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)

Characterization of the distribution of the peptide-degrading enzyme neutral endopeptidase-24.11 (E.C. 3.4.24.11; NEP; enkephalinase) in the rat brainstem was examined by means of a unique fluorescent histochemical method. Enzyme staining was completely blocked by three potent NEP inhibitors (thiorphan, phosphoramidon, and JHF-26) at a concentration of 50 nM, supporting the specificity of this method to visualize sites of NEP activity selectively. At all levels of the brainstem, NEP was localized to cell bodies, cell processes or terminal-like fields and was localized to more than 90 distinct nuclei or subnuclei. In the mesencephalon these included the central gray, cuneiform n., dorsal and lateral tegmental n., inferior colliculus, interpeduncular n., lateral and medial geniculate n., central linear raphe n., mesencephalic n. of the trigeminal nerve, mammillary nuclei, occulomotor n., red n., superior colliculus, ventral n. of the lateral lemniscus, substantia nigra-ventral tegmental area, and the zona incerta. In the pons, NEP staining was restricted to fewer regions or nuclei, including the dorsal and ventral cochlear n., facial n., motor trigeminal n., principal sensory trigeminal n., parabrachial nuclei, pontine n., the oral and caudal pontine reticular n., pontine olivary nuclei, several pontine tegmental nuclei, pontine raphe nuclei, and the trapezoid n. In the cerebellum, staining was localized largely to the granule cell layer of the cerebellar cortex. Scattered staining was observed in the molecular cell layer. The medulla contained extensive NEP staining localized to nuclei that included the ambiguous n., dorsal motor n. of the vagus, hypoglossal n., inferior olivary n., prepositus hypoglossus n., solitary tract n., nuclei of the spinal tract of the trigeminal n., and the lateral, medial, and superior vestibular nuclei. Nuclei of the medullary reticular formation that were also richly stained for NEP included the raphe magnus n., raphe obscurus n., raphe pallidus n., dorsal, lateral, and ventral reticular nuclei of the medulla, and the gigantocellular, lateral paragigantocellular, linear, paramedian and parvicellular reticular nuclei. The widespread distribution of NEP in the brainstem suggests the existence of a number of functional systems, including the pathways involved in the mechanisms of pain and analgesia, which are potential targets of NEP inhibitors. In most regions, the distribution of NEP closely overlapped with that reported for the enkephalins, and showed a more restricted overlap with the reported distribution of substance P.
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PMID:Fluorescent histochemical localization of neutral endopeptidase-24.11 (enkephalinase) in the rat brainstem. 169 88

The activation or interruption of the responses induced by regulatory peptides are ensured by ectoenzymes, the most important of them belonging to the group of zinc metallopeptidases. Thus angiotensin converting enzyme (ACE) forms the hypertensive peptide angiotensin II from its inactive precursor AI. This also the case for aminopeptidase N (APN) and neutral endopeptidase 24.11 (NEP, CALLA) which together inactivate the endogenous opioid peptides, enkephalins, whereas only NEP is involved in the metabolism of the atrial natriuretic factor (ANP) at the kidney and vascular levels. The pharmacological effects resulting from the inhibition of these enzymatic processes will appear only in tissues where the peptide substrate is tonically or phasically released. This promising approach is expected to avoid, or at least to minimize, the side effects resulting from excessive and ubiquitous stimulation of peptide receptors by exogenously administered agonists or antagonists. The essential amino acids known to be present in the active site of the bacterial endopeptidase thermolysin from crystallographic studies, have also been found in NEP by using a new program of sequence comparison associated with mutagenesis experiments. Several classes of selective inhibitors of NEP, APN and ACE have been rationally designed by taking into account the structural differences in the active site of these peptidases. Thus, the retro-inversion of the amide bond of the NEP inhibitor thiorphan resulted in the elimination of a residual interaction with ACE. Moreover, we have proposed to associate inhibitory potencies towards two peptidases in the same compound. Thus kelatorphan HONH-CO-CH2-CH(CH2 phi)-CONH-CH(CH3)-COOH and other systemically-active mixed NEP/APN inhibitors were shown capable of completely blocking enkephalin metabolism in vivo. This concept has been extended to mixed NEP/ACE inhibitors with compounds such as HS-CH2-CH(CH2 phi)-CONH-CH(CH2R)-COOH where R = CH-(CH3)2 (ES 34) or -OCH2 phi (ES 37). Only mixed inhibitors of NEP and APN are able to produce potent analgesia after intracerebroventricular or systemic administration without the major side effects of morphine (tolerance and dependence). Thiorphan or its prodrugs acetorphan or sinorphan lead to a increase in natriuresis and diuresis by protection of ANP degradation, but without any significant antihypertensive effect. Contrastingly mixed NEP/ACE inhibitors such as ES34 induce decreases in blood pressure higher than those that produced by the association of selective NEP and ACE inhibitors.
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PMID:[New approach in the research of analgesics and antihypertensive agents]. 184 70

