Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake".

Zhang et al. (Research Articles, 11 November 2005, p. 996) reported that obestatin, a peptide derived from the ghrelin precursor, activated the orphan G protein-coupled receptor GPR39. However, we found that I125-obestatin does not bind GPR39 and observed no effects of obestatin on GPR39-transfected cells in various functional assays (cyclic adenosine monophosphate production, calcium mobilization, and GPR39 internalization). Our results indicate that obestatin is not the cognate ligand for GPR39.

Desensitization of type 1 angiotensin II receptor subtypes in the rat kidney.

Differences involving serine residues in the sequence of the carboxyl-terminal tail of type 1 angiotensin II (Ang II) receptor subtypes AT(1A) and AT(1B) suggest differences in desensitization ability. We examined the Ang II-induced homologous desensitization patterns of both receptor subtypes in freshly isolated renal structures: glomerulus (Glom), afferent arteriole, and cortical thick ascending limb (CTAL), whose content in each subtype mRNA is different, by measuring variations in intracellular calcium concentration. A preexposure to a maximal dose of Ang II, followed by a second application of the same concentration, induced: 1) a complete desensitization in Glom, where AT(1A) and AT(1B) mRNAs were expressed in similar proportions, and 2) no or partial desensitization in afferent arteriole and CTAL, where AT(1A) mRNA was predominant. In the absence of nephron structure containing only AT(1B) mRNA, we studied rat anterior pituitary cells that exhibit high content in this subtype and observed that desensitization was not complete. In Glom, CTAL, and pituitary cells, desensitization proceeded in a dose-dependent manner. In Glom and CTAL, desensitization occurred via a PKC-independent mechanism. These results suggest that desensitization does not depend on the nature of Ang II receptor subtype but either on the proportion of each subtype in a given cell and/or on cell specific type. This could allow adaptive biological responses to Ang II appropriate to the specific function of a given cell type.

Physiological role of a novel neuropeptide, apelin, and its receptor in the rat brain.

Apelin, a peptide recently isolated from bovine stomach tissue extracts, has been identified as the endogenous ligand of the human orphan APJ receptor. We established a stable Chinese hamster ovary (CHO) cell line expressing a gene encoding the rat apelin receptor fused to the enhanced green fluorescent protein, to investigate internalization and the pharmacological profile of the apelin receptor. Stimulation of this receptor by the apelin fragments K17F (Lys1-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and pE13F (pGlu5-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) resulted in a dose-dependent inhibition of forskolin-induced cAMP production and promoted its internalization. In contrast, the apelin fragments R10F (Arg8-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe17) and G5F (Gly13-Pro-Met-Pro-Phe17) were inactive. The physiological role of apelin and its receptor was then investigated by showing for the first time in rodent brain: (i) detection of apelin neurons in the supraoptic and paraventricular nuclei by immunohistochemistry with a specific polyclonal anti-apelin K17F antibody; (ii) detection of apelin receptor mRNA in supraoptic vasopressinergic neurons by in situ hybridization and immunohistochemistry; and (iii) a decrease in vasopressin release following intracerebroventricular injection of K17F, or pE13F, but not R10F. Thus, apelin locally synthesized in the supraoptic nucleus could exert a direct inhibitory action on vasopressinergic neuron activity via the apelin receptors synthesized in these cells. Furthermore, central injection of pE13F significantly decreased water intake in dehydrated normotensive rats but did not affect blood pressure. Together, these results suggest that neuronal apelin plays an important role in the central control of body fluid homeostasis.

Aminopeptidase A, which generates one of the main effector peptides of the brain renin-angiotensin system, angiotensin III, has a key role in central control of arterial blood pressure.

Overactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several experimental animal models. We have recently reported that, in the murine brain RAS, angiotensin II (AngII) is converted by aminopeptidase A (APA) into angiotensin III (AngIII),which is itself degraded by aminopeptidase N (APN), both peptides being equipotent to increase vasopressin release and arterial blood pressure when injected by the intracerebroventricular (i.c.v.) route. Because AngII is converted in vivo into AngIII, the exact nature of the active peptide is not precisely known. To delineate their respective roles in the central control of cardiovascular functions, specific and selective APA and APN inhibitors are needed to block the metabolic pathways of AngII and AngIII respectively. In the absence of such compounds for APA, we first explored the organization of the APA active site by site-directed mutagenesis. This led us to propose a molecular mechanism of action for APA similar to that proposed for the bacterial enzyme thermolysin deduced from X-ray diffraction studies. Secondly, we developed a specific and selective APA inhibitor, compound EC33 [(S)-3-amino-4-mercaptobutylsulphonic acid], as well as a potent and selective APN inhibitor, PC18 (2-amino-4-methylsulphonylbutane thiol). With these new tools we examined the respective roles of AngII and AngIII in the central control of arterial blood pressure. A central blockade of APA with the APA inhibitor EC33 suppressed the pressor effect of exogenous AngII, suggesting that brain AngII must be converted into AngIII to increase arterial blood pressure. Furthermore, EC33, injected alone i.c.v. but not intravenously, caused a dose-dependent decrease in arterial blood pressure by blocking the formation of brain AngIII but not systemic AngIII. This is corroborated by the fact that the selective APN inhibitor PC18 administered alone via the i.c.v. route increased arterial blood pressure. This pressor response was blocked by prior treatment with the angiotensin type 1 receptor antagonist losartan, showing that blocking the action of APN on AngIII metabolism leads to an increase in endogenous AngIII levels, resulting in arterial blood pressure increase through an interaction with angiotensin type 1 receptors. These results demonstrate that AngIII is a major effector peptide of the brain RAS, exerting a tonic stimulatory control over arterial blood pressure. Thus APA, the enzyme responsible for the formation of brain AngIII, represents a potential central therapeutic target that justifies the development of APA inhibitors, crossing the blood-brain barrier, as central anti-hypertensive agents.

Potent and selective inhibition of zinc aminopeptidase A (EC 3.4.11.7, APA) by glutamyl aminophosphinic peptides: importance of glutamyl aminophosphinic residue in the P1 position.

Through the development of a new chemical strategy, aminophosphinic peptides containing a pseudoglutamyl residue (Glu Psi(PO2-CH2)Leu-Xaa) in the N-terminal position were synthesized and evaluated as inhibitors of aminopeptidase A (APA). The most potent inhibitor developed in this study, Glu Psi(PO2-CH2)Leu-Ala, displayed a Ki value of 0.8 nM for APA, but was much less effective in blocking aminopeptidase N (APN) (Ki = 31 microM). The critical role of the glutamyl residue in this phosphinic peptide, both in potency and selectivity, is exemplified by the P1 position analogue, Ala Psi(PO2-CH2)Leu-Ala, which exhibited a Ki value of 0.9 microM toward APA but behaved as a rather potent inhibitor of APN (Ki = 25 nM). Glu Psi(PO2-CH2)Leu-Xaa peptides are poor inhibitors of angiotensin converting enzyme (Ki values higher than 1 microM). Depending on the nature of the Xaa residue, the potency of these phosphinic peptides toward neutral endopeptidase 24-11 varied from 50 nM to 3 microM. In view of the in vivo role of APA in the formation of brain angiotensin III, one of the main effector peptides of the renin angiotensin system in the central nervous system, highly potent and selective inhibitors of APA may find important therapeutic applications soon.

Cloning, pharmacological characterization and brain distribution of the rat apelin receptor.

