Faculty Bibliography

1. The stereospecificity of the enantiomers of LY253352, a potent and selective alpha 1-adrenoceptor antagonist, were studied in the human prostate and canine brain using radioligand receptor binding methods. 2. The mean equilibrium dissociation constant (KD) in the canine brain and human prostatic adenoma was 84.4 pM and 65.4 pM, respectively. 3. The alpha 1-adrenoceptor density in the canine brain was approximately eight fold greater than in the human prostatic adenoma. 4. The mean Ki values of (-)-LY253352 and (+)-LY253352 in the prostate were 0.19 nM and 5.79 nM, respectively. 5. The mean Ki values of (-)-LY253352 and (+)-LY253352 in the brain were 0.29 nM and 34.7 nM, respectively. 6. This study indicates that the stereochemical specificity of the optical isomers of LY253352 is a manifestation of differential affinities of the enantiomers for alpha 1-adrenoceptor binding sites. 7. The differential affinities of (+)-LY253352 in the brain and prostate are suggestive of subtle unique properties of adrenoceptor binding sites in these tissues

The purpose of this study was to determine whether resection of the prostate with electrocautery alters the binding properties of various neurotransmitter ligands. Prostate glands were removed from four adult male dogs. The prostates were divided in the midsaggital plane and one half of the prostate was resected using a resectoscope. Saturation experiments were performed on the resected and control prostatic tissue using 3H-NMS, 125I-Heat, and 3H-rauwolscine. The mean equilibrium dissociation constants (Kd) and the mean densities of 3H-NMS, 125I-Heat, and 3H-Rauwolscine binding sites were similar in tissue homogenates obtained from control and resected portions of the prostate (p greater than 0.05). Resection of the prostate using electrocautery did not alter the binding properties of various neurotransmitter ligands for characterizing and quantifying muscarinic cholinergic, alpha 1 adrenergic, and alpha 2 adrenergic binding sites in the canine prostate. Approximately 90% of prostatectomies for symptomatic BPH (benign prostatic hyperplasia) are performed transurethrally. The ability to accurately measure neurotransmitter receptor densities in prostate tissues obtained following transurethral resection is imperative for our future studies designed to elucidate the role of alpha adrenergic receptors in the development of bladder outlet obstruction in men with BPH

Clinical trials are currently underway to evaluate the efficacy of terazosin for the treatment of symptomatic benign prostatic hyperplasia (BPH). Terazosin is a potent and selective alpha 1 adrenergic blocking agent structurally similar to prazosin. The alpha adrenergic binding properties of terazosin were studied in human prostate adenomas and canine brains using radioligand receptor binding methods. Saturation analyses were performed at varying concentrations of [125I]-Heat and [3H]rauwolscine [( 3H]Ra) in human prostate adenomas and canine brains. The binding of [125I]-Heat and [3H]Ra in the human prostates and canine brains was consistently saturable and of high affinity. The equilibrium dissociation constant (Kd) for [125I]-Heat binding in the canine brains and human prostate adenomas was 84.4 +/- 4.3 pM and 65.4 +/- 19.2 pM, respectively (p greater than 0.05). The (Kd) for [3H]Ra binding in the human prostate adenomas and canine brains was 1.21 +/- 0.23 nM and 1.52 +/- 0.28 nM, respectively (p greater than 0.05). The density of alpha 1 (0.37 +/- 0.15 fmol/mg. wet wt.) and alpha 2 (0.29 +/- 0.09 fmol/mg. wet wt. adrenergic binding sites in the human adenomas were similar (p greater than 0.05). The IC50 corrected (IC50 corr) of terazosin for [125I]-Heat and [3H]Ra binding sites in the human prostate was 2.5 nM and 1.0 micron., respectively. The IC50 corr of terazosin for [125I]-Heat and [3H]Ra binding sites in the canine brain was 2.0 nM and 0.8 microM, respectively. The competitive binding assays indicate that terazosin binds selectively to alpha 1 adrenergic binding sites in the human prostate and canine brain

