RESUMEN
This first-in-dog study evaluates the use of the PET-radioligand [11C]DASB to image the density and availability of the serotonin transporter (SERT) in the canine brain. Imaging the serotonergic system could improve diagnosis and therapy of multiple canine behavioural disorders. Furthermore, as many similarities are reported between several human neuropsychiatric conditions and naturally occurring canine behavioural disorders, making this tracer available for use in dogs also provide researchers an interesting non-primate animal model to investigate human disorders. Five adult beagles underwent a 90 minutes dynamic PET scan and arterial whole blood was sampled throughout the scan. For each ROI, the distribution volume (VT), obtained via the one- and two- tissue compartment model (1-TC, 2-TC) and the Logan Plot, was calculated and the goodness-of-fit was evaluated by the Akaike Information Criterion (AIC). For the preferred compartmental model BPND values were estimated and compared with those derived by four reference tissue models: 4-parameter RTM, SRTM2, MRTM2 and the Logan reference tissue model. The 2-TC model indicated in 61% of the ROIs a better fit compared to the 1-TC model. The Logan plot produced almost identical VT values and can be used as an alternative. Compared with the 2-TC model, all investigated reference tissue models showed high correlations but small underestimations of the BPND-parameter. The highest correlation was achieved with the Logan reference tissue model (Y = 0.9266 x + 0.0257; R2 = 0.9722). Therefore, this model can be put forward as a non-invasive standard model for future PET-experiments with [11C]DASB in dogs.
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Bencilaminas/análisis , Química Encefálica , Tomografía de Emisión de Positrones/veterinaria , Animales , Bencilaminas/sangre , Perros , Femenino , Ligandos , Masculino , Neuroimagen/veterinaria , Ensayo de Unión Radioligante/veterinaria , Valores de Referencia , Proteínas de Transporte de Serotonina en la Membrana Plasmática/análisis , Proteínas de Transporte de Serotonina en la Membrana Plasmática/sangre , Distribución TisularRESUMEN
OBJECTIVE: To evaluate anti-inflammatory effects of several novel adenosine receptor agonists and to determine their specificity for various adenosine receptor subtypes on neutrophils, cells heterologously expressing equine adenosine receptors, or equine brain membranes. SAMPLE POPULATION: Neutrophils isolated from 8 healthy horses. PROCEDURES: Radioligand binding experiments were performed to compare binding affinities of adenosine receptor agonists to equine adenosine A(1), A(2A), and A(3) receptor subtypes. Effects of these agonists on endotoxin-induced production of reactive oxygen species (ROS) by equine neutrophils and roles of specific adenosine receptor subtypes and cAMP production in mediating these effects were determined. RESULTS: Radioligand binding experiments yielded a ranked order of affinity for the brain equine A(2A) receptor on the basis of 50% inhibitory concentrations (IC(50)) of the agonists as follows: ATL307 (IC(50) = 1.9nM) and ATL313 > ATL309 and ATL310 > ATL202 > 2-([p-2- carboxyethyl] phenylethylamino)-5'-N-ethylcarboxyamidoadenosine > 5'-N-ethylcarboxamidoadenosine. Furthermore, ATL313 had approximately 100-fold greater selectivity for A(2A) over A(1) and A(3) receptors. In functional assays with equine neutrophils, the compounds inhibited endotoxin-induced ROS production and stimulated production of cAMP with the same ranked order of potency. Results of experiments performed with selective adenosine receptor antagonists indicated that functional effects of ATL313 were via stimulation of A(2A) receptors. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that activation of A(2A) receptors exerted anti-inflammatory effects on equine neutrophils and that stable, highly selective adenosine A(2A) receptor agonists may be developed for use in management of horses and other domestic animals with septic and nonseptic inflammatory diseases.
Asunto(s)
Agonistas del Receptor de Adenosina A2 , Caballos/inmunología , Neutrófilos/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Unión Competitiva , AMP Cíclico/inmunología , Concentración 50 Inhibidora , Cinética , Lipopolisacáridos/inmunología , Neutrófilos/inmunología , Fenetilaminas/farmacología , Piperidinas/farmacología , Ensayo de Unión Radioligante/veterinaria , Especies Reactivas de Oxígeno/inmunología , Receptor de Adenosina A2A/inmunología , Receptor de Adenosina A2A/metabolismo , Xantinas/farmacologíaRESUMEN
The aim of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA), for determination of serum thymidine kinase 1 (sTK1) activity in dogs with malignant lymphoma (ML) and compare it with a thymidine kinase (TK) radioenzymatic assay (TK-REA). The TK-REA has recently been shown to be useful in determining the clinical stage and prognosis in canine ML. In addition, serum lactate dehydrogenase (LDH) was measured. Forty-five dogs were included in the study. Sixty serum samples from these dogs, stored in a tumour serum sample bank (stored at -20 degrees C), were analysed. Apart from 37 dogs with ML, four normal dogs as well as two dogs with mammary carcinomas, one dog with bladder carcinoma, and one dog with malignant fibrous histiocytoma were included. Staging of ML was based on the modified World Health Organization (WHO) staging system for canine ML. The diagnosis of all tumours was verified by histopathology. The TK activity (units per litre [U/L]) ranged from 1.0 to 607.9 in the TK-REA analysis and from 1.1 to 510 in the TK-ELISA (normal reference value <7U/L). The range for LDH was between 12 and 1194 U/L (normal reference value <228 U/L). There was a significant correlation between the TK-REA and the TK-ELISA. The correlation coefficient (CC) was 0.97 and the standard error of the estimate (SEE) was 3.7 U/L. There was no correlation between LDH and either the TK-REA or the TK-ELISA (CC=0.53 for both assays; SEE=26.7 and 12.7 U/L, respectively). Most of the variation in LDH was still within the normal reference range. The mean LDH in dogs with high-stage (stage IV+V) disease was 201.9 U/L. The corresponding values for the TK-REA and TK-ELISA were 109 and 109.9 U/L, respectively. The significant relation between the TK-REA and the TK-ELISA was confirmed by Bland-Altman analysis. The TK-ELISA assay, because of its relative simplicity, will permit measurement of TK in cases of ML in dogs to become a routine procedure.
