Mary Baumann

Macrophage Elastase (Matrix Metalloproteinase-12) Suppresses Growth of Lung Metastases

Matrix metalloproteinases (MMP) have been implicated in virtually all aspects of tumor progression. However, the recent failure of clinical trials employing synthetic MMP inhibitors in cancer chemotherapy has led us to hypothesize that some MMPs may actually serve the host in its defense against tumor progression. Here we show that mice deficient in macrophage elastase (MMP-12) develop significantly more gross Lewis lung carcinoma pulmonary metastases than their wild-type counterparts both in spontaneous and experimental metastasis models. The numbers of micrometastases between the two groups are equivalent; thus, it seems that MMP-12 affects lung tumor growth, and not metastasis formation, per se. MMP-12 is solely macrophage derived in this model, being expressed by tumor-associated macrophages and not by tumor or stromal cells. The presence of MMP-12 is associated with decreased tumor-associated microvessel density in vivo and generates an angiostatic>angiogenic tumor microenvironment that retards lung tumor growth independent of the production of angiostatin. These data define a role for MMP-12 in suppressing the growth of lung metastases and suggest that inhibitors designed to specifically target tumor-promoting MMPs may yet prove effective as cancer therapeutics.

Autonomic receptors in human prostate adenomas.

Radioligand receptor binding techniques were used to characterize alpha 1 adrenergic, alpha 2 adrenergic and muscarinic cholinergic (MCh) binding sites in human prostate adenomas obtained from men with symptomatic and asymptomatic benign prostatic hyperplasia (BPH). Prostate adenoma specimens were obtained from nine men with asymptomatic BPH undergoing cystoprostatectomy, 11 men with symptomatic BPH undergoing open prostatectomy, and 11 men with symptomatic BPH undergoing transurethral resection of the prostate (TURP). A quantitative symptoms score analysis and urinary flow rate determinations documented the absence of bladder outlet obstruction in men undergoing cystoprostatectomy and confirmed the presence of bladder outlet obstruction in men undergoing prostatectomy. The mean equilibrium dissociation constants (Kd) and the mean densities of 125I-Heat (alpha 1 adrenergic) and 3H-NMS (MCh) binding sites were similar in tissue homogenates obtained from men with asymptomatic and symptomatic BPH. The mean Kd of 3H-Rauwolscine (3H-Ra) was significantly greater in the prostatectomy specimens obtained from men with symptomatic BPH compared to the specimens obtained from men with asymptomatic BPH (p less than 0.05). The density of 3H-Ra (alpha 2 adrenergic) binding sites was significantly greater in the prostate adenomas obtained from men with symptomatic BPH compared to the prostate adenomas obtained from men with asymptomatic BPH (p less than 0.05). The difference in alpha 2 adrenoceptor density was accounted for by an increased receptor density in the open prostatectomy specimens. There was no significant correlation between alpha 2 adrenergic, alpha 1 adrenergic, and MCh receptor densities and prostate weight or patient age. This study indicates that the development of infravesical obstruction in men with BPH is not related to upregulation or altered binding affinity of alpha 1 adrenergic or MCh receptor binding sites. The significance of the observed upregulation of alpha 2 adrenoreceptors in the prostate adenomas obtained from men undergoing open prostatectomy is unknown, and requires further investigation.

The alpha adrenergic binding properties of terazosin in the human prostate adenoma and canine brain.

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.

Muscarinic Cholinergic Receptors in Normal Pediatric and Myelodysplastic Bladberi

Radioligand receptor binding experiments and in vitro muscle contractile studies were performed to determine the binding and functional properties of detrusor muscarinic cholinergic receptors in control and myelodysplastic bladders. Control bladder tissue was obtained from 8 children with primary vesicoureteral reflux undergoing ureteral reimplantation and 1 child at the time of organ transplant harvesting. Bladder specimens also were obtained from 10 children with myelomeningocele undergoing augmentation cystoplasty. Preoperative cystograms revealed that all children with vesicoureteral reflux had a smooth-walled bladder with normal capacity, whereas those with myelomeningocele undergoing augmentation cystoplasty had a small capacity bladder with trabeculations. Experiments were performed on detrusor tissue obtained from the bladder body in all cases. Radioligand receptor binding experiments with the 3H-N-methylscopolamine revealed that the equilibrium dissociation constant in control and myelodysplastic bladders was 0.44 +/- 0.09 and 0.40 +/- 0.10 nM., respectively. The equilibrium dissociation constant was similar in control and myelodysplastic bladders. The muscarinic cholinergic receptor density (Bmax) in control and myelodysplastic bladders was 0.66 +/- 0.12 and 0.24 +/- 0.03 fmol. per micrograms, protein, respectively. The significantly lower density of muscarinic cholinergic receptors in the myelodysplastic bladders may be explained by either a down regulations or by the histologically observed development of fibrosis. Concentration response experiments were performed on 7 control and 6 myelodysplastic bladders using carbachol and potassium chloride. The carbachol and potassium chloride concentrations producing half of the maximal response were similar in the control and myelodysplastic bladders, suggesting that detrusor dysfunction in myelodysplasia is not associated with detrusor supersensitivity. The maximal response (Emax) for potassium chloride was less in the myelodysplastic bladders than in the control bladders but the carbachol Emax was not significantly different. Concentration inhibitory experiments with oxybutynin and imipramine demonstrated that the myelodysplastic and control bladders were identically inhibited by these antagonists. Radioligand receptor binding studies and in vitro contractile experiments indicate that the detrusor dysfunction associated with myelomeningocele is not mediated by changes in the binding or functional properties of detrusor muscarinic cholinergic receptors.

