Further characterization of a neurotensin-degrading neutral metalloendopeptidase from rat brain

A peptidase inactivating neurotensin at the Pro¹⁰-Tyr¹¹ peptidyl bond, leading to the biologically inactive fragments neurotensin_1–10 and neurotensin_11–13 was purified from rat brain homogenate. The peptidase was characterized as a 70 kDa monomer and could be classified as a metaliopeptidase with respect to its sensitivity to o-phenanthroline, EDTA and divalent cations. The enzyme was also strongly inhibited by dithiothreitol but appeared totally insensitive to thiol-blocking agents, acidic and serine protease inhibitors. Experiments performed with a series of highly specific peptidase inhibitors clearly indicated that the peptidase was a novel enzyme distinct from previously purified cerebral peptidases. The enzyme displayed a rather high affinity for neurotensin (Km = 2.3 itM). Studies on its specificity indicated that: (i) neurotensin_9–13 was the shortest neurotensin fragment with full inhibitory potency of [³H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule progressively led to inactive analogs; (ii) the peptidase exhibited a strong stereospecificity towards the residues in positions 8, 9 and 11. By contrast, neither introduction of a steric hindrance in position 11 nor amidation of the C-terminal end of the neurotensin molecule affected the ability of the corresponding analog to inhibit [³H]neurotensin degradation; (iii) Pro-Phe was the most potent dipeptide to compete for [³H]neurotensin degradation; (iv) the peptidase could not be described as an exclusive “neurotensinase” activity since, in addition to the neurotensin natural analogs (neuromedin N and xenopsin), non related natural peptides such as angiotensins I and II, dynorphins 1–8 and 1–13, atriopeptin III and bradykinin potently inhibited [³H]neurotensin degradation. Most of these peptides behaved as substrates for the enzyme.     

Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes

A peptidase that cleaved neurotensin at the Pro10-Tyr11 peptide bond, leading to the formation of neurotensin-(1-10) and neurotensin-(11-13), was purified nearly to homogeneity from rat brain synaptic membranes. The enzyme appeared to be monomeric with a molecular weight of about 70,000-75,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography filtration. Isoelectrofocusing indicated a pI of 5.9-6. The purified peptidase could be classified as a neutral metallopeptidase with respect to its sensitivity to pH and metal chelators. Thiol-blocking agents and acidic and serine protease inhibitors had no effect. Studies with specific peptidase inhibitors clearly indicated that the purified enzyme was distinct from enzymes capable of cleaving neurotensin at the Pro10-Tyr11 bond such as proline endopeptidase and endopeptidase 24-11. The enzyme was also distinct from other neurotensin-degrading peptidases such as angiotensin-converting enzyme and a recently purified rat brain soluble metalloendopeptidase. The peptidase displayed a high affinity for neurotensin (Km = 2.6 microM). Studies on its specificity revealed that neurotensin-(9-13) was the shortest neurotensin partial sequence that was able to fully inhibit [3H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule as well as substitutions in positions 8, 9, and 11 by D-amino acids strongly decreased the inhibitory potency of neurotensin. Among 20 natural peptides, only angiotensin I and the neurotensin-related peptides (xenopsin and neuromedin N) were found as potent as unlabeled neurotensin.     

Neurotensin metabolism in various tissues of central and peripheral origins: ubiquitous involvement of a novel neurotensin degrading metalloendopeptidase

The metabolism of neurotensin in vitro, in various membrane preparations and cell lines of central and peripheral origins was studied. Neurotensin degradation products were separated by HPLC and identified by either amino acid analysis or by their retention times. Peptidases responsible for the cleavages were identified by means of specific fluorigenic substrates or inhibitors. Although the patterns of neurotensin inactivation varied according to the tissue source in all cases, a major primary cleavage occurred at the Pro10-Tyr11 bond, leading to the biologically inactive fragments NT1-10 and NT11-13. A novel neurotensin-degrading metallopeptidase was responsible for this cleavage. Interestingly, it was the only peptidase that was ubiquitously detected. In addition, endopeptidase 24.11 (EC 3.4.24.11) contributed to this cleavage in rat brain synaptic membranes as well as in circular and longitudinal smooth muscle plasma membranes from dog ileum.     

