Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The endothelium regulates smooth muscle tone in blood vessels through the release of endothelium-deprived relaxing factor (EDRF). We hypothesized that the lymphatics possess endothelium-dependent relaxant properties analogous to those in blood vessels. Fresh porcine tracheobronchial lymphatic vessel rings were mounted in organ baths and connected to force-displacement transducers. Rings were submaximally precontracted with 10(-5) M histamine and exposed to cumulative addition of acetylcholine (ACH; 10(-7)-3 x 10(-4) M) or bradykinin (BRD; 10(-8)-3 x 10(-6) M), both of which are known to promote release of EDRF. ACH caused concentration-dependent relaxation (maximum effect = -2.3 +/- 2.6% of initial histamine-induced active tension), while a similar but less pronounced effect was seen with BRD (39.6 +/- 5.4%). The relaxant effects of ACH and BRD were inhibited by collagenase pretreatment and mechanical endothelial denudation. The results confirm our hypothesis that lymphatic vessels possess endothelium-dependent relaxant properties and suggest that lymph vessels can regulate lymph flow through processes similar to those found in blood vessels.
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PMID:Modulation of lymphatic smooth muscle contractile responses by the endothelium. 131 82

Neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is a cell membrane associated zinc metalloprotease, which cleaves peptides like atrial natriuretic peptide (ANP) on the amino-side of hydrophobic amino acids. Although NEP is mainly located in reabsorptive epithelia (kidney proximal tubule), it is also present in non-epithelial cells like neuronal cells. As the renal NEP cannot account for the entire ANP metabolism, other locations were postulated. The present experiments show its expression in endothelial cells (EC) from arterial (bovine pulmonary, porcine and human aorta) and venous (human umbilical, rabbit ear marginal) origins. Three different methods were used to demonstrate the presence of the protein and its mRNA: 1) NEP enzymatic activity was estimated using both a synthetic ([D-Ala2, Leu5] enkephalin) and a natural substrate (bradykinin). Using the synthetic substrate, the enzymatic activity in EC was completely blocked by thiorphan, a specific NEP inhibitor with an IC50 value in the nM range. In contrast, captopril, bestatin, GEMSA, inhibitors of angiotensin-converting enzyme, aminopeptidases and carboxypeptidases, respectively, were 10,000 times less active, revealing an inhibition profile similar to that of the purified enzyme. Bradykinin, a natural substrate of NEP, was in part metabolized by NEP, in presence of captopril, since 50% of the formation of the major metabolite bradykinin 1-7 was inhibited by thiorphan. 2) Immunoreactive NEP was detected on the plasma membrane of rabbit EC using a monoclonal antibody directed against the homologous renal enzyme. 3) NEP mRNA was detected by Northern blot analysis on rabbit EC as a major transcript of 3.9 kb. Reverse transcriptase PCR amplification showed the presence of a specific transcript in all EC tested. Therefore, endothelial NEP could play an important role in the inactivation of ANP, bradykinin and endothelins by its localization facing the circulating vasoactive peptides.
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PMID:[Identification and characterization of neutral endopeptidase in endothelial cells of arterial or venous origin]. 133 90