We have recently utilized radioligand receptor binding methods to characterize muscarinic cholinergic and alpha adrenergic receptors in human prostate adenomas. The primary advantages of radioligand receptor binding methods are that neurotransmitter receptor density is quantitated, the affinity of unlabelled drugs for receptor sites is determined, and receptors can be localized using autoradiography on slide-mounted tissue sections. Recently, [125I]-Heat, a selective and high affinity ligand with high specific activity (2200 Ci/mmole) has been used to characterize alpha 1 adrenergic receptors in the brain. In this study alpha 1 adrenergic receptors in the dog prostate were characterized using [125I]-Heat. The Scatchard plots were linear indicating homogeneity of [125I]-Heat binding sites. The mean alpha 1 adrenergic receptor density determined from these Scatchard plots was 0.61 +/- 0.07 fmol/mg. wet wt. +/- S.E.M. The binding of [125I]-Heat to canine prostate alpha 1 adrenergic binding sites was of high affinity (Kd = 86 +/- 19 pM). Steady state conditions were reached following an incubation interval of 30 minutes and specific binding and tissue concentration were linear within the range of tissue concentrations assayed. The specificity of [125I]-Heat for alpha 1 adrenergic binding sites was confirmed by competitive displacement assays using unlabelled clonidine and prazosin. Retrospective analysis of the saturation experiments demonstrated that Bmax can be accurately calculated by determining specific [125I]-Heat binding at a single ligand concentration. [125I]-Heat is an ideal ligand for studying alpha 1 adrenergic receptors in the prostate and its favorable properties should facilitate the autoradiographic localization of alpha 1 adrenergic receptors in the prostate

Radioligand receptor binding methods were used to characterize the alpha 1-adrenergic receptor in the bladder body, bladder base, prostate and urethra of the male dog. Saturation experiments were performed in tissue homogenates using [125iodine]-Heat, an alpha 1-adrenergic antagonist of high specific activity (2,200 Ci. per mmol.). The equilibrium dissociation constant Kd for [125iodine]-Heat binding in the bladder body (0.56 pM.), bladder base (0.81 +/- 0.11 pM.), prostate (0.86 +/- 0.19 pM.) and urethra (0.55 pM.) was similar, suggesting homogeneity of alpha 1-adrenergic binding sites in lower genitourinary tissues. The receptor density in the bladder body, bladder base, prostate and urethra, expressed as fmol. per mg. wet weight, was 0.22 +/- 0.02, 0.82 +/- 0.09, 0.55 +/- 0.06 and 0.27 +/- 0.06, respectively (mean +/- standard error of mean). Competitive binding experiments with [125iodine]-Heat and unlabeled prazosin and clonidine confirmed the selectivity of Heat for alpha 1-adrenergic binding sites. Anatomical dissections have revealed that a major component of the smooth muscle of the bladder base and prostate originates from the ureter, whereas a major component of the smooth muscle of the urethra originates from the bladder. The measured alpha 1-adrenergic receptor densities support these developmental theories

The sympathetic innervation of human prostate adenomas has been previously demonstrated using fluorescence microscopy and in vitro isometric studies. A clinical implication of these observations is that bladder outlet obstruction in men with benign prostatic hypertrophy may be subject to pharmacologic manipulation using adrenergic drugs. Randomized clinical trials have demonstrated the efficacy of alpha adrenergic antagonists for symptomatic BPH. We have previously characterized the alpha1 and alpha2 adrenergic receptors in the human prostate using [3H]prazosin and [3H]rauwolscine, respectively. The mean alpha1 and alpha2 receptor densities in the adenomas studied were equivalent. The effect of alpha2 adrenergic drugs on prostatic urethral pressure has not been examined in the human or in an animal model. In this study a canine model was used to define the effect of alpha2 drugs on prostatic urethral pressure. Intravenous administration of clonidine, a selective alpha2 agonist, resulted in a dose dependent increase in prostatic urethral pressure. The maximal increase in urethral pressure ranged between 18 to 30 cm. H2O. The maximal response to clonidine was approximately 50% less than the response to epinephrine, indicating that clonidine acts as a partial agonist. Pretreatment with yohimbine, a selective alpha2 adrenergic antagonist, abolished the effects of clonidine and epinephrine. The alpha2 adrenergic receptors were then studied in the canine prostates using [3H]rauwolscine. The equilibrium dissociation constant, Kd, ranged between 0.68 to 1.80 nM and the receptor density ranged between 14.8 to 69.3 fmol./mg. protein. The receptor density was homogeneous in specimens obtained from the proximal, midportion, and distal canine prostate suggesting that the effect of alpha2 drugs is not sphincter mediated. These in vitro and in vivo studies provide the basis for investigating the effects of alpha2 antagonists in men with symptomatic BPH