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Enfermedades de los Perros/sangre , Enfermedades de los Perros/enzimología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Linfoma/sangre , Linfoma/veterinaria , Ensayo de Unión Radioligante/veterinaria , Timidina Quinasa/sangre , Animales , Biomarcadores de Tumor/sangre , Perros , Femenino , Linfoma/enzimología , Masculino , Estadificación de NeoplasiasRESUMEN
The objective of this study was to characterize porcine beta1- and beta2-adrenergic receptors (beta1-AR and beta2-AR) in heart, skeletal muscle, and adipose tissue by measuring the binding of a radioligand to cell membrane fragments. In skeletal muscle (LM), [3H]CGP12177 labeled a homogeneous population of beta2-AR as evidenced by the rank order of affinity of catecholamines [(-)isoproterenol > (-)epinephrine > (-)norepinephrine], a high affinity of the binding site for the beta2-AR-agonist clenbuterol (equilibrium dissociation constant, Kd = 16 nM), and a low affinity of the binding site for the beta1-AR-antagonist CGP20712A (Kd = 21 microM). The affinity of ICI118551, a ligand selective for beta2-AR in other species, was uncharacteristically low in porcine LM (Kd = 441 nM), but was consistent with a value reported for the cloned porcine beta2-AR. In heart ventricle, ligand binding revealed a predominant population of beta1-AR, judged by the rank order of affinity of catecholamines [(-)isoproterenol > (-)epinephrine > or = (-)norepinephrine] and high-affinity binding to CGP20712A (Kd = 40 nM). The Kd for ICI118551 (731 nM) was close to that observed at beta2-AR in LM, confirming that ICI118551 is not subtype-selective in the pig. Displacement studies using (-)propranolol, clenbuterol, and (-)isoproterenol revealed a second high-affinity binding site in the heart that was not a beta2-AR and could not be eliminated by guanosine 5'-triphosphate or guanylyli-midodiphosphate. In adipose tissue, an equal number of beta1- and beta2-AR was identified through the binding of clenbuterol and CGP20712A, whereas ICI118551 could not discriminate between these sites. In further experiments, we used 10 microM CGP20712A to eliminate beta1-AR binding and allow accurate Kd values to be determined at beta2-AR for nonselective ligands. Under these conditions, another binding site was observed that had a high affinity for (-)propranolol (Kd = 20 pM), which is inconsistent with beta3- or beta4-AR binding reported elsewhere. Our results indicate that porcine adipose tissue contains beta1-AR, beta2-AR, and an atypical binding site in the proportions 50, 34, and 16%, respectively, of the total binding sites labeled by [3H]CGP12177.
