Phosphorylation events during Müllerian duct regression.

Regression of the fetal rat Müllerian duct in vitro was stimulated by sodium fluoride in the absence of Müllerian inhibiting substance. The action of Müllerian inhibiting substance was inhibited by sodium vanadate, adenosine 5'-triphosphate, and several related nucleotides in the presence of manganese ions. Epidermal growth factor specifically inhibited the substance, but only with manganese ions present. Insulin, platelet-derived growth factor, and nerve growth factor had no effect. These results suggest that dephosphorylation of membrane proteins mediates the action of Müllerian inhibiting substance.

Cyclic adenosine 3',5'-monophosphate modulation of mullerian duct regression.

(Bu)2cAMP inhibits the action of testicular Mullerian inhibiting substance (MIS) in vitro, but it is unknown whether the intracellular nucleotide diminishes production of MIS by the testis or interferes with its action at the Mullerian duct. When added to the 14 1/2-day old rat embryo Mullerian duct in organ culture, (Bu)2cAMP (0.1 or 1.0 mM) inhibited regression caused by biologically active exogenous MIS fractions, as well as that produced by the fetal testis (1.0 mM). Dibutyryl cyclic guanosine 3',5'-monophosphate was ineffective against exogenous MIS fractions or the fetal testis. The phosphodiesterase inhibitor, methyl-isobutyl-xanthine, had the same inhibitory effect on the MIS-fraction (0.1 or 1.0 mM), as well as against MIS secreted from the fetal testis (1.0 mM). Theophylline at 1.0 mM inhibited the action of the MIS fraction. The presence of (Bu)2cAMP in the medium was required for at least the first 24 h of the 72-h incubation to significantly inhibit MIS action. These results indicate that intracellular cAMP inhibits the action of MIS at the Mullerian duct itself by a potentially reversible change in the cells. We speculate that cAMP may act by altering the state of differentiation in the cells, perhaps by mediating phosphorylation of intracellular (and extracellular) proteins.

Androgen stimulation of nucleotide pyrophosphatase during mullerian duct regression.

Mullerian inhibiting substance (MIS) causes regression of the embryonic Mullerian duct. In the fetal rat urogenital ridge, extracellular nucleotide pyrophosphatase (NPPase) can be detected by histochemical staining on the regressing male Mullerian duct, with no corresponding enzyme localization on developing female Mullerian ducts. In vivo results in male embryos can be confirmed in vitro by incubating 14.5-day-gestation female urogenital ridges with MIS and testosterone for 72 h before enzyme localization. Since the addition of testosterone to MIS is obligatory to detect NPPase activity in vitro, and certain steroids enhance Mullerian duct regression, additional steroids were tested in vitro alone or in combination with MIS for their abilities to stimulate NPPase. NPPase induction occurred only with the combinations of MIS and testosterone or MIS and medroxyprogesterone acetate. Neither MIS alone nor any steroid used alone stimulated NPPase activity. The effect of exogenous NPPase added alone to the developing urogenital ridge was also assessed. Incubation of the female urogenital ridge for 72 h with exogenous NPPase caused marked hyperplasia of the Mullerian duct epithelial cells and early mesenchymal cell condensation, without the basement membrane breakdown normally seen in regression. Since NPPase activity is present in the Mullerian duct only during regression, these findings suggest that MIS and fetal androgens are synergistically modulating the activity of this enzyme. Its role in the Mullerian duct, as suggested by its cytological effects, may be to stimulate cellular responses before migration during regression.

Mullerian inhibiting substance inhibits colony growth of a human ovarian carcinoma cell line.

Mullerian inhibiting substance (MIS), a fetal testicular product which causes regression of the Mullerian duct in the male mammalian embryo, is being evaluated as an inhibitor of genital tract tumors. The present study demonstrates in a clonogenic soft agar that biologically active MIS inhibits the growth of a human ovarian carcinoma cell line (HOC-21) in a dose-dependent manner when compared to phosphate-buffered saline medium controls, heat-inactivated MIS, or biochemical fractions which lack biological activity (P less than 0.05).

Small atrial natriuretic peptide analogues: design, synthesis, and structural requirements for guanylate cyclase activation.