[3H]Rauwolscine ([3H]Ra), a selective ligand for the alpha 2 adrenergic receptor, was used to identify and characterize alpha 2 adrenergic receptors in prostate glands of men with benign prostatic hyperplasia. Specific binding of [3H]Ra to prostatic tissue homogenates was rapid and readily reversible by addition of excess unlabelled phentolamine. Scatchard analysis of saturation experiments demonstrates a single, saturable class of high affinity binding sites (Bmax = 0.31 +/- 0.04 fmol./microgram. DNA, Kd = 0.9 +/- 0.11 nM.). The relative potency of alpha adrenergic drugs (clonidine, alpha-methylnorepinephrine and prazosin) in competing for [3H]Ra binding sites was consistent with the order predicted for an alpha 2 subtype. The role of alpha 2 adrenergic receptors in normal prostatic function and in men with bladder outlet obstruction secondary to BPH requires further investigation

The surgical management of classical bladder exstrophy (functional bladder closure or urinary diversion) should be influenced by the inherent detrusor function of the exstrophied bladder. Cystometrograms performed previously on individuals with successful exstrophy closures demonstrate normal bladder function. The biochemical and neurophysiological properties of the exstrophied bladder have otherwise not been investigated. In this study radioligand receptor binding techniques were used to compare the density and equilibrium dissociation constant of muscarinic cholinergic receptors in control and exstrophy bladders. The density of muscarinic cholinergic receptors in the control and exstrophy groups was 1.97 plus or minus 0.29 and 1.44 plus or minus 0.21 fmol. per microgram deoxyribonucleic acid (mean plus or minus standard error of mean), respectively. The dissociation constant of the control and exstrophy groups was 0.15 plus or minus 0.02 and 0.14 plus or minus 0.02 nM. (mean plus or minus standard error of mean), respectively. These data show that the muscarinic receptor density and binding affinity in control and exstrophy bladders are similar. Therefore, the neurophysiological composition of the exstrophied bladder is not grossly altered during the anomalous development

Prostatic secretion is dependent upon the integrity of the endocrine and autonomic nervous systems and is dramatically influenced by muscarinic cholinergic analogs. In this study, we have used radioligand receptor binding methods on whole tissue homogenates and slide mounted tissue sections of rat prostate to determine whether androgens regulate the density of muscarinic cholinergic receptors in the prostate. The muscarinic cholinergic receptor binding affinities (Kd) of [3H] N-methylscopolamine in prostatic homogenates obtained from intact, castrate, and castrate rats receiving testosterone replacement (castrate + T) were similar (0.07 to 0.10 nM). The muscarinic cholinergic receptor binding capacity decreased 73 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in castrate rats to intact levels. In order to ensure that the loss of receptor density was not due to a decrease in the epithelial: stromal cell ratio, the number of muscarinic cholinergic receptors per unit area of epithelium was determined in the 3 treatment groups using autoradiography on slide mounted tissue sections. The density of muscarinic cholinergic receptors in a unit area of epithelium was decreased 91 per cent following castration. Testosterone administration restored the density of muscarinic cholinergic receptors in the castrate rats to intact levels. The modulation of neurotransmitter receptors by steroid hormones may be a mechanism by which sex steroids regulate biological responsiveness of target tissues