Mechanism of degradation of LH-RH and neurotensin by synaptosomal peptidases

The products of degradation of LH-RH and neurotensin by synaptosomes isolated from rat hypothalamus and cortex have been identified. LH-RH is cleaved at Tyr5-Gly6 and Pro9-Gly10 giving rise to LH-RH (1-5), LH-RH (6-10) and LH-RH (1-9). Neurotensin is cleaved at Arg8-Arg9, Pro10-Tyr11 and Ile12-Leu13, giving neurotensin (1-8), neurotensin (1-10), neurotensin (1-12) and neurotensin (9-13) as major products. While most of the peptidase activity is localized in the cytoplasmic fraction, a small but significant proportion is membrane bound. For LH-RH, the specificity of the membrane-bound activity is similar to that in the cytosol fraction; for neurotensin, the membrane fraction preferentially gives rise to the (1-10) and (1-11) peptides. The most potent inhibitors of the LH-RH and neurotensin degrading enzymes in synaptosomes are heavy metal ions (mercury and copper), p-chloromercuribenzoate and 1,10 phenanthroline.     

Preparation and receptor binding affinities of cyclic C-terminal neurotensin (8-13) and (9-13) analogues.

Cyclic analogues of neurotensin (NT) C-terminal fragments NT(8-13) and NT(9-13) were produced via intramolecular nucleophilic substitution of the Tyr(11) phenoxide anion on a 6-bromohexanoyl side chain substituted at position 8 or 9 and tested for NT receptor binding affinity.     

Hydrolysis of substance p and neurotensin by converting enzyme and neutral endopeptidase

Angiotensin I converting enzyme (ACE) and neutral endopeptidase ("enkephalinase"; NEP), were purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln6-Phe7,-Phe8, and Gly9-Leu10 and neurotensin (NT) at Pro10-Tyr11 and Tyr11-Ile12. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (mumol/min/mg): SP1-11 = 7.8, SP4-11 = 11.7, SP5-11 = 15.4, SP6-11 = 15.6, SP8-11 = 6.7, NT1-13 = 2.9, and NT8-13 = 4.0. Purified ACE rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe8-Gly9 and Gly9-Leu10 to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl- dependent and inhibited by captopril. ACE released mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80-93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr11-Ile12 to release Ile12-Leu13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (mumol/min/mg): SP1-11 = 1.2, SP methyl ester = 0.7, SP free acid = 8.5, SP4-11 = 2.4, SP5-11 = 0.9, SP6-11 = 1.4, SP8-11 = 0, NT1-13 = 0.2, and NT8-13 = 1.3. Peptide substrates were used as inhibitors of ACE (substrate = FA-Phe-Gly-Gly) and NEP (substrate = Leu5-enkephalin).(ABSTRACT TRUNCATED AT 250 WORDS)     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.     

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).     

Novel activity of angiotensin-converting enzyme. Hydrolysis of cholecystokinin and gastrin analogues with release of the amidated C-terminal dipeptide.

ACE (angiotensin-converting enzyme; peptidyl dipeptidase A; EC 3.4.15.1), cleaves C-terminal dipeptides from active peptides containing a free C-terminus. We investigated the hydrolysis of cholecystokinin-8 [CCK-8; Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2] and of various gastrin analogues by purified rabbit lung ACE. Although these peptides are amidated at their C-terminal end, they were metabolized by ACE to several peptide fragments. These fragments were analysed by h.p.l.c., isolated and identified by comparison with synthetic fragments, and by amino acid analysis. The initial and major site of hydrolysis was the penultimate peptide bond, which generated a major product, the C-terminal amidated dipeptide Asp-Phe-NH2. As a secondary cleavage, ACE subsequently released di- or tri-peptides from the C-terminal end of the remaining N-terminal fragments. The cleavage of CCK-8 and gastrin analogues was inhibited by ACE inhibitors (Captopril and EDTA), but not by other enzyme inhibitors (phosphoramidon, thiorphan, bestatin etc.). Hydrolysis of [Leu15]gastrin-(14-17)-peptide [Boc (t-butoxycarbonyl)-Trp-Leu-Asp-Phe-NH2] in the presence of ACE was found to be dependent on the chloride-ion concentration. Km values for the hydrolysis of CCK-8, [Leu15]gastrin-(11-17)-peptide and Boc-[Leu15]gastrin-(14-17)-peptide at an NaCl concentration of 300 mM were respectively 115, 420 and 3280 microM, and the catalytic constants were about 33, 115 and 885 min-1. The kcat/Km for the reactions at 37 degrees C was approx. 0.28 microM-1.min-1, which is approx. 35 times less than that reported for the cleavage of angiotensin I. These results suggest that ACE might be involved in the metabolism in vivo of CCK and gastrin short fragments.     