Structure/activity studies on atrial natriuretic peptide ANP (1-28) have highlighted three portions of the native molecule as necessary for its biological responses. We have linked these three regions and excised the remaining segments to produce a family of small analogues (less than half the size of the parent) which demonstrate the full range of ANP's actions. Importantly, these compounds act at both major types of ANP receptor. Two critical modifications lead to more potent analogues; both involve expanding the cyclic portion of the molecule. Further optimization of one of these modified structures leads to A68828, a full ANP agonist which shows promise as a preventative agent against acute renal failure.

Regional differences in endothelin converting enzyme activity in rat brain: inhibition by phosphoramidon and EDTA.

1. It has been demonstrated previously that conversion of big endothelin-1 (bET-1) to endothelin-1 (ET-1) is inhibited in vitro and in vivo by phosphoramidon. In addition, ET-1 binding sites and mRNA have been shown within the brain. Here we expand upon our previous observation that rat brain contains phosphoramidon-inhibitable endothelin converting enzyme (ECE) and show that this activity is not uniformly distributed throughout the brain. 2. ECE activity was detected by a bioassay which depended upon the 10,000 fold difference in potency between bET-1 and ET-1 as stimulants of guanosine 3':5'-cyclic monophosphate (cyclic GMP) accumulation in kidney epithelial (PK1) cells of the pig. Data were confirmed by specific enzyme-linked immunosorbent assay (ELISA) employing antibody directed against ET-1/3(17-21). 3. Following homogenization of the whole brain and ultracentrifugation the 100,000 g pellet contained greater than 4 times more ECE activity than the cytosol. Washing of the pellet with KCl (1 M) and extraction with the detergent CHAPS (20 mM) revealed a phosphoramidon-inhibitable ECE within the residual particulate fraction (nominally classified as the cytoskeletal fraction). Phosphoramidon (IC50, approx. 5 microM) or EDTA inhibited the conversion of bET-1 to ET-1 by the cytoskeletal fraction of rat brain by more than 60%.2+ 4. Following dissection of rat brain into olfactory bulb, cerebral cortex, striatum, hippocampus, cerebellum, midbrain (including thalamus), hypothalamus and medulla oblongata (including pons) the greatest ECE was detected in the hypothalamus and medulla oblongata.After fractionation, the ECE-activities in the cytoskeletal fractions prepared from the hypothalamus or medulla oblongata were inhibited concentration-dependently by phosphoramidon or EDTA, with maximum inhibitions of>80% and >70%, respectively.5. These data show that rat brain contains a phosphoramidon- and EDTA-inhibitable ECE which maybe similar to that present in endothelial cells. The localization of this enzyme correlates with published reports of immunoreactive-ET-l, ET-1-binding sites, and messenger RNA for ET-1 in the rat brain, and suggests the presence of the entire synthetic pathway for ET-1.

Effect of cell density on endothelin release from endothelial cells and phosphoramidon dependent inhibition.

The modulation of endothelin (ET) release from endothelial cells was investigated as a function of cell density. The present study examined the release of ET from bovine pulmonary artery endothelial cells (BPAEC) and bovine aortic endothelial cells (BAEC) at various cell densities, as well as the effects of phosphoramidon, thiorphan and pepstatin on ET release at different densities. ET release from BPAEC and BAEC decreased as cell density increased. This cell density effect was not observed with prostacyclin release from either BPAEC or BAEC. Phosphoramidon (1 mM) inhibited ET release at every density examined for both BPAEC and BAEC. Thiorphan (1 mM) inhibited ET release from BPAEC weakly at low density and had no effect on ET release from BAEC. Pepstatin (1 mM) slightly inhibited ET release in BPAEC at the lowest density and had no effect at any other cell density for either cell type. These protease inhibitors had no effect on cell viability as determined by trypan blue exclusion and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide conversion assay. This study supports the concept that ET release is modulated by endothelial cell density. In addition, these data demonstrate that phosphoramidon, which presumably inhibits the endothelin converting enzyme, can inhibit ET release over a range of cell densities without affecting cell viability.

Müllerian inhibiting substance blocks autophosphorylation of the EGF receptor by inhibiting tyrosine kinase.

