Faculty Bibliography

Molecular cloning studies have revealed the existence of three subtypes of alpha 1-adrenergic receptors. However, the link between any individual subtype and its functional role in the body has remained elusive. In an effort to bridge the gap between molecular biology and pathophysiology, we have chosen a model smooth muscle system, the human prostate, and investigated the role of alpha 1 subtypes in this tissue. To determine which alpha 1-adrenergic receptor subtype mediates the contractile response of the human prostate, we first studied the pharmacological properties of three cloned human alpha 1 subtypes (alpha 1a/d, alpha 1b, and alpha 1c). Prazosin, terazosin, doxazosin, alfuzosin, and abanoquil showed no selectivity for the human alpha 1 subtypes. WB-4101 and 5-methylurapidil showed a rank order of potency of alpha 1c > alpha 1a/d >> alpha 1b. Indoramin and (+)-niguldipine were selective for the alpha 1c-adrenergic receptor, with at least 10-fold lower affinity at either alpha 1a/d or alpha 1b subtypes. SK&F104856 was found to be 6-fold more potent at the alpha 1a/d receptor subtype than at alpha 1b- or alpha 1c-adrenergic receptors. We next determined the potency of these antagonists to inhibit the phenylephrine-induced contraction of human prostatic tissue in vitro. The potencies of indoramin, 5-methylurapidil, and SK&F104856 to inhibit the contractile response and to displace [3H]prazosin from the cloned human alpha 1c subtype were similar. Our data suggest that the alpha 1 receptor that mediates the contraction of human prostate smooth muscle has the pharmacological properties of the cloned human alpha 1c-adrenergic receptor. The findings of the present study suggest that selective alpha 1c-adrenergic receptor antagonists may be clinically more efficacious and better tolerated agents for the treatment of symptomatic benign prostatic hyperplasia

The objective of the present study was to localize endothelin receptors in the human prostate using quantitative autoradiography. Slide-mounted tissue sections 20 microns. in thickness were obtained from the transition zones of seven patients undergoing radical prostatectomies for low volume prostate cancer. Sarafotoxin (S6C) and BQ123 have been used to distinguish endothelin receptor subtypes (ETA and ETB). The prostatic tissue sections were incubated in four different stock solutions containing the following: 0.1 nM. 125I-endothelin-1 (125I-ET-1) (total ET-1 binding); 0.1 nM. 125I-ET-1 and 100 nM. S6C (total ETA binding); 0.1 nM. 125I-ET-1 and 1 microM. BQ123 (total ETB binding); and 0.1 nM. 125I-ET-1 and 1 microM. ET-1 (nonspecific ET-1 binding). Nonspecific binding accounted for only 12 and 15% of total 125I-ET-1 binding in the stroma and glandular epithelium. Autoradiograms were quantitatively analyzed using a computerized image analysis system. Specific radioactive densities (nCi/mg.) were determined for the stromal and glandular epithelial elements of the prostate. The specific radioactive densities of ETA and ETB binding sites in the stroma were 7.57 +/- 0.65 and 2.98 +/- 0.81. The specific radioactive densities of ETA and ETB binding sites in the glandular epithelium were 1.59 +/- 0.15 and 7.87 +/- 1.35. The present study demonstrates that the predominant endothelin receptors in the stroma and glandular epithelium are the ETA and ETB subtypes, respectively

The objective of the present study was to characterize the binding and functional properties of endothelin (ET) receptor subtypes in the human prostate. Human prostatic tissue was obtained from male subjects undergoing radical prostatectomy for low-volume prostate cancer. The optimal assay conditions for characterizing human prostatic ET-1 binding sites on slide-mounted tissue sections were defined. Maximal specific 125I-ET-1 binding was achieved after a 10-min preincubation, a 120-min incubation, and a washing procedure that consisted of a brief rinse and a 1-min wash. The mean equilibrium dissociation constant (Kd) and density (Bmax) of ET-1 binding sites determined from six saturation studies were 0.72 +/- 0.13 nM and 40.4 +/- 6.9 fmol/mg of wet weight, respectively. The mean Hill coefficient was 0.99 +/- 0.01, indicating that 125I-ET-1 identifies a single population of binding sites. The pharmacology of 125I-ET-1 binding sites was characterized using competitive binding experiments. The competition plots for ET-1 were best fit by a one-binding site model, whereas the plots for sarafotoxin 6C (S6C) and BQ123 were consistently best fit by a two-site model. The mean Ki value of ET-1 was 0.34 +/- 0.12 nM. The mean Ki values for the high and low affinity S6C binding sites were 0.50 +/- 0.09 nM and 0.84 +/- 0.28 microM, respectively. The mean Ki values for the high and low affinity BQ123 binding sites were 5.51 +/- 1.05 nM and 24.9 +/- 6.5 microM, respectively. The ratio of ETA to ETB binding sites was approximately 2:1. The ET receptor subtype mediating prostatic smooth muscle tension was investigated using agonist-antagonist competition studies. ET-1, a nonselective ET agonist, elicited a potent contraction of prostate smooth muscle. The pA2 of BQ123 for inhibiting ET-1-mediated contraction was 6.84. S6C, a selective ETB agonist, also elicited a potent contraction of prostate smooth muscle. BQ123 at concentractions between 0.1 and 10 microM did not shift the S6C dose-response curve. These functional studies suggest that both ETA and ETB receptors mediate the tension of prostate smooth muscle. Endogenous ETS may be involved in the pathophysiology of bladder outlet obstruction in men with benign prostatic hyperplasia. If this is the case, then ET antagonists may represent effective treatment for benign prostatic hyperplasia

