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C Llorens-Cortes

Publications and source records attributed to C Llorens-Cortes.

18 recordsLinked to original sources
PubMed1992

Identification and characterization of neutral endopeptidase in endothelial cells from venous or arterial origins.

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.

Animals↗
PubMed1992

[Identification and characterization of neutral endopeptidase in endothelial cells of arterial or venous origin].

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.

Animals↗
PubMed1991

Dopaminergic regulation of enkephalin release.

The effects of dopamine receptor stimulation on enkephalin release were evaluated in vitro and in vivo by measuring the changes in the levels of [Met5]enkephalin (YGGFM) and Tyr-Gly-Gly (YGG), a characteristic extracellular enkephalin metabolite produced under the action of enkephalinase. In rat striatal slices, D1-receptor agonists or antagonists did not modify enkephalin release. By contrast, D2-receptor agonists enhanced the potassium-induced release of YGGFM and YGG without affecting spontaneous release from nondepolarized slices. This response was prevented by the D2-receptor antagonists haloperidol and RIV 2093, the latter compound being more potent, which suggested the involvement of a putative D2-receptor subtype. Acute administration of apomorphine or selective D2-receptor agonists, but not that of a D1-receptor agonist, enhanced the steady-state level of YGG without affecting the YGGFM level in rat striatum. The effect was blocked selectively by D2-receptor antagonists which, administered alone, had no effect. These observations indicate that D2-receptor stimulation in vitro or in vivo facilitates enkephalin release from striatal neurons, but that endogenous dopamine does not exert any tonic influence upon the opioid peptide neuron activity under basal conditions. However, chronic administration of haloperidol resulted in increases in striatal YGGFM and YGG, an effect presumably reflecting a long-term adaptive process.

Animals↗
PubMed1990

Enkephalin biosynthesis and release in mouse striatum are inhibited by GABA receptor stimulation: compared changes in preproenkephalin mRNA and Tyr-Gly-Gly levels.

In order to assess changes in enkephalin release and biosynthesis, the levels of the tripeptide Tyr-Gly-Gly (YGG), a characteristic extracellular metabolite of enkephalins, and of the proenkephalin mRNA in mouse striatum were evaluated after a single administration of GABAergic agents. Significant and long-lasting decreases in steady state YGG levels were elicited by muscimol, a gamma-aminobutyric acid-A (GABAA) receptor agonist, diazepam, a benzodiazepine receptor agonist, or aminooxyacetic acid, a GABA-transaminase inhibitor. In addition, muscimol offset the elevation of striatal YGG elicited by bestatin, an aminopeptidase inhibitor, which entirely drives the released enkephalins into the metabolic pathway operated by enkephalinase (EC 3.4.24.11). Diazepam potentiated the effect of muscimol so that the YGG decrease induced by the combination of these two drugs was maximal after 30 min (-60%) and still significant (-40%) after 6 h, this potentiation being antagonized by pre-treatment with Ro 15-1788, a specific benzodiazepine receptor antagonist. By contrast [Met5]enkephalin steady-state levels were marginally affected by GABAergic agents, being only slightly reduced 6 h after the combination of muscimol and diazepam. After 3 h the same treatment also reduced by about 30% the level of proenkephalin mRNA, this change being maximal after 6 h (-45%) and still present after 24 h. These compared changes in various indexes of enkephalin neuron activity suggest that stimulation of GABAA receptors depresses enkephalin release immediately and for several hours, whereas preproenkephalin gene expression is decreased in a somewhat delayed and longer lasting manner. These patterns of temporal changes in biosynthesis and release of the neuropeptide presumably account for the limited changes in its steady state levels.

Aminooxyacetic Acid↗
PubMed1990

A novel potential metallopeptidase derived from the enkephalinase gene by alternative splicing.