The isolation of a peptidyl dipeptidase from mouse brain cytosol that cleaves adrenocorticotropic hormone-(7-10) and des-tyrosine-enkephalins

An enzyme present in mouse brain cytosol cleaves C-terminal dipeptides from substrates including ACTH-(7-10) (Phe-Arg-Trp-Gly), and des-Tyr-[Met]- and des-Tyr-[Leu]enkephalin. By means of ion-exchange chromatography and gel filtration, the peptidase was purified to a specific activity of 1570 times that of brain homogenate. At this purification, a second peptidase, which hydrolyzes Trp-Gly and other peptides [M. E. A. Reith and A. Neidle (1979) Biochem. Biophys. Res. Commun. 90, 794-800] was still present, but could be removed by preparative polyacrylamide gel electrophoresis. The des Tyr-enkephalin-cleaving enzyme has a molecular weight of about 85,000 and a pH optimum of 7.8. It is inhibited by metal-chelating and sulfhydryl reagents. The enzyme has a strong preference for substrates with an aromatic residue in the position adjacent to the C-terminal amino acid, although some peptides meeting this criterion were competitive inhibitors rather than substrates. Peptides with less than four residues were inactive and, in general, tetrapeptides were found to be more reactive than larger analogs, when peptides with common C-terminal sequences were compared. The peptidyl dipeptidase, which has not been described previously, can be readily distinguished from angiotensin-converting enzyme (EC 3.4.15.1) and from neutral endopeptidase (EC 3.4.24.11) by its subcellular localization, substrate specificity, and response to inhibitors. It was suggested that peptidyl dipeptidase-B (PDP-B, EC 3.4.15.-) would be an appropriate name for the enzyme. PDP-B is widely distributed among mouse tissues.     

In vitro degradation of neurotensin in human plasma

To study the degradation of neurotensin in plasma in vitro, fresh human plasma was incubated with neurotensin in the presence and absence of the peptidase inhibitors pepstatin A, EDTA, PMSF and aprotinin. The half-time of disappearance of neurotensin at 37 degrees C was calculated to be 226 min in vitro as opposed to 1.4 min in vivo when measured by radioimmunoassay with a C-terminally directed neurotensin antiserum. Both gel filtration and reversed phase high-pressure liquid chromatography (HPLC) showed that the main degradation product of neurotensin in human plasma in vitro was chromatographically and immunologically identical to neurotensin 1-8 and HPLC also demonstrated the formation of neurotensin 1-11. The loss of neurotensin incubated in human plasma in vitro was greatly reduced by EDTA but not by the other peptidase inhibitors tested. In this respect peptidase(s) responsible for the degradation of neurotensin in plasma differ from those present in brain homogenates. EDTA may be of importance in the preservation of neurotensin in plasma samples.     

Involvement of endopeptidase 24.15 in the inactivation of bradykinin by rat brain slices

The effect of peptidase inhibitors on the degradation of [3H]-bradykinin by rat hypothalamic slices was studied using HPLC to separate and identify the products. The degradation appears to be mainly mediated by an enzyme which cleaves the peptide at the Phe5-Ser6 bond and is inhibited by 1,10-phenanthroline, dynorphin(1-13) and carboxyphenylethyl-Ala-Ala-Phe-p-aminobenzoate. This suggest the involvement of a membrane bound variant of the soluble metalloendopeptidase (EC3.4.24.15) isolated from rat brain which degrades neurotensin, angiotensin and other neuropeptides as well as bradykinin.     

Effects of ICV administration of neurotensin and analogs on EEG in rats

The electroencephalographic (EEG) effects of the ICV administration of neurotensin (NT 1-13), NT 1-8 (an inactive neurotensin fragment) and D TYR-11 NT (a long-lasting analog of neurotensin) were studied in rats. In awake rats, NT 1-13 (30 micrograms) and D TYR-11 NT (10 micrograms) induced an increase of the power spectrum in the theta range activity (4-7 Hz). In rats recorded during the sleep-wakefulness cycles, NT 1-13 (10 and 30 micrograms) and D TYR-11 NT (10 micrograms) had an awakening effect and also induced an increase of latency to the first episode of the different sleep stages (intermediate stage and slow wave sleep). NT 1-8 (30 and 90 micrograms in awake rats, 10 and 90 micrograms for sleep-wakefulness cycles) was inactive in all these experiments. Thus, it seems that all these effects can be linked to neurotensin receptors; indeed only fragments which recognize receptors possess an EEG activity.     

Specific inhibition of endopeptidase 24.16 by dipeptides.

