Donna Haynes-Johnson

Identification and functional study of phosphodiesterases in rat urinary bladder

Abstract Cyclic nucleotides are important secondary messengers involved in modulating the contractility of various smooth muscles. Phosphodiesterases (PDE) play important roles in this process by modulating the levels of cyclic nucleotides and their duration of action. This study was designed to identify and characterize the PDE isoenzymes in rat urinary bladder and to evaluate their roles in regulating bladder smooth muscle tone. The involvement of cAMP and cGMP pathways in this process was also assessed. The studies were carried out with tissues from male and female rats and no significant sex-related difference was found in the results. Utilizing the unique pharmacological properties of different isoenzymes, PDE1, 2, 3, 4, and 5 were identified in rat bladder. Organ bath experiments showed that forskolin was most potent in relaxing pre-contracted rat bladder strips while sodium nitroprusside was moderately effective, suggesting the relaxation was mainly mediated by the cAMP pathway and that the cGMP pathway is moderately involved. For PDE inhibitors, the non-specific inhibitor papaverine was most effective in relaxing pre-contracted bladder strips. Among isoenzyme-selective inhibitors, vinpocetine, EHNA, and sildenafil induced more relaxation than milrinone and rolipram.

Nonsteroidal progesterone receptor ligands with unprecedented receptor selectivity

We have characterized a series of nonsteroidal progesterone receptor ligands, the tetrahydropyridazines. Compounds in this series, exemplified by RWJ 26819, demonstrate high affinity and unprecedented specificity for the progesterone receptor relative to other steroid hormone receptors. Like steroidal progestins, RWJ 26819 induces binding of the receptor to a progesterone response element in vitro, and stimulates gene expression in and proliferation of T47D human breast cancer cells. When administered to rabbits orally or subcutaneously, the compound induces histological changes in the uterine lining comparable to those induced by levonorgestrel. It also inhibits ovulation in monkeys. Though less potent in cells and in animal models than would be predicted from binding affinity alone, their enhanced selectivity suggests that they could be effectively used in a clinical setting. Most of the tetrahydropyridazines synthesized are progestin agonists or mixed agonists and antagonists in vitro; however, one compound with antagonist activity in the rabbit uterine transformation assay has been identified.

Cyclic nucleotide phosphodiesterases in rabbit detrusor smooth muscle

OBJECTIVES To identify the phosphodiesterase (PDE) isoenzymes in the rabbit detrusor and to evaluate their roles in regulating detrusor muscular tone. Cyclic nucleotides are important secondary messengers involved in modulating the contractility of various smooth muscles. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are synthesized by their respective cyclases and degraded by various PDEs. METHODS PDE isoenzymes from male and female rabbit detrusor were isolated by the Mono-Q anion exchange column and identified with various inhibitors. Detrusor strips from both sexes were precontracted with carbachol and relaxed with PDE inhibitors and adenylate and guanylyl cyclase activators in a tissue bath. Cyclic nucleotide concentrations in strips from male rabbits were determined after the compound treatment. RESULTS Similar results were obtained from both sexes in the experiments in which both sexes were used. The activities of PDE1, 2, 3, 4, and 5 were identified. Forskolin induced a dramatic rise in the cAMP levels and was the most effective relaxant. Papaverine generated moderate increases in the cAMP and cGMP levels and induced very good relaxation. Vinpocetine produced no detectable changes in the cyclic nucleotide levels but elicited good relaxation. Sildenafil caused an increase in the cGMP levels and had a similar relaxation effect as vinpocetine. Sodium nitroprusside induced some increase in cGMP and had no relaxation effect. Rolipram raised the cAMP levels significantly, yet had a moderate effect on relaxation. CONCLUSIONS Our results demonstrated the presence of PDE1 through 5 in rabbit detrusor muscle and supported their involvement in regulating detrusor muscle tone. The relaxation of rabbit detrusor was mainly mediated by the cAMP pathway.

Increased expression of stromelysin 3 mRNA in leiomyomas (uterine fibroids) compared with myometrium

Objective: To determine the level of expression of selected matrix metalloproteinases in uterine leiomyoma compared with unaffected myometrium in an effort to explain the abnormal accumulation of extracellular matrix in the leiomyoma. Methods: The levels of matrix metalloproteinase (MMP) mRNA in leiomyoma and myometrium weer measured in samples from 22 patients during either proliferative (n = 6) or secretory phases (n = 16) of the menstrual cycle. Relative amounts of collagenase (MMP-1) and stromelysin (MMP-3) mRNAs were measured by Northern blot analysis, and amounts of stromelysin 3 (MMP-11) and matrilysin (MMP-7) mRNA from each sample were determined by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and normalized to β-actin mRNA. Results: The levels of MMP-1 and MMP-3 mRNAs were similar in both leiomyoma and unaffected myometrium. The levels of MMP-11 mRNA were twofold greater in leiomyoma compared with myometrium throughout the menstrual cycle, and the differences in the levels of MMP-11 were significantly different during the secretory phase. The level of MMP-7 mRNA expression was similar in leiomyoma, myometrium, and endometrium. Conclusions: Among the metalloproteinases examined in this study, only the levels of MMP-11 mRNA were elevated in leiomyoma compared with myometrium. The increased expression of MMP-11 in uterine leiomyoma compared with myometrium is analagous to previously reported elevations of MMP-11 mRNA in dermatofibromas compared with unaffected skin. The increased expression of MMP-11 mRNA in fibroid tumors suggests that this MMP may be involved in the formation of a more fibrous extracellular matrix in leiomyoma relative to unaffected myometrium.

Fluorescence Polarization Assay and SDS-PAGE Confirms Matrilysin Degrades Fibronectin and Collagen IV whereas Gelatinase: A Degrades Collagen IV but not Fibronectin

Matrilysin and gelatinase A are hypothesized to have significant roles in uterine and ovarian function. However, proteolytic activity assays for these enzymes are limited. We describe the development of simple and rapid assays for the proteolysis of fluorescein-labeled full-length substrates, collagen IV (Col-IV) and fibronectin (FN), and demonstrate the selectivity of matrilysin (MMP-7) compared to gelatinase A (MMP-2) for fibronectin. Changes in fluorescence intensity (FIU) and fluorescence polarization (mP) resulting from the protease activity of matrilysin and gelatinase A were measured. These studies show that the fluorescently labeled substrates, Col-IV and FN, are as reliable and amenable to rapid in vitro assay as peptide substrates. In addition, they are easier to use than previously described, non-fluorescent methods. The results demonstrate that assays using full-length, biological matrix proteins are more sensitive indicators of MMP-specific substrate activity than peptide based assays.

Synthesis and progesterone receptor binding affinity of substituted 1-phenyl-7-benzyl-4,5,6,7-tetrahydro-1H-indazoles

Abstract Research directed toward the discovery of non-steroidal ligands for steroid receptors led to the preparation of a series of substituted 1-phenyl-7-benzyltetrahydroindazole-3-carboxaldehydes. Appropriately substituted 3-formyl analogs (4) were found to bind with high affinity to progesterone receptors and showed agonist activity in human T47D cells but were inactive in several in vivo models for progestational activity.