Tyrosine hydroxylase (TH) is phosphorylated at four sites in situ and in vivo, and the protein kinases that phosphorylate three of these sites (Ser8,Ser19,Ser40) have been identified. In intact cells, the phosphorylation of the fourth site (Ser31) is increased in response to phorbol esters or nerve growth factor (NGF). Here, we show that Ser31 is phosphorylated by ERK1 and ERK2, two myelin basic protein and microtubule-associated protein kinases. Extracts of NGF- or bradykinin-treated PC12 rat pheochromocytoma cells were fractionated on Mono Q columns. Protein kinase activity toward Ser31 in TH was present in two peaks corresponding to myelin basic protein kinase activities previously identified as ERK1 and ERK2. Phosphorylation of purified TH in vitro by both kinases was selective for Ser31 up to at least 0.6 mol of phosphate per mol of TH subunit. Treatment of intact PC12 cells with bradykinin or NGF increased both the phosphorylation of TH-Ser31 in situ and the catalytic activity of ERKs (measured subsequently in vitro with myelin basic protein as substrate). Pretreatment of the cells with genistein (a protein-tyrosine kinase inhibitor) decreased the bradykinin- but not the NGF-induced changes in both TH-Ser31 phosphorylation and ERK activity. Genistein also inhibited the increases in Ser31 phosphorylation produced by phorbol dibutyrate, muscarine, and Ba2+. The data indicate that ERK activity is responsible for phosphorylating TH at Ser31 in intact cells and suggest that TH-Ser31 phosphorylation may be regulated by multiple signaling pathways that converge at or prior to the activation of the ERKs.
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PMID:ERK1 and ERK2, two microtubule-associated protein 2 kinases, mediate the phosphorylation of tyrosine hydroxylase at serine-31 in situ. 134 49

Kinins and substance P have been implicated in the pathogenesis of inflammatory arthritis by virtue of their abilities to induce vasodilation, edema, and pain. The relative biological potencies of these peptides in vivo would depend at least in part upon their rates of catabolism in the joint. We hypothesized that human synovial lining cells may regulate intraarticular levels of kinins and neuropeptides via degradation by cell surface-associated peptidases. We exposed intact human synovial fibroblasts to kinins and substance P, in the presence or absence of specific peptidase inhibitors, and measured the amount of intact substrate remaining and degradation product(s) generated over time. Aminopeptidase M (AmM; EC 3.4.11.2), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) were identified on the cell surface of synovial cells. Bradykinin degradation was due entirely to NEP-24.11 (1.39 +/- 0.29 nmol/min per well). Lysylbradykinin was also degraded by NEP-24.11 (0.80 +/- 0.19 nmol/min per well); however, in the presence of phosphoramidon, AmM-mediated conversion to bradykinin (3.74 +/- 0.46 nmol/min per well) could be demonstrated. The combined actions of NEP-24.11 (0.93 +/- 0.15 nmol/min per well) and DAP IV (0.84 +/- 0.18 nmol/min per well) were responsible for the degradation of substance P. AmM (2.44 +/- 0.33 nmol/min per well) and NEP-24.11 (1.30 +/- 0.45 nmol/min per well) were responsible for the degradation of the opioid peptide, [Leu5]enkephalin. The identity of each of the three peptidases was confirmed via synthetic substrate hydrolysis, inhibition profile, and immunological identification. The profiles of peptidase enzymes identified in cells derived from rheumatoid and osteoarthritic joints were identical. These data demonstrate the human synovial fibroblast to be a rich source of three specific peptidases and suggest that it may play a prominent role in regulating peptide levels in the joint.
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PMID:Cultured human synovial fibroblasts rapidly metabolize kinins and neuropeptides. 138 26

Neutral endopeptidase (NEP; enkephalinase, EC 3.4.24.11) is a cell membrane-associated zinc metalloprotease, which cleaves peptides like atrial natriuretic peptide (ANP) on the amino side of hydrophobic amino acids. Although NEP is mainly located in reabsorptive epithelia (kidney proximal tubule), it is also present in non-epithelial cells such as neuronal cells. As the renal NEP cannot account for the entire ANP metabolism, other locations were postulated. The present experiments show its expression in endothelial cells (EC) from arterial (bovine pulmonary, porcine, and human aorta) and venous (human umbilical, rabbit ear marginal) origins. Three different methods were used to demonstrate the presence of the protein and its mRNA. 1) NEP enzymatic activity was estimated using both a synthetic ([D-Ala2,Leu5]enkephalin) and a natural substrate (bradykinin). Using the synthetic substrate, the enzymatic activity in EC was completely blocked by thiorphan, a specific NEP inhibitor with an IC50 value in the nanomolar range. In contrast, captopril, bestatin, [2-guanidinoethylmercapto]succinic acid, inhibitors of angiotensin-converting enzyme, aminopeptidases, and carboxypeptidases, respectively, were 10,000 times less active, revealing an inhibition profile similar to that of the purified enzyme. Bradykinin, a natural substrate of NEP, was in part metabolized by NEP, in the presence of captopril, since 50% of the formation of the major metabolite bradykinin 1-7 was inhibited by thiorphan. 2) Immunoreactive NEP was detected on the plasma membrane of rabbit EC using a monoclonal antibody directed against the homologous renal enzyme. 3) NEP mRNA was detected by Northern blot analysis of rabbit EC as a major transcript of 3.9 kilobases. Reverse transcriptase polymerase chain reaction amplification showed the presence of a specific transcript in all EC tested. Therefore, endothelial NEP may play an important role in the inactivation of ANP, bradykinin, and endothelins by its localization facing the circulating vasoactive peptides.
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PMID:Identification and characterization of neutral endopeptidase in endothelial cells from venous or arterial origins. 162 99

