Distribution of Brush-Border Membrane Peptidases Along the Rat Intestine

The longitudinal distribution of brush-border endopeptidase-24.11, endopeptidase-2, aminopeptidase W, angiotensin-converting enzyme (ACE), dipeptidyl peptidase IV (DPP IV), carboxypeptidase P, and aminopeptidase P in the rat intestine was determined. The jejunum has the highest activities of endopeptidase-24.11 and ACE while the ileum has the highest activities of aminopeptidase W and carboxypeptidase P, and the jejunoileal junction has the highest activity of aminopeptidase P. The jejunum and ileum have similar activities of DPP IV. The profiles of differential hydrolysis of neurotensin and acetylneurotensin (8–13) along the intestine agree with distribution of endopeptidase-24.11 and ACE, suggesting that amino acid sequences of peptides and the substrate specificity of enzymes will determine site-dependent hydrolysis. There is substantial similarity in the intestinal distribution of peptidases in the human, rat, and rabbit.     

Effects of spinorphin and tynorphin on synaptic transmission in rat hippocampal slices

Spinorphin has been isolated from the bovine spinal cord as an endogenous inhibitor of enkephalin-degrading enzymes (aminopeptidase, dipeptidyl aminopeptidase III, angiotensin-converting enzyme and enkephalinase), and tynorphin has been synthesized as a more potent inhibitor of dipeptidyl aminopeptidase III. In this study, the effects of spinorphin and tynorphin on synaptic transmission were studied in rat isolated hippocampal slices. Field potentials were recorded from the CA1 region after stimulation of Schaffer collaterals. Spinorphin (1 microM), which alone had no effect, potentiated the facilitatory effects of enkephalin on the filed potentials at a stimulation interval of 15 s. At a stimulation interval of 10--4 s, spinorphin alone frequency dependently inhibited the field potential. On the other hand, tynorphin (1 microM), which alone had no effect at any stimulus interval, tended to potentiate the facilitatory effects of enkephalin. Spinorphin blocked long-term potentiation induced by tetanic stimulation (100 Hz, 1 s), whereas tynorphin had no effect on long-term potentiation. These results suggest that, at a low stimulation frequency, spinorphin potentiates the facilitatory effects of enkephalin by preventing degradation of enkephalin, whereas at a high stimulation frequency spinorphin use dependently inhibits synaptic transmission independently of enkephalin. On the other hand, tynorphin tends to potentiate the facilitatory effects of enkephalin without use-dependent inhibition.     

The role of bestatin-sensitive aminopeptidase, angiotensin converting enzyme and thiorphan-sensitive "enkephalinase" in the potency of enkephalins in the guinea-pig ileum

The role of each enkephalin-hydrolyzing peptidase in the inhibitory potency of exogenously added enkephalins in the myenteric plexus-longitudinal muscle preparation of guinea-pig ileum was studied by using the relatively specific inhibitor of each enzyme. Results showed that three distinct enzymes, bestatin-sensitive aminopeptidase(s), angiotensin converting enzyme, and thiorphan-sensitive "enkephalinase", played a critical role in the inactivation of enkephalins. Additionally, these enzymes are likely to be located close to opioid receptors, since they produce a significant concentration difference of enkephalin between the surrounding organ bath and the vicinity of opioid receptors. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D-phenylalanine-sensitive carboxypeptidase are indicated not to be involved significantly in the degradation of exogenously added enkephalins in the guinea-pig ileum.     

The enhancing effects of amastatin, phosphoramidon and captopril on the potency of [Met5]-enkephalin in rat vas deferens

The enkephalin-inactivating enzymes in rat vas deferens were studied by using the relatively specific inhibitor of each enzyme. The results showed that the rat vas deferens, like the other three preparations, guinea-pig ileum, mouse vas deferens and striatal membranes of guinea-pig brain, which had been investigated previously, contained three distinct enkephalin-hydrolyzing peptidases. Additionally, the enkephalin-hydrolyzing aminopeptidase, endopeptidase-24.11 and peptidyl dipeptidase A in rat vas deferens were found to be inhibited maximally with 1 microM of amastatin, 1 microM of phosphoramidon and 1 microM of captopril, respectively. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D-phenylalanine-sensitive carboxypeptidase were suggested not to be involved significantly in the inactivation of exogenously given enkephalin in rat vas deferens. The characteristics of the enkephalin-degradative enzymes in rat vas deferens were discussed in terms of their similarities to and differences from those in the other preparations.     