5 beta-Methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone (MET-CAMO) and its corresponding N-cyclopropylmethyl analog, N-cyclopropylmethylnor-5 beta-methyl-14 beta-(p-nitrocinnamoylamino)- 7,8-dihydromorphinone (N-CPM-MET-CAMO) were tested in opioid receptor binding assays and in the mouse tail-flick test in order to characterize the affinity, selectivity and antinociceptive properties of these two compounds. Incubating bovine striatal membranes with either MET-CAMO or N-CPM-MET-CAMO produced a wash-resistant, concentration- and time-dependent inhibition of the binding of the mu-selective ligand, [3H]-[D-Ala2,MePhe4,Gly(ol)5]enkephalin, but with no change in delta or kappa binding. Preincubating membranes with N-CPM-MET-CAMO decreased the maximum binding value for [3H]-[D-Ala2,MePhe4,Gly(ol)5]enkephalin binding without changing the Kd value. In the mouse tail-flick assay, MET-CAMO and N-CPM-MET-CAMO did not produce any antinociception up to a dose of 100 nmol after i.c.v. administration. However, pretreatment of mice with either compound produced a time- and dose-dependent antagonism of morphine-induced antinociception. Analgesia mediated by delta or kappa opioids was not altered by either MET-CAMO or N-CPM-MET-CAMO at a dose of up to 100 nmol. The mu antagonistic effect of 1 nmol of MET-CAMO and N-CPM-MET-CAMO appeared at 8 hr and lasted up to 72 hr, with a maximal effect at 16 to 24 hr after i.c.v. administration. Pretreatment of mice with 1 nmol of MET-CAMO or N-CPM-MET-CAMO, given by i.c.v. administration at -24 hr, produced a rightward and downward shift of dose-response line of i.c.v. morphine.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:5 beta-Methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone and its corresponding N-cyclopropylmethyl analog, N-cyclopropylmethylnor-5 beta-methyl-14 beta-(p-nitrocinnamoylamino)- 7,8-dihydromorphinone: mu-selective irreversible opioid antagonists. 751 Nov 63

Irreversible opioid antagonists, when administered at small doses, require several hours to display their antagonism of antinociception mediated by opioid receptors. However, most opioid affinity ligands only need a few minutes to produce wash-resistant inhibition of opioid binding to brain membranes. Our study investigated whether the irreversible antagonists, beta-funaltrexamine (beta-FNA), 14 alpha, 14'beta-[dithiobis[(2-oxo-2,1-ethanediyl)imino]]-7,8-dihydro-N- (cyclopropylmethyl)normorphine (N-CPM-TAMO), and N-cyclopropylmethyl-5 beta-methyl- beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone (N-CPM-MET-CAMO) had any effect on morphine-induced antinociceptive tolerance before the appearance of their antagonism in the mouse tail-flick assay. All opioids were given by i.c.v. administration. The irreversible antagonists, beta-FNA (20 nmol), N-CPM-TAMO (0.5 nmol) and N-CPM-MET-CAMO (1 nmol) did not produce any antagonism of morphine-induced analgesia until at least 8 hr after administration. Pretreatment with morphine (3 nmol, -140 min) produced acute antinociceptive tolerance as demonstrated by a 45-fold rightward shift of the morphine dose-response curve. When coadministered with morphine, beta-FNA, N-CPM-TAMO and N-CPM-MET-CAMO completely prevented the development of morphine tolerance 140 min after administration in a dose-dependent manner. This preventive effect lasted for up to 420 min, during which time, morphine was given repeatedly up to four times. This antinociception produced by morphine after coadministration with irreversible antagonists was antagonized by naloxone, demonstrating that morphine-induced analgesia was still mediated by opioid receptors. The kappa- and delta-selective opioid antagonists, nor-binaltorphimine and ICI 174,864, respectively, did not block the preventive effect produced by the irreversible antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Preventing morphine antinociceptive tolerance by irreversible mu opioid antagonists before the onset of their antagonism. 775 70

