- 0.46% for Beagles and 1.71 +/- 0.56% for Cocker Spaniels. The hair follicle basal cell-labeling index was 1.46 +/- 0.78 and 1.07 +/- 0.42%, respectively. Calculated epidermal cell-renewal time for viable layers of the epidermis was 23.38 +/- 5.93 days for Beagles and 20.97 +/- 4.92 days for Cocker Spaniels. Differences between cell kinetic data for the 2 breeds were not significant (P greater than 0.05). The basal cell-labeling index for the sebaceous gland was significantly (P = 0.009) lower for Cocker Spaniels (0.40 +/- 0.18%) than for Beagles (1.81 +/- 1.08%). Seemingly, epidermal and follicular cell proliferation kinetics in healthy dogs was similar between the 2 breeds, whereas sebaceous gland basal cells were less proliferative in healthy Cocker Spaniels than in healthy Beagles.     

The bovine preoptic area and median eminence: sites of opioid inhibition of luteinizing hormone-releasing hormone secretion.

Autoradiography was used to quantify opioid receptors in the median eminence (ME) and preoptic area (POA) of the brain of eight heifers, and in vitro perifusion of ME and POA tissue from seven cows and heifers was used to examine the release of LHRH after administration of naloxone (NAL). For quantitative receptor autoradiography, [3H]NAL was used as the radioligand and NAL or morphine as competitors. Specific binding of [3H]NAL in POA and ME resulted in linear Scatchard plots with similar equilibrium dissociation constants (Kd = 4.2 +/- 1.1 nM) and mean binding site densities in the POA and ME (POA: 80.3 +/- 5.8; ME 67.5 +/- 8.0 fmol/mm2). There were no differences between mean binding site densities of zonas externa and interna of the ME; however, between various regions of the POA within individual animals, binding site densities varied threefold (47.6 to 165.1 fmol/mm2). During in vitro perifusions of isolated POA and ME, basal LHRH secretion from ME decreased (P less than .001) from 15.9 +/- 1.8 to 7.3 +/- .8 pg/10 min fraction (500 microliters) but remained constant for POA (3.1 +/- .4 pg/fraction). Injections of medium alone did not affect LHRH secretion. Although there was no significant dose (10(-9) to 10(-7) M) effect, NAL increased (P less than .05) LHRH efflux from the ME and POA when administered at 110 min from the initiation of perfusion and again at 200 min for ME but not for POA. All tissues responded to KCl (30 mM) administered at 290 min of perifusion with increased (P less than .001) LHRH efflux. Both immunoreactive-LHRH and immunoreactive-beta-endorphin were immunocytochemically localized in neurons from some of these perifused tissues. We suggest that endogenous opioids suppress LHRH secretion by actions on specific opioid receptors located within the POA and ME of the brain.     

Density and binding characteristics of beta-adrenoceptors in the normal and failing equine myocardium

Beta-adrenoceptors are important regulators of cardiac function and their characteristics are known to change in human and canine diseased myocardium. This study aimed to determine the density and subtypes of beta-adrenoceptors in the normal and failing equine ventricular myocardium. Membrane preparations of the left papillary muscles were incubated with increasing concentrations of the nonselective beta-adrenoceptor antagonist [3H]-CGP12177. Saturable and reversible binding of [3H]-CGP12177 to myocardial membranes was demonstrated with Kd values (+/- s.d.) of 0.49 +/- 0.40 and 0.43 +/- 0.22 nmol/l and Bmax values of 93.4 +/- 20.5 and 110.0 +/- 21.2 and fmol/mg protein for normal (n = 19) and heart failure (n = 10) tissues, respectively. Heart failure had no significant effect on the density of ventricular beta-adrenoceptors. The cardiac beta-adrenoceptors were further characterised by studying displacement of [3H]-CGP12177 (0.6 nmol/l) with the beta1-selective antagonists CGP20712A and the beta2-selective antagonist ICI118.551. In normal ventricular muscle, CGP20712A was 26 times more potent than ICI118.551 (Ki values 30.4 +/- 24.8 and 814.1 +/- 485.2 nmol/l, respectively). In heart failure cases, CGP 20712A curves were monophasic with a Ki value of 45.6 +/- 39.7 nmol/l. ICI 118.551 curves were biphasic in 5 horses where 11-31% of the cardiac beta-adrenoceptors had a high affinity for ICI 118.551. These data suggest that the normal equine ventricular myocardium possesses predominately beta1-adrenoceptors, with no evidence for co-existence of a significant population of beta2-adrenoceptors. The density of beta-adrenoceptors did not appear to change in heart failure, but the appearance of receptors with a high affinity for ICI118.551 may suggest that, in some cases, heart failure increases the expression of beta2-adrenoceptors in equine ventricular myocardium. This study provides an insight into the role of the adrenergic system in heart disease in the horse. Further studies in this area are warranted.     