The fetal regressor Müllerian inhibiting substance (MIS), in concentrations as low as picomolar, inhibited the growth of A-431 cells and the autophosphorylation of its epidermal growth factor (EGF) receptor. The inhibition of membrane phosphorylation was due neither to the reduction of the total number of EGF receptor binding sites, nor to stimulation of intrinsic phosphates, but rather to inhibition of tyrosine kinase activity. MIS control of EGF receptor autophosphorylation by tyrosine kinase may be one mechanism by which Müllerian duct regression in the embryo and the inhibition of A-431 proliferation is initiated. In addition, MIS as an inhibitor of phosphorylation may furnish a tool to probe the role of membrane phosphorylation in growth control.

Atrial peptides induce mast cell histamine release.

Human atrial natriuretic peptide [ANF(1-28)] contains five arginine residues and carries an overall positive change of four. It was hypothesized that atrial peptides may induce mast cell histamine release. In vitro, three atrial peptides [ANF(1-28), (3-28) and (5-28)] were demonstrated to induce dose-dependent histamine release from isolated rat peritoneal mast cells. In vivo, ANF(3-28) produced a dose-dependent increase in rat skin permeability which was blocked by antagonists of histamine and serotonin. The results indicate atrial peptides are capable of inducing mast cell degranulation in a manner similar to that described for other positively charged peptides.

Effects of methotrexate on nucleotide pools in normal human T cells and the CEM T cell line.

It has been proposed that the clinical utility of methotrexate (MTX) in the treatment of rheumatoid arthritis may be due, in part, to inhibition of 5-amino imidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) by polyglutamated forms of MTX. AICARFT is the second folate dependent enzyme in de novo purine biosynthesis. In this study, the effects of MTX on de novo purine biosynthesis as well as total nucleotide pools were evaluated in both the human T cell line, CEM, and phytohemagglutinin-activated normal human T lymphocytes. De novo synthesized purines were metabolically labeled with 14C-glycine after MTX treatment and analyzed by HPLC. In normal T cells, MTX produced a dose-dependent reduction in de novo adenosine and guanosine pools with maximal effects (>50%) at 1 microM MTX. In CEM cells, de novo purine synthesis was almost completely blocked by 1 microM MTX. Total purine pools were also reduced in both cell types after MTX treatment. Since 1 microM MTX caused almost complete growth inhibition in CEM cells, we evaluated whether growth could be reconstituted with exogenous purine bases and pyrimidine nucleosides which can be utilized via salvage pathways. The combination of hypoxanthine and thymidine substantially reversed growth inhibition with 1 microM MTX in CEM cells. Taken together, these results demonstrate that MTX inhibits de novo nucleotide synthesis in T cells and suggest that AICARFT inhibition may be one aspect of the multi-site mechanism of MTX action in the treatment of rheumatoid arthritis.

Steroid enhancement of Mullerian duct regression.

The biologic activity of the glycoprotein, Mullerian Inhibiting Substance (MIS), was enhanced by added testosterone, medroxyprogesterone acetate, and progesterone, although these steroids did not cause Mullerian duct regression when added alone. Statistically significant augmentation by testosterone and medroxyprogesterone was observed at concentrations of 10(-7) and 10(-8) M. Progesterone enhanced MIS activity at a concentration of 10(-6) M. By contrast, dihydrotestosterone, estradiol, and corticosterone at concentrations of 10(-9) to 10(-5) M produced no significant effect on MIS activity. Since MIS does not bind directly to the steroids studied, we suspect that the enhancement effect occurs at the receptor level, probably at the mesenchyme as observed in other developmental systems.

Subrenal capsule assay to test the viability of parenterally delivered müllerian inhibiting substance.

