In vitro alpha-adrenoceptor autoradiography of the urethra and urinary bladder of the female pig, cat, guinea-pig and rat

OBJECTIVE: The aim of this study was to investigate the distribution of alpha1- and alpha2-adrenoceptors in the urethra and urinary bladder of the female pig, cat, guinea-pig and rat. MATERIALS AND METHODS: The binding distributions of an alpha1-adrenoceptor ligand (3H-prazosin) and an alpha2-adrenoceptor ligand (3H-rauwolscine) were determined using in vitro autoradiography. Autoradiograms were analysed by combining computer-based image analysis and light microscopy. RESULTS: In the pig, guinea-pig and rat urethra 3H-prazosin binding was highest in the muscle layer. In the cat urethra 3H-prazosin binding could not be analysed due to a negative chemography artefact. In the pig, cat and guinea-pig urethra 3H-rauwolscine binding was highest in the urothelium, followed by the sub-mucosa, with low levels in muscle. Little 3H-rauwolscine binding was observed in the rat urethra. In the urinary bladder of all species 3H-prazosin binding was low. In the rat bladder, binding was higher in the trigone than in the dome. In the pig, cat and guinea-pig bladder 3H-rauwolscine binding was highest in the mucosa. with little binding in muscle or lamina propria. In the rat bladder, there was little binding and no regional differences. CONCLUSIONS: Alpha1-adrenoceptors were predominantly located in urethral smooth muscle, indicating their contractile importance in maintaining continence. Alpha2-Adrenoceptors were present in the urethral submucosa and bladder mucosa, but not in muscle, suggesting a role in regulation of blood flow, urethral lubrication and tumescence, but not in contraction.     

Autoradiographic localization of α1-adrenoceptors in the dog prostate and urethra with 3H-prazosin

The regional distribution of α₁-adrenoceptors in the dog urethra has been studied by quantitative autoradiography using the specific α₁-antagonist ³H-prazosin as a ligand. The binding of ³H-prazosin to urethral tissue sections was saturable, reversible, and of high affinity (Kd = 0.7 nM); it occurred at a single population of sites and possessed the pharmacological features of the α₁-adrenoceptor. The binding parameters of ³H-prazosin on urethral tissue sections were found similar to those obtained on urethral membranes. Autoradiographic results revealed that the α₁-adrenoceptors are heterogeneously distributed along the dog urethral axis. The highest densities of ³H-prazosin binding sites were found in the preprostatic urethra. Comparison between autoradiographic and histological slides revealed that the ³H-prazosin binding sites were only localized on the smooth muscle fibres of the dog urethra.     

Study of the effects of dopamine hydrochloride on alpha 1-adrenoceptors in rat kidney

We studied the number and affinity of catecholamine receptors in SD rat kidney by radioreceptor technique. The following conclusions were obtained: 1) By using 3H-prazosin, the numbers of alpha 1-receptor (Bmax) in rat renal cortex were greater than those in rat kidney medulla. As for affinity (Kd), the significance was not recognized between the two. Bmax of the rat renal cortex to 3H-prazosin binding was 96.1 fmol/mg protein, and Kd was 0.17 nM, and for the rat renal medullar these values were 44.5 fmol/mg protein and 0.13 nM, respectively. 2) By measurement of D1-receptor using 3H-SKF38393 in the rat renal cortex in the Scatchard plot analysis, positive cooperativity was observed under the low concentration of hot ligand which was less than 1 nM. But at the concentration of hot ligand over 1 nM, the plots showed a straight line. Bmax of the rat renal cortex to 3H-SKF38393 was 2.5 pmol/mg protein and Kd was 5.3 nM. 3) Based on displacement by dopamine for 3H-prazosin binding to rat renal cortex, it was surmised that high concentration of dopamine had an affinity to alpha 1-adrenoceptors. 4) There was no change in the Kd and Bmax of alpha 1-receptor in the rat renal cortex after incubation of samples with low concentration of dopamine. However, in the case of high concentration of dopamine, a remarkable decrease of the affinity (Kd) of alpha 1-adrenoceptor was observed.     