Serum concentrations of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in clinically normal dogs and dogs with acute and chronic renal failure

OBJECTIVE: To compare serum concentrations of 1,25-dihydroxycholecalciferol (1,25-[OH]2D3) and 25-hydroxycholecalciferol (25-[OH]D3) in healthy control dogs and dogs with naturally occurring acute renal failure (ARF) and chronic renal failure (CRF). ANIMALS: 24 control dogs, 10 dogs with ARF, and 40 dogs with CRF. PROCEDURE: Serum concentrations of 1,25-(OH)2D3 were measured by use of a quantitative radioimmunoassay, and serum concentrations of 25-(OH)D3 were measured by use of a protein-binding assay. RESULTS: Mean +/- SD serum concentration of 1,25-(OH)2D3 was 153 +/- 50 pmol/L in control dogs, 75 +/- 25 pmol/L in dogs with ARF, and 93 +/- 67 pmol/L in dogs with CRF. The concentration of 1,25-(OH)2D3 did not differ significantly between dogs with ARF and those with CRF and was in the reference range in most dogs; however, the concentration was significantly lower in dogs with ARF or CRF, compared with the concentration in control dogs. Mean +/- SD concentration of 25-(OH)D3 was 267 +/- 97 nmol/L in control dogs, 130 +/- 82 nmol/L in dogs with ARF, and 84 +/- 60 nmol/L in dogs with CRF. The concentration of 25-(OH)D3 was significantly lower in dogs with ARF or CRF, compared with the concentration in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The concentration of 1,25-(OH)2D3 was within the reference range in most dogs with renal failure. Increased serum concentrations of parathyroid hormone indicated a relative deficiency of 1,25-(OH)2D3. A decrease in the serum concentration of 25-(OH)D3 in dogs with CRF appeared to be attributable to reduced intake and increased urinary loss.     

Aspects of pharmacodynamics and biotransformation of the glucocorticoid resocortol butyrate

Aspects of the biotransformation and pharmacodynamics of the novel glucocorticoid resocortol butyrate (RCB) and its metabolites were assessed in vitro and in vivo in comparison with selected reference compounds. The main route of biotransformation of ((3)H)-RCB in the skin and the liver was 5alpha-reduction of the A-ring followed by reduction of the 3-carbonyl group. In the liver, metabolism was much more rapid than in the skin and 5beta-reduction also occurred. RCB had a relative binding affinity for the glucocorticoid receptor similar to that of triamcinolone acetonide, about 1.5 times that of dexamethasone, three times that of betamethasone valerate (BMV) and 10-14 times that of cortisol. The metabolites of RCB displayed only low to very low affinities for the receptor. The suppression of the hypothalamic-pituitary-adrenal axis was investigated in placebo- and positive-controlled studies in dogs by measurement of basal and corticotrophin-releasing hormone (CRH) stimulated plasma cortisol concentrations. The AUC of the plasma cortisol vs. time curve following CRH stimulation, a measure of adrenal suppression, was reduced significantly after topical application of BMV compared with the pretreatment values. The AUC in the RCB group was not reduced significantly. Adrenocorticotrophic hormone concentrations were not affected. Oral administration of RCB did not suppress adrenocortical function, whereas BMV induced almost complete suppression of basal and CRH-induced cortisol concentrations. The pharmacodynamics of RCB makes it a relatively safe glucocorticosteroid for topical application.     

Canine plasma cortisol (hydrocortisone) measured by radioimmunoassay: clinical absence of diurnal variation and results of ACTH stimulation and dexamethasone suppression tests.