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Tejido Adiposo/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Porcinos/metabolismo , Agonistas Adrenérgicos/farmacología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/fisiología , Células CHO , Cricetinae , Cricetulus , Masculino , Propanolaminas/metabolismo , Ensayo de Unión Radioligante/veterinaria , Receptores Adrenérgicos beta 1/efectos de los fármacos , Receptores Adrenérgicos beta 2/efectos de los fármacosRESUMEN
We report the production of recombinant possum prolactin (posPrl), and its use in the development and validation of a highly specific homologous radioimmunoassay for the measurement of prolactin (Prl) in brushtail possums. This enabled the subsequent investigation of some basic mechanisms involved in the regulation of Prl secretion in this species. Recombinant posPrl spanning the entire coding region was expressed in Escherichia coli, resulting in a 199 amino acid protein with a molecular weight approximately 23 kDa. The potency of posPrl was 45.3 +/- 4.8% that of ovine Prl in a radioreceptor assay using possum mammary gland receptors and induced a 3.4 +/- 0.8-fold increase in progesterone secretion in primary possum granulosa cells. Antiserum (G27) was raised against recombinant posPrl and was highly specific for possum Prl (approximately 30% binding at 1:60,000 final dilution), and exhibited negligible cross-reactivity (<0.0001%) with possum growth hormone. Serial dilutions of pituitary gland extracts, and plasma samples from male and female possums gave parallel inhibition curves to recombinant posPrl standards in the assay. Biological validation of the RIA included treating possums with drugs known to alter Prl secretion in other mammals. In seasonally anoestrous female possums, administration of 20 microg thyrotropin-releasing hormone (TRH) resulted in a 15-fold increase (P < 0.01) in plasma Prl concentrations. In mid-late lactating female possums, a bolus of cabergoline (dopamine agonist; 75 microg) reduced (P < 0.05) plasma Prl levels to baseline for 24 h, while repeated administration (6 x 75 microg at 12 h intervals) suppressed (P < 0.01) plasma Prl concentrations until 24h after the last injection. Prolonged inhibition of Prl levels subsequently caused marked (P < 0.01) attenuation in rate of bodyweight increase of pouch young. The amplitude of the Prl surge in response to a bolus of TRH (15 microg) was 5-fold lower in cabergoline-treated, compared to control mid-late lactating possums. In conclusion, we report the development and validation of a robust and sensitive RIA for measuring Prl concentrations in the plasma of brushtail possums.
Asunto(s)
Zarigüeyas/fisiología , Prolactina/fisiología , Radioinmunoensayo/veterinaria , Animales , Bioensayo/veterinaria , Western Blotting/veterinaria , Cabergolina , Agonistas de Dopamina/farmacología , Ergolinas/farmacología , Femenino , Células de la Granulosa , Masculino , Nueva Zelanda , Zarigüeyas/metabolismo , Progesterona/análisis , Prolactina/análisis , Prolactina/química , Prolactina/genética , ARN/química , ARN/genética , Radioinmunoensayo/métodos , Ensayo de Unión Radioligante/veterinaria , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Hormona Liberadora de Tirotropina/farmacologíaRESUMEN
In order to study the effect of estradiol-17beta (E2) on estrogen (E) and progesterone (P) receptors expression in oviduct and cervix of lambs, their respective transcripts (ERalpha mRNA and PR mRNA) were determined by solution hybridization and the receptor proteins (ER and PR) by binding assays after E2 treatments. Lambs (n=4 in each group) were not treated or treated with one, two or three i.m. injections of E2 (1 microg/kg) at 24 h of interval. Tissues were obtained 12 or 24 h after the last E2 injection. Estradiol treatments increased ERalpha mRNA and PR mRNA concentrations in an organ-dependent manner: transitory in the oviduct while maintained in the cervix. The E2 effect on the oviductal and cervical ER and PR concentrations were biphasic, with an initial reduction of receptors content that was followed by restoration. The ER restoration in oviduct was earlier than in the cervix. In summary, this study shows that E2 treatments may exert an inductive effect in ERalpha mRNA and PR mRNA levels and a biphasic effect in ER and PR concentrations in oviduct and cervix of immature ewe. These E2 effects varied in timing and strength depending on the organ of the reproductive tract.
Asunto(s)
Cuello del Útero/efectos de los fármacos , Estradiol/farmacología , Receptor alfa de Estrógeno/biosíntesis , Trompas Uterinas/efectos de los fármacos , Receptores de Progesterona/biosíntesis , Ovinos/metabolismo , Animales , Animales Lactantes , Cuello del Útero/metabolismo , Receptor alfa de Estrógeno/genética , Trompas Uterinas/metabolismo , Femenino , Modelos Lineales , Hibridación de Ácido Nucleico , Tamaño de los Órganos/fisiología , ARN Mensajero/genética , Ensayo de Unión Radioligante/veterinaria , Distribución Aleatoria , Receptores de Progesterona/genéticaRESUMEN
Serum thymidine kinase (sTK) activity was evaluated as a tumor marker for canine malignant lymphoma (ML). The objective was to investigate if sTK, as in humans, could be used as a prognostic marker for survival time in dogs with ML and if sTK could identify early signs of progression of disease in treated dogs. Serum samples from 52 dogs with ML were tested for initial TK activity. Samples from 21 normal dogs and 25 dogs with nonhematologic neoplasms were used for comparison. Forty-four dogs with ML were treated. Serum TK activity was measured in treated dogs before each treatment and every 4 weeks thereafter until relapse. Dogs with ML had 2-180 times higher TK activity (TK 5-900 U/L) than normal dogs (TK <7 U/L) based on the mean + 2 standard deviations. In the group of other neoplasms, only 2 dogs had a moderate increase (6.4 and 7.5 U/L) compared with the controls. Mean sTK activities in the dogs with ML that had gone into complete remission (CR) were not significantly different from activities in healthy controls (P = .68). Mean sTK at least 3 weeks before and at the time of relapse was significantly higher than activity measured at CR (P < .0001). Dogs with ML that initially had sTK >30 U/L had significantly shorter survival times (P < .0001). Furthermore, sTK activity reflected the clinical staging of ML. Measuring sTK can be used as a powerful objective tumor marker for prognosis and for predicting relapse before recurrence of clinically detectable disease in dogs with ML undergoing chemotherapy.
