RESUMEN
OBJECTIVE: To investigate the effects of castration on the expression of endothelins (ETs), ET receptors and ET converting enzyme-1 (ECE-1) in the rat seminal vesicle (RSV). MATERIALS AND METHODS: Sprague-Dawley rats (3 months old) were surgically castrated or sham-operated, and then killed 7 days after surgery. Biochemical and pharmacological properties and the location of ET receptors in the RSV were determined by a series of binding experiments with [125I]ET-1, using membrane particulates and slide-mounted frozen sections of RSV. Expression levels of ETA and ETB receptor subtypes, ET-1, ET-3 and ECE-1 mRNAs were assessed by relative multiplex reverse-transcription polymerase chain reaction (RT-PCR). RESULTS: The density of total ET receptors increased significantly in the seminal vesicle of the castrated rat. The predominance of the ETA receptor subtype in the RSV did not change with castration. Autoradiographic studies showed the presence of ET receptors on the smooth muscle and epithelium of the RSV. In addition, RT-PCR showed an up-regulation in the expression of ETA and ETB receptor subtypes, ET-1 and ECE-1 mRNAs in the seminal vesicle of the castrated rat. However, castration caused no significant change in the expression levels of ET-3 mRNA. CONCLUSION: These findings suggest a regulatory role for testosterone in the expression of the ET receptor system in the RSV.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Castración , Endotelinas/metabolismo , Receptores de Endotelina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Vesículas Seminales/metabolismo , Animales , Autorradiografía/métodos , Enzimas Convertidoras de Endotelina , Masculino , Metaloendopeptidasas , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Endothelins (ETs) mediate paracrine control of vascular tone and secretion of steroids and catecholamines in the adrenal gland through two ET receptor subtypes, ETA and ETB. The differential distribution and function of these subtypes are responsible for the multiplicity of endothelin actions in this tissue. This study examines the regulatory effects of experimental diabetes on the gene expression, subtype specificity and localization of ET receptor subtypes, ET isopeptides, and endothelin-converting enzyme-1 (ECE-1) in the rat adrenal gland. The densities, pharmacological properties and distribution of ET receptor subtypes ETA and ETB in adrenal glands from streptozotocin-induced diabetic, insulin-treated diabetic and age-matched control rats were investigated, using radioligand receptor binding and autoradiographic techniques. The gene expression of ETA and ETB receptors ET-1, ET-3 and ECE-1 was evaluated using relative multiplex reverse transcription/PCR. The induction of diabetes caused a marked reduction in body weight but no significant change in adrenal gland size. The density of ET receptors was significantly increased in the diabetic rat adrenal gland, mainly because of an increase in the expression of ETB receptors. Insulin treatment normalized the diabetes-induced changes in the expression levels of ET receptor subtypes to control levels. The expression level of ET-1 mRNA was up-regulated, whereas ET-3 mRNA was down-regulated in the diabetic adrenal gland compared with the controls. The ECE-1 mRNA level in the adrenal gland was not altered by the induction of diabetes. Autoradiographic studies showed that ETA and ETB are the predominant receptor subtypes in the adrenal medulla and cortex respectively. These results suggest that ETA and ETB receptors are differentially distributed and regulated in the diabetic rat adrenal gland.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Diabetes Mellitus Experimental/metabolismo , Endotelina-1/metabolismo , Receptores de Endotelina/metabolismo , Animales , Autorradiografía , Unión Competitiva , Endotelina-3/metabolismo , Expresión Génica , Masculino , Unión Proteica , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores de Endotelina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
OBJECTIVE: To examine the effect of age on the biochemical and functional properties, and regional distribution of endothelin (ET) receptors in the rabbit renal pelvis. MATERIALS AND METHODS: The properties of ET receptors in 6-week-old and 6-month-old male rabbit renal pelves were examined using isolated muscle-bath and radioligand receptor-binding techniques. RESULTS: ET-1 caused a significant increase in the contractile force in muscle strips from all regions of the renal pelvis from both age groups, with the following rank order: upper=middle>lower. The magnitude of the ET-1-induced contractile responses were similar in the lower pelvic regions in both ages, but the responses in the upper and middle regions were significantly greater in younger rabbits. ET-1 increased the frequency of spontaneous activity in a concentration-dependent manner in the upper and middle pelvic regions in both age groups, with significantly smaller ED50 values in the younger than in the older rabbits. In both age groups the lower pelvic region lacked spontaneous activity. The density of total ET receptors was higher in the upper and middle regions of the renal pelvis than in the lower renal pelvis of both ages, with the density in the upper and middle regions being greater in older than in younger rabbits. In all regions, ET subtype selective compounds inhibited [125I]ET-1, binding consistent with the predominance of the ETA receptor subtype, except in the lower region of the older rabbits, in which the densities of ETA and ETB subtypes were similar. In all regions, the younger renal pelvis contained a higher proportion of ETA receptors than in older tissues. Light microscopic autoradiographic data indicated the presence of ETA and ETB receptors in smooth muscle and epithelial cells, respectively. CONCLUSION: These data indicate the presence of regional differences in the density of ET receptors and in the contractile responses to ET-1 in rabbit renal pelvis, and that although older rabbit renal pelvis contains more total ET receptors than younger renal pelvis, the latter had a higher portion of the ETA receptor subtype and the younger tissues were more responsive to ET-1.