Effects of three peptidase inhibitors, amastatin, captopril and phosphoramidon, on the hydrolysis of [Met5]-enkephalin-Arg6-Phe7 and other opioid peptides

The contents of [Met⁵]-enkephalin-Arg⁶-Phe⁷ (met-enk-RF) and its six hydrolysis products: Y, YG, YGG, YGGF, YGGFM, and YGGFMR were estimated after incubating met-enk-RF with either a guinea-pig ileal or striatal membrane fraction for various times at 37° C. After 45 min incubation with either ileal or striatal membranes, met-enk-RF was completely hydrolyzed, yielding Y as the major product. Incubation with either membrane preparation for 60 min in the presence of the aminopeptidase inhibitor amastatin hydrolyzed 90 or 92% of met-enk-RF, respectively, with YGG being the major product. If the dipeptidyl carboxypeptidase I inhibitor captopril is also included in the incubation, met-enk-RF hydrolysis decreases by about half for both membranes, with YGG remaining the major product. Inclusion of three peptidase inhibitors, amastatin, captopril, and phosphoramidon (inhibition of endopeptidase-24.11) further reduced met-enk-hydrolysis, with 87% or more remaining intact. This shows that met-enk-RF was mainly hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, estimations of [Leu⁵]-enkephalin (leu-enk), α- and β-neoendorphins (α- and β-neoends), and dynorphin B (dyn B) contents after incubating the individual peptides with striatal membrane for 60 min in the presence of the three peptidase inhibitors showed that 98, 32, 5, and 23%, respectively, remained intact. Our previous studies together with the data obtained here show that one group of endogenous opioid peptides: met-enk, leu-enk, met-enk-RF, met-enk-RGL, and dyn A-(1-8) are largely or almost exclusively hydrolyzed by the three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I, and phosphoramidon-sensitive endopeptidase-24.11, and indicate that an unidentified fourth enzyme(s) is involved in the hydrolysis of another group of peptides: α-neoend, β-neoend, and dyn B. Received: 15 July 1997 / Accepted: 20 November 1997     

Nociception,enkephalin content and dipeptidyl carboxypeptidase activity in brain of mice treated with exopeptidase inhibitors

Thiorphan (60 μg intracerebrally) increased the met⁵-enkephalin content of mouse striatum by 30% in 30 min. This increase was no longer evident at 1 hr. If the dipeptidyl carboxypeptidase, inhibited by thiorphan, were located extraneuronally as suggested by De La Baume, Patey and Schwartz (1981), the met⁵-enkephalin accumulation represents the rate at which the pentapeptide is released extraneuronally. The increase in met⁵-enkephalin content was accompanied by an inhibition, greater than 80%, of the dipeptidyl carboxypeptidase that degrades striatal met⁵-enkephalin. Such an inhibition lasted longer than 2 hr. Thiorphan, given to mice intracerebrally, prolonged the latency time to jump off a 54° plate. The effects of thiorphan on brain met⁵-enkephalin content and hot plate latencies were significantly potentiated by bestatin, which inhibits aminopeptidase B and leucine aminopeptidase.     

The inactivation of [Met5]-enkephalin by bestatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A and thiorphan-sensitive endopeptidase-24.11 in mouse vas deferens

The enkephalin-hydrolyzing peptidases in mouse vas deferens were studied and compared with those in guinea pig ileum which had been characterized in the previous study. The present results showed that three distinct peptidases, bestatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A, and thiorphan-sensitive endopeptidase-24.11, played a critical role in the inactivation of enkephalin in mouse vas deferens, being consistent with the previous results obtained with guinea pig ileum. However, the data in both previous and present studies showed that the activity of the bestatin-sensitive aminopeptidase relative to that of either the captopril-sensitive peptidyl dipeptidase A or the thiorphan-sensitive endopeptidase-24.11 in guinea pig ileum was higher than that in mouse vas deferens, while the activity of either peptidyl dipeptidase A or endopeptidase-24.11 relative to that of aminopeptidase in mouse vas deferens was higher than that in guinea-pig ileum. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D-phenylalanine-sensitive carboxypeptidase were suggested not to be involved significantly in the inactivation of enkephalin in mouse vas deferens as well as guinea pig ileum.     