Production of bovine müllerian inhibiting substance (MIS) has been increased to allow generation of large quantities of biologically active purified material. The limited MIS previously available allowed only pretreatment of tumors prior to colony inhibition or implanting in nude mice. In preparation for posttransplantation tumor treatment, a subrenal capsule assay, which was first used against human tumors heterotransplanted into nude mice and subsequently against those heterotransplanted into immunocompetent mice, was adapted to determine (1) if MIS preparations could traverse the bloodstream without degradation and (2) the optimal dose required to produce a biologic effect. Urogenital ridges from female 14-day-old rat embryos were transferred atraumatically to small pouches beneath the renal capsule of the immunocompetent male CDF1 mice. The cranial-caudal orientation of the ridge with its müllerian duct was maintained. Over the next 72 hours, the mice were injected via the tail vein with 0.1 mL of an MIS-containing solution over a 100-fold concentration range. After three days, the kidneys were removed and shaved just below the ridge, which was then placed in soft agar for orientation and subsequent serial sectioning. After fixation, dehydration, and paraffin embedding, sections were stained and regression of the müllerian duct was graded and compared according to concentration and number of MIS doses administered. Regression diminished from almost complete (4+) at the highest dose, to minimal (1 to 2+) at 1/100 of that dose. Heat-inactivated and vehicle controls caused no regression of the müllerian ducts.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of monoclonal antibodies for affinity purification of bovine mullerian inhibiting substance activity.

Two monoclonal antibodies (IG8 and IG10) specific for Mullerian inhibiting substance (MIS) were obtained from the fusion between myeloma cell line SP2/0 and spleen cells from an A/J mouse immunized with partially purified MIS. The resulting hybridomas were screened by a solid-phase RIA and two lines were selected and cloned. Both MAbs IG8 and IG10 subsequently demonstrated specificity for MIS by their ability to inhibit biologically active MIS by precipitation with a second antibody, directly block MIS activity in the organ culture assay, and adsorb and elute active MIS when coupled to a solid support. SDS-polyacrylamide gel electrophoresis of affinity purified MIS demonstrated a major band at 140 kD in unreduced gels and two bands with approximate molecular weights of 70 and 74 KD following reduction. Protein bands were localized either directly by silver staining or on immunoblots developed with radiolabeled anti-MIS MA.

Mullerian inhibiting substance: a fetal hormone with surgical implications.

Mullerian Inhibiting Substance (MIS) is secreted from the fetal (and postnatal) testis and is known to cause regression of the Mullerian ducts, the anlage of the fallopian tubes, uterus and upper vagina. It is a large glycoprotein hormone, the action of which appears to be modulated by sex steroids: mainly testosterone in mammals and oestrogen in birds. Recent evidence has raised the possibility that its action may be to diminish cell surface phosphorylation and thereby change the direction of differentiation of the Mullerian duct towards regression. Other suspected functions for MIS include control of testicular descent and inhibition of malignant tumours of the female genital tract.

Characterization and partial purification of endothelin-converting enzyme in rat lung.

Endothelin-1 (ET-1) was originally identified in the culture supernatant of porcine aortic endothelial cells. From the deduced amino acid sequence, a biosynthetic pathway has been proposed that a prepro- form of porcine ET-1 is initially processed by dibasic pair proteolysis to a 39-amino acid intermediate form (big ET), which is then converted to ET-1 by specific proteolytic cleavage between Trp21 and Val22. We have identified an enzyme activity that converts human big endothelin[1-38] to endothelin[1-21] and a C-terminal fragment (CTF, 22-38) in a homogenate fraction from rat lung. The conversion activity was enriched threefold in a plasma membrane fraction. Metal ions activated the activity by about 1.5- to 2.5-fold, in the order of Mn2+ greater than Zn2+ = Ca2+ greater than Mg2+ greater than Ba2+. The conversion activity was optimal at pH 4.0, was inhibited by pepstatin-A (IC50 = 20 nmol), but not affected by TLCK, aprotinin, PMSF, E-64, bestatin, phosphoramidon, or thiorphan at 40 microM. The converting enzyme was partially purified from rat lung plasma membranes by sequential HPLC on Mono Q, Superose 12, and Mono P. The enzyme has an apparent molecular mass of 90 kDa as estimated by SDS-PAGE or gel filtration and appears to be a single peptide protein. The enzyme may exist as isozymes with isoelectric point (pI) values at 6.2 and 6.3.

Detection by bioassay and specific enzyme-linked immunosorbent assay of phosphoramidon-inhibitable endothelin-converting activity in brain and endothelium.

