Activation of the human neutrophil nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase by protein kinase C.

A variety of phagocytosable and soluble agonists stimulate the human neutrophil respiratory burst enzyme, NADPH-oxidase, an activity required for normal microbicidal function. Of these agonists, the phorbol esters, which stimulate diverse systems by their ability to substitute for diacylglycerol to activate protein kinase C (the major phorbol ester receptor), have now been shown to directly stimulate NADPH-oxidase through this same receptor. Almost 90% of the specific receptors for phorbol 12,13-dibutyrate (PDBu) were found in the cytosol upon subcellular fractionation. The dissociation constant for [3H]PDBu was 1.2 nM. No significant difference was found in the distribution of the receptor between subcellular fractions from resting as compared with phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils. On the basis of these binding studies, we were able to establish a reconstituted system in which PMA activated dormant NADPH-oxidase in a light membrane fraction when cytosol, NADPH, phosphatidylserine, or phosphatidylinositol and ATP were added. The calcium chelator, EGTA, inhibited the activation, which suggested a requirement for calcium at low concentrations. The half-maximally effective PMA dose was 1.1 nM, as predicted from the receptor content in these preparations. Reconstitution of oxidase activity was rapid, peaking within 1 min of incubation. Purified protein kinase C was able to substitute for the cytosol fraction, and accounted for 80% of the cytosol activity. These studies demonstrate that phorbol esters stimulate the neutrophil respiratory burst through activation of cytosolic protein kinase C, which in turn activates either a regulatory constituent or the NADPH-oxidase directly in the plasma membrane to generate an active O-2-generating system.

The hemodynamic and metabolic profiles of Zucker diabetic fatty rats treated with a single molecule triple vasopeptidase inhibitor, CGS 35601.

CGS 35601 is a triple vasopeptidase inhibitor (VPI) of angiotensin converting enzyme (ACE), neutral endopeptidase (NEP), and endothelin (ET) converting enzyme-1 (ECE-1), with respective IC(50) values of 22, 2, and 55 nM. The aim of the present study was to establish the hemodynamic profile of Zucker diabetic fatty (Zdf)-Fatty rats, a high-fat diet gene-prone model developing spontaneous Type 2 diabetes (T2D) and the effects of CGS 35601. Male Zdf-Fatty (14 weeks, n = 17-23), Zdf-Lean (14 weeks, n = 8-10), and Wistar (14 weeks, n = 9-10) rats on distinct diets were implanted with a catheter in the left carotid and placed individually in a metabolic cage for 30 days. The hemodynamic profile and some metabolic biomarkers were assessed daily. After a 7-day stabilization period, the Zdf-Fatty rats were divided into two groups: Group 1, controls (n = 7-10) receiving vehicle-saline (250 microl/hr) and Group 2, (n = 10-13) receiving increasing doses of CGS 35601 (0.1, 1, and 5 mg/kg/day x 6 days each, intra-arterially) followed by a 5-day washout period. Mean arterial blood pressure (MABP) of young Zdf-Fatty rats was compared with age-matched Zdf-Lean and Wistar rats, which were found similar. MABP decreased by 5.9% (from baseline at 102 +/- 5 to 96 +/- 4 mmHg), 12.7% (to 89 +/- 6 mmHg) and 21.6% (to 80 +/- 4 mmHg), at 0.1, 1, and 5 mg/kg/day, respectively, in CGS 35601-treated Zdf-Fatty rats. Systolic and diastolic blood pressures were similarly reduced. The heart rate was not affected. Hyperglycemic status and insulin-resistance were not modulated by short-term treatment. CGS 35601 presented an excellent short-term safety profile. This novel molecule and class of VPI may be of interest for lowering vascular tone. Further long-term studies, once cardiovascular and renal complications have developed in this T2D rat model are warranted to define the efficacy of this class of VPI.

Triple VPI CGS 35601 reduces high blood pressure in low-renin, high-salt Dahl salt-sensitive rats.