The peptide apelin, recently isolated from bovine stomach tissue extracts, has been identified as an endogenous ligand of the human putative receptor protein related to the angiotensin receptor AT(1) (APJ). In this article, we report cloning of the rat apelin receptor cDNA. The sequence shares 90% identity with the human APJ receptor and 31% with the rat AT(1A) angiotensin receptor. Subsequently a stable CHO cell line expressing the receptor fused at its C-terminal part with the enhanced green fluorescent protein (EGFP) was established, allowing to verify its cell surface distribution and to determine the affinity of various apelin and angiotensin fragments on the cloned receptor. As shown for the human APJ receptor, the rat apelin receptor expressed in the cell line was negatively coupled to adenylate cyclase. The apelin fragment K17F (Lys(1)-Phe-Arg-Arg-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe(17)) inhibited forskolin-stimulated cAMP production at sub-nanomolar concentrations whereas angiotensin II and angiotensin III were inactive. N-terminal elongation of K17F with a tyrosine or the N-terminal deletion of the first four amino acids did not modify the inhibitory action of K17F on cAMP production. In contrast, deletion of the first seven amino acids of K17F or substitution of phenylalanine by an alanine residue at the C-terminus completely abolished the activity of the peptide. In situ hybridization analysis of apelin receptor mRNA expression in the adult rat brain showed intense labeling in the hypothalamus, especially in the supraoptic and the paraventricular nuclei. The anterior and intermediate lobes of the pituitary were also highly labeled, as well as the pineal gland. Labeling was also found in extrahypothalamic structures such as the piriform cortex, the nucleus of the lateral olfactory tract, the central grey matter, the pars compacta of the substantia nigra, the dorsal raphe nucleus, the entorhinal cortex, the dentate gyrus and the Ammon's horn. The hypothalamic and hypophyseal distribution of the receptor suggests an involvement of apelin in the control of neuro- and adenohypophyseal hormone release, whereas its presence in the pineal gland and in discrete higher brain structures points out to possible roles in the regulation of circadian rhythms and of water and food intake behavior.

Investigation of subsite preferences in aminopeptidase A (EC 3.4.11.7) led to the design of the first highly potent and selective inhibitors of this enzyme.

The study of the physiological roles of the membrane-bound zinc-aminopeptidase A (glutamyl aminopeptidase, EC 3.4.11.7) needs the design of efficient and selective inhibitors of this enzyme. An acute exploration of aminopeptidase A active site was performed by a combinatorial approach using (3-amino-2-mercapto-acyl)dipeptides able to fit its S(1), S(1)', and S(2)' subsites. This analysis confirmed that the S(1) subsite is optimally blocked by a glutamate or isosteric residues and demonstrated that the S(1)' subsite is hydrophobic whereas the S(2)' subsite recognizes preferentially negatively charged residues derived from aspartic acid. The optimization of these structural parameters led to the synthesis of nanomolar and subnanomolar inhibitors of aminopeptidase A such as H(3)N(+)CH(CH(2)CH(2)SO(3)(-))CH(SH)CO-Ile-(3-COOH)Pro that exhibits a K(i) of 0.87 nM. The best compounds were synthesized by a stereochemically controlled route. These first described highly potent inhibitors could allow studies about the role of physiological substrates of APA such as angiotensin II and cholecystokinin CCK(8) in the central nervous system.

Identification of endocrine cell populations expressing the AT1B subtype of angiotensin II receptors in the anterior pituitary.

Angiotensin II (Ang II) participates in the regulation of anterior pituitary hormone secretion by acting either directly on the anterior pituitary or indirectly on the hypothalamus. When applied directly on pituitary cells, Ang II increases both ACTH and PRL secretion and has also been reported to affect GH secretion. Three distinct subtypes of Ang II receptors (AT1A, AT1B, and AT2) have been identified; they are unequally distributed and differently regulated in various tissues. We have previously demonstrated that only AT1A receptors are present in the hypothalamus while anterior pituitary cells express predominantly the AT1B subtype. Using in situ hybridization in combination with immunohistochemistry, the aim of the present study was to identify the phenotype of the endocrine cell expressing AT1B receptor messenger RNA (mRNA) in the anterior pituitary of adult male Sprague-Dawley rats. Expression of AT1B receptor mRNA was present in 33.9 +/- 1.0% of anterior pituitary cells. AT1B mRNA is predominantly expressed by lactotropes (78.2 +/- 2.1% of AT1B mRNA-expressing cells) and to a lower degree by corticotropes (18.3 +/- 2.1%) and is not detectable in somatotropes, mammosomatotropes, gonadotropes, or thyrotropes. These results indicate that in adult male rats, Ang II, which has been shown to be synthesized in gonadotropes, can directly stimulate PRL and ACTH release from lactotropes and corticotropes through activation of AT1B receptors. As only 53.8 +/- 2.7% of lactotropes and 23.6 +/- 2.8% of corticotropes expressed AT1B mRNA, our findings suggest a functional heterogeneity of both cell types regarding their sensitivity to Ang II.