Asunto(s)
Biomarcadores de Tumor/sangre , Enfermedades de los Perros/enzimología , Linfoma/veterinaria , Timidina Quinasa/sangre , Animales , Enfermedades de los Perros/diagnóstico , Perros , Femenino , Linfoma/diagnóstico , Linfoma/enzimología , Masculino , Pronóstico , Ensayo de Unión Radioligante/veterinaria , Inducción de RemisiónRESUMEN
Estrogens are important regulators of physiological functions. Although environmental contaminants (xenoestrogens) which interfere with estrogen signaling are of increasing concern, there is only limited information about their ability to interact with estrogen-binding proteins (SHBG) or receptors (ER). Recombinant ERalpha and beta were obtained after transient transfection of COS-7 cells with channel catfish ER cDNA. Plasma from adult female channel catfish was the source of SHBG. Tritiated estradiol (3H-E2) was used in standard radioligand-binding assays to characterize the binding properties of channel catfish SHBG (ccfSHBG) and to estimate the inhibition constants for various estrogenic compounds. Binding of 3H-E2 to ccfSHBG was saturable and of high affinity with a Kd (+/-SE) of 1.9+/-0.14 nM and a Bmax of 14.3+/-2.4 pmol/mg protein ( n = 3 assays). Additionally, ccfSHBG displayed binding specificity for androgens and estrogens. Endosulfan, 4-nonylphenol, and 4-octylphenol displaced 3H-E2 binding to ccfSHBG albeit only at very high concentrations, whereas dieldrin and atrazine showed little displacement activity even at the highest concentrations used. The synthetic estrogen ethynylestradiol had higher affinity than E2 for ccfSHBG. This finding differs from results with human and rainbow trout SHBG. The alkylphenolic compounds (4-octylphenol and 4-nonylphenol) displayed some ability to displace 3H-E2 binding from ERalpha and beta at high concentrations, but dieldrin and atrazine had little binding activity for both ER subtypes and endosulfan for ERbeta. The xenobiotics tested generally showed equivalent or greater affinity for ERalpha than ERbeta, whereas natural estrogens had much greater affinity for ERbeta than ERalpha. These observations suggest that results of studies using fish tissue ER extracts must be interpreted with caution, since both ER subtypes may be present, and that the binding of xenoestrogens to SHBG must be taken into account for proper assessment of endocrine disruption caused by environmental contaminants.
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Ictaluridae/metabolismo , Receptores de Estrógenos/metabolismo , Globulina de Unión a Hormona Sexual/metabolismo , Xenobióticos/farmacología , Animales , Atrazina/farmacología , Unión Competitiva , Células COS , Chlorocebus aethiops , Dieldrín/farmacología , Endosulfano/farmacología , Receptor alfa de Estrógeno , Receptor beta de Estrógeno , Etinilestradiol/farmacología , Femenino , Ictaluridae/sangre , Fenoles/farmacología , Unión Proteica , Ensayo de Unión Radioligante/veterinaria , Proteínas RecombinantesRESUMEN
OBJECTIVE: To test the hypothesis that the distribution, density, and subtype of opioid and alpha (alpha)-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 alpha-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 degrees C. Receptor autoradiography was performed using established techniques. [3H]DAMGO, [3H]U-69593, and [3H]RX821002 were used for mu ( micro )-opioid, kappa (kappa)-opioid, and alpha-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 micro -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 kappa-opioid receptors in the frontal cortex of dogs compared to horses, whereas binding to kappa-opioid receptors in the cerebellum was higher in horses. Binding to alpha-2 adrenergic receptors in the mid-brain was significantly higher in dogs than in horses. There was higher binding of alpha-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 alpha-2 adrenergic agonists in these species.
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Encéfalo/metabolismo , Perros/metabolismo , Caballos/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Receptores Opioides/metabolismo , Animales , Autorradiografía/veterinaria , Femenino , Masculino , Estudios Prospectivos , Ensayo de Unión Radioligante/veterinaria , Factores SexualesRESUMEN
Xylazine is an alpha2-adrenoceptor agonist sedative with a much higher interspecies variability in effect than detomidine, another alpha2-agonist used in veterinary practice. In the present study, we have used radioligand binding in brain tissue to investigate if the high species variation in sensitivity to xylazine could be explained in terms of receptor interactions. Species known to be more (cattle) or less (swine and rats) sensitive to xylazine were used. There was no variation in the density or the subtype pattern of the alpha2-adrenoceptors that could explain the species variation recorded in vivo, as a homogenous population of the alpha2A/D-subtype (200-300 fmol/mg protein) was found in all species. The species differences in the affinities of xylazine and detomidine were minor and similar for the two drugs. The only parameter investigated where a significant species difference was found for xylazine but not for detomidine was the slope of the inhibition binding curve when the G-protein coupling was diminished. For xylazine this slope was considerably lower than unity (i.e. 0.77 +/- 0.075) using cattle preparations compared with 0.92 +/- 0.037 (mean +/- SE) and 0.90 +/- 0.028, respectively for swine and rats, while for detomidine this parameter was close to unity in all species (cattle, swine, rat). This finding indicates that the species variation in effect for xylazine could be due to differences at the G-protein level or further down-stream in the effect cascade.
