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.

The nitric oxide synthase inhibitor, L-NG-monomethylarginine, reduces carrageenan-induced pleurisy in the rat.

Nitric oxide (NO) is a biological mediator that, when produced by the type II (inducible) nitric oxide synthase (NOS), has been implicated in the pathophysiology of inflammatory diseases. To examine this putative role of NO, pleural inflammation was elicited in rats by the intrapleural injection of carrageenan (1 mg). A pleural exudate and cellular influx developed, which peaked at 24 h and generally resolved by 72 h. The cellular influx was primarily composed of polymorphonuclear cells during the first 24 h, followed by macrophages during the subsequent 24 h. Inflammatory cell-associated NOS activity and pleural exudate nitrite (NO2-) + nitrate (NO3-) (NOx) also increased, peaking at 6 h and 24 h, respectively. Cell-associated NOS activity was calcium-independent, indicating the presence of the type II NOS isoform; NOS activity in the pleural cavity and polymorphonuclear cells influx were temporally correlated. Administration of L-NG-monomethylarginine (L-NMA) (200 mg/kg/day) attenuated the pleural exudation, cellular influx, pleural exudate NOx, and cell-associated NOS activity. The relative composition of the pleural cavity cellular infiltrate was not changed by L-NMA, indicating the influx of individual cell types were affected equally. L-Arginine (500 mg/kg/day) completely prevented the effects of L-NMA on pleural exudation and cellular influx and partially prevented the inhibition of pleural exudate NOx accumulation by L-NMA. These data implicate NO as a modulator of the pleural inflammatory response and support a future clinical role for NOS inhibitors in the treatment of inflammatory disease.

Novel potent and selective inhibitors of inducible nitric oxide synthase.

We have identified two novel potent and selective inhibitors of inducible nitric oxide synthase, S-ethylisothiourea and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine. Ki values of 14.7 nM for S-ethylisothiourea and 4.2 nM for 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine were obtained with partially purified preparations of inducible nitric oxide synthase. These compounds demonstrate about 1000-fold greater potency than prototypical inhibitors, and the inhibitions are 10-40-fold more selective for murine inducible nitric oxide synthase, compared with the rat neuronal and bovine endothelial isoforms of nitric oxide synthase. These compounds also potently inhibit the nitric oxide synthase activity in intact J774 mouse macrophages. The inhibition is competitive with the substrate L-arginine and reversible in both enzymatic and intact cell assays. These potent and selective inhibitors of inducible nitric oxide synthase may have potential therapeutic applications in the treatment of inflammatory and autoimmune diseases.

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.

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.

A ring-reversed analog of atrial natriuretic peptide retains receptor binding, guanylate cyclase stimulation.

We have prepared an atrial natriuretic peptide analog, ANP[13-27][1-12], in which the connectivity of the disulfide-linked ring has been reversed by formally cleaving the ring and cyclizing the N- and C-terminal tails. This analog, which retains many of the spatial relationships of the native molecule, binds to both ANP-A and ANP-C receptor subtypes, and triggers the production of cyclic-GMP by ANP-A. ANP-C binding of ANP[13-27][1- 12] is roughly equipotent to that of ANP itself, although the ring cleavage falls within the putative ANP-C binding domain. ANP[13-27][1-8], a truncated analog in which much of this binding domain has been removed, surprisingly maintains a high affinity for ANP-C; however, this peptide has lost the ability to activate the ANP-A-linked guanylate cyclase.

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.

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.

Truncated atrial natriuretic factor analogs retain full agonist activity.

The synthesis, receptor binding, and agonist activity of a series of truncated atrial natriuretic analogs (ANF) are described. These analogs incorporate two portions of the native 28 amino peptide, the eight amino acids C-terminal to Cys7, and two amino acids from the C-terminus (phenylalanine and arginine), into disulfide-bonded cyclic peptides. The inclusion of the C-terminal amino acids converted the ANF analogs from receptor ligands to full agonists, as measured by several methods, including the stimulation of cGMP biosynthesis in endothelial cells, inhibition of aldosterone biosynthesis in rat adrenal cells, and natriuretic-hypotensive activity in vivo. The most potent analogs have cyclohexylalanine (Cha) at position 8. The lead compound (Arg6,Cha8 ANF 6-15 Phe-Arg-Cys-NH2) is a tridecapeptide that integrates the C-terminal amino acids inside the disulfide ring. This peptide, designated as A-68828, has a binding affinity of IC50 = 120 nM, approximately 1/400 of ANF 1-28. However, this analog, in vivo, is only slightly less natriuretic (1/20-1/50) than ANF 1-28. Unlike the native peptide, A-68828 is only mildly hypotensive and at the highest concentration tested reduced blood pressure less than 15 mmHg (1 mmHg = 133.322 Pa). A-68828 inhibited ACTH-induced aldosterone release to a greater extent than ANF 1-28: 100 vs. 50%. The selective natriuretic activity of A-66828, relative to ANF, suggests clinical utility for the treatment of acute renal failure.