Age-related changes in alpha1-adrenoceptors in rat prostate

The age-related changes in the density of alpha₁-adrenoceptors in the dorsolateral lobe of the rat prostate were evaluated in Wistar rats at 8, 52, and 104 weeks of age. [³H]YM617, a newly senthesized alpha₁-adrenergic blocker, was used as the ligand. The mean maximum number of binding sites, or alpha₁-adrenoceptor density (B_max)±SE of 104-week-old rats (11.0±1.2 fmol/mg protein) was significantly lower than that in the 8-week-old rats (37.0±4.3 fmol/mg protein) and 52-week-old rats (37.2±3.4 fmol/mg protein) respectively, P<0.01. In contrast, mean affinity (K_d) values±SE of these groups showed no significant differences (8-week-old rats, 115.8±9.1; 52-week-old rats, 100.5±5.8; and 104-week-old rats, 116.4±9.8 pM). Mean volumes±SE of muscle cells of the prostate were 3.7±1.1x10³ m³ at 8 weeks, 30.0±6.2x10³ m³ at 52 weeks, and 18.6±8.2x10³ m³ at 104 weeks. Volumes for 8-week-old rats were significantly smaller than those for 52-week-old (P<0.01) and 104-week-old rats (P<0.05). However, the mean area density of the muscle cells showed no difference among the three groups: 20.1±2.2% at 8 weeks, 27.3±2.9% at 52 weeks and 20.3±3.4% at 104 weeks. In conclusion, the density of YM617-binding sites (alpha₁-adrenoceptors) in 104-week-old rats was lower than in 8- and 52-week-old rats. Muscle volume in the rat prostate was larger in rats aged 52 and 104 weeks than in 8-week-old rats, but no correlation was found between alpha₁-adrenoceptor density and the muscle volume or muscle density in aging.     

Dopamine receptors in the developing sheep kidney

These studies were designed to characterize dopamine receptor density and affinity in kidneys removed from sheep of varying ages (fetal, newborn, and adult) using radioligand binding methods. Three different radioligands were used: the specific dopamine-1 antagonist³H-SCH 23390, the dopamine-1/dopamine-2 antagonist³H-haloperidol, and the dopamine-2 antagonist³H-spiroperidol. The specific binding of³H-haloperidol and³H-spiroperidol was saturable with time and ligand concentration, being indicative of dopamine receptors. The specific binding of the dopamine-1 selective radioligand³H-SCH 23390 was also saturable with time but displayed several points of saturation with increasing ligand concentration. The specific binding of³H-haloperidol, which had a low affinity and is indicative of dopamine-1 receptors, showed no age-related changes in maximum receptor density or affinity. On the other hand, the maximum receptor density of dopamine-2 receptors measured by³H-spiroperidol decreased with age. The observations that renal dopamine-1 receptor density or affinity do not change with maturation are in agreement with our pervious studies that showed no age-related changes in dopamine-receptor-mediated renal vasodilatation in sheep. The significance of the decrease in renal dopamine-2 receptor density with age remains to be determined.     

Muscarinic cholinergic receptors in human gastric mucosa

Muscarinic cholinergic receptor sites in human gastric mucosa were analyzed directly by using radioligand binding techniques with the specific muscarinic antagonist³H-quinuclidinyl benzilate (QNB) as ligand. Specific binding of³H-QNB to membrane preparations from human gastric mucosa was saturable, of high affinity (Kd=4.17±1.94 nM, Bmax=0.37±0.04 pmol/mg protein) and selectively inhibited by muscarinic antagonists (atropine, scopolamine) and agonists (acetylcholine, pilocarpine). These findings provide direct evidence for the existence of muscarinic cholinergic receptors in human gastric mucosa. The specific³H-QNB binding to its receptor was blocked by atropine but not by histamine, cimetidine, pentagastrin, or synthetic human gastrin. The muscarine and histamine H₂-receptor, or muscarine and gastrin receptor, probably do not share the same locus.     