Asunto(s)
Agonistas alfa-Adrenérgicos/farmacología , Encéfalo/efectos de los fármacos , Imidazoles/farmacología , Receptores Adrenérgicos alfa 2/efectos de los fármacos , Xilazina/farmacología , Agonistas alfa-Adrenérgicos/administración & dosificación , Animales , Encéfalo/metabolismo , Tronco Encefálico/efectos de los fármacos , Tronco Encefálico/metabolismo , Bovinos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Imidazoles/administración & dosificación , Masculino , Ensayo de Unión Radioligante/veterinaria , Ratas/metabolismo , Especificidad de la Especie , Porcinos/metabolismo , Xilazina/administración & dosificaciónRESUMEN
Goldfish prolactin cDNA was subcloned into a pRSET A vector and expressed in Escherichia coli. Recombinant goldfish prolactin was expressed mainly as insoluble inclusion bodies in the form of N-terminal 6x His-tagged fusion protein. This fusion protein was purified, refolded, and (125)I-labeled to generate a radioligand for receptor binding and validation of a radioimmunoassay for goldfish prolactin. Using goldfish gill membrane as the substrate for prolactin receptor binding, both recombinant and native forms of goldfish prolactin were effective in displacing the specific binding of the radioligand in a similar dose range, suggesting that the fusion protein was refolded properly and could be recognized by goldfish prolactin receptors. To quantify prolactin contents in biological samples from the goldfish, a radioimmunoassay using the (125)I-labeled recombinant prolactin as a tracer was established. This assay was shown to be selective for goldfish prolactin without cross-reactivity with mammalian prolactin and pituitary hormones from other fish species (e.g., growth hormone and gonadotropin II). This newly validated assay system was used to investigate neuroendocrine and signal transduction mechanisms regulating prolactin release in the goldfish. In this case, the Ca(2+) ionophore A23187 and protein kinase C activator TPA were effective in elevating basal levels of prolactin secretion in perifused goldfish pituitary cells. In parallel studies using a static incubation approach, somatostatin and dopamine, but not vasoactive intestinal polypeptide, were inhibitory to basal prolactin release in goldfish pituitary cells. These results suggest that somatostatin and dopamine may serve as negative regulators of basal prolactin secretion and that extracellular Ca(2+) influx and protein kinase C activation may be important signaling events mediating prolactin release in the goldfish.
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Carpa Dorada/metabolismo , Prolactina/biosíntesis , Radioinmunoensayo/veterinaria , Ensayo de Unión Radioligante/veterinaria , Animales , Calcimicina/farmacología , Células Cultivadas , Escherichia coli/genética , Ionóforos/farmacología , Cinética , Prolactina/análisis , Prolactina/genética , Radioinmunoensayo/métodos , Ensayo de Unión Radioligante/métodos , Proteínas Recombinantes , Reproducibilidad de los Resultados , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The equilibrium dissociation constant (Kd) and the maximum binding capacity (Bmax) of receptors for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) and calcitonin (CT) in the membrane fraction of the calvaria and the kidney of chickens were examined from 1 d after the hatch up to 24 wk of age by the use of radioligand binding assays. The Kd values of the PTH/PTHrP receptor in both tissues were decreased at 10 and 24 wk in female birds, whereas the values were increased at 24 wk in male birds. The Bmax values of the PTH/PTHrP receptor in both tissues were decreased at 10 wk and returned to baseline at 24 wk in female birds. The values were increased at 24 wk in male birds. The Kd and Bmax values of CT receptors in the both tissues were constant during the experimental period in female and male birds. The results suggest that the binding properties of PTH/PTHrP receptor and of CT receptor may be influenced by gonadal hormones relating to sexual maturation.