We previously reported that CGS 35601, a potent triple inhibitor of angiotensin-converting enzyme, neutral endopeptidase, and endothelin-converting enzyme 1, completely normalized mean arterial blood pressure (MABP) in 36-week-old spontaneously hypertensive rats, a normal renin model. The aim of the present study was to determine the effects of this triple vasopeptidase inhibitor (VPI) on the hemodynamic profile of instrumented, conscious, and unrestrained Dahl salt-sensitive (DSS) rats, a gene-prone, high-salt diet-induced low-renin hypertension model. Male DSS rats (mean weight [+/-SEM], 385 +/- 10 g) were fed a normal diet (Group 1) or a high-salt diet (Groups 2 and 3; 8% NaCl in food) for 6 weeks and then instrumented with a carotid catheter and placed individually in metabolic cages for 30 days. The hemodynamic, hematological, and biochemical profiles were assessed daily. Dose-dependent treatment started after a 7-day stabilization period in Groups 1 and 2 (vehicle dosage, 250 microl/hr) and Group 3 (CGS 35601 dosages of 0.1, 1, and 5 mg/kg/day for 6 days per dose by means of constant intra-arterial infusion), followed by a 5-day washout period. Two additional groups included normotensive Wistar rats (Group 4) and DSS rats that received a double high-salt solid (8% NaCl) and liquid (1% NaCl) diet (Group 5). The MABP in rats receiving CGS 35601 decreased in a dose-dependent fashion toward the baseline level observed in DSS rats receiving a normal diet. The heart rate was unaffected. The hemodynamic profile returned to normal during the washout period. This novel triple VPI is a potent and effective antihypertensive agent with a safe short-term profile that may be of interest for treating hypertension and other cardiovascular diseases. Other hypertensive rat models are being tested.

Purification of stable protein kinase C from mouse brain cytosol by specific ligand elution using fast protein liquid chromatography.

The Ca2+- and phospholipid-dependent protein kinase (protein kinase C) has been purified to electrophoretic homogeneity from mouse brain cytosol. [20-3H]phorbol 12,13-dibutyrate binding activity was found to coelute quantitatively with the protein kinase activity throughout the purification procedure. The crude extract was first run over a DE52 column. Fractions containing peak activities were then chromatographed using a fast protein liquid chromatography system with a Mono Q column followed by chromatography on the same column run in the presence of 1 mM adenosine triphosphate. The adenosine triphosphate specifically shifted the elution position of the Ca2+- and phospholipid-dependent protein kinase, providing a powerful step in the purification procedure. The remaining minor contaminants were removed by hydrophobic chromatography on a TSK-phenyl-5-PW column. This purification procedure required less than 2 days after the initial large batch DE52 column chromatography. The molecular weight of the purified receptor was estimated to be 81,000 by its mobility on sodium dodecyl sulfate/polyacrylamide gels, in agreement with the published values. Optimal conditions for the storage of the purified receptor were sought. Both protein kinase and phorbol ester binding activities were stable for 2 mo when stored in the presence of 0.01% Triton X-100 at -70 degrees C. Polyclonal antibodies to the purified receptor have been prepared from rabbits. These antibodies recognized the purified receptor in electroblotting assays and were able to immunoprecipitate the purified receptor.

Heterogeneity of [3H]phorbol 12,13-dibutyrate binding in primary mouse keratinocytes at different stages of maturation.