Comparison of distribution of brush-border exo- and endopeptidases in rat and rabbit intestine

Abstract— The distribution of brush-border endopeptidase-2, aminopeptidase W, carboxypeptidase P, and aminopeptidase P along the rat and rabbit intestine was examined. In both species, aminopeptidases P and W increased distally and reached the highest in the ileum; their activities in the ileo-caecal junction were the lowest. Endopeptidase-2 had a uniform intestinal distribution in both species with the highest activity in the ileum and little activity in the ileo-caecal junction or caecum. With a distribution similar to that of endopeptidase-2, carboxypeptidase P also had high activity in the ileum in rats and rabbits.     

Investigations of the in-vitro metabolism of three opioid tetrapeptides by pancreatic and intestinal enzymes

The metabolism of three opioid tetrapeptides, Tyr-D-Arg-Phe-Nva-NH2, Tyr-D-Arg-Phe-Phe-NH2 and Tyr-D-Ala-Phe-Phe-NH2, was investigated in the presence of pure pancreatic enzymes (trypsin, chymotrypsin, elastase, carboxypeptidase A and carboxypeptidase B), as well as in the presence of pure carboxylesterase and aminopeptidase N. The cleavage patterns of the pure pancreatic enzymes were then compared with those found in rat and human jejunal fluid. Metabolism was also studied in homogenates from different intestinal regions (duodenum, jejunum, ileum and colon) and in enterocyte cytosol from rats. The effect of various protease inhibitors was investigated in the jejunal homogenate. The parent peptides were assayed by high-performance liquid chromatography and metabolites were identified by means of liquid chromatography-mass spectrometry. Of the pure enzymes, the quickest hydrolysis of the peptides was observed for the pancreatic enzymes chymotrypsin, trypsin and carboxypeptidase A. In most cases they formed the corresponding deamidated tetrapeptides (chymotrypsin and trypsin) or tripeptides with a missing C-terminal amino acid (carboxypeptidase A). Regional differences in intestinal metabolism rates were found for all three peptides (P < 0.001), with the highest rates observed in jejunal and/or colonic homogenates. The deamidated tetrapeptides were formed both in rat intestinal homogenates and in enterocyte cytosol. Metabolism in the jejunal homogenate was markedly inhibited by some serine and combined serine and cysteine protease inhibitors. In conclusion, the C-terminal amide of these tetrapeptides did not fully stabilise them against intestinal deamidase and carboxypeptidase activities. The significant hydrolysis of the peptides by pure chymotrypsin, trypsin and carboxypeptidase A showed that lumenal pancreatic proteases might be a clear metabolic obstacle in oral delivery even for small peptides such as these tetrapeptides.     

In vivo effect of tunicamycin on the expression of rat small intestinal brush border membrane glycoproteins and glycoenzymes

Tunicamycin, a known inhibitor of the lipid-dependent glycosylation of proteins, was used in vivo to study the biosynthesis of rat intestinal brush border membrane aminopeptidase N and dipeptidyl aminopeptidase IV. The incorporation of [3H]glucosamine into newly synthesized total protein of mucosal cell homogenates was inhibited by 60%, whereas incorporation of [3H]leucine was decreased only 21% by tunicamycin. This effect was much more pronounced in the brush border membrane fraction isolated from intestinal mucosal cells where incorporation of radiolabled leucine and glucosamine was reduced to 50 and 82% of control values respectively. An examination of the brush border membrane protein profile by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that there was a marked selective decrease in the amount of glycoproteins of molecular weights greater than 130 kD. In addition, there were decreased levels of assayable aminopeptidase N, dipeptidyl aminopeptidase IV and disaccharidase activity in intestinal mucosal cell homogenates and brush border membranes of tunicamycin-treated rats. Though tunicamycin decreased incorporation of newly synthesized aminopeptidase N and dipeptidyl aminopeptidase IV protein into brush border membranes by 70-75%, the newly synthesized enzyme that was incorporated was indistinguishable from that of controls. Further, non-glycoslyated forms of both enzymes were not detected in any other subcellular fractions. These results show that tunicamycin, an inhibitor of glycosylation, significantly affected the expression of brush border membrane glycoproteins, suggesting that both polypeptide synthesis and degradation of these proteins may be altered in the presence of this drug.     