Asunto(s)
Envejecimiento/metabolismo , Pelvis Renal/metabolismo , Receptores de Endotelina/metabolismo , Animales , Autorradiografía/métodos , Endotelina-1/farmacología , Pelvis Renal/crecimiento & desarrollo , Masculino , Contracción Muscular/efectos de los fármacos , Conejos , Ensayo de Unión Radioligante/métodosRESUMEN
Streptozotocin (STZ)-induced diabetes causes an upregulation in the expression of endothelin (ET) receptors in the rat prostate (Eur J Pharmacol 310:197, 1996). We examined the effects of insulin treatment, started 8 weeks after the induction of diabetes, on the expression and distribution of ET receptors and their respective mRNAs in the rat prostate. The densities, pharmacological properties and distribution of ET receptors in the rat prostate were examined using radioligand receptor binding and autoradiographic studies, and gene expression of ET receptors was evaluated utilizing the reverse transcription-polymerase chain reaction (RT-PCR). STZ-injected rats had smaller prostates and reduced serum testosterone levels than control and insulin treated diabetic animals. ET receptor density was shown to be significantly higher in the prostate from diabetic rats than those from either control or insulin treated diabetic animals. The pharmacological profile of prostatic ET receptors was similar in all groups (approximately 80% ET(A); 20% ET(B) subtype). ET receptors were predominantly localized to the prostatic stroma. Induction of diabetes increased the expression of mRNA levels of ET(A) and ET receptors, and insulin treatment reversed this upregulation to control levels. These results indicate that (1) ET receptor subtypes are expressed in the rat prostate as transcription and translation products; (2) insulin can normalize the diabetes-induced upregulation in the expression of ET receptors and their respective mRNAs; and (3) diabetes-induced regression of the prostate may involve an alteration in ET receptors.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Insulina/farmacología , Próstata/metabolismo , Receptores de Endotelina/metabolismo , Animales , Autorradiografía , Glucemia/análisis , Peso Corporal , Diabetes Mellitus Experimental/tratamiento farmacológico , Antagonistas de los Receptores de Endotelina , Endotelina-1/metabolismo , Insulina/administración & dosificación , Insulina/sangre , Radioisótopos de Yodo , Masculino , Tamaño de los Órganos , Próstata/efectos de los fármacos , Isoformas de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/agonistas , Receptores de Endotelina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Testosterona/sangre , Regulación hacia ArribaRESUMEN
PURPOSE: As there are significant amounts of endothelin (ET) receptors in the mammalian urinary tract, we investigated the pharmacological properties and localization of ET receptors in the rabbit lower urinary tract as a function of age. MATERIALS AND METHODS: The characteristics of ET receptors in bladder dome, trigone and urethra of 6 weeks and 6 months old male rabbits were determined using muscle bath and autoradiographic techniques. RESULTS: ET-1 produces significant contractile responses in smooth muscle strips from bladder dome, trigone, and urethra in both 6 weeks and 6 months old rabbits. Although there was no significant difference in the maximum contractile response of urethral muscle strips to ET-1 between 6 weeks and 6 months old rabbits, the maximum responses to ET-1 were higher in both bladder dome and trigone of 6 weeks than 6 months old rabbits. A selective ETA receptor antagonist, BQ 610, shifted the concentration response curve to ET-1 to the right without decreasing maximal contractile responses in all regions from both age groups, whereas a selective ETB receptor antagonist, IRL 1038, had no significant effect on the contractile response in these tissues. Autoradiographic studies indicate that both ET receptor subtypes are expressed in bladder dome, trigone, and urethra with the ETA subtype being located only in the smooth muscle layers and the ETB subtype being located in both the urothelial and smooth muscle layers. CONCLUSION: Our data indicate the presence of region- and age-dependent differences in the contractile properties of ET receptors in the male rabbit lower urinary tract. Although both ETA and ETB receptor subtypes are present in the smooth muscle layers, the ETA receptor is the sub-type that is primarily involved in the mediation of contractions.