Mouse keratinocytes respond heterogeneously to phorbol esters with distinct subpopulations stimulated to proliferate or induced to differentiate. The maturation state of the epidermal cell at the time of exposure may determine its response. The binding of phorbol esters to primary mouse keratinocytes was studied under culture conditions selecting for proliferating cells or differentiating cells. [20-3H]-12-Deoxyphorbol 13-isobutyrate ([3H]-DPB) bound to both types of cells at one class of binding sites. The dissociation constant (Kd) for [3H]DPB in the proliferative cells [epidermal cells grown in Eagle's minimal essential medium containing 8% fetal calf serum (Chelex treated) and 0.05 mM CaCl2] was 69 nM and the binding at saturation (Bmax) was 1.3 pmol/mg of protein. The corresponding values in the differentiative cells (epidermal cells grown in Eagle's minimal essential medium containing 8% fetal calf serum and 1.2 mM CaCl2) were 96 nM and 1.5 pmol/mg of protein, respectively. In contrast to the results obtained with [3H]DPB, [20-3H]phorbol 12,13-dibutyrate ([3H]PDBU) bound to both cell types in a heterogeneous fashion. Analyzed by the Ligand Program with a two-site model, the Kd values for the two binding sites in the cells grown in the medium containing 0.05 mM CaCl2 were 5.5 and 100 nM and the Bmax values were 0.9 and 1.7 pmol/mg of protein, respectively. The site for [3H]DPB binding seemed to correspond to the higher affinity [3H]PDBU binding site. The major difference in the cells grown in the medium containing 1.2 mM CaCl2 was an increase (approximately two-fold, depending on details of the analysis) in the Bmax of the lower affinity binding site with the other three parameters remaining similar. The state of epidermal differentiation thus appears to modulate the amount of the lower affinity binding sites for phorbol esters. Three Ca2+-resistant cell lines have been developed from carcinogen-treated primary keratinocytes. These cells are not tumorigenic and demonstrate characteristics consistent with their being initiated cells. In the medium containing 1.2 mM CaCl2, [3H]PDBU bound to these cells at one class of binding sites with affinities similar to that of the higher affinity sites for [3H]PDBU in cells grown in the medium containing either 0.05 or 1.2 mM CaCl2.

Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells.

Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, we have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with [3H]arachidonic acid. Treatment with phospholipase C at 0.05 units/ml for 30 min led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C at 0.1 enzyme unit/ml yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding. Several diacylglycerols at concentrations of 250 microM and above effectively competed for phorbol-12,13-dibutyrate binding, reduced epidermal growth factor binding, and to a lesser extent induced ornithine decarboxylase and transglutaminase. These results support the hypothesis that diacylglycerols can act through the phorbol ester receptors and thus produce biological and biochemical responses similar to those of the phorbol esters.

Contrasting actions of staurosporine, a protein kinase C inhibitor, on human neutrophils and primary mouse epidermal cells.

Staurosporine, a recently described microbial alkaloid, is uniquely potent as an inhibitor of protein kinase C in vitro, being active at nM concentrations rather than the microM concentrations typical of other inhibitor classes. Like these other inhibitors, however, staurosporine exhibits only limited selectivity among different protein kinases. We report here that, in intact human neutrophils, nM concentrations of staurosporine blocked the action of the phorbol ester tumor promoters. In mouse primary epidermal cells, on the other hand, staurosporine failed to block the effects of phorbol 12,13-dibutyrate on epidermal growth factor binding and on induction of ornithine decarboxylase and epidermal transglutaminase. Unexpectedly, staurosporine induced morphological changes in keratinocytes to a dendritic shape resembling that induced by the phorbol esters. It also induced epidermal transglutaminase and cornified envelope production, markers of the differentiative pathway in the epidermal cells. We conclude that the effectiveness of staurosporine as a protein kinase C inhibitor in intact cells may depend markedly on the cell system. Other actions of staurosporine may predominate, and, in keratinocytes, its activity is suggestive of a tumor promoter rather than of an inhibitor of tumor promotion.

Multiple forms of peptidyl alpha-amidating enzyme: purification from rat medullary thyroid carcinoma CA-77 cell-conditioned medium.

Conditioned medium from cultures of rat medullary thyroid carcinoma CA-77 cells was used as a source for purification of the secreted forms of peptidyl alpha-amidating enzyme. The alpha-amidating enzyme activity was partially purified using a combination of weak anion exchange and gel filtration chromatography. Subsequent strong anion exchange chromatography at pH 6.0 resolved this partially pure enzyme into four distinct peaks of activity, termed Ia, Ib, II, and III. Peaks Ia and Ib exhibited broad pH optima between pH 6.0-8.5, whereas peaks II and III both exhibited pH optima at approximately pH 5.0. The peak III activity was further purified to electrophoretic homogeneity using hydrophobic interactive chromatography followed by strong anion exchange chromatography at pH 8.0. The enzyme exhibited an apparent molecular mass of 75K, a pH optimum of approximately pH 5.0, and a maximal turnover number of 580 min-1 in the presence of L-ascorbate. Kinetic studies demonstrated that the enzyme probably functions through a ping-pong mechanism with respect to the binding of the glycine-extended peptide substrate and the L-ascorbate cofactor. The peak III enzyme exhibits several distinctive characteristics compared to amidating enzymes isolated and characterized by other laboratories.