Identification and Characterization of Alpha1 Adrenergic Receptors in the Canine Prostate Using [125I]-Heat

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.

Muscarinic Cholinergic Receptors in the Normal and Neurogenic Human Bladder

Bladder dysfunction secondary to neurologic conditions occurs in all age groups and is associated with significant morbidity. The role of neuroreceptors in the development of detrusor dysfunction has not been studied previously. Control bladder tissue specimens were obtained from eight children with ureterovesical reflux undergoing ureteral reimplantation and 14 adults with bladder carcinoma undergoing cystectomy. Neurogenic bladder specimens were obtained from 10 children with myelomeningocele and five adults with neurogenic bladder dysfunction undergoing augmentation cystoplasty. Saturation experiments using 3H-N-methylscopolamine (3H-NMS) were performed in these control and neurogenic bladder homogenates. The mean equilibrium dissociation constants in the neurogenic and control bladders were 0.41 nM and 0.55 nM, respectively. The mean density of muscarinic cholinergic (MCh) receptor binding sites in the neurogenic and control bladders was 0.34 fmol/mg wet wt. and 0.65 fmol/mg. wet wt., respectively. Competitive binding experiments with 3H-NMS and various unlabelled MCh antagonists indicated that the pharmacology of MCh binding sites was similar in neurogenic and control bladders. Age was not significantly correlated with MCh receptor density in the control and neurogenic bladders. Muscarinic cholinergic binding sites are homogeneous in neurogenic and control bladders. The lower density of MCh receptors in the neurogenic bladders may represent down regulation of MCh receptors or a replacement of smooth muscle by fibrosis.

The stereospecificity of LY253352 for α1-adrenoceptor binding sites in the brain and prostate

1 The stereospecificity of the enantiomers of LY253352, a potent and selective α1‐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 α1‐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 α1‐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 Effect of Electrocautery on Neurotransmitter Receptor Binding Assays in the Canine Prostate

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.

Comparison of muscarinic cholinergic and alpha adrenergic receptors in canine ileum, colon, intestinal urinary reservoirs and bladder.

The muscarinic cholinergic (MCh) and alpha 2 adrenergic receptor densities in canine ileum, colon, ileal and colonic urinary reservoirs and bladder were determined using radioligand receptor binding methods in order to provide a rational basis for pharmacologic management of urinary incontinence following bladder replacement with intestinal segments. Muscarinic cholinergic and alpha 2 adrenergic receptor binding sites were studied in these tissues using saturation experiments with 3H-NMS and 3H-rauwolscine, respectively. The mean equilibrium dissociation constants for 3H-NMS binding (0.13 to 0.17 nM) in these tissues were similar (p greater than 0.05) indicating homogeneity of muscarinic cholinergic binding sites. The mean equilibrium dissociation constants for 3H-rauwolscine binding (1.27 to 1.98 nM) in these tissues were also similar (p greater than 0.05). A substantial density of MCh (1.06 to 1.22 fmol/mg. wet wt.) and alpha 2 adrenergic (0.47 to 1.11 fmol/mg. wet wt.) binding sites was identified in the intestinal tissues assayed. The density of ileal and colonic MCh and alpha 2 adrenergic binding sites was not altered following construction of urinary intestinal reservoirs. The presence of a substantial density of MCh and alpha 2 adrenergic binding sites in the intestinal tissues suggests that MCh and alpha 2 adrenergic analogs may be utilized for the management of urinary incontinence following bladder replacement with intestinal urinary reservoirs.