In vitro assessment of intestinal IGF-I stability.

Insulin-like growth factor (IGF-I) is a 7648-Da polypeptide consisting of 70 amino acids. Clinically, IGF-I might be used in type II diabetes, which requires a life-long treatment. Therefore, delivery routes other than parenteral injections are highly desirable. For convenience, the peroral route is the most attractive. Therefore, in an attempt to answer the feasibility of oral delivery of IGF-I we examined the metabolism of this polypeptide in the gut in the presence of crude porcine pancreatic enzymes (CPPE) and flushings of the small and large intestine from pig, rat, and dog. Moreover, incubation studies with purified pancreatic enzymes that are present in the intestine were performed to determine the most active enzymes responsible for the intestinal cleavage of IGF-I. IGF-I was mainly degraded by chymotrypsin (t(1/2) = 2.7 min) and trypsin (t(1/2) = 34.6 min), whereas in the presence of aminopeptidase M and carboxypeptidase A IGF-I was stable up to 90 min. IGF-I was degraded in flushings from the jejunum, ileum, and colon. However, there were no significant differences in the stability of IGF-I between the examined intestinal segments. The addition of serine protease inhibitors such as a combination of aprotinin, soybean trypsin inhibitor, and Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), as well as casein profoundly improved the stability. Because we were able to improve the stability of IGF-I in vitro in all species at the same degree we speculate that a similar extension of half-life might also be possible in the human intestinal system.     

OF4949, new inhibitors of aminopeptidase B. IV. Effects of OF4949 and its derivatives on enzyme systems

OF4949-I and II inhibited aminopeptidase B from Ehrlich ascites carcinoma in a competitive way and the Ki value for both against L-arginine-beta-naphthylamide was 8 X 10(-9) M. Inhibition by I and II of various exopeptidases and endopeptidases was examined. OF4949-I and II both strongly inhibited leucine aminopeptidase and enkephalin-degrading aminopeptidase; I also inhibited enkephalinase B. The inhibitory effects of various derivatives of I and II on aminopeptidase B activity, showed that the terminal amino and carboxamide groups are essential for activity.     

Stabilization of methionine enkephalin in various rabbit mucosal extracts by enzyme inhibitors

The inhibition of the enzymatic degradation of methionine enkephalin (Met-Enk) was investigated kinetically in nasal, rectal, and vaginal extracts of rabbits with and without inhibitors, such as puromycin (PM), amastatin (AM), thiorphan (TP), Na₂EDTA, and thimerosal (TM), alone or in combination, by analyzing the parent peptide and its hydrolytic fragments by HPLC. The effects of variation of pH in the nasal extracts, and the addition of 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CyD) on the stabilization of Met-Enk were also studied. The degradation of Met-Enk was found to be fastest at around pH 7, indicating that the activity of enkephalin-degrading enzymes is optimal at this pH. Addition of 2-HP-β-CyD (10%) to the nasal, rectal, and vaginal extracts was noted to reduce the first-order degradation rate constants for Met-Enk by 2.5-2.8-fold, compared to the control. AM alone inhibited the enzymatic degradation of Met-Enk with IC₅₀ values of 3.5 and 0.22 μM for the rectal and vaginal extracts, respectively, whereas PM was found to be approx. 14.2- and 26.8-fold less potent than AM, respectively. The effects of both aminopeptidase inhibitors in the nasal extracts were smaller. Even at 50 μM, TP (a potent enkephalinase A inhibitor) alone revealed only a small increase of Met-Enk stability in the various mucosal extracts, however, EDTA (5 μM) was observed to inhibit enzymatic hydrolysis considerably by blocking both enkephalinase A and B and, to some extent, aminopeptidase. On the other hand, TM (0.05%) was found to be a new and potent inhibitor for enkephalinase B and aminopeptidases, which was more potent than AM (50 μM) in inhibiting the degradation of Met-Enk in various mucosal extracts. Furthermore, the addition of TM (0.01%) to a combination of AM (50 μM) and EDTA (5 μM) was observed to protect Met-Enk from enzymatic degradation in nasal, rectal, and vaginal extracts by more than 90%, after 24 h of incubation, by inhibiting almost completely all the enkephalin-degrading enzymes present in the incubation mixtures.     