Phosphorylation of neurofilament proteins by protein kinase C.

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional alpha-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [32P]orthophosphate in vivo.

The design, structure, and therapeutic application of matrix metalloproteinase inhibitors.

Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes involved in the degradation and remodeling of extracellular matrix proteins. The activities of these enzymes are well regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). Chronic stimulation of MMP activities due to an imbalance in the levels of MMPs and TIMPs has been implicated in the pathogenesis of a variety of diseases such as cancer, osteoarthritis, and rheumatoid arthritis. Thus, MMP inhibitors are expected to be useful for the treatment of these disorders. This article reviews briefly the biochemistry of MMPs and evidence for their pathogenic roles using molecular biology approaches. Biomolecular structures used in the design of MMP inhibitors are thoroughly covered. Major emphasis is on recently published potent, small molecular weight MMP inhibitors and their pharmacological properties. Finally, available clinical results of compounds in development are summarized.

Inhibition of peptidylglycine alpha-amidating monooxygenase by N-substituted homocysteine analogs.

C-terminal amidation is a posttranslational modification found in many neuropeptides. Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the synthesis of the biologically essential C-terminal amide from a glycine-extended precursor peptide. Reported herein are the first potent inhibitors of PAM. Dipeptides containing a C-terminal homocysteine and an N-acylated hydrophobic amino acid were found to inhibit PAM with IC50s in the low nanomolar range. Inhibition potency was dependent on both the carboxylate and the thiolate functionalities of the homocysteine and on the hydrophobic groups of the second amino acid. The thiolate was postulated to produce high binding affinities through coordination with the active-site copper. The compound series also exhibited potent inhibition of PAM in rat dorsal root ganglion cells as demonstrated by a dose-dependent increase in the substance P-Gly/substance P ratio. These results indicate that the compounds have sufficient potency and intracellular bioavailability to aid future studies focused on neuropeptide function and the contributions of neuropeptides to various disease processes.

Mercaptoacyl dipeptides as orally active dual inhibitors of angiotensin-converting enzyme and neutral endopeptidase.

Dual inhibitors of the two zinc metallopeptidases, neutral endopeptidase (NEP, EC 3.4.24.11) and angiotensin-I-converting enzyme (ACE, EC 2.4.15.1), have been the focus of much clinical interest for the treatment of hypertension and congestive heart failure. We have previously reported that compound 2 (N-[[1-[(2(S)-mercapto-3-methyl-1-oxobutyl) amino]-1-cyclopentyl]-carbonyl]-L-tyrosine) was a potent dual inhibitor in vitro (IC50 (ACE) = 7.0 nM, IC50 (NEP) = 1.5 nM) (Fink et al. J. Med. Chem. 1995, 38, 5023-5030). This compound was found to have oral activity; however, its duration of effect was short. A series of thioacetate carboxylic acid ester analogs of compound 2 was prepared. Modifications were also made to the tyrosine phenol. These compounds were evaluated for their ability to inhibit plasma ACE activity when administered orally to conscious normotensive rats. Most of the compounds prepared were found to be orally active with longer durations of effect than compound 2. Compound 38 (N-[[1-[(2(S)-(acetylthio)-3-methyl-1-oxobutyl) amino]-1-cyclopentyl]carbonyl]-O-methyl-L-tyrosine ethyl ester), administered at 11.7 mg/kg po, was found to be more efficacious than captopril at 10 mg/kg po. This compound was also found to inhibit plasma NEP activity following oral administration to conscious rats and was more efficacious than acetorphan. Compound 38 was found to lower blood pressure in the aorta-ligated rat and the spontaneously hypertensive rat when administered orally. The synthesis and biological activity of these dual inhibitors are discussed.