Alpha 1 adrenoceptor binding sites have been characterized in prostatic tissue homogenates using radioligand receptor binding studies. The objective of the present study was to characterize and localize prostatic alpha 1 adrenoceptor binding sites using slide-mounted tissue sections and the ligand 3H-prazosin. The present study demonstrated that preincubation is not required; the optimal incubation interval is 40 minutes; and a 1-minute wash (once or twice) maximizes the proportion of specific 3H-prazosin binding. Saturation studies were performed at 8 different concentrations of 3H-prazosin ranging between 0.0625 nM. to 8.0 nM. The binding of 3H-prazosin was consistently saturable and of high affinity. The mean Kd and Bmax determined from 6 saturation studies was 4.16 x 10(-10) M. and 1.30 fmol./mg. wet weight, respectively. The pharmacology of these 3H-prazosin binding sites was characterized using competitive displacement experiments. The mean IC50 corrected for prazosin, phentolamine and yohimbine was 7.8 x 10(-10) M., 6.0 x 10(-9) M. and 2.1 x 10(-6) M. The rank order of the IC50 corrected values indicates that alpha 1 binding sites were measured under the assay conditions. In the present study, the mean values for Kd, Bmax and IC50 corrected are similar to values previously reported using prostatic tissue homogenates. Prostatic tissue sections were apposed to x-ray film after being incubated with 3 nM. 3H-prazosin (total prazosin binding) and 3 nM. 3H-prazosin + 8 microM. prazosin (nonspecific prazosin binding). The autoradiograms were analyzed using a computerized analyzing system. The specific radioactive densities of 3H-prazosin in the stroma and glandular epithelium were 1099 +/- 48 pCi/mg. and 163 +/- 42 pCi/mg. The present study validates the technique of assaying alpha 1 adrenoceptor binding sites on slide-mounted prostatic tissue sections and provides further evidence that alpha 1 adrenoceptor binding sites are localized primarily to the stromal elements of the prostate

The clinical controversy regarding the timing of surgery for asymptomatic newborns with obstructed hydronephrosis was addressed using a model of reversible partial ureteral obstruction in the newborn rabbit. The histomorphometric changes in the ureteropelvic junction complex (for example, pelvis, ureteropelvic junction and upper ureter) and kidney in 44 normal cases were determined and compared with the effects of 47 cases of ongoing partial obstruction and timed reversal of partial obstruction at 1 week in 9 cases, at 2 weeks in 10 or at 4 weeks in 10 (end of the study at age 8 weeks). After partial obstruction hydronephrosis appeared by 1 week postoperatively. There were progressive increases in the thickness of the lamina muscularis and mass index of smooth muscle and collagen (all p < 0.001). However, since the per cent surface area of smooth muscle did not change significantly in comparison to normal, there was disproportionately more collagen. For reversals at 1 week the muscle and collagen in the lamina muscularis were not significantly different from normal. For reversals at 2 weeks the mass index of collagen was greater than normal (p < 0.05) and reversal at 1 week (p < 0.05). For reversals at 4 weeks the lamina muscularis was thicker, and the mass index of collagen and muscle was greater than the earlier reversal groups and normal (all p < 0.05). In conclusion, partial ureteral obstruction causes progressive thickening of the lamina muscularis by collagen and muscle with a disproportionately greater increase in collagen than muscle. The earlier the obstruction can be reversed, the more normal is the ureteropelvic junction complex histology. The functional significance of these changes needs to be determined

Endothelins mediate contractile responses in many types of vascular and nonvascular smooth muscle. The present study represents the first detailed characterization of endothelins in the human prostate. The objectives of this study were to determine the tissue levels and source of endogenous endothelin-1 (ET1) in the human prostate. The contractile effects of ET1 were also investigated using in vitro isometric tension studies. The mean tissue level of ET1 was 0.58 +/- 0.08 pg./mg. tissue wet weight. Endothelin-like activity was markedly prominent in the glandular epithelium of the human prostate, whereas minimal endothelin-like activity was observed in the prostatic stroma. Strips of human prostatic tissue were suspended in isolated tissue chambers and challenged to a concentration response of ET1. The mean EC50 and Emax for ET1 was 3.2 x 10(-8) M. and 0.12 +/- 0.02 gm. force per mm.2 cross-sectional area (CSA), respectively. Preincubation with indomethacin, terazosin, or nifedipine did not alter the concentration-dependent response to ET1. A calcium-free buffer abolished the contractile response to ET1. Thus, ET1 mediates a potent contraction of human prostatic smooth muscle that is not mediated via alpha 1 adrenergic or dihydropyridine sensitive calcium channels or prostaglandin synthesis. The presence of marked endothelin-like immunoreactivity strongly suggests a biological significance for endogenous endothelins in the human prostate