Inhibition of the enzyme neutral metalloendopeptidase potentiates responses to atrial natriuretic factor and elicits reductions of blood pressure in desoxycorticosterone acetate sodium hypertensive rats. The present study evaluated the role of atrial natriuretic factor and bradykinin in the antihypertensive response to neutral metalloendopeptidase inhibition through the use of antibodies and antagonists, respectively. In addition, the pharmacokinetic mechanism by which neutral metalloendopeptidase inhibition interferes with atrial natriuretic factor metabolism was explored. The antihypertensive response to the neutral metalloendopeptidase inhibitor SCH 34826 was abruptly reversed by i.v. injection of a polyclonal antiserum to atrial natriuretic factor. In contrast, the antihypertensive response to SCH 34826 was unaffected by injection of the bradykinin antagonist Thi5,8-D-Phe7 bradykinin. The renal response to atrial natriuretic factor, SCH 34826, and phosphoramidon was inhibited by the bradykinin antagonist. The NEP inhibitor SCH 39370 significantly delayed the disappearance of TCA precipitable radioactivity from plasma following i.v. bolus dosing with 125I-labelled ANF 99-126. The effects were enhanced in the presence of the C receptor ligand. The results indicate that atrial natriuretic factor, but not bradykinin, plays an important role in the antihypertensive response to SCH 34826. Bradykinin plays a permissive role in the diuretic responses to atrial natriuretic factor and inhibitors of neutral metalloendopeptidase. Lastly, neutral metalloendopeptidase inhibition significantly alters the clearance and metabolism of tracer quantities of atrial natriuretic factor.
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PMID:Neutral metalloendopeptidase inhibitors as ANF potentiators: sites and mechanisms of action. 183 29

The effect of peptidase inhibitors on neuropeptide release from peripheral endings of capsaicin-sensitive sensory neurons was studied in cerebral superior sagittal and transverse sinuses of guinea-pig. Capsaicin (1 microM)-evoked release of substance P-like immunoreactivity (SP-LI) was increased in a concentration-dependent manner by thiorphan (0.1-10 microM). Captopril (10 microM) or a mixture of bestatin (10 microM), leupeptin (10 microM) and bacitracin (10 microM) did not affect the capsaicin-evoked SP-LI release. Thiorphan (10 microM) increased also the capsaicin-evoked release of neurokinin A-like immunoreactivity (TK-LI) and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) by 228% and 172%, respectively, while captopril (10 microM) was without effect. Thiorphan (10 microM), but not captopril (10 microM), enhanced by 239% CGRP-LI release induced by bradykinin (10 microM). In the cerebral venous vessels neutral endopeptidase (EC 3.4.24.11, NEP)-like activity was 58.8 +/- 6.1 pmol/mg protein/min, while angiotensin converting enzyme-like activity was below the detection limit of the assay. A thiorphan-sensitive mechanism, putatively attributable to NEP, plays a major role in the inactivation of peptides released from or acting on capsaicin-sensitive sensory fibres of cerebral venous sinuses of guinea-pig.
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PMID:The effect of thiorphan on release of sensory neuropeptides from guinea-pig cerebral venous sinuses. 206 52