Asunto(s)
Envejecimiento/fisiología , Endotelina-1/farmacología , Contracción Muscular/efectos de los fármacos , Uretra/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacos , Vasoconstrictores/farmacología , Análisis de Varianza , Animales , Autorradiografía , Antagonistas de los Receptores de Endotelina , Endotelina-1/antagonistas & inhibidores , Endotelinas/farmacología , Masculino , Músculo Liso/efectos de los fármacos , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Conejos , Receptores de Endotelina/clasificación , Receptores de Endotelina/efectos de los fármacos , Urotelio/efectos de los fármacosRESUMEN
PURPOSE: Transforming growth factor-beta (TGF-beta), a potent inhibitor of cell growth, plays an important role in the androgen-dependent processes of the prostate through a complex network of growth factors. TGF-beta expression in the prostate is under negative regulatory control of androgen. As experimental diabetes causes a regression of the prostate and decrease in serum testosterone levels in rats, we examined TGF-beta alterations at the mRNA and protein levels in the diabetic rat prostate. MATERIALS AND METHODS: The expression of TGF-beta1 and TGF-beta2 and their respective mRNAs in prostates from streptozotocin (STZ)-induced diabetic, insulin-treated diabetic and age-matched control rats were investigated, using relative multiplex RT-PCR, semi-quantitative Western blotting, and immunohistochemistry. RESULTS: Induction of diabetes caused a significant reduction in prostatic weight and in serum testosterone levels in rats. Both mRNA and protein levels of TGF-beta1, and mRNA level of TGF-beta2 were up-regulated in the diabetic rat prostate. Insulin-treatment normalized changes observed in prostatic weight and serum testosterone levels, and reversed the alterations in the TGF-beta1 and TGF-beta2 expression at the gene transcript and protein levels to control levels. Immunohistochemical studies demonstrated that TGF-beta1 is localized to prostatic stromal cells, whereas TGF-beta2 is located in both epithelial and stromal cells. CONCLUSION: These results suggest that TGF-beta1 and TGF-beta2 may be involved in the diabetes-induced regression of the prostate gland.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Próstata/crecimiento & desarrollo , Próstata/metabolismo , Factor de Crecimiento Transformador beta/biosíntesis , Animales , Masculino , Próstata/química , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Factor de Crecimiento Transformador beta/genéticaRESUMEN
OBJECTIVE: To assess fibroblast growth factor-2 (FGF2/bFGF), which is important in the development and maintenance of the normal prostate and in the development of human benign prostatic hyperplasia (BPH) and prostatic carcinoma, in an animal model of experimentally induced diabetes. Materials and methods Using Western blotting and immunohistochemical analyses, the expression of FGF2 in prostates from several groups of rats was investigated. Rats had diabetes for 8 or 16 weeks (induced by intravenous injection with 65 mg/kg streptozotocin); rats were also treated with insulin (starting 8 weeks after the induction of diabetes, for 8 weeks), and two further groups acted as age-matched control rats. Immunohistochemical markers for smooth muscle (alpha-actin) and epithelium (cytokeratin) were used to distinguish different cell types in adjacent prostatic sections. RESULTS: Diabetic rats had smaller prostates and lower serum testosterone levels than their controls; insulin treatment of diabetic rats increased prostatic size and testosterone levels. As shown by Western blotting, diabetes caused greater FGF2 expression than in controls, whereas reverse-transcriptase polymerase chain reaction studies showed similar levels of prostatic FGF-2 mRNA in all groups. Immuno-histochemical studies showed that FGF-2 was expressed in both stromal and epithelial components of the rat prostate. Furthermore, although the expression of FGF2 was higher in epithelial than stromal cells in control prostates, it was distributed uniformly in the diabetic prostate. CONCLUSION: The differences in the level of expression and pattern of distribution of FGF2 suggests a potential role for FGF2 in the changes observed in prostatic growth in diabetic rats.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Próstata/metabolismo , Animales , Western Blotting , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