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Pollos/crecimiento & desarrollo , Riñón/metabolismo , Receptores de Calcitonina/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Cráneo/metabolismo , Factores de Edad , Animales , Calcitonina , Pollos/metabolismo , Femenino , Cinética , Masculino , Hormona Paratiroidea/metabolismo , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/metabolismo , Ensayo de Unión Radioligante/veterinaria , Receptor de Hormona Paratiroídea Tipo 1 , Maduración Sexual/fisiologíaRESUMEN
Estrogen receptors (ER) were determined by both the biochemical dextran-coated charcoal (DCC-ER) and the immunohistochemical Avidin biotin-peroxidase complex (IHC-ER) methods in proliferative mammary lesions collected from 37 cats: 20 malignant tumors without metastasis at first presentation, seven malignant tumors with lung and/or lymph node metastases and 10 benign tumors and dysplasias. Total number of samples analyzed by both methods was 44. The DCC-ER method was applied to frozen tissue samples and the IHC-ER method was applied to neutral buffered formalin-fixed, paraffin wax-embedded tissue samples by using the NCL-6F11 monoclonal antibody. Biochemically, 21 (47.7%) cases had equal or more than 5 fmol/mg of protein (standard positivity threshold). Immunohistochemically, 11 (25%) cases were scored positive, the percentage of positive nuclei being statistically linked to the intensity of immunostaining. Normal mammary gland tissue (13 cases) and/or dysplastic areas (5 cases) found in the surroundings of the main lesion were IHC-ER positive in 76.9% and 40% of the cases, respectively. Concordance between DCC-ER and IHC-DCC was 72.7% and the results of the DCC and the IHC-ER methods were significantly correlated (P < 0.05) by the chi2 test. Specificity (true negatives) and sensitivity (true positives) of the ICH-ER method were 95.6% and 47.6%, respectively. One out of eleven DCC-ER positive and IHC-ER negative discordant cases (9.09%) was a DCC-ER false positive, because the surrounding normal mammary gland tissue was IHC-ER positive. The remaining 10 cases had ER content values equal or lower than 23 fmol/mg of protein, a figure that could represent the sensitivity threshold of the immunohistochemical method employed.
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Enfermedades de los Gatos/diagnóstico , Neoplasias Mamarias Animales/diagnóstico , Receptores de Estrógenos/análisis , Adenocarcinoma/diagnóstico , Adenocarcinoma/secundario , Adenocarcinoma/veterinaria , Animales , Anticuerpos Monoclonales , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/secundario , Carcinoma Papilar/veterinaria , Gatos , Dilatación Patológica/diagnóstico , Dilatación Patológica/veterinaria , Femenino , Hiperplasia/diagnóstico , Hiperplasia/veterinaria , Inmunohistoquímica , Neoplasias Pulmonares/secundario , Metástasis Linfática , Glándulas Mamarias Animales/química , Neoplasias Mamarias Animales/química , Neoplasias Mamarias Animales/patología , Ensayo de Unión Radioligante/veterinaria , Receptores de Estrógenos/química , Sensibilidad y EspecificidadRESUMEN
This study characterizes the alpha2-adrenergic receptors present in canine brainstem. Radioligand binding and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were performed in canine brainstem to identify the receptors present and determine the pharmacological properties of these receptors. The pKi values derived from radioligand competition curves for a number of adrenergic receptor agents at the four alpha2-adrenergic receptor subtypes were compared to the canine brainstem. The pKi values at the canine brainstem alpha2-adrenergic receptor were consistent with the presence of the alpha2A-adrenergic receptor. To determine whether the canine brainstem expressed the message for the alpha2A-adrenergic receptor, RT-PCR was performed with specific primers for the four subtypes of alpha2-adrenergic receptors. In the canine brainstem, only the primers corresponding to a region in the human alpha2A-adrenergic receptor produced a PCR product. No bands were detected in the canine brainstem lanes with the alpha2B-, alpha2C-, or alpha2D-receptor primers. These data suggest that the canine brainstem contains the alpha2A-adrenergic receptor.
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Antagonistas de Receptores Adrenérgicos alfa 2 , Tronco Encefálico/metabolismo , Perros/metabolismo , Receptores Adrenérgicos alfa 2/clasificación , Animales , Animales Recién Nacidos , Tronco Encefálico/citología , Técnicas de Cultivo de Célula , Cartilla de ADN , Pulmón/citología , Ensayo de Unión Radioligante/veterinaria , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa 2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinariaRESUMEN
The efficiency of superovulating mares with an enriched fraction of equine follicle-stimulating hormone (feFSH) and an equine pituitary extract (EPE) with similar FSH content but differing in the LH amount was compared. Mares were randomly assigned to an feFSH (n = 5) or EPE (n = 5) treatment. The experimental period was of 2 successive estrous cycles, with the first cycle as the control. At Days 6 and 7 of the estrous cycle, the mares received 250 micrograms i.m. cloprostenol. The treatments consisted of daily injections of 25 mg feFSH or EPE beginning on Day 6 post ovulation. Mares were inseminated every other day until the last ovulation was detected. When the mares in the control and treatment cycles developed at least 1 or 2 > or = 35-mm follicle, respectively, the treatment was interrupted, and a single injection of EPE (25 mg, i.v.) was administered to induce ovulation(s). Nonsurgical embryo recovery was performed 6 or 7 d after ovulation in both control and treatment cycles. The number of ovulations per mare was not significantly different (P > 0.05) between feFSH and EPE groups, but both were higher (P < 0.05) than that of the control cycle. The number of recovered embryos per ovulation was similar (P > 0.05) for control, feFSH and EPE groups. The high amount of LH presented in EPE did not affect the superovulatory response of the mares. Superovulatory treatments increased the ovulation rate of mares but did not affect the embryo recovery rate per ovulation.