Amplification of rat intestine mRNAs was performed by the reverse transcriptase-polymerase chain reaction (RT-PCR) using various oligonucleotide primers mainly corresponding to the translated region of the enkephalinase (EC 3.4.24.11, membrane metalloendopeptidase, MME I) gene. In addition to the expected transcript, a shorter one was identified and its sequence indicated that it corresponds to an alternatively spliced mRNA from which exons 5-18 of MME I are deleted. It encodes a deduced 255 amino acid protein, MME II, instead of the 742 amino acid sequence of enkephalinase. The deduced structure of MME II is consistent with its being a membrane-bound, zinc-containing glycoprotein with a modified peptidase activity. MME II mRNA is also expressed, together with MME I mRNA, in brain and thyroid in a tissue-specific manner.

Animals↗
PubMed1987

Enkephalinase (EC 3.4.24.11) is highly localized to a striatonigral pathway in rat brain.

Autoradiographic visualization of enkephalinase (membrane metalloendopeptidase, EC 3.4.24.11) in sagittal sections of rat brain using a 125I-labelled monoclonal antibody showed the presence of a dense immunoreactivity in a tract joining the striatum to the substantia nigra. Unilateral kainate injections into the striatum elicited a strong ipsilateral decrease in enkephalinase activity and immunoreactivity in both the injected area and substantia nigra, particularly its pars compacta. This demonstrates the presence of enkephalinase all along fibers of a striatonigral pathway.

Animals↗
PubMedDate not supplied

Changes in levels of the tripeptide Tyr-Gly-Gly as an index of enkephalin release in the spinal cord: effects of noxious stimuli and parenterally-active peptidase inhibitors.

The tripeptide Tyr-Gly-Gly (YGG), representing the product of enkephalin hydrolysis by enkephalinase (EC 3.4.24.11), was characterized and its levels measured in spinal cord perfusates of halothane-anaesthetized rats. During noxious pinching of the muzzle, which is known to trigger enkephalin release, YGG levels were enhanced more markedly and for longer than were those of [Met5]enkephalin (YGGFM), in the same samples. By contrast, neither YGG nor YGGFM levels were affected by pinching the tail. Treatment with carbaphethiol, a parenterally-active aminopeptidase inhibitor, markedly increased YGG levels and lengthened the duration of the increase produced by pinching the muzzle. Treatment with acetorphan, a parenterally-active enkephalinase inhibitor, given alone or in combination with carbaphethiol, completely prevented the rise in YGG triggered by noxious stimulation. By contrast, [Met5]enkephalin levels in the perfusates were increased by the combined administration of the two peptidase inhibitors but these levels were not further enhanced by noxious stimulation. Thus, spinal cord YGG appears to be formed under the influence of enkephalinase and to constitute a sensitive index of enkephalin release.

Analgesics↗
PubMedDate not supplied

125I-[Tyr0,D-Trp8]somatostatin-14 binding sites in the locus coeruleus of the rat are located on both ascending and descending projecting noradrenergic cells.

Radioautographic determinations of 125I-[Tyr0,D-Trp8]somatostatin-14 (125I-SRIF) binding sites were performed on frozen serial sections of the locus coeruleus (LC) of control rats and of rats subjected to either bilateral microinjections of 6 hydroxydopamine (6-OHDA) into the LC or unilateral microinjection into the ascending noradrenergic bundles. These experiments were performed in order to determine whether 125I-SRIF binding was localized to noradrenergic-containing cells and in which regions the cells which contain the binding sites are projecting. The extent of the lesions was assessed by measuring norepinephrine (NE) levels in the hippocampus (88% decrease as compared to sham-operated animals) for bilateral LC lesions and in the frontal cortex (87% reduction vs. contralateral side) for unilateral bundle lesions. In control rats, 125I-SRIF binding sites were restricted to the boundaries of the LC and followed closely the distribution of tyrosine hydroxylase-labeled cells. Three weeks after bilateral injections of 6-OHDA, 125I-SRIF binding decreased by 79% in all regions of the LC. In contrast, unilateral destruction of the ascending noradrenergic bundles resulted in a moderate decrease only in the middle part of the LC with a more important effect in the dorsal (55%) than in the ventral (24%) portion of the nucleus. These data demonstrate that: 1) most SRIF receptors in the LC are located in the vicinity of NE-containing cell bodies and 2) NE-containing cells bearing SRIF receptors project to the forebrain as well as to other terminal areas located more caudally in the brain. These data suggest a general role for SRIF in the control of the multiple functions of the LC.

Animals↗