Design and synthesis of potent, selective inhibitors of endothelin-converting enzyme.

Endothelin-1 is the most potent peptidic vasoconstrictor discovered to date. The final step of posttranslational processing of this peptide is the conversion of its precursor by endothelin-converting enzyme-1 (ECE-1), a metalloprotease which displays high amino acid sequence identity with neutral endopeptidase 24.11 (NEP) especially at the catalytic center. A series of potent and selective arylacetylene-containing ECE-1 inhibitors have been prepared. (S, S)-3-Cyclohexyl-2-[[5-(2, 4-difluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]amino] propio nic acid (47), an arylacetylene amino phosphonate dipeptide, was found to inhibit ECE-1 and NEP with IC50 values of 14 nM and 2 microM, respectively. Similarly, (S)-[[1-[(2-biphenyl-4-ylethyl)carbamoyl]-4-(2-fluorophenyl)but-3- yny l]amino]methyl]phosphonic acid (56), an arylacetylene amino phosphonate amide, had IC50's of 33 nM and 6.5 microM for ECE-1 and NEP, respectively. Slight modification of the aryl moiety was found to have dramatic effects on ECE-1/NEP selectivity. The 2-fluoro dipeptide analogue, (S, S)-2-[[5-(2-fluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]+ ++amin o]-4-methylpentanoic acid (40), showed a 72-fold selectivity for ECE-1 over NEP, while the 3-fluoro dipeptide analogue, (S, S)-2-[[5-(3-fluorophenyl)-2-[(phosphonomethyl)amino]pent-4-ynoyl]+ ++amin o]-4-methylpentanoic acid (22), was equipotent for ECE-1 and NEP. Several of these inhibitors were shown to be potent in blocking ET-1 production in vivo as demonstrated by the big ET-1-induced pressor response in rats. These potent inhibitors are the most selective for ECE-1 reported to date and are envisaged to have a variety of therapeutic applications.

Potent and selective non-peptidic inhibitors of endothelin-converting enzyme-1 with sustained duration of action.

Potent and selective non-peptidic inhibitors of human endothelin-converting enzyme-1 (ECE-1) have been designed as potential modulators of endothelin (ET-1) production in vivo. Because of its unique structural characteristics and long duration of action in vivo, the dual ECE-1 and neutral endopeptidase 24.11 (NEP) inhibitor, CGS 26303, was selected as an attractive lead for further optimization of potency and selectivity. Replacement of the P(1)' biphenyl substituent of CGS 26303 by a conformationally restricted 3-dibenzofuranyl group led to more potent and more selective ECE-1 inhibitors, such as the tetrazole 27. The remarkable effect of this P(1)' modification allowed for the first time phosphonomethylcarboxylic acids, such as 29, to display both potent (IC(50) = 22 nM) and selective (104-fold vs NEP) ECE-1 inhibition. Chemoenzymatic syntheses of the new alpha-amino acid (S)-3-dibenzofuran-3-ylalanine intermediate were developed, and improved procedures to generate substituted alpha-aminoalkylphosphonic acids were devised to support the production of various analogues. Although additional gains in intrinsic ECE-1 inhibitory potency could occasionally be achieved by addition of a P(1) side chain, these compounds (e.g. 43a) showed poor functional activity in vivo in the big ET-1 pressor test. Phosphonoalkyl dipeptides featuring 3-dibenzofuranyl groups in both the P(1)' and P(2)' positions were also very potent ECE-1 inhibitors, albeit lacking the desired selectivity against NEP. Functionally, 27and 29 were the two most efficacious compounds from this study, producing sustained inhibition of ECE-1 activity in rats, as measured by their ability to block the hypertensive effects induced by big ET-1. This profile was similar to that of a potent ET(A)/ET(B) dual receptor antagonist, SB 209670. Due to their favorable in vitro and in vivo profiles, 27 (CGS 34043) and 29 (CGS 35066) constitute new pharmacological tools useful in assessing the role of ECE-1 in pathological conditions.