The inhibitory effect of various dipeptides on the neurotensin-degrading metallopeptidase, endopeptidase 24.16, was examined. These dipeptides mimick the Pro10-Tyr11 bond of neurotensin that is hydrolyzed by endopeptidase 24.16. Among a series of Pro-Xaa dipeptides, the most potent inhibitory effect was elicited by Pro-Ile (Ki approximately 90 microM) with Pro-Ile greater than Pro-Met greater than Pro-Phe. All the Xaa-Tyr dipeptides were unable to inhibit endopeptidase 24.16. The effect of Pro-Ile on several purified peptidases was assessed by means of fluorigenic assays and HPLC analysis. A 5 mM concentration of Pro-Ile does not inhibit endopeptidase 24.11, endopeptidase 24.15, angiotensin-converting enzyme, proline endopeptidase, trypsin, leucine aminopeptidase, pyroglutamyl aminopeptidase I and carboxypeptidase B. The only enzyme that was affected by Pro-Ile was carboxypeptidase A, although it was with a 50-fold lower potency (Ki approximately 5 mM) than for endopeptidase 24.16. By means of fluorimetric substrates with a series of hydrolysing activities, we demonstrate that Pro-Ile can be used as a specific inhibitor of endopeptidase 24.16, even in a complex mixture of peptidase activities such as found in whole rat brain homogenate.     

Inactivation of Neurotensin by Rat Brain Synaptic Membranes. Cleavage at the Pro10-Tyr11 Bond by Endopeptidase 24.11 (Enkephalinase) and a Peptidase Different from Proline-Endopeptidase

It was shown previously that the tridecapeptide neurotensin is inactivated by rat brain synaptic membranes and that one of the primary inactivating cleavages occurs at the Pro10-Try11 peptide bond, leading to the formation of NT1-10 and NT11-13. The present study was designed to investigate the possibility that this cleavage was catalyzed by proline endopeptidase and/or endopeptidase 24.11 (enkephalinase). Purified rat brain synaptic membranes were found to contain a N-benzyloxycarbonyl-Gly-Pro-4-methyl-coumarinyl-7-amide-hydrolyzin g activity that was markedly inhibited (93%) by the proline endopeptidase inhibitor N-benzyloxycarbonyl-Pro-Prolinal and partially blocked (25%) by an antiproline endopeptidase antiserum. In contrast, the cleavage of neurotensin at the Pro10-Tyr11 bond by synaptic membranes was not affected by N-benzyloxycarbonyl-Pro-Prolinal and the antiserum. When the conversion of NT1-10 to NT1-8 by angiotensin converting enzyme was blocked by captopril and when the processing of NT11-13 by aminopeptidase(s) was inhibited by bestatin, it was found that thiorphan, a potent endopeptidase 24.11 inhibitor, partially decreased the formation of NT1-10 and NT11-13 by synaptic membranes. In conclusion: (1) proline endopeptidase, although it is present in synaptic membranes, is not involved in the cleavage of neurotensin at the Pro10-Tyr11 bond; (2) endopeptidase 24.11 only partially contributes to this cleavage; (3) there exists in rat brain synaptic membranes a peptidase different from proline endopeptidase and endopeptidase 24.11 that is mainly responsible for inactivating neurotensin by cleaving at the Pro10-Tyr11 bond.     

Tripeptidyl carboxypeptidase activity of kininase II (angiotensin-converting enzyme)

The degradation of des-Arg9-brady kinin and its analogues by highly purified preparations of hog lung and kidney kininase II (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) was studied. The degradative peptides fragments were separated and isolated by high performance liquid chromatography and identified by amino acid analysis. Both enzymes released C-terminal tripeptides from des-Arg9-bradykinin, des-Arg9-(Leu8)-bradykinin, Pro-Pro-Gly-Phe-Ser-Pro-Phe, Pro-Gly-Phe-Ser-Pro-Phe, Gly-Phe-Ser-Pro-Phe, Bz-Gly-Ser-pro-Phe and Bz-Gly-Ala-Pro-Phe. Hydrolysis of Phe-Ser-Pro-Phe, Bz-Gly-His-Pro-Phe, Bz-Gly-Phe-Pro-Phe and Bz-Gly-Gly-Pro-Phe by both enzymes was negligible. These data indicate that kininase II can release C-terminal tripeptides of substrates having a proline residue in the penultimate position such as des-Arg9-bradykinin and its analogues, and that this enzyme is able not only to act as a dipeptidyl carboxypeptidase but also acts as a tripeptidyl carboxy-peptidase. The tripeptidyl carboxypeptidase enzyme was sensitive to inhibition by kininase II inhibitors.     