As there are significant amounts of functional endothelin receptors in the mammalian urinary tract, we examined the effect of experimental diabetes on the expression of endothelin receptors and their mRNAs in the rat bladder dome. The density of endothelin receptors in the rat bladder dome was higher (8 and 16 weeks following the onset of diabetes) than in age-matched controls. Insulin treatment, started 8 weeks after the induction of diabetes, partially reversed the endothelin receptor alterations. The pharmacological profile of the endothelin receptors in the bladder dome was similar in all groups and was consistent with the predominance of the endothelin ET(A) receptor subtype (ET(A):ET(B)=approximately 4:1). Autoradiographic studies demonstrated that the endothelin receptors were located in all tissue components of the bladder, including epithelial and muscular layers. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) data indicated that diabetes increased the expression level of gene transcripts for both endothelin receptor subtypes and that insulin treatment reversed the mRNA upregulation.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , ARN Mensajero/análisis , Receptores de Endotelina/análisis , Vejiga Urinaria/química , Animales , Autorradiografía , Endotelina-1/metabolismo , Masculino , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Endotelina/genética , EstreptozocinaRESUMEN
Since evidence of development/age-related alterations of endothelin receptors in circulation and respiration systems has been increasing, we attempted to investigate the pharmacological characterization of endothelin receptors in neonatal, premature, and mature male rabbit lower urinary tract. The biochemical properties of ET receptors were examined in the lower urinary tracts of 1-day (neonatal)-, 6-week (premature)-, and 1-year(mature)-old male rabbits with binding technique utilizing [(125)I]ET-1. The rank orders of the densities (B(max) values) of endothelin receptors in the bladder dome, bladder base, and urethra of different aged rabbits were bladder dome, 1 day > 6 week &vbar;Ls 1 year, bladder base, 1 day > 6 week &vbar;Ls 1 year, and urethra, 1 day > 6 week > 1 year. The pharmacological profiles of these binding sites inhibited by various kinds of endothelin receptor compounds showed similar K(i) values and similar proportions of endothelin receptor subtypes in the same regions of 1-day-, 6-week-, and 1-year-old animals. Our data clearly demonstrated the presence of regional difference and development-related changes in the density and subtype specificity of endothelin receptors in the lower urinary tract of the male rabbit. Neurourol. Urodynam. 19:71-85, 2000.
Asunto(s)
Envejecimiento/metabolismo , Animales Recién Nacidos/metabolismo , Receptores de Endotelina/metabolismo , Sistema Urinario/metabolismo , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Sitios de Unión , Unión Competitiva , Endotelina-1/metabolismo , Endotelina-3/metabolismo , Endotelinas/agonistas , Endotelinas/antagonistas & inhibidores , Endotelinas/metabolismo , Masculino , Oligopéptidos/metabolismo , Tamaño de los Órganos , Fragmentos de Péptidos/metabolismo , Conejos , Uretra/anatomía & histología , Uretra/crecimiento & desarrollo , Vejiga Urinaria/anatomía & histología , Vejiga Urinaria/crecimiento & desarrolloRESUMEN
BACKGROUND: The present investigation was undertaken to examine the effect of insulin treatment on diabetes-induced alterations in endothelin (ET) receptors in rat ureters. METHODS: The biochemical properties of ET receptors were examined in rat ureters from the following groups: 8 weeks diabetic (D8); 8 weeks age-matched control (C8); 16 weeks diabetic (D16); 16 weeks diabetic-insulin treated (insulin started 8 weeks after the onset of diabetes) (DI16); and 16 weeks age-matched control (C16). Diabetes was induced by the i.v. injection of 65 mg/kg streptozotocin (STZ). RESULTS: The densities of ET receptors (Bmax values), as determined by saturation experiments with [125I]-ET-1, in the ureteral plasma membranes of D8, C8, D16, DI16 and C16 were 91.7 +/- 10.1, 42.1 +/- 7.2, 71.1 +/- 2.4, 51.5 +/- 6.3 and 45.1 +/- 3.3 fmol/mg of protein, respectively. [125I]-ET-1 binding to the ET receptors in rat ureteral membrane particulates was inhibited by ET-1 (non-selective), ET-3 (ET(B/C selective), BQ610 (ET(A) selective) and IRL 1620 (ET(B) selective) with the following rank order of Ki values: ET-1 < BQ 610 < ET-3 << IRL 1620. The pharmacological profile of the ET receptors was similar in all groups examined and was consistent with the predominance of the ET(A) receptor subtype in the ureteral membrane particulates. The subtype specificity of ET receptors in the ureteral tissues is confirmed with inhibition data obtained from similar binding studies in cloned human ET(A) and ET(B) receptors. CONCLUSION: The data indicate that diabetes results in an up-regulation of ET receptors in the rat ureter, which is normalized by insulin treatment.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Receptores de Endotelina/biosíntesis , Uréter/metabolismo , Animales , Membrana Celular/metabolismo , Cricetinae , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Antagonistas de los Receptores de Endotelina , Estudios de Seguimiento , Humanos , Masculino , Tamaño de los Órganos , Ratas , Ratas Sprague-Dawley , Estreptozocina/toxicidad , Regulación hacia Arriba , Uréter/patologíaRESUMEN