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human renal membranes. NEP hydrolyzed substance P (SP) at Gln6-Phe7, Phe7-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was dependent on chloride ion and inhibited by captopril. Modification of arginine residues in ACE with cylcohexanedione or butanedione inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE cleaved NT at Tyr11-Ile12 to release Ile12-Leu13. These studies indicate that ACE and NEP, two enzymes which are widely distributed in the body, may be involved in the metabolism of SP and NT.
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PMID:Characterization of the metabolism of substance P and neurotensin by human angiotensin I converting enzyme and "enkephalinase". 241 54

In order to clarify the significance of NEP in human renal kallikrein-kinin system, an assay system was developed for the simultaneous determination of kininase I, II and NEP activities in human. Each kininase activity was determined by measuring the hydrolysis of bradykinin in the presence of specific inhibitors of kininase I (2-mercaptomethyl-3-guanidinoethylthiopropanoic acid), kininase II (captopril) and NEP (phosphoramidon) in 8 normal subjects. The effects of the different assay buffers on kininase activities were also investigated by using a phosphate buffer. Total kininase, kininase I, II and NEP activities were 499 +/- 65 ng/min/ml (mean +/- S.E.), 55 +/- 8, 141 +/- 21 and 299 +/- 42, respectively in our method using a tris buffer, while a phosphate buffer brought about activities of 358 +/- 43, 45 +/- 5, 156 +/- 21 and 135 +/- 25 ng/min/ml. The relative contributions of kininase I, II and NEP to total kininase activity were 11, 29 and 59% in our assay system, while they were 13, 44 and 35% when a phosphate buffer was used. From these results it was suggested that 1) phosphate may inhibit urinary NEP activity, so that a tris buffer should be used as the incubation buffer, 2) NEP is the major component of human urinary kininases, and 3) NEP may play an important role in the renal kallikrein-kinin system.
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PMID:A sensitive method for differential determination of kininase I, II and neutral endopeptidase (NEP) in human urine. 255 8

The effects of epidermal growth factor (EGF) on membrane potential were investigated in suspensions of the following three cell types endowed with a large complement of specific receptors: EGFR-T17 (a clone of mouse NIH-3T3 fibroblasts overexpressing EGF receptors); A431 and KB (two human carcinoma lines). In all these lines EGF induced a rapid and marked hyperpolarization constituted by an initial peak (in all three cell lines) and a subsequent sustained plateau phase, concomitant with the well-known increase of [Ca2+]i. The time course and phorbol ester inhibitability of the membrane potential effects were the same as for the [Ca2+]i response. Experiments with Na+-free and chloride-free media excluded a major role of the latter ions in the EGF-induced hyperpolarization. In contrast, experiments with high K+ media, with the monovalent cation ionophore gramicidin and with Ca2+-free media together with either a Ca2+ ionophore (ionomycin, in A431 and EGFR-T17), or an agonist (bradykinin, in A431) addressed to a receptor coupled to phosphoinositide hydrolysis, were consistent with the involvement of Ca2+-activated K+ channels. The EGF-induced hyperpolarization was completely blocked by the K+ channel blocker, quinidine, and unaffected by a variety of other drugs. Patch clamping of individual EGFR-T17 cells confirmed the initial hyperpolarization (from approximately -30 mV, the resting potential, to -60, -80 mV) was due to activation of an outward current. This initial hyperpolarization was followed by fluctuations (period approximately 1 min) persisting as long as the cells could be analyzed. Thus, the changes of membrane potential appear to be not only novel members of the group of early events triggered by EGF in target cells but also long-lasting effects of the growth factor, which continue for unexpectedly long periods of time after EGF application.
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PMID:The effect of epidermal growth factor on membrane potential. Rapid hyperpolarization followed by persistent fluctuations. 278 95


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