The affinity, selectivity and antinociceptive properties of 5 beta-methyl-14 beta-(p-chlorocinnamoylamino)-7,8-dihydromorphinone (MET-Cl-CAMO) and N-cyclopropyl-methyl-5 beta-methyl-14 beta-(p-chlorocinnamoylamino)-7, 8-dihydronormorphinone (N-CPM-MET-Cl-CAMO) for the multiple opioid receptors were characterized. In competition binding assays using bovine striatal membranes, both compounds inhibited the binding of 0.25 nM [3H][D-Ala2, (Me)-Phe4,Gly(ol)5]enkephalin (DAMGO) with IC 50 values of less than 2 nM. Preincubation of membranes with MET-CI-CAMO and N-CPM-MET-Cl-CAMO produced a concentration-dependent, wash-resistant inhibition of mu-opioid receptor binding. Saturation binding experiments with N-CPM-MET-Cl-CAMO showed a reduction in the number of mu-opioid binding sites without a change in affinity. In the mouse 55 degrees C warm-water tail-flick assay, neither MET-Cl-CAMO nor N-CPM-MET-Cl-CAMO at doses up to 100 nmol produced antinociception after intracerebroventricular administration, but morphine-induced antinociception was antagonized in a time- and dose-dependent manner by both compounds. The antagonism produced by 1 nmol of either MET-Cl-CAMO or N-CPM-MET-Cl-CAMO reached a maximal effect after 24 h, and lasted up to 48 h. Analgesia mediated by delta- or kappa-opioids was not altered by either compound. In summary, the data suggest that MET-Cl-CAMO and N-CPM-MET-Cl-CAMO are long-term, mu-opioid receptor antagonists, devoid of agonist properties in the mouse tail-flick assay, and that N-CPM-MET-Cl-CAMO may produce its antagonistic effects by binding irreversibly to the mu-opioid receptor.
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PMID:14 beta-Chlorocinnamoylamino derivatives of metopon: long-term mu-opioid receptor antagonists. 905 44

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.
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PMID:Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene. 1110 52

Tolerance to opiates reduces their effectiveness in the treatment of severe pain. Although the mechanisms are unclear, overactivity of pro-nociceptive systems has been proposed to contribute to this phenomenon. We have reported that the development of morphine tolerance significantly increased calcitonin-gene-related-peptide-like immunoreactivity (CGRP-IR) in primary sensory afferents of the spinal dorsal horn, suggesting that changes in pain-related neuropeptides in the dorsal root ganglion (DRG) neurons may be involved (Menard et al., 1996, J. Neurosci., 16, 2342-2351). Recently, we have shown that repeated morphine treatments induced increases in CGRP- and substance P (SP)-IR in cultured DRG, mimicking the in vivo effects (Ma et al., 2000, Neuroscience, 99, 529-539). In this study, we investigated the intracellular signal transduction pathways possibly involved in morphine-induced increases in CGRP- and SP-IR in DRG neurons. Repeated morphine exposure (10-20 microm) for 6 days increased the number of neurons expressing phosphorylated (p) mitogen-activated protein (MAP) kinases, including the extracellular signal-regulated kinase (pERK), c-jun N-terminal kinase (pJNK) and P38 (pP38 MAPK). The number of neurons expressing phosphorylated cAMP responsive element binding protein (pCREB) was also markedly increased in morphine-exposed cultured DRG neurons. pERK-, pP38-, pJNK- and pCREB-IR were colocalized with CGRP-IR in cultured DRG neurons. Naloxone effectively blocked these actions of morphine, whereas a selective MEK1 inhibitor, PD98059, inhibited the morphine-induced increase in the phosphorylation of ERK and CREB, and the expression of CGRP and SP. Moreover, in morphine-tolerant rats, the number of pCREB-, CGRP- and SP-IR neurons in the lumbar DRG was also significantly increased. These in vitro and in vivo data suggest that the phosphorylation of MAP kinases and CREB plays a role in the morphine-induced increase in spinal CGRP and SP levels in primary sensory afferents, contributing to the development of tolerance to opioid-induced analgesia.
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PMID:Chronic morphine exposure increases the phosphorylation of MAP kinases and the transcription factor CREB in dorsal root ganglion neurons: an in vitro and in vivo study. 1168 1