PURPOSE: The subtype specificity, localization and distribution of urethral alpha1-adrenoceptors were studied in the male rabbit urethra. MATERIALS AND METHODS: The properties of the urethral alpha1-adrenoceptors were investigated using radioligand receptor binding and light microscopic autoradiography with [125I]iodo-2-[b-(4-hydroxyphenyl)-ethylaminomethyl]tetralone (HEAT), and immunohistochemistry with monoclonal anti-alpha smooth muscle actin and anti-alpha sarcomeric actin antibodies. RESULTS: Saturation experiments with [125I]HEAT demonstrated the presence of significant amounts of a single high affinity binding site for alpha1 adrenoceptors in the male rabbit urethra. The pharmacological profile of the alpha1 adrenoceptors in rabbit urethra, determined by inhibition experiments with subtype selective alpha1 adrenoceptor antagonists, was characterized by the following rank order of potency of inhibition constants (Ki values): prazosin < or = WB 4101 < spiperone < 5-methylurapidil < BMY 7378. The pKi values for the rabbit urethra were correlated with the pKi values for rat spleen, submaxillary glands, and vas deferens and for those reported for cloned alpha1d receptors with correlation coefficients of 0.68, 0.929, 0.909, and 0.523, respectively. CONCLUSIONS: The pharmacological characterization demonstrates the predominance of alpha1A or alpha1A + alpha1B adrenoceptor subtype(s) in male rabbit urethral smooth muscle. Furthermore, the autoradiographic and immunohistochemical studies show a heterogeneous distribution of alpha1 adrenoceptors along the longitudinal axis of the urethra, within the smooth muscle fibers, with the receptors being localized more densely in the proximal than in the distal urethra.
Asunto(s)
Receptores Adrenérgicos alfa 1/análisis , Uretra/química , Antagonistas de Receptores Adrenérgicos alfa 1 , Animales , Autorradiografía , Dioxanos/farmacocinética , Inmunohistoquímica , Masculino , Piperazinas/farmacocinética , Prazosina/farmacocinética , Conejos , Espiperona/farmacocinéticaRESUMEN
We studied the effects of 8 weeks of streptozotocin (STZ)-induced diabetes on the density and the pharmacological properties of alpha1-adrenoceptors in the rat prostate using receptor-binding experiments with [125I]iodo-2[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone [125I]HEAT. Saturation experiments showed the presence of specific [125I]HEAT-binding sites in the control and diabetic rat prostate and that the induction of diabetes significantly decreased the density of [125I]HEAT-binding sites in the rat prostate. [125I]HEAT-binding sites in the prostate of both groups were inhibited by prazosin (nonselective), spiperone (alpha1B-selective), WB4101 and 5-methylurapidil (alpha1A-selective) and BMY7378 (alpha1D-selective) with the following rank order of Ki values: prazosin < WB4101 < 5-methylurapidil < spiperone < BMY7378, indicating a similar pharmacological profile of alpha1-adrenoceptor in the 2 groups. Comparing the Ki values of the rat prostate with those obtained from the rat submaxillary gland (alpha1A), rat spleen (alpha1B), rat vas deferens (alpha1A + alpha1B) and those reported for cloned alpha1D, indicates the predominance of the alpha1A + alpha1B or the alpha1A subtype in the rat prostate. The present study demonstrates that STZ-induced diabetes downregulates the expression of alpha1-adrenoceptor in the rat prostate, without significantly affecting the receptor subtype specificity.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Próstata/metabolismo , Receptores Adrenérgicos alfa/clasificación , Receptores Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Análisis de Varianza , Animales , Sitios de Unión , Técnicas de Cultivo , Diabetes Mellitus Experimental/inducido químicamente , Modelos Animales de Enfermedad , Regulación hacia Abajo , Modelos Lineales , Masculino , Próstata/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Adrenérgicos alfa/efectos de los fármacos , Valores de Referencia , Bazo/metabolismo , Estreptozocina , Glándula Submandibular/metabolismo , Conducto Deferente/metabolismoRESUMEN