Endothelin converting enzyme-1-, endothelin-1-, and endothelin-3-like immunoreactivity in the rat brain.

Neurons likely to use endothelin as a neurotransmitter/neurohormone were mapped in the rat brain using polyclonal antibodies directed against endothelin-converting enzyme-1, endothelin-1, and endothelin-3. Anti-endothelin-converting enzyme-1 antibodies produced the most robust staining, permitting the best visualization of the distribution and morphology of neurons. Labeled neurons were found in the dorsal thalamic nuclei and reticular thalamic nuclei, medial preoptic area, pontine nucleus, and locus coeruleus. Localization of endothelin-converting enzyme-like immunoreactivity in the locus coeruleus and in the reticular nucleus of the thalamus suggests that endothelin is co-localized with norepinephrine and GABA, respectively. Additionally, endothelin-converting enzyme-like immunoreactivity was found in the globus pallidus, septal nuclei, and in both the vertical and horizontal limbs of the nucleus of the diagonal band of Broca, and the ventrolateral area of the caudate-putamen. Strong endothelin-converting enzyme-like immunoreactivity was found in a continuous band of pyramidal neurons throughout the neocortex primarily in layer V, extending into the cingulate gyrus and piriform cortex. Motor nuclei, including oculomotor, facial, and trigeminal nuclei, were also endothelin-converting enzyme-immunoreactive. In the cerebellum, Purkinje cells were stained. Non-neuronal cells such as oligodendroglia, microglia, and astrocytes generally were not endothelin-converting enzyme-immunoreactive, although astrocytes were rarely stained. Endothelin-converting enzyme-, endothelin-1-, and endothelin-3-like immunoreactivities were generally found co-existing in given nuclei. The diversity of neurons immunostained for endothelin suggests multiple roles of endothelin in the CNS.

Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action.

The effects of inhibitors of protein kinase C on the activities of the intact enzyme, the proteolytically-generated catalytic domain, and [3H]phorbol 12,13-dibutyrate (PDBu) binding were compared in an effort to evaluate this approach for clarifying mechanisms of inhibitor action. Staurosporine, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], and quercetin inhibited the catalytic fragment with similar potencies as for the intact enzyme while having little or no effect on binding, consistent with reports that they are competitive with ATP. Adriamycin, trifluoperazine, and tamoxifen, suggested to disrupt hydrophobic interactions between the regulatory domain of protein kinase C and phospholipid, were all most effective on the intact enzyme. They appear to possess a mixed mechanism, however, inhibiting activity of the catalytic domain with approximately 3-fold lower potencies. Gossypol inhibited intact enzyme, catalytic fragment, and PDBu binding with similar potencies. In light of multiple apparent sites of action for such protein kinase C inhibitors, comparison of their activities on the individual functional domains of the kinase may provide a useful complement to studies with the intact enzyme.

Effects of metalloprotease inhibitors on smooth muscle endothelin-converting enzyme activity.

The enzyme responsible for the conversion of exogenous big endothelin-1 to endothelin-1 by porcine coronary arterial smooth muscle has been shown to be a metalloprotease. The potencies of eight metalloprotease inhibitors for this endothelin-converting enzyme were determined. CGS 25015, CGS 26129, and thiorphan inhibited the enzyme activity monophasically with IC50 values of 2.6, 2.4, and 190 microM, respectively. In contrast, the data obtained using phosphoramidon as an inhibitor were best fit by a two-site model. The biphasic concentration-response curve had IC50 values of 4.6 microM and 2.2 mM. Three analogs of phosphoramidon were also tested for enzyme inhibition. Removal of the rhamnose moiety of phosphoramidon reduced the potency (IC50 = 15 microM), whereas substitution of the rhamnose by N-[2-(2-naphthyl)ethyl] improved the potency (IC50 = 2.0 microM). These results identify a thiol and a phosphonyl series of compounds as smooth muscle endothelin-converting enzyme inhibitors. The structure-activity relationships revealed that an aromatic or aliphatic group in the P2' position or an aromatic group in the P1 position of the inhibitor significantly increased the potency.