Autoradiographic quantification of adrenergic receptors in rat kidney

The adrenergic nervous system is active in kidney function, and the kidney has large numbers of adrenergic receptor subtypes. Because of the cellular complexity of the kidney, it is difficult to obtain direct assessments of adrenergic receptor binding characteristics over specific tissue compartments. Qualitative autoradiography allows the localization of adrenergic receptors over tissue types in the kidney, but quantitative autoradiography allows direct comparison of adrenergic receptor number over different cellular compartments. The purpose of this study was to obtain direct assessments of alpha 1, alpha 2, and beta adrenergic receptor numbers over different tissue compartments of the kidney using quantitative autoradiography. Sections of Sprague-Dawley rat kidney were incubated in several concentrations of 3H-dihydroalprenolol to label beta receptors, 3H-prazosin to label alpha 1 receptors and 3H-rauwolscine to label the alpha 2 receptors. Sections of rat heart incubated in 3H-dihydroalprenolol were included as standards. The sections were then prepared for receptor autoradiography. After processing, the grains were then quantified on an image analysis system, and binding curves constructed from the specific binding. In some animals, the proximal tubules were stained to localize the proximal convoluted tubules. Significant Scatchard analyses were obtained in the glomeruli with dihydroalprenolol (5.18 X 10(9) receptors/mm3) and with rauwolscine (2.48 X 10(9) receptors/mm3). Significant Scatchard analyses were obtained in the cortex with rauwolscine (9.47 X 10(9) receptors/mm3) and with prazosin (3.9 X 10(9)). In addition, specific binding was seen with rauwolscine and prazosin to the kidney arterioles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications of glomerular fibrinolysis in human renal graft rejection

The glomerular fibrinolytic activity (GFA) was studied using the fibrin slides technique for isolated glomeruli in normal human cortex (N = 8) and in renal allografts (N = 11), in order to evaluate whether fibrinolysis is modified after rejection. In normal cases all glomeruli showed a lytic zone when incubated at 37°C during 150 min; the mean GFA value was 0.0313 ± 0.0021 min^?1. A highly significant decrease of the number of glomeruli showing a lytic zone was noted in rejected renal allografts; however, in the glomeruli which retain their fibrinolytic capacity, the GFA did not increase. This study indicates that the GFA can be quantitatively estimated in human glomeruli and suggests that the normal fibrinolytic capacity of numerous glomeruli is lost in rejected renal allografts.     

Direct evidence of alpha-adrenoceptor binding in rat and human adrenal glands

In order to determine whether or not alpha-adrenoceptors are present in adrenal glands, radioligand receptor binding assay was performed in both Sprague-Dawley (SD) rat and human adrenal gland membranes. Radioligand binding assay using 3H-prazosin as an alpha 1-adrenoceptor ligand and 3H-yohimbine as an alpha 2-adrenoceptor ligand, clearly demonstrated alpha 1 and alpha 2 receptors present in both rat and human adrenal gland membranes. Maximal binding capacity (Bmax) and dissociation constant (Kd) of 3H-prazosin binding to the rat adrenal gland were 12.5 fmol/mg protein, and 0.11 nM, respectively. Those for the membrane preparations from adrenal cortex and medulla of the normal human were 16.3 fmol/mg protein, 0.34 nM and 16.3 fmol/mg protein, 0.27 nM, respectively. And those of the human pheochromocytoma were 25.6 fmol/mg protein, 0.15 nM, respectively. On the other hand, Bmax and Kd of 3H-yohimbine binding in the rat adrenal gland to were 22.9 fmol/mg protein, and 4.28 nM, respectively. Those for the membrane preparations from adrenal cortex and medulla of the normal human were 40.4 fmol/mg protein, 5.15 nM and 12.2 fmol/mg protein, 5.39 nM, respectively. And those of the human pheochromocytoma were 35.8 fmol/mg protein, and 1.08 nM, respectively. Bmax (35.8 fmol/mg protein) of 3H-yohimbine binding in the pheochromocytoma was significantly (p less than 0.01) greater than that (12.2 fmol/mg protein) in the human normal adrenal medulla, while Kd (1.08 nM) of this binding in the human pheochromocytoma was significantly (p less than 0.01) lower than that (5.39 nM) in the human normal adrenal medulla. Our data suggest that the alpha 2 receptor had greater affinity and binding site density to its agonist in the human pheochromocytoma than in the human normal adrenal medulla.     