To elucidate the subtype specificity of muscarinic cholinergic receptors in mediating contractile responses in the lower urinary tract, we investigated contractile and biochemical properties of muscarinic receptors in bladder dome, bladder base and urethra of the rabbit. Isometric contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype selective muscarinic antagonists. Bladder dome, bladder base, and urethra demonstrate different characteristics in terms of efficacy and potency with respect to carbachol-induced contractile responses. Emax values are significantly larger and ED50 values are significantly smaller in bladder dome and bladder base than in urethra. Calculation of the pA2 values, the negative logarithm of the antagonist affinity constant (KB), for a series of muscarinic antagonists, i.e., atropine (nonselective), pirenzepine (M1 selective), methoctramine (M2 selective), and 4-DAMP (M1/M3 selective) indicate that the carbachol-induced contractile response in bladder dome and bladder base is mediated through the M3 receptor subtype whereas the carbachol-induced contractile response in urethra is probably mediated through the M1 and/or M3 and possibly M2 subtypes. Muscarinic cholinergic antagonists inhibit [3H]quinulidinyl benzilate binding to bladder dome, bladder base and urethra with the following rank order of affinities: atropine > 4-DAMP > methoctramine > pirenzepine. The binding data indicate the predominance of the M2 receptor subtype in all three regions.
Asunto(s)
Receptores Muscarínicos/fisiología , Uretra/fisiología , Vejiga Urinaria/fisiología , Animales , Atropina/farmacología , Carbacol/farmacología , Diaminas/farmacología , Femenino , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Parasimpatolíticos/farmacología , Pirenzepina/farmacología , Conejos , Receptores Muscarínicos/efectos de los fármacos , Especificidad de la Especie , Uretra/química , Uretra/efectos de los fármacos , Vejiga Urinaria/química , Vejiga Urinaria/efectos de los fármacosRESUMEN
We investigated the binding characteristics of endothelin (ET) receptors in the ureters of rats with experimentally induced diabetes and diuresis. Receptor binding experiments demonstrated an upregulation in the expression of [125I]ET-1 binding sites in the diabetic rat ureter but not in the diuretic rat ureter. ET-1, ET-3, IRL 1620, and BQ 610 inhibited [125I]ET-binding to the rat ureter consistent with the predominance of ETA receptors in these tissues. The subtype specificity of ET receptors in ureteral tissues was confirmed with inhibition data obtained from cloned human ETA and ETB receptors.
Asunto(s)
Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Receptores de Endotelina/genética , Uréter/metabolismo , Animales , Células CHO , Clonación Molecular , Cricetinae , Endotelina-1/metabolismo , Endotelina-3/metabolismo , Endotelinas/metabolismo , Humanos , Masculino , Oligopéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Ratas , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/clasificación , Receptores de Endotelina/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: The purpose of this study was to evaluate the muscarinic receptor subtypes expressed in rat bladder smooth muscle and characterize the muscarinic receptor-coupled phosphatidylinositol (PI) hydrolysis in order to clarify the first step of bladder smooth muscle contraction. METHODS: Expressions of mRNAs of muscarinic receptor subtypes were examined by Northern blot analysis. Changes in the mass of inositol 1,4,5-trisphosphate (IP3) and the inhibitory effects of muscarinic subtype specific antagonists on PI hydrolysis were determined after carbachol stimulation. RESULTS: mRNAs of m2 and m3 genes, encoding M2 and M3 receptors, were expressed in rat bladder smooth muscle. Carbachol produced a rapid increase of IP3, which returned to the basal level within 30 seconds. 4-Diphenylacetoxyl-N-methylpiperidine methiodide (4-DAMP; M1 and M3 antagonist) strongly inhibited the PI hydrolysis, but methoctramine (M2 antagonist) partially inhibited it at 10(-4) mol/L. The IC50 value for atropine was 9.5 x 10(-9) mol/L, for pirenzepine 6.4 x 10(-6) mol/L, and for 4-DAMP 1.5 x 10(-7) mol/L. CONCLUSION: M2 and M3 receptors are expressed in rat urinary bladder. Only M3 receptor was involved in the production of IP3, which might induce the initial phase of contractile response in rat bladder smooth muscle after carbachol stimulation.