Expression of angiotensin II receptor subtypes AT(1A) and AT(1B) in enriched fractions of dispersed rat pituitary cells.

Differential evaluation of angiotensin II (Ang II) receptors (AT1A, AT1B and AT2) expression was performed in dispersed adenohypophyseal cells fractionated by unit gravity sedimentation. Binding of [125I-Sar1-Ile8]-Ang II and its displacement by specific nonpeptidic AT1 (DuP753) and AT2 (PD123319) antagonists was monitored throughout the gradient. Quantification of mRNA levels corresponding to both AT1 receptor subtypes (AT1A and AT1B) was achieved by reverse transcriptase polymerase chain reaction (RT-PCR) amplification in the presence of an AT1 receptor mutant cRNA as internal standard. Fractions were characterised by radioimmunoassay for the five major anterior pituitary hormones and by counting immunocytochemically labelled cells. Quantification of AT1 receptor subtype mRNA levels was also performed in four hypophyseal cell lines secreting prolactin, growth hormone, corticotropin and a gonadotropin subunit. As already described for the whole pituitary, AT1B receptor mRNA is predominantly expressed (80% of total AT1A + AT1B receptor mRNA content), whereas AT1A is expressed at lower level (20%) in dispersed pituitary cells. Most AT1 receptor mRNA and binding co-elute with fractions enriched in lactotropes and corticotropes. In contrast to AT1B, AT1A receptor mRNA is not present in heavier populations of lactotropes or in somatomammotropes. Low AT1B mRNA levels are detected in GH4C1 and in GC cells, two clones which secrete respectively prolactin and growth hormone. In contrast, no AT1 receptor mRNA expression was found in two other cell lines, AtT20 and alphaT3-1, which produce pro-opiomelanocortin and gonadotropin. It is concluded that expression of AT1 receptor subtypes is heterogeneous in different populations of lactotropes and corticotropes.

A tyrosine residue essential for catalytic activity in aminopeptidase A.

Aminopeptidase A (EC 3.4.11.7; APA) is a 130 kDa membrane-bound zinc enzyme that contains the consensus sequence HEXXH (residues 385-389) conserved among the zinc metalloprotease family. In this motif, both histidine residues and the glutamic residue were shown to be involved respectively in zinc co-ordination and catalytic activity. Treatment of APA with N-acetylimidazole results in a loss of enzymic activity; this is prevented by the competitive aminopeptidase inhibitor amastatin, suggesting the presence of an important tyrosine, lysine or cysteine residue at the active site of APA. A tyrosine residue was previously proposed to be involved in the enzymic activity of aminopeptidase N. Furthermore sequence alignment of mouse APA with other monozinc aminopeptidases indicates the presence of a conserved tyrosine (Tyr-471 in APA). The functional role of Tyr-471 in APA was investigated by replacing this residue with a phenylalanine (Phe-471) or a histidine (His-471) residue by site-directed mutagenesis. Kinetic studies showed that the Km values of both mutants were similar to that of the wild-type enzyme, whereas kcat values were decreased by three orders of magnitude and corresponded to a variation in free energy of the rate-limiting step by 4.0 and 4.2 kcal/mol (0.96 and 1.00 kJ/mol) for the Phe-471 and His-471 mutants respectively. The mutation did not modify the inhibitory potency of a thiol-containing inhibitor that strongly chelates the active-site zinc ion, whereas that of a putative analogue of the transition state presumed to mimic the reaction intermediate was reduced. Taken together, these results strongly suggest that the Tyr-471 hydroxy group participates in catalysis by stabilizing the transition state complex through interaction with the oxyanion.

Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control.

Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1 beta (IL-1 beta) at 10 U.ml-1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 microM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

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.

[Identification and characterization of neutral endopeptidase in endothelial cells of arterial or venous origin]. / Identification et caractérisation de l'endopeptidase neutre dans les cellules endothéliales d'origine artérielle ou veineuse.

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.

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.

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.

Inhibition of enkephalin degradation by phelorphan: effects on striatal [Met5]enkephalin levels and jump latency in mouse hot plate test.

The effect of phelorphan (mercaptoacetyl-Phe-Phe), an inhibitor of various enkephalin-degrading enzymes, was tested in the mouse hot plate test and its efficacy to prevent endogenous enkephalin degradation in mouse striatum was determined. The i.c.v. injection of phelorphan (50 micrograms) increased the [Met5]enkephalin immunoreactivity by 41% in the extrasynaptosomal fraction of mouse striatum compared to a 43% increase induced by i.c.v. administration of thiorphan (50 micrograms) + bestatin (75 micrograms). Jump latency in the mouse hot plate test was significantly prolonged by phelorphan (25 micrograms i.c.v.). The analgesic effect of phelorphan was the same as that of thiorphan (25 micrograms i.c.v.) + bestatin (25 micrograms i.c.v.) and was antagonized by pretreatment with naloxone. These results suggest that phelorphan induces naloxone-reversible analgesia by elevation of [Met5]enkephalin levels in the synaptic cleft.

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.

Steady-state level and turnover rate of the tripeptide Tyr-Gly-Gly as indexes of striatal enkephalin release in vivo and their reduction during pentobarbital anesthesia.

Tyr-Gly-Gly (YGG) was recently shown to be an extraneuronal metabolite of opioid peptides derived from proenkephalin A, formed in brain by the action of "enkephalinase" (membrane metalloendopeptidase, EC 3.4.24.11) and degraded by aminopeptidases. The dynamic state of YGG in mouse striatum was studied by evaluating the changes in its level elicited by inhibitors of these peptidases. Inhibition of YGG synthesis by Thiorphan or acetorphan reduced YGG levels with a t1/2 (mean +/- SEM) of 12 +/- 2 min, indicating an apparent turnover rate (mean +/- SEM) of 18 +/- 2 pmol/mg of protein per hr. An apparent turnover rate of 18 +/- 2 pmol/mg of protein per hr was derived from the rate of YGG accumulation elicited by the aminopeptidase inhibitor bestatin. In addition, accumulation of Tyr-Gly-Gly-Phe-Met (YGGFM) in an extrasynaptosomal fraction after blockade of its degradation by Thiorphan and bestatin occurred at a rate of 18 +/- 3 pmol/mg of protein per hr, which is likely to reflect the rate of enkephalin release in vivo. Hence, the three series of data suggest that striatal enkephalins rapidly turn over--e.g., with a t1/2 in the 1-hr range. Pentobarbital anesthesia reduced by about 60% the rate of YGG accumulation elicited by bestatin and the extrasynaptosomal YGGFM accumulation elicited by Thiorphan and bestatin. This suggests that the activity of striatal enkephalin neurons is depressed during anesthesia. Pentobarbital (and chloral hydrate) did not affect the steady-state level of YGGFM but rapidly reduced that of YGG. Hence, the steady-state levels of YGG seem a reliable index of changes in enkephalin release, and measuring levels of characteristic fragments might therefore provide a general means of evaluating neuropeptide release in vivo.