Mechanism of action of the phorbol ester tumor promoters: specific receptors for lipophilic ligands.

Cells and tissue preparations specifically bind the phorbol ester tumor promoters. The agreement in structure-activity relationships between binding and biological response strongly argues that these binding sites function as phorbol ester receptors. Upon subcellular fractionation, the phorbol ester binding activity is particulate. In addition, a phorbol ester apo-receptor can be detected in cytosol which requires phospholipids for reconstitution. This apo-receptor appears to correspond to protein kinase C. Diacylglycerols, the probable natural activators of protein kinase C, competitively inhibit phorbol ester binding, consistent with their being the postulated endogenous phorbol ester analogs. In certain systems, heterogeneity of phorbol ester binding is found. An outstanding issue therefore is whether protein kinase C is the phorbol ester receptor or whether it is only the most abundant class of receptor. Although this question remains unresolved, we can demonstrate heterogeneity of phorbol ester binding by reconstitution of apo-receptor into a heterogeneous lipid environment.

A soluble protease identified from rat kidney degrades endothelin-1 but not proendothelin-1.

Endothelin-1 (ET-1) is a potent peptidic vasoconstrictor. This peptide has been shown to be cleared rapidly by the kidney. The purpose of the present study was to assess the involvement of renal proteolytic enzymes in the clearance/degradation of ET-1. Incubation of ET-1 with the cytosolic fraction of rat kidney homogenate resulted in a decrease of contractile activity on rabbit aortic rings when compared to the untreated ET-1. This cytosolic fraction was chromatographed by anion-exchange and concanavalin A columns. The partially purified enzyme cleaved off the C-terminal tryptophan of ET-1 rapidly, resulting in a peptide which is three orders of magnitude weaker in potency than ET-1 in causing smooth muscle contraction. In contrast, proendothelin-1 was not degraded by this endothelin degradation enzyme (EDE). The effects of EDE on other vasoactive peptides were also examined. The C-terminal tyrosine of atrial natriuretic peptide was cleaved by EDE, but the biological activity of the resulting peptide was not significantly changed. Angiotensin II was not a substrate for EDE. The EDE was shown to be different from both carboxypeptidases A and B based on the HPLC analysis of the degradation products of ET-1 produced by these enzymes. In addition, these enzymes displayed different sensitivities toward a carboxypeptidase inhibitor from potato tuber. These results suggest that this previously unidentified enzyme inactivates ET-1 effectively and that it may play a role in modulating the levels of ET-1 in the kidney.

Mouse macrophage metalloelastase expressed in bacteria absolutely requires zinc for activity.

Mouse macrophage metalloelastase was expressed in Escherichia coli. This recombinant enzyme (rMME) was present in the inclusion bodies that were solubilized in 7 M guanidine HCl. After removal of guanidine HCl, rMME was purified with a Q-Sepharose column. Degradation of [3H]elastin by rMME absolutely required Ca2+; the optimal Ca2+ concentration was 5 mM. NaCl stimulated the enzyme activity; maximal stimulation was obtained at 400 mM. The rMME activity was inhibited by metalloprotease inhibitors, but not by serine, aspartyl, or thiol protease inhibitors. Among the divalent cations tested, only Ba2+ and Sr2+ exhibited marginal stimulation of rMME activity in the absence of Ca2+. Cu2+, Zn2+, or Cd2+ strongly inhibited rMME activity with IC50 values between 68 and 180 microM, while Mg2+, Ba2+, Mn2+, Co2+, and Sr2+ had no effect. The requirement of Zn2+ for rMME activity was determined. Significant enzyme activity was present in rMME treated with EDTA followed by Q-Sepharose column chromatography. Only when the inclusion bodies were solubilized in the presence of 20 mM EDTA, did an enzyme preparation which was absolutely dependent on exogenous Zn2+ for activity result. The optimal Zn2+ concentration for rMME activation was 100 microM. These results indicate that Zn2+ is tightly bound to rMME.