Asunto(s)
Músculo Liso/metabolismo , Fosfatidilinositoles/metabolismo , ARN Mensajero/metabolismo , Receptores Muscarínicos/metabolismo , Vejiga Urinaria/metabolismo , Animales , Northern Blotting , Cartilla de ADN/química , Expresión Génica , Hidrólisis , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Contracción Muscular/fisiología , Músculo Liso/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/genética , Vejiga Urinaria/citologíaRESUMEN
As there is increasing evidence that diabetes induces changes in the plasma levels of endothelins (ETs) and in the properties of ET receptors in peripheral tissues, and as there are reports indicating the presence of significant amounts of ET receptors in mammalian vasa deferentia, we studied possible alterations in ET receptor characteristics in the vasa deferentia of the following groups of rats: 8 weeks diabetic (D8), 8 weeks age-matched control (C8), 16 weeks diabetic (D16), 16 weeks diabetic-insulin-treated (started 8 weeks after the onset of diabetes) (DI16), and 16 weeks age-matched control (C16). Diabetes was induced by the i.v. injection of 65 mg/kg streptozotocin (STZ). Diabetic rats had hyperglycemia, hypoinsulinemia, glucosuria, polydipsia, and polyuria and had smaller vasa deferentia than control and diabetic-insulin-treated animals. Receptor binding experiments with [125I]ET-1 demonstrated that the densities of ET receptors in vasa deferentia from D8, C8, D16, DI16, and C16 animals were 377 +/- 11, 255 +/- 24, 315 +/- 18, 210 +/- 12, and 214 +/- 7 fmol/mg of protein, respectively. [125I]ET-1 binding to the ET receptors was inhibited by ET-1 (non-selective), BQ 610 (ETA selective), ET-3 (ETC selective), and IRL 1620 (ETB selective) with the following rank order of Ki values: ET-1 < BQ 610 < ET-3 < < IRL 1620. The pharmacological profile of the ET receptors was similar in all groups and was consistent with the predominance of the ETA receptor subtype in the rat vasa deferentia. Our data indicate that experimental diabetes up-regulates the density of ET receptors in the rat vasa deferentia and that the receptor up-regulation is reversed by insulin treatment.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Receptores de Endotelina/metabolismo , Conducto Deferente/metabolismo , Animales , Diabetes Mellitus Experimental/tratamiento farmacológico , Endotelina-1/antagonistas & inhibidores , Endotelina-1/metabolismo , Endotelina-1/farmacología , Endotelinas/farmacología , Insulina/uso terapéutico , Cinética , Masculino , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Endotelina A , Receptor de Endotelina B , Receptores de Endotelina/efectos de los fármacos , Factores de Tiempo , Regulación hacia Arriba , Conducto Deferente/efectos de los fármacosRESUMEN
We studied the properties of endothelin receptors in the prostate of 8-week streptozotocin-diabetic and control rats. The density of endothelin receptors, as determined by saturation experiments with [125I]endothelin-1, were 95.8 +/- 5.4 and 171.3 +/- 16.7 fmol/mg of protein in control and diabetic rat prostates, respectively. The pharmacological profile of the endothelin receptors was similar in both groups and was consistent with the predominance of the endothelin ETA receptor subtype in the prostate. Thus, the induction of diabetes upregulates the expression of endothelin receptors in the rat prostate, but does not alter the pharmacological profile of the receptors in these tissues.