Inhibition of substance P degradation in rat brain preparations by peptide hydroxamic acids

A peptidase activity of rat diencephalon membranes, which acts on the C-terminal hexapeptide sequence of substance P, was characterized using the radiolabeled substrate N alpha-[( 125I]iododesaminotyrosyl)-substance P (6-11)-hexapeptide. This activity presents certain characteristics similar to those of the substance-P-degrading enzyme purified from human brain by Lee et al. [Eur. J. Biochem. 114, 315-327 (1981)]. It is inhibited by metal chelators and some thiol reagents, but is insensitive to inhibitors of serine proteases and aminopeptidases. The activity is different from angiotensin-converting enzyme and enkephalinase, since it is not affected by specific inhibitors of these enzymes. Substance P and substance P C-terminal fragments longer than the pentapeptide inhibited the degradation of the radiolabeled substrate with inhibition constants around 200 microM. Short fragments of the substance P sequence, such as Boc-Phe-Phe-OMe and Boc-Phe-Phe-Gly-OEt, were also found to inhibit the degradation of the substrate. When the metal-chelating hydroxamic acid moiety was attached to the carboxyl terminus of these short peptides, potent inhibitors of the substance-P-degrading activity were obtained, with inhibition constants in the micromolar range. The most potent of these compounds, iododesaminotyrosyl-Phe-Phe-Gly-NHOH (IBH-Phe-Phe-Gly-NHOH), is a competitive inhibitor, with a Ki value of 1.9 microM. The degradation of substance P by rat diencephalon slices was inhibited to the same extent (40-50%) by IBH-Phe-Phe-Gly-NHOH (20 microM) and by phosphoramidon (1 microM). A combination of both reagents reduced the degradation rate by 75-80%, suggesting that both enkephalinase and the substance-P-degrading activity are involved in the metabolism of substance P in this preparation. IBH-Phe-Phe-Gly-NHOH seems to be quite specific for the latter enzyme, since at a high concentration (0.1 mM) it did not affect the degradation of the radiolabeled substrate by alpha-chymotrypsin, papain, or thermolysin.     

Peptidases involved in the catabolism of neurotensin: inhibitor studies using superfused rat hypothalamic slices

In order to identify which peptidases are involved in the catabolism of neurotensin in the CNS, [3H-Tyr3,11]-neurotensin was superfused over rat hypothalamic slices in the presence and absence of peptidase inhibitors. The degree of degradation of the peptide was determined by reverse phase HPLC separation of 3H-labelled neurotensin from 3H-labelled products. Very little degrading activity was released from the slice into the medium during the superfusion. In the absence of inhibitors, 20 to 50% of 3H-neurotensin was degraded giving mainly 3H-Tyr along with other unidentified 3H-labelled products. Inhibitors of endopeptidase 24.11 (phosphoramidon) and proline endopeptidase (antibody) had no effect on the degradation. Captopril, an inhibitor of angiotensin converting enzyme, had a small inhibitory effect. In contrast, dynorphin(1-13), an inhibitor of a soluble, thiol dependent metallopeptidase which hydrolyses neurotensin at Arg8-Arg9, gave greater than 80% inhibition of 3H-neurotensin degradation in the slice preparation. 1,10-Phenanthroline, an inhibitor of metallopeptidases, was also an effective inhibitor. The dynorphin sequence responsible for the inhibition contains the Arg6-Arg7 bond. Other peptides (bradykinin and angiotensin) which are substrates of the soluble metallopeptidase also inhibited neurotensin breakdown by the slice. This evidence suggests that this thiol dependent metalloendopeptidase is the major neurotensin catabolizing enzyme in hypothalamic slices.     

Neutral endopeptidase modulates neurotensin-induced airway contraction

To determine the role of endogenous neutral endopeptidase (NEP) (also called enkephalinase, EC 3.4.24.11) in regulating neurotensin-induced airway contraction, we used phosphoramidon, a specific NEP inhibitor, in the guinea pig. In studies in vitro, neurotensin and the COOH-terminal fragment neurotensin-(8-13) contracted strips of bronchial smooth muscle in a concentration-dependent fashion (P less than 0.001). In contrast, the NH2-terminal fragment neurotensin-(1-11) and the COOH-terminal fragment neurotensin-(12-13), the main fragments of neurotensin hydrolysis by NEP, had no effect. Phosphoramidon (10(-5) M) did not change resting tension but shifted the concentration-response curves to neurotensin to lower concentrations (P less than 0.001), whereas inhibitors of kininase II, aminopeptidases, serine proteases, and carboxypeptidase N were without effect. Removing the epithelium increased the contractile response to neurotensin (P less than 0.001), and phosphoramidon further increased the response to neurotensin in these tissues (P less than 0.001). Similar results were obtained in studies in vivo using aerosolized neurotensin and phosphoramidon. These results suggest that endogenous NEP in the airways modulates the effects of neurotensin on airway smooth muscle contraction by inactivating the peptide.