A radioimmunoassay for plasma cortisol (hydrocortisone) was developed and validated for sensitivity, specificity, accuracy, precision, and parallelism. Steroids were extracted with ethyl ether, and cortisol was purified by gel column chromatography prior to assay. [1,2-3H] cortisol and a commercially available sheep antibody to cortisol-21-hemisuccinate were used. Free steriods were separated from bound steroids by centrifugation after adsorption to dextran-coated charcoal. Plasma cortisol was measured by this technique in 6 normal dogs. Circadian rhythm of cortisol secretion was not detected in samples obtained by venipuncture at 8 different hours on 3 separate days, suggesting that adrenal function tests may be started in clinical patients at any time of day. Resting plasma cortisol concentrations averaged 19.4+/-3.0 (SD) ng/ml and ranged from nondetectable (less than 3 ng/ml) to 77.5 ng/ml. Of 144 canine plasma samples, 95% contained less than 50 ng of cortisol/ml. Intramuscular injection of 2.2 units of adrenocorticotropic hormone/kg of body weight caused detectable increase in plasma cortisol concentrations; maximum response (68.3 to 111.6 ng/ml) occurred 1 to 2 hours after injection. Oral administration of dexamethasone suppressed plasma cortisol to nondetectable concentrations for 32 hours in all 6 dogs.     

Antagonism of adenosine receptors by caffeine and caffeine metabolites in equine forebrain tissues

OBJECTIVE: To determine the presence of adenosine receptor subtypes A1 and A2a in equine forebrain tissues and to characterize the interactions of caffeine and its metabolites with adenosine receptors in the CNS of horses. SAMPLE POPULATION: Brain tissue specimens obtained during necropsy from 5 adult male research horses. PROCEDURE: Membrane-enriched homogenates from cerebral cortex and striatum were evaluated by radioligand binding assays with the A1-selective ligand [3H]DPCPX and the A2a-selective ligand [3H]ZM241385. Functional responses to adenosine receptor agonists and antagonists were determined by a nucleotide exchange assay using [35S]-guanosine 5'-(gamma-thio) triphosphate ([35S]GTPgammaS). RESULTS: Saturable high affinity [3H]DPCPX binding (A1) sites were detected in cerebral cortex and striatum, whereas high-affinity [3H]ZM241385 binding (A2a) sites were detected only in striatum. Caffeine and related methylxanthines had similar binding affinities at A1 and A2a sites with rank orders of drug binding affinities (theophylline > paraxanthine > or = caffeine > theobromine) similar to other species. [35S]GTPgammaS exchange revealed that caffeine and its metabolites act as pure adenosine receptor antagonists at concentrations that correspond to A1 and A2a receptor binding affinities. CONCLUSIONS AND CLINICAL RELEVANCE: Results of our study affirm the presence of guanine nucleotide binding protein linked adenosine receptors (ie, high-affinity A1 and A2a adenosine receptors) in equine forebrain tissues and reveal the antagonistic actions by caffeine and several biologically active caffeine metabolites. Antagonism of adenosine actions in the equine CNS by these stimulants may be responsible for some central actions of methylxanthine drugs, including motor stimulation and enhanced racing performance.     

Qualification of the Stewart variables for the assessment of the acid-base status in healthy dogs and dogs with different diseases