Expression of a monoclonal antibody (3G5) defined ganglioside antigen in the renal cortex.

The monoclonal antibody (mAb) 3G5 was found, by indirect immunofluorescence, to bind to renal cortical structures in frozen sections of human, rat and calf kidneys. Double indirect immunofluorescence studies on frozen sections of rat kidneys showed that 3G5 stained only the glomerulus and the distribution of the 3G5 antigen on the glomerulus was more extensive than the staining observed with antibodies to Factor VIII antigen. 3G5 stained the proximal convoluted tubules and collecting tubules in bovine renal sections but glomeruli did not stain with 3G5. The 3G5 mAb did not stain tissue cultured bovine glomerular endothelial cells or mesangial cells, but did stain bovine glomerular epithelial cell cultures. 3G5 did not stain MDCK cell cultures. The binding of mAb 3G5 to glomeruli was investigated by immunoelectron microscopy of rat renal tissue. In contrast to the podocyte specificity on bovine glomerular cells in vitro, it was found that the specificity of 3G5 expression on rat glomerular cells in vivo was broader. No binding of mAb 3G5 was found outside the glomerulus in the rat renal cortex. Podocytes, endothelial cells and capsular epithelial cells expressed the 3G5 antigen most strongly. A lesser amount of binding was found in the glomerular basement membrane. The mesangium showed a little binding of mAb 3G5 and no binding at all was found to other cortical structures. The 3G5 antigen in rat renal tissue was found to be a glycolipid that migrated between the ganglioside markers GM2 and GM1 by immunostaining of thin layer chromatograms.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-affinity specific [3H]tamsulosin binding to α1 in human prostates with benign prostatic hypertrophy

The binding of a novel radioligand, [³H]tamsulosin, to human prostatic membranes with benign prostatic hypertrophy (BPH) has been characterized. [³H]Tamsulosin rapidly associated with its binding sites in human prostatic membranes with BPH, and the binding reached steady state by 30 min at 25°C. The rate constants for association and dissociation of [³H]tamsulosin binding were calculated to be 0.21±0.05/nM per minute and 0.01±0.004/min, respectively. The specific binding of [³H]tamsulosin in human prostatic membranes was saturable and of high affinity (K _d=0.04±0.01 nM). The density of [³H]tamsulosin-binding sites (B _max) was 409±28 fmol/mg protein. The K _d and B _max values for [³H]tamsulosin binding in human prostates were significantly lower than those for [³H]prazosin binding. [³H]tamsulosin binding was remarkable for its significantly lower degree of nonspecific binding. Six -adrenoceptor antagonists competed with [³H]tamsulosin for the binding sites in the rank order: tamsulosin>WB4101>prazosin>S-(+)-isomer>naftopidil>yohimbine. The binding affinities (pK_i) of these antagonists for [³H]tamsulosin binding in human prostates closely correlated with their pharmacological potencies (pA₂) in prostates. In conclusion, [³H]tamsulosin selectively labels ₁-adrenoceptors in human prostates, and thus may become a useful radioligand for the further analysis of these receptors.     

CHARACTERIZATION, LOCALIZATION AND DISTRIBUTION OF alpha1 ADRENOCEPTOR SUBTYPE IN MALE RABBIT URETHRA

Purpose

The subtype specificity, localization and distribution of urethral alpha_{1-adrenoceptors} were studied in the male rabbit urethra.

Materials and Methods

The properties of the urethral alpha_{1-adrenoceptors} were investigated using radioligand receptor binding and light microscopic autoradiography with [(¹²⁵) I]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 [(¹²⁵) I]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 (K_i values): prazosin <or= to 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 alpha_1d receptors with correlation coefficients of 0.68, 0.929, 0.909, and 0.523, respectively.

Conclusions

The pharmacological characterization demonstrates the predominance of alpha_1A or alpha_1A + alpha_1B 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.     