Neutral endopeptidase (EC3.4.24.11, NEP, enkephalinase) is a zinc-metalloendopeptidase, cleaving a variety of substrates like enkephalins, substance P, and bradykinin. In the brain, NEP is a key enzyme in the degradation of enkephalins. Pharmacological inhibition of NEP-activity causes analgesia resulting from enhanced extracellular enkephalin concentrations. Recently, transgenic mice lacking the enzyme NEP have been developed (Lu, 1995). The present study was designed to investigate the nociceptive behavior of these NEP-knockout mice. Interestingly, NEP-deficient mice did not respond with decreased pain perception, but exhibited hyperalgesia in the hot-plate jump, warm-water tail-withdrawal, and mostnotablyin theacetic-acid writhing test. Inhibition of aminopeptidase N by bestatin reduced writhing in both strains, whereas NEP-inhibition by thiorphan reduced writhing selectively in wild-type mice. Naloxone increased writhing in wild-type but not in knockouts, whereas the bradykinin B2-receptor antagonist HOE140 reduced writhing selectively in NEP-knockouts. Similarly, the nitric oxide synthase inhibitor L-NAME reduced writhing in NEP-knockouts. These results indicate that genetic elimination of NEP, in contrast to pharmacological inhibition, leads to bradykinin-induced hyperalgesia instead of enkephalin-mediated analgesia. Nitric oxide (NO) is suggested to be involved in this process.
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PMID:Neutral endopeptidase knockout induces hyperalgesia in a model of visceral pain, an effect related to bradykinin and nitric oxide. 1193 42

Sialorphin is an exocrine and endocrine signaling mediator, which has been identified by a genomic approach. It is synthesized predominantly in the submandibular gland and prostate of adult rats in response to androgen steroids and is released locally and systemically in response to stress. We now demonstrate that the cell surface molecule to which sialorphin binds in vivo in the rat kidney is the membrane-anchored neutral endopeptidase (neprilysin; NEP, EC 3.4.24.11). NEP plays an important role in nervous and peripheral tissues, as it turns off several peptide-signaling events at the cell surface. We show that sialorphin prevents spinal and renal NEP from breaking down its two physiologically relevant substrates, substance P and Met-enkephalin in vitro. Sialorphin inhibited the breakdown of substance P with an IC50 of 0.4-1 microM and behaved as a competitive inhibitor. In vivo, i.v. sialorphin elicited potent antinociceptive responses in two behavioral rat models of injury-induced acute and tonic pain, the pin-pain test and formalin test. The analgesia induced by 100-200 mcicrog/kg doses of sialorphin required the activation of mu- and delta-opioid receptors, consistent with the involvement of endogenous opioid receptors in enkephalinergic transmission. We conclude that sialorphin protects endogenous enkephalins released after nociceptive stimuli by inhibiting NEP in vivo. Sialorphin is a natural systemically active regulator of NEP activity. Furthermore, our study provides evidence that it is a physiological modulator of pain perception after injury and might be the progenitor of a new class of therapeutic molecules.
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PMID:Sialorphin, a natural inhibitor of rat membrane-bound neutral endopeptidase that displays analgesic activity. 1283 17

In order to study the development of the delivery device of long-acting local anaesthetics for post-operative analgesia and control of chronic pain of cancer patient, fentanyl loaded poly(l-lactide-co-glycolide) (PLGA, molecular weight; 5000, 8000, 20,000, and 33,000 g/mole) microspheres (FMS) were studied. FMS were prepared by an emulsion solvent-evaporation method. The influence of several preparation parameters such as initial drug loading, PLGA concentrations, emulsifier concentrations, oil phase volume and mole ratio and molecular weight has been investigated on the fentanyl release patterns. Generally, the drug showed the biphasic release patterns, with an initial diffusion followed by a lag period before the onset of the degradation phase, but there were no lag times in the device. Fentanyl was slowly released from FMS over 10 days in vitro, with a quasi-zero order property. The release rate increased with increasing drug loading as well as increasing polymer concentration with a relatively small initial burst effect. From the results, FMS may be a good formulation to deliver the anaesthesia for the treatment of chronic pain.
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PMID:Study on in vitro release patterns of fentanyl-loaded PLGA microspheres. 1290 42


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