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.

Atrial natriuretic peptide antagonists: biological evaluation and structural correlations.

A collection of analogues of atrial natriuretic peptide (ANP) were screened for their ability to inhibit ANP-induced cGMP stimulation. The antagonists revealed through this screen are structurally related; almost all are substituted at either aspartate-13 or phenylalanine-26. This tendency is consistent throughout several families of small ANP analogues, suggesting that these two amino acid residues are involved in the process of ANP/cGMP signal transduction. One compound, A74186, was studied in some detail. A74186 is a potent inhibitor of the activation of guanylate cyclase by ANP; it acts with a pA2 of 7.12 in rat vascular smooth muscle cells and shifts the ANP/cGMP dose-response curve by 3 orders of magnitude at a 10 microM concentration. It also inhibits cGMP release in vivo, and at an infusion rate of 10 micrograms/kg-min it completely abolishes ANP-induced natriuresis and diuresis. A74186 does not, however, antagonize the hypotensive or vasorelaxant effects of ANP; in fact it acts as an agonist in these assays. It thus appears that cGMP, although it may mediate the renal responses to ANP, is not responsible for the vascular and hemodynamic effects that result from the action of the hormone.

Characterization of endothelin converting enzyme in rat lung.

An enzyme activity which converts human big endothelin (1-38) to endothelin (1-21) and a C-terminal fragment (CTF, 22-38) was identified in a plasma membrane fraction prepared from rat lung. The conversion activity was optimal at pH 4.0, was inhibited by Pepstatin-A (IC50 = 20 nM), but was not affected by TLCK, Aprotinin, PMSF, E-64, Bestatin, Phosphoramidon or Thiorphan at 40 microM. Metal ions activated the activity by 1.5 - 2.5 fold in the order of Mn+2 greater than Zn+2 = Ca+2 greater than Ba+2. These data suggest that a Pepstatin-A inhibitable, metal ion related aspartic protease may be involved in the conversion of big endothelin to endothelin in rat lung.

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.

Divergence of ANF analogs in smooth muscle cell cGMP response and aorta vasorelaxation: evidence for receptor subtypes.

ANF analog potencies in stimulating smooth muscle cell cGMP were compared with the ability to relax histamine-constricted rabbit aorta in vitro. ANF[1-28], [5-28], [5-27] and Lys-11[5-28] elevated cGMP and were potent vasorelaxants. ANF[7-23] and Lys-11[7-23] were potent cGMP stimulators but 1000-fold weaker relaxants. Tyr-8[5-27] did not stimulate cGMP synthesis or antagonize the response of the other peptides, yet was a potent vasorelaxant. Crosslinking with 125I-ANF identified bands at 150 and 65 KD by SDS-PAGE. ANF[1-28], Lys-11[7-23] and Tyr-8[5-27] blocked crosslinking at low concentration despite disparate activities. These data support the existence of ANF receptor subtypes and suggest that cGMP elevation alone is not sufficient to promote atrial peptide-induced vasorelaxation.

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.

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.

Mullerian inhibiting substance reduction of colony growth of human gynecologic cancers in a stem cell assay.

Mullerian Inhibiting Substance (MIS), a fetal testicular product that causes regression of the Mullerian duct in the male mammalian embryo, was evaluated for its antitumor effect on the premise that a substance active against this genital precursor in the fetus might also be active against tumors derived from these tissues. Increasingly pure fractions of biologically active MIS, prepared from newborn calf testes, were tested in the soft agar colony inhibition assay against single cell suspensions of fresh tumors derived in ascitic or solid form from patients with gynecologic malignancies. Twenty-eight tumor specimens placed in soft agar culture have provided sufficient growth to assess an MIS effect. Twenty-five of these 28 tumors showed significant colony inhibition after incubation with MIS. Increased antitumor response correlated with increased purification of MIS when the same tumor was treated with preparations of different purity. Samples obtained from the same patient at different times, from both ascites and solid tumor sources, produced nearly identical responses to MIS. MIS preparations, previously shown to be active in microcytotoxicity and colony inhibition assays against established human ovarian and endometrial carcinoma lines demonstrate consistent antitumor activity against fresh human gynecologic cancers removed at surgery.

Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review.

Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.

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)