The endothelin-converting enzyme (ECE) activity present in endothelial cells and rat and human brains was characterized using a selective and rapid bioassay for endothelin-1 (ET-1) or endothelin-3 (ET-3) together with a sensitive enzyme-linked immunosorbent assay. We found that ECE activity was predominantly in the membrane fraction of endothelial cells from which it could be extracted by treatment with detergent. In rat brain tissue, the ECE activity was in the membrane fraction and was not solubilized by detergent treatment. Further dissection of the brain revealed that there was a strong localization of ECE activity in the hypothalamus, midbrain, and medulla oblongata in agreement with earlier observations of ET-like immunoreactivity and binding sites. Experiments with human brain tissue also showed the presence of ECE activity. In conclusion, our studies confirmed the presence of ECE activity within endothelial cells, and showed ECE to be localized in brain tissue in sites consistent with the selective distribution of the ET-1 synthetic pathway. In all tissues studied, the ECE activity was significantly inhibited by phosphoramidon or ethylenediaminetetra-acetate.

The role of nucleotide pyrophosphatase in Mullerian duct regression.

Mullerian inhibiting substance (MIS), a glycoprotein from the fetal testis causing regression of the embryonic Mullerian duct, can be inhibited in vitro in the presence of Mn2+ by a wide range of nucleotides including GTP, NAD, ATP, AMP, and several nonhydrolyzable synthetic ATP analogs. Extracellular nucleotide pyrophosphatase (NPPase), an enzyme able to hydrolyze the wide variety of the nucleotides and analogs found to inhibit Mullerian duct regression, was studied by histochemical staining (H. Sierakowska and D. Shugar (1963). Biochem. Biophys. Res. Commun. 11, 70-74) to determine if NPPase localized in or around the Mullerian duct during regression. Frozen sections of urogenital ridges from 14 1/2- to 17 1/2-day rat fetuses (n = 77) were incubated with a-naphthyl thymidine-5'-phosphate (naphthyl TMP) and Fast Red TR. Nucleotide pyrophosphatase hydrolyzes naphthyl TMP, releasing naphthol, which then reacts with Fast Red to produce color at the enzyme site. Nucleotide hydrolysis was detected around regressing male (n = 16) Mullerian duct cells at 16 1/2 days of gestation, but no hydrolysis was detected around female (n = 17) Mullerian duct cells at any stage. Controls (n = 24) incubated without substrate did not stain. Addition of exogenous ATP (n = 20) to the histochemical incubation medium inhibited nucleotide hydrolysis on male Mullerian ducts, suggesting that this staining is specific for pyrophosphatase activity. Results in vivo were confirmed in vitro by incubating 14 1/2 day female rat urogenital ridges with MIS for 72 hr prior to histochemical staining. The addition of testosterone to MIS was obligatory to detect staining in vitro (n = 10). The localized NPPase activity around the regressing Mullerian duct suggests that NPPase may appear as a consequence of duct regression and may act to control the degree of membrane phosphorylation by degrading excess trinucleotides.

Enhanced purification of Mullerian inhibiting substance by lectin affinity chromatography.

Mullerian inhibiting substance (MIS), a secreted testicular product responsible for regression of the Mullerian ducts in the male mammalian embryo, was purified 7000 fold, exploiting the glycoprotein nature of this important fetal regressor to achieve enhanced purification. The present procedure employs media incubation of newborn calf testis, passage through DEAE Bio-Gel A and CM Bio-Gel A and sequential lectin affinity chromatography on wheat germ lectin (WGL)-Sepharose 6MB and concanavalin A (Con A)-Sepharose 4B. Strongly bioactive MIS was released from both lectin columns in the bound glycoprotein fraction only after elution with lectin-specific sugar. Carbohydrate analysis of the highly purified glycoprotein fraction eluted from Con A indicated the presence of both N-acetyl glucosamine and mannose, as would be expected from its sequential lectin affinity, as well as of galactose, galactosamine and N-acetyl neuraminic acid. Electrophoresis of this fraction on polyacrylamide-SDS gels showed an identical band pattern after staining with either Coomassie blue or periodic acid-Schiff reagent, further indicating that MIS is a glycoprotein.