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Fertilización In Vitro/veterinaria , Hormona Folículo Estimulante/fisiología , Caballos/fisiología , Superovulación/fisiología , Animales , Cloprostenol/uso terapéutico , Estro , Femenino , Hormona Folículo Estimulante/análisis , Hormona Luteinizante/análisis , Hormona Luteinizante/fisiología , Masculino , Folículo Ovárico/fisiología , Ovario/diagnóstico por imagen , Fotoperiodo , Hipófisis/química , Hipófisis/metabolismo , Ensayo de Unión Radioligante/veterinaria , Distribución Aleatoria , UltrasonografíaRESUMEN
OBJECTIVE: To identify beta-adrenoceptor subtypes involved in motility inhibition of circular and longitudinal smooth muscle layers of equine ileum. SAMPLE POPULATION: Isolated strips of equine ileum circular smooth muscle and membrane preparations from circular and longitudinal muscle layers. PROCEDURE: Functional assays of circular muscle preparations and radioligand binding assays and measurements of cAMP production in smooth muscle membranes from circular and longitudinal layers. RESULTS: Selective beta-adrenergic agonists exerted inhibitory effects on circular muscle preparations. Binding studies of cell membranes indicated that the density and distribution of 3 beta-adrenoceptor subtypes did not differ between longitudinal and circular muscle layers. Measurement of cAMP production in membrane preparations of longitudinal and circular muscle after selective beta-stimulation confirmed presence of the 3 adenylate cyclase-coupled beta-adrenoceptor subtypes; however, preparations from the 2 layers had differing cAMP production efficacy. CONCLUSIONS: The data may partly explain the differing functional responses between circular and longitudinal muscle preparations. CLINICAL RELEVANCE: Findings support the important role of beta-atypical adrenoceptors in the inhibitory regulation of equine ileum motility.
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Motilidad Gastrointestinal/fisiología , Caballos/fisiología , Íleon/fisiología , Músculo Liso/fisiología , Receptores Adrenérgicos beta/fisiología , Agonistas Adrenérgicos/farmacología , Animales , Membrana Basal/química , Membrana Basal/metabolismo , Membrana Basal/fisiología , Clenbuterol/farmacología , AMP Cíclico/biosíntesis , Dobutamina/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Caballos/metabolismo , Íleon/química , Íleon/metabolismo , Isoproterenol/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Músculo Liso/química , Músculo Liso/metabolismo , Ensayo de Unión Radioligante/veterinaria , Receptores Adrenérgicos beta/análisis , Receptores Adrenérgicos beta/efectos de los fármacosRESUMEN
OBJECTIVE: To identify beta-adrenergic receptor subtypes in ileum smooth muscle of the horse. SAMPLE POPULATION: Isolated strips of equine longitudinal ileum smooth muscle and membrane preparations from smooth muscle of the intestinal wall. PROCEDURE: Functional assays and radioligand binding assays. RESULTS: Relaxation of ileum longitudinal smooth muscle proved to be mainly caused by stimulation of beta-atypical and beta 2-adrenergic receptors. Binding studies on cell membranes indicated that the total beta-adrenergic receptors population consists of 54% beta-atypical, 34% beta 2- and 12% beta 1-subtypes. CONCLUSIONS: The data suggest that sympathetic relaxation of equine ileum smooth muscle depends mainly on beta-atypical receptor subtypes activation, with a minor contribution by beta 2-subtypes. CLINICAL RELEVANCE: The important role of beta-atypical adrenergic receptor subtypes in the relaxation of equine ileum suggests possible clinical use of selective beta-atypical receptor agonists to control intestinal disturbances.
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Caballos/fisiología , Íleon/fisiología , Relajación Muscular/fisiología , Músculo Liso/fisiología , Receptores Adrenérgicos beta/fisiología , Agonistas Adrenérgicos beta/metabolismo , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/metabolismo , Antagonistas Adrenérgicos beta/farmacología , Animales , Unión Competitiva , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Femenino , Íleon/química , Íleon/citología , Masculino , Músculo Liso/química , Músculo Liso/ultraestructura , Propanolaminas/metabolismo , Propanolaminas/farmacología , Unión Proteica , Ensayo de Unión Radioligante/métodos , Ensayo de Unión Radioligante/veterinaria , Receptores Adrenérgicos beta/análisis , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta 1/análisis , Receptores Adrenérgicos beta 1/metabolismo , Receptores Adrenérgicos beta 1/fisiología , Receptores Adrenérgicos beta 2/análisis , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/fisiología , TritioRESUMEN
OBJECTIVE: The purpose of this study was to determine whether an endogenous benzodiazepine receptor ligand (EBZ) was present in the arterial and portal blood of dogs with congenital portosystemic shunts (CPSS). STUDY DESIGN: The presence or absence of an EBZ was determined by the collection of systemic and portal blood from dogs with CPSS. ANIMALS: Fifteen client-owned dogs with a confirmed CPSS. All dogs had historical signs compatible with hepatic encephalopathy. Eight healthy dogs were used as controls. METHODS: In all dogs, systemic blood samples were collected after they were anesthetized. Portal blood samples were collected intraoperatively. EBZ was measured by radioreceptor assay. RESULTS: In 10 of 15 dogs, the portal blood concentration of EBZ was significantly elevated compared with normal dogs (mean, 13.2 +/- 18.55 ng/mL). Five dogs had elevated systemic blood EBZ levels (mean, 8.2 +/- 16.08 ng/mL). Eleven of 15 dogs had a higher portal than systemic blood concentration of EBZ. In contrast, control dogs had extremely low EBZ concentrations detected in their portal blood (mean, 0.16 +/- 0.3 ng/mL) and systemic blood (0 ng/mL). The mean portal and systemic blood concentrations in dogs with CPSS were significantly greater than in control dogs (P < .05). CONCLUSIONS: Elevated blood levels of EBZ were found in dogs with CPSS. The portosystemic gradient noted in 11 dogs suggests the gastrointestinal tract as a possible source for the endogenous ligand. CLINICAL RELEVANCE: Generalized motor seizures have been reported in dogs after surgical correction of CPSS. If the presence of a CPSS results in stimulation of brain receptors for benzodiazepines, post-CPSS ligation seizures may result from a withdrawal of EBZ after ligation of the portosystemic shunt.