Increased angiotensin-converting enzyme and type 1 angiotensin receptors in cortical vasculature and tubulointerstitium of chronically rejected human kidney allografts

SUMMARY: Chronic rejection of human renal allografts after transplantation is characterized by interstitial infiltration, arteriosclerosis and glomerulosclerosis in the grafts. Apart from tissue HLA compatibility, angiotensin II (AII) may be implicated in accelerating the processes of chronic rejection. In the present study, the cellular distribution of angiotensin‐converting enzyme (ACE) and type 1 AII (AT₁) receptors was mapped and compared in non‐rejected human kidneys (n = 8) and chronically rejected renal allografts (n = 9) using complementary immunohistochemistry and quantitative in vitro autoradiography. Chronically rejected allografts showed typical histopathological characteristics of tissue rejection, including concentric intimal thickening of intrarenal arteries, extensive or focal glomerulosclerosis, tubular atrophy and severe tubulointerstitial fibrosis. In rejected allografts, total ACE binding in the cortex was decreased to 46% of that in non‐rejected kidneys (P < 0.01), whereas AT₁ receptor binding in the glomeruli and the inner stripe of the outer medulla was maintained. However, ACE and AT₁ receptor binding were increased in the cortical tubulointerstitium of chronically rejected allografts. In non‐rejected kidneys, strong ACE immunostaining occurred in proximal tubules and vascular endothelium, whereas AT₁ receptors occurred in vascular smooth muscle cells, as expected. In rejected allografts, intense ACE and AT₁ receptor immunostaining were detected not only in the same sites as those non‐rejected kidneys, but also in cortical tubulointerstitium between atrophied tubules and surrounding the glomeruli. AT₁ receptors were markedly up‐regulated in vascular smooth muscle cells of thickened atherosclerotic vessels. These results provide novel morphological evidence that increased expression and/or altered distribution of ACE and AT₁ receptors in cortical tubulointerstitium and in the neointima of intrarenal arteries may play an important role in the progression of chronic rejection of human renal allografts after transplantation.     

Protective Effect of Verapamil in Cyclosporin-Incubated Human and Murine Isolated Glomeruli

Cyclosporin A (CsA)-induced nephrotoxicity is churucterized by a decrease in the glomerular filtration rate (GFR) which is associated with a large increase in renal vascular resistance (RVR). Using a video image analyzer, we have demonstrated CsA-induced glomerular vasoconstriction in rat isolated glomeruli as assessed by a significant reduction of glomerular area. This vasoactive response explains in part the renal hemodynamic changes and the development of CsA-induced reversible decline in renal function. To confirm the direct vasoactive effect of CsA on glomeruli and to rietermine if calciumblocking agents modified this response, we compared the changes in area of isolated rat and human glomeruli incubated either with CsA alone or with CsA plus verapamil. The area of the isolated glomeruli was quantitatively evaluated by a camera video image analyzer; euch glomerulus served as its own control. Area kinetics were studied at 5 min intervals oves 30 min. CsA-induced glomerular size reduction is dose dcpendent (-4.2% for 10-¹⁰ M crnd -10.2% for 10^?6 M) and time dependent (-2.3% at 5 min, -4.7% at 10 min, and -12.1% at 30 min for 10^?6 M). With verapamil pretreatment, CsA-induced reduction in glomerular size was reduced (-0.6% and -3.6%, respectively, for 10^?6 M and 10^?7 M verapamil). Thus, verapamil can be considered as a protective agent against CsA-induced vasoconstriction in rat and human isolated glomeruli.     

The effect of aging on cholinergic receptor binding in the rat urinary bladder

Binding sites with high affinity and specificity for [³H]quinuclidinyl benzilate (QNB), a cholinergic muscarinic receptor antagonist, were found in homogenates of rat urinary bladder. Atropine and scopolamine inhibited 50% of the specifically bound [³H]QNB (IC₅₀) at a concentration of approximately 2 nM, whereas the agonist oxotremorine had an IC₅₀ of 580 nM. By contrast, the IC₅₀ for the ganglionic nicotinic antagonist mecamylamine was greater than 100,000 nM. These values are similar to those reported for [³H]QNB binding sites in rat brain. [³H]QNB binding was characterized in rat bladder obtained from animals aged 6, 16, and 26 months. No differences in either the number of binding sites or the affinity constant for the radioligand were noted between these groups, suggesting that there are no agerelated alterations in muscarinic receptor recognition sites in this organ.     

The muscarinic receptor antagonist propiverine exhibits α(1)-adrenoceptor antagonism in human prostate and porcine trigonum

Purpose

Combination therapy of male lower urinary tract symptoms with ??₁-adrenoceptor and muscarinic receptor antagonists attracts increasing interest. Propiverine is a muscarinic receptor antagonist possessing additional properties, i.e., block of L-type Ca²⁺ channels. Here, we have investigated whether propiverine and its metabolites can additionally antagonize ??₁-adrenoceptors.

Methods

Human prostate and porcine trigone muscle strips were used to explore inhibition of ??₁-adrenoceptor-mediated contractile responses. Chinese hamster ovary (CHO) cells expressing cloned human ??₁-adrenoceptors were used to determine direct interactions with the receptor in radioligand binding and intracellular Ca²⁺ elevation assays.

Results

Propiverine concentration-dependently reversed contraction of human prostate pre-contracted with 10???M phenylephrine (?log IC₅₀ [M] 4.43?±?0.08). Similar inhibition was observed in porcine trigone (?log IC₅₀ 5.01?±?0.05), and in additional experiments consisted mainly of reduced maximum phenylephrine responses. At concentrations ??1???M, the propiverine metabolite M-14 also relaxed phenylephrine pre-contracted trigone strips, whereas metabolites M-5 and M-6 were ineffective. In radioligand binding experiments, propiverine and M-14 exhibited similar affinity for the three ??₁-adrenoceptor subtypes with ?log?K _i [M] values ranging from 4.72 to 4.94, whereas the M-5 and M-6 did not affect [³H]-prazosin binding. In CHO cells, propiverine inhibited ??₁-adrenoceptor-mediated Ca²⁺ elevations with similar potency as radioligand binding, again mainly by reducing maximum responses.

Conclusions

In contrast to other muscarinic receptor antagonists, propiverine exerts additional L-type Ca²⁺-channel blocking and ??₁-adrenoceptor antagonist effects. It remains to be determined clinically, how these additional properties contribute to the clinical effects of propiverine, particularly in male voiding dysfunction.     

Muscarinic cholinergic and alpha 2-adrenergic receptors in the epithelium and muscularis of the human ileum

The aim of this study was to characterize the binding and functional properties of muscarinic cholinergic (MCh) and alpha 2-adrenergic receptors in the human ileum to provide insight into pharmacologic strategies for managing urinary and fecal incontinence after bladder and rectal replacement with intestinal segments. MCh and alpha 2-adrenergic binding sites were characterized in the epithelium and muscularis of eight human ileal segments with 3H-N-methylscopolamine and 3H-rauwolscine, respectively. The dissociation constant for 3H-N-methylscopolamine in the epithelium and muscularis was 0.32 +/- 0.07 nmol/L and 0.45 +/- 0.10 nmol/L, respectively (p = 0.32). The MCh receptor content was approximately eightfold greater in the muscularis compared with the epithelium (p = 0.008). The dissociation constant for 3H-rauwolscine in the muscularis and epithelium was 2.55 +/- 0.42 nmol/L and 2.03 +/- 0.19 nmol/L, respectively (p = 0.29). The alpha 2-adrenoceptor density was twofold greater in the epithelium compared with the muscularis (p = 0.05). Noncumulative concentration-response experiments were performed with carbachol, an MCh agonist, and UK-14304, a selective alpha 2-adrenergic agonist. The epithelium did not contract in the presence of high concentrations of carbachol and UK-14304. The muscularis preparations were responsive only to carbachol. The muscularis contains primarily MCh receptors mediating smooth muscle contraction. The alpha 2-adrenoceptors are localized primarily to the epithelium and may regulate water secretion in the intestine. The distribution and functional properties of ileal MCh and alpha 2-adrenergic receptors provide a theoretic basis for the treatment of incontinence after bladder and rectal replacement with intestinal segments.     