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Diabetes Mellitus Experimental/metabolismo , Próstata/metabolismo , Receptores de Endotelina/metabolismo , Animales , Peso Corporal , Masculino , Tamaño de los Órganos , Próstata/fisiopatología , Ratas , Ratas Sprague-DawleyRESUMEN
We previously have shown an increase in muscarinic receptor density in streptozotocin (STZ)-induced diabetic and sucrose-fed diuretic rat detrusor that correlates with an increase in the contractile response to muscarinic agonist (J Pharmacol Exp Ther 248:81, 1989; Diabetes 40: 265, 1991). To investigate the signal transduction pathway involved in this altered functional response, we examined muscarinic receptor-coupled phosphatidylinositol metabolism in STZ-diabetic, sucrose-fed diuretic and age-matched control rat bladders. [3H]myo-inositol uptake was similar in all groups, but incorporation of myo-inositol into phosphatidylinositol (PI) was significantly increased in the diabetic bladder compared to the sucrose-fed and control rat bladders. Carbachol-induced increase in inositol phosphate (IPs) production was higher in the diabetic bladder than in bladders from control and sucrose-fed animals although the EC50 values were similar for all groups. Enhanced inositol phosphate production after muscarinic agonist stimulation may be due not only to the upregulation of muscarinic receptors but also the increased incorporation of myo-inositol into PI in the STZ-induced diabetic bladder.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Fosfatidilinositoles/metabolismo , Receptores Muscarínicos/metabolismo , Transducción de Señal , Vejiga Urinaria/metabolismo , Animales , Hidrólisis , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
We investigated the binding characteristics of endothelin (ET) receptors in rabbit ureter, bladder dome, bladder base, and urethra and compared the observed receptor properties with those of cloned human ETA and ETB receptors expressed in Chinese hamster ovary K-1 (CHO) cells. Receptor binding experiments with [125I]ET-1 revealed the presence of a single class of specific, saturable, high affinity [125I]ET-1 binding sites in all of the regions of the studied urinary tract. The rank order of the densities (Bmax values) of [125I]ET-1 binding sites was: ureter "bladder dome > bladder base = urethra. ET-1 and ET-2 inhibited [125I]ET-1 binding to the membrane particulates from the various regions of the urinary tract with single high affinity constants. A selective ETA receptor antagonist, BQ 123, and selective ETB agonists, ET-3 and sarafotoxin S6c (STXc), inhibited [125I]ET-1 binding to bladder dome, bladder base, and urethra with high and low affinity constants indicating the presence of both ETA and ETB receptor subtypes in these tissues. The subtype specificity of ET receptors in the rabbit tissues is confirmed with inhibition data obtained from similar binding studies in cloned human ETA and ETB receptors. The proportions of high affinity binding sites for ET-3, representing ETB receptors, were approximately 25%, 27%, and 46% in bladder dome, bladder base, and urethra, respectively. Corresponding values for STXc were approximately 17%, 28%, and 43% in bladder dome, bladder base, and urethra, respectively. In contrast to the findings for ET-3 and STXc, the proportions of high affinity binding sites for BQ 123, representing ETA receptors, in bladder dome, bladder base, and urethra were approximately 84%, 74%, and 60%, respectively. In ureter, these selective compounds inhibited [125I]ET-1 binding with either a low (ET-3 and STXc) or a high binding affinity (BQ 123), suggesting the presence of only a single receptor subtype (ETA) in this tissue. These data indicate that there are regional differences in the density and subtype specificity of ET receptors in the rabbit urinary tract.
Asunto(s)
Receptores de Endotelina/metabolismo , Sistema Urinario/metabolismo , Animales , Sitios de Unión , Células CHO , Clonación Molecular , Cricetinae , Endotelinas/metabolismo , Femenino , Radioisótopos de Yodo , Conejos , Ensayo de Unión Radioligante , Receptores de Endotelina/genéticaRESUMEN
The relaxant effects of norepinephrine (NE, 10(-7) to 10(-4) M.) and isoproterenol (ISO, 10(-9) to 10(-4) M.) on maximal KCl-induced tonic contractions and the relaxant effects of ISO on contractions induced by electrical field stimulation (EFS) were measured in detrusor muscle strips obtained from 22-25 day, 90-95 day and 22-month-old male Fischer 344 rats. The maximum relaxant response to NE and ISO on KCl-induced tonic contractions decreased significantly with increasing age. The ED50 values for ISO, but not NE, increased with age. The maximum relaxant response to ISO on EFS-induced contractions also was reduced significantly in the old bladders. The relaxation effects of forskolin (10(-6) to 3 x 10(-5) M.), dibutyryl cyclic AMP (DBcAMP, 10(-4) to 3 x 10(-3) M.) and cholera toxin (10 micrograms/ml.) were examined on maximal KCl-induced contractions of the muscle strips obtained from the three age groups. The relaxant responses to forskolin decreased significantly with increasing age, whereas DBcAMP relaxed the muscle strips from the three age groups equally. Cholera toxin (10 micrograms) attenuated KCl-induced phasic contractions, and this effect was impaired in the aged rat detrusor. The density of beta-adrenergic receptors, as determined by radioligand binding with [125I]iodopindolol ([125I]-PIN) decreased with increasing age. These data demonstrate an age-related decrease in the responsiveness of the bladder detrusor to beta-adrenergic stimulation that may be related to the decreased density of beta-adrenergic receptors and decreased cyclic AMP (cAMP) production.