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Benzodiazepinas/sangre , Enfermedades de los Perros/sangre , Enfermedades de los Perros/congénito , Perros/anomalías , Sistema Porta/anomalías , Animales , Benzodiazepinas/análisis , Química Encefálica , Anomalías Congénitas/sangre , Anomalías Congénitas/patología , Anomalías Congénitas/veterinaria , Enfermedades de los Perros/patología , Femenino , Masculino , Sistema Porta/química , Ensayo de Unión Radioligante/veterinaria , Receptores de GABA-A/análisisRESUMEN
Methyl farnesoate (MF) binding proteins were identified in the hemolymph of male crabs, Cancer magister, using a tritium-labeled photoaffinity analog of MF, farnesyl diazomethyl ketone (FDK). Crab hemolymph was incubated with [3H]FDK in the presence of increasing amounts of unlabeled MF and the proteins were separated using SDS-PAGE. The associated fluorogram revealed the presence of two specific MF binding proteins with apparent molecular masses of 34 and 44 kDa. MF binding proteins were not detected in other tissues including testes, eyestalks, hepatopancreas, heart, muscle, epidermis, and Y-organs. Unlabeled MF and FDK were capable of displacing [3H]FDK from hemolymph MF binding proteins in a dose-dependent way. The apparent dissociation constant (Kd) of each binding protein for MF and FDK was approximately 65 and 100 nM, respectively, as determined by saturation binding studies. A ligand binding assay followed by Scatchard analysis was used to determine a more accurate apparent Kd value of 145 +/- 10 nM. A single MF binding peak was demonstrated when hemolymph samples incubated with [3H]FDK were electrophoresed under nondenaturing conditions.
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Braquiuros/metabolismo , Proteínas Portadoras/metabolismo , Ácidos Grasos Insaturados/metabolismo , Hemolinfa/metabolismo , Marcadores de Afinidad/análisis , Marcadores de Afinidad/metabolismo , Animales , Proteínas Portadoras/sangre , Diazometano/análogos & derivados , Diazometano/análisis , Diazometano/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida/veterinaria , Farnesol/análogos & derivados , Farnesol/análisis , Farnesol/metabolismo , Ácidos Grasos Insaturados/química , Hemolinfa/química , Masculino , Ensayo de Unión Radioligante/veterinaria , TritioRESUMEN
Recent findings in the African elephant (Loxodonta africana) indicate that the major progestins contained within and biosynthesized by corpora lutea are 5alpha-reduced metabolites and that progesterone is quantitatively of minor importance. The specific gestagenic action within the reproductive tract of elephants was determined by measurement of relative binding affinity of the respective progestins to the gestagen receptor extracted from elephant endometrium. The cytosol was incubated with 40 nmol/liter [3H]ORG-2058 and increasing concentrations of the tested progestin. Progesterone (P4), 5alpha-pregnane-3,20-dione (DHP), and 5alpha-pregnane-3alpha-ol-20-one (5alpha-P-3OH) were used. The competition for binding sites on the progestin receptor was shown by decreasing counts measured after extraction with scintillation fluid. The progestin concentration which induced a 50% reduction of measured counts was estimated (C50) and relative binding affinity of progesterone to other progestins was calculated (RBA = C50progestin/C50p4). The relative binding affinity of DHP to P4 at the gestagen receptor of elephant endometrium was equivalent. The other 5alpha-reduced progestin (5alpha-P-3OH) showed no competition to the [3H]ORG-2058 receptor binding. We conclude that the biological significance of P4 and DHP at the receptor level is very similar. The higher quantitative levels of DHP in corpus luteum and serum support the hypothesis that this progestin is the major gestagen in the elephants, whereas 5alpha-P-3OH is an inactive metabolite.