Proline specific peptidases and their specific inhibitors]

Several proline specific peptidases were newly isolated from mammalian organs, plants and microorganisms, and their enzymatic properties characterized. The genes of prolyl endopeptidase, aminopeptidase P and proline iminopeptidase from some microorganisms were cloned and their nucleotide sequences determined. By high expression of these genes in Escherichia coli, the enzymes became possible to be used for the industrial application as well as its basic research. Novel inhibitors specific for the prolyl endopeptidase and dipeptidyl aminopeptidase IV were synthesized and some inhibitors for prolyl endopeptidase found to show an anti-amnesic effect.     

Composite effects of actinonin when inhibiting enkephalin-degrading enzymes

Actinonin which has been found to be an inhibitor of aminopeptidase M (EC 3.4.11.2) also inhibited enkephalinase A (EC 3.4.24.11) and enkephalin aminopeptidase which were partially purified from the corpus striatum membrane of guinea-pig brain. The IC50 values were 5.6 microM for enkephalinase A and 0.39 microM for enkephalin aminopeptidase. Actinonin also inhibited with an IC50 value of 1.1 microM dipeptidyl aminopeptidase tested on whole brain homogenate of rats in the presence of thiorphan and bestatin. Analgesia was assessed by measuring the tail-flick latency of mice. The analgesic effect of [Met5]enkephalin injected intracisternally (i.cist., 50 micrograms) was potentiated by an intraperitoneal (i.p., 100 and 300 mg/kg) as well as an i.cist. (25 micrograms) injection of actinonin. Actinonin was found to inhibit all three enzymes of enkephalin metabolism and, when given peripherally, to potentiate enkephalin analgesia.     

Immunoelectrophoretic analysis of vascular, membrane-bound angiotensin I converting enzyme, aminopeptidase M, and dipeptidyl(amino)peptidase IV

Antisera raised against specific renal brush border peptidases have been used to characterize vascular surface membrane angiotensin I converting enzyme (ACE; EC 3.4.15.1), aminopeptidase M (AmM; EC 3.4.11.2), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) by techniques of differential solubilization, fused-rocket immunoelectrophoresis and crossed immunoelectrophoresis. The vascular membrane-bound enzymes are immunologically indistinguishable from their brush border counterparts and can be solubilized by treatment with detergent and/or papain. The electrophoretic mobilities of the papain-treated forms of each enzyme were greater than those of the detergent-treated forms. This increased mobility is associated with the removal of small, hydrophobic, non-antigenic components of the enzymes. Regardless of the method of solubilization, the electrophoretic mobilities of the vascular enzymes were greater than those of the brush border enzymes. However, after treatment with neuraminidase to remove sialic acid, their respective mobilities were similar. The mobilities of serum AmM and DAP IV were identical to the respective papain-solubilized vascular enzymes both before and after neuraminidase. Thus, like the brush border enzymes, the data presented are consistent with the model that vascular ACE, AmM and DAP IV are intrinsic membrane peptidases bound to their surface membranes by small, non-antigenic, hydrophobic anchors associated with the lipid bilayer. In addition, these vascular surface membrane peptidases are similar to and may be a source of the circulating enzymes.     

