Regional differences in the density and subtype specificity of endothelin receptors in rabbit urinary tract

Abstract

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.

Pharmacology of the Lower Urinary Tract: Basis for Current and Future Treatments of Urinary Incontinence

“…The main role of ET A receptors in the contractile effects of ETs in the detrusor has been confirmed by several other investigators in animal as well as human bladders (Donoso et al, 1994;Latifpour et al, 1995;Okamoto-Koizumi et al, 1999;Calvert et al, 2002;Yoshida et al, 2003). In human detrusor, OkamotoKoizumi et al (1999), using isometric contraction experiments and RT-PCR, demonstrated that the ET-1-induced contractions were mediated mainly by the ET A receptor and not by the ET B receptor.…”

Regulatory effect of experimental diabetes on the expression of endothelin receptor subtypes and their gene transcripts in the rat adrenal gland

“…300 nM BQ 123, and 30 nM STXc. These concentrations of BQ 123 and STXc, which are approximately 100 times greater than that of their high-affinity constants, K iH , (Latifpour et al 1995) occupy virtually all of the endothelin ETA and ETB receptor subtypes, respectively, in the rat adrenal sections.…”

“…The membrane particulates from adrenal glands were prepared as described previously (Latifpour et al 1995, Saito et al 2000a. In brief, the adrenal specimens were homogenized on ice in 20 mM Hepes (pH 7·4) containing 100 mM NaCl, 3 mM EDTA, 1 mM EGTA and the following protease inhibitors: 0·1 mM phenyl methyl sulfonyl fluoride and 10 µg/ml each of aprotinin, leupeptin, pepstatin A, and soybean trypsin inhibitor.…”

“…The density and pharmacological properties of ET receptors in rat adrenal glands were examined by saturation and inhibition experiments (Latifpour et al 1995, Saito et al 2000a. In the saturation experiments, the membrane suspensions of the rat adrenal glands prepared in 50 mM Tris-HCl (pH 7·4) containing 154 mM NaCl, 25 mM MnCl 2 , 1 mM EDTA, 1 mM N-acetyl--methionine, 0·25% bovine serum albumin and 0·14% bacitracin were incubated, in triplicate, for 2 h with increasing concentrations (10-300 pM) of 125 I-labeled ET-1 (2200 Ci/ mmol; New England Nuclear, Boston, MA, USA) in a total volume of 0·25 ml at 23 C. At the end of the incubation period, using a Brandel Cell Harvester (Model M-24R; Brandel Instruments, Gaithersburg, MD, USA), the reaction mixtures were filtered rapidly under a vacuum through Whatman GF/B glass-fiber filters that had been treated previously with a 1% solution of bovine serum albumin (to reduce non-specific binding to the filter papers).…”

Expression of the ET_A and ET_B Endothelin Receptor Subtype mRNA in Human Detrusor Cultured Smooth Muscle Cells

“…In the urinary tract, several investigators have investigated the physiological effects of ET and ET receptors using pharmacological, histochemical and receptor binding technique both in bladder [13][14][15][16][17][18][19][20][21][22] and prostate [23][24][25][26]. ET receptors have been shown to mediate contraction of isolated human bladder tissue strips [19][20][21].…”