IN VIVO EFFECT OF A SELECTIVE ENDOTHELIN RECEPTOR ANTAGONIST, BQ-123, ON RENAL FUNCTION IN CYCLOSPORIN A-TREATED RATS

To inveslgate the possible involvement of endogenous endothelin (ET) in cyclosporin A (CsA) nephrotoxicity, we examined the renal effects of the selective [¹²⁵I]ET-1 binding to porcine aortic membrane (ET_A) receptor antagonist, BQ-123 (BQ), on CsA-treated rats. An osmotic minipump filled with BQ or its vehicle (saline), was subcutaneously implanted and animals were treated with CsA (50mg/kg/d, i.p.) for 4 d. In both BQ-and its vehicle-treated rats, the 24-hour urine volume, urinary N-acetyl-D-glucosaminidase (NAG) excretion, urinaryCa/creatinine (Cr) and urinaryNAG/Cr ratios were significantly increased compared with those before administration of CsA. In contrast, after administration of CsA, urinary Cr excretion was significantly decreased in both rat groups. Although urinary NAG excretion and the NAG/Cr ratio in the rats treated with BQ were significantly increased compared with those treated with saline, the serum Cr levels in BQ-treated rats were significantly lower than those in saline-treated rats. Urinary immunoreactive ET-1 (irET-1) excretion in all animals was significantly increased after administration of CsA. There were no significant differences in urinary irET-1 excretion in both BQ-treated and saline-treated rats with and without CsA. Expression of irET-1 was seen in the cytoplasm of renal tubules, but the degree of expression was similar in the BQ-treated and saline-treated rats on CsA. In the BQ-treated rats, small round areas with high grain densities over the glomeruli in the cortex and vesa recta bundles in the outer medulla were masked by the antagonist action of BQ. On microscopic examination of autoradiograms, the numbers of autoradiographic grains were clearly decreased in the glomeruli and vessels, which confirmed the presence of ET_A. It was concluded that: (1) BQ had no effect on the products and release of ET-1; (2) BQ could not entirely prevent CsA nephrotoxicity in rats; (3) BQ increased the tubular damage caused by CsA.     

Upregulation of endothelin A receptor sites in the rabbit diabetic kidney: potential relevance to the early pathogenesis of diabetic nephropathy.

BACKGROUND/AIM: Nephropathy is an important complication of diabetes mellitus (DM). The plasma endothelin 1 (ET-1) levels are increased in DM, and ET-1 may cause deleterious effects on renal function. We, therefore, investigated whether changes in ET receptors occur in the DM rabbit kidney. METHODS: Nine adult New Zealand White rabbits were injected with alloxan, of which 6 became diabetic; the other 3 acted as alloxan-treated controls. Six age-matched healthy rabbits served as controls. At 6 months, following cervical dislocation, the kidneys were removed, and sections (cortex and medulla) were incubated with ET(A) and ET(B) radioligands to produce low- and high-resolution autoradiographs. Immunohistochemical localization of ET-1 immunoreactivity was also performed. RESULTS: There was greater ET(A) and ET(B) receptor binding in the control (ET(A) p = 0.0003; ET(B) p < 0.0001) and DM (ET(A) p = 0.001; ET(B) p < 0.0001) rabbits in the medulla as compared with the cortex. DM kidneys showed a significant increase in ET(A), but not ET(B), binding in the cortex (p < 0.0001) and in the medulla (p < 0.0001). High-resolution autoradiographs revealed striking [(125)I]-ET-1 receptor binding predominantly to the glomeruli. Immunohistochemistry revealed dense ET-1 immunoreactivity associated with the renal tubules, but the glomeruli exhibited no staining. Alloxan-treated controls had similar results to age-matched controls. CONCLUSION: There are regional differences in both ET(A) and ET(B) binding in control and DM kidneys. ET(A) receptor binding sites are increased in the DM kidney (cortex and medulla). ET-1 may act in a paracrine fashion on the glomeruli. These changes may contribute to the pathogenesis of diabetic nephropathy.