Peter Stewart criticized the traditional theory of the acid-base status by Henderson-Hasselbalch as too simple and incomplete. He developed a new model with 3 independent variables: (1) pCO2, (2) SID (strong ion difference) and (3) Atot (Acid total). In healthy and ill dogs the diagnostic usefulness of both acid-base models were compared. This study included n=58 healthy dogs and 3 clinical cases of sick dogs.The age of the healthy dogs was 5.0 (2.0-7.0) years (= median (1.-3. quartil)).The 3 clinical cases included (1) a dog with septic shock, (2) with acute renal insufficiency, and (3) with hypovolaemic shock due to gastric torsion.Venous blood was taken of all dogs and the acid-base parameters were determined within < or =30 minutes. Electrolytes and albumin were determined in blood serum and used for calculation of the Stewart variables. Limits of reference intervals (x+/-1.96 - s) were determined for the healthy dogs yielding pCO2 = 3.6-6.5 kPa, [SID3] = 33.1-50.9 mmol/l resp. [SID4] = 31.8-49.6 mmol/l and [Al = 8.5-13.1 mmol/l. In Case 1 the Henderson-Hasselbalch parameters demonstrated the presence of a strong metabolic acidosis with mild respiratory influence (pH, [HCO3-], [BE] and PCO2 at upper range of normal). Analysis of the Stewart variables [SID3] resp. [SID4] revealed an electrolyte imbalance with [Cl-] and [lactate-] as the reason for metabolic acidosis. Case 2 showed a metabolic acidosis with respiratory compensation (pH, [HCO3-], [BE] and PCO2). Analysis of the Stewart variables with [SID3] resp. [SID4 caused by [K+], [Na+] and [lactate-]demonstrated the acidotic metabolism due to a renal malfunction. Case 3 had a metabolic acidosis (pH-value in the lower range) caused by electrolyte imbalances ([SID4]. The Stewart variables allow a better understanding of the causes of acid-base-disturbances in animals with implications for successful therapy via infusion.     

Effects of colostrum feeding and glucocorticoid administration on insulin-dependent glucose metabolism in neonatal calves

Colostrum feeding and glucocorticoid administration affect glucose metabolism and insulin release in calves. We have tested the hypothesis that dexamethasone as well as colostrum feeding influence insulin-dependent glucose metabolism in neonatal calves using the euglycemic-hyperinsulinemic clamp technique. Newborn calves were fed either colostrum or a milk-based formula (n=14 per group) and in each feeding group, half of the calves were treated with dexamethasone (30 microg/[kg body weight per day]). Preprandial blood samples were taken on days 1, 2, and 4. On day 5, insulin was infused for 3h and plasma glucose concentrations were kept at 5 mmol/L+/-10%. Clamps were combined with [(13)C]-bicarbonate and [6,6-(2)H]-glucose infusions for 5.5h (i.e., from -150 to 180 min, relative to insulin infusion) to determine glucose turnover, glucose appearance rate (Ra), endogenous glucose production (eGP), and gluconeogenesis before and at the end of the clamp. After the clamp liver biopsies were taken to measure mRNA levels of phosphoenolpyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC). Dexamethasone increased plasma glucose, insulin, and glucagon concentrations in the pre-clamp period thus necessitating a reduction in the rate of glucose infusion to maintain euglycemia during the clamp. Glucose turnover and Ra increased during the clamp and were lower at the end of the clamp in dexamethasone-treated calves. Dexamethasone treatment did not affect basal gluconeogenesis or eGP. At the end of the clamp, dexamethasone reduced eGP and PC mRNA levels, whereas mitochondrial PEPCK mRNA levels increased. In conclusion, insulin increased glucose turnover and dexamethasone impaired insulin-dependent glucose metabolism, and this was independent of different feeding.     

Phagocytosis and intracellular killing of Staphylococcus aureus by bovine blood polymorphonuclear leukocytes after migration in vitro

Phagocytic activity and intracellular killing of opsonized staphylococci (Smith strain) by bovine blood polymorphonuclear leukocytes (PMNL) was studied after their migration in vitro in blind well chambers. The results indicate that migration of PMNL to RPMI-1640 medium without chemotactic factor significantly (P less than 0.01) increased the percentage of rosette-forming PMNL (from 11 +/- 2 to 49 +/- 4%), as well as phagocytic activity mediated through Fc receptors (FcRs) (from 25 +/- 3 to 81 +/- 4% phagocytizing PMNL and from 151 +/- 22 to 942 +/- 54 number of phagocytized staphylococci/100 PMNL), and intracellular killing of bacteria (from 13 +/- 2 to 59 +/- 7%). On the other hand, PMNL migration to RPMI-1640 medium with the chemotactic factor (serum activated with zymosan [AS] or lipopolysaccharide [LPS] or formyl-L-methionyl-L-leucyl-L-phenylalanine [fMLP]) did not significantly (p greater than 0.05) increase either the FcRs number on the PMNL surface or the phagocytic and bactericidal activity connected with these receptors. The possible mechanisms are discussed.