Vascular, plasma membrane aminopeptidase M. Metabolism of vasoactive peptides

Aminopeptidase M (EC 3.4.11.2), an enzyme present on the cell surface of vascular endothelium and/or smooth muscle, rapidly hydrolyzes leucyl- and arginyl-2-naphthylamides and a number of vasoactive peptides at physiologic pH. Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was found that vascular aminopeptidase M converted kallidin to bradykinin and inactivated des(Asp1)angiotensin I, angiotensin III, hepta(5-11)substance P and hexa(6-11)substance P. Aminopeptidase M did not, however, hydrolyze bradykinin, angiotensin I, angiotensin II, saralasin, vasopressin, oxytocin or any form of substance P containing a component of the Arg-Pro-Lys-Pro sequence. Both the naphthylamidase and peptidase activities were inhibited similarly by known amino-peptidase M inhibitors including o-phenanthroline, amastatin, bestatin and puromycin. However, inhibitors of angiotensin I converting enzyme (captopril), carboxypeptidase N (MERGETPA), neutral endopeptidase (phosphoramidon), post proline cleaving enzyme and dipeptidyl(amino)peptidase IV (diisopropylphosphofluoridate, DFP) were without effect. These results demonstrate that vascular, cell surface aminopeptidase M can selectively metabolize vasoactive peptides and may play a role in modulating their levels in the circulation and/or within the vessel wall.     

Renal and macrophage aminopeptidase activities in cyclosporin-treated mice

Cyclosporin, an immunosuppressive drug, is known to affect macrophage and to exert a nephrotoxic effect. Aminopeptidases play important roles for renal and macrophage functions. In this work, we attempt to test the hypothesis that the aminopeptidases participate within macrophage and renal effects induced by cyclosporin. Macrophage and renal aminopeptidase activities of cyclosporin-treated and control mice were evaluated, as well as renal caspase 3 activity, hematocrit, urinary protein and plasma osmolality, creatinine and uric acid concentrations. Cyclosporin treatment increased caspase 3 activity, hematocrit and osmolality, while urinary protein, creatinine and uric acid were unaltered. Soluble and particulate aminopeptidases in resident and elicited macrophages were unaffected by cyclosporin. The treatment with cyclosporin increased neutral, basic, cystyl, prolyl imino and pyroglutamyl soluble aminopeptidase activities in the renal cortex. Acid and basic soluble aminopeptidase activities increased in the renal medulla. Increased levels of particulate form in the cortex were detected for acid and pyroglutamyl aminopeptidase activities. Cyclosporin increased cortical soluble while decreased medullar particulate prolyl dipeptidyl aminopeptidase IV activity. With the exception of prolyl dipeptidyl aminopeptidase IV, particulate aminopeptidase activities returned to levels similar to controls after fifteen days of cyclosporin withdrawal, and soluble aminopeptidase activities did not regress. Our data indicate that the adopted regimen of cyclosporin treatment produced mild renal impairment with consistent changes on the levels of renal but not macrophage aminopeptidase activities. The obtained profiles of macrophage and renal aminopeptidase activities should be considered into the elaboration of new potential strategies for preventing nephrotoxicity during the treatment with cyclosporin.     

Representative aminopeptidases and prolyl endopeptidase from murine macrophages: comparative activity levels in resident and elicited cells

Macrophages are considered the main effector cells of immune system. Under stimulation these cells are known to be activated by a process involving morphological, biochemical and functional changes. Since altered peptidase activities could be among the factors leading to the differentiation and activation of these cells, in the present work seven naphthylamide derivative substrates were employed to assess representative aminopeptidase and prolyl endopeptidase activities in resident and elicited macrophages of mice. Soluble basic aminopeptidase and prolyl endopeptidase and soluble and particulate neutral and prolyl dipeptidyl aminopeptidase IV activities were present at measurable levels while particulate prolyl endopeptidase and basic aminopeptidase, and particulate and soluble cystyl and pyroglutamyl aminopeptidases were not detectable. Kinetic parameters, chloride activation and the inhibitory effects of puromycin, bestatin, amastatin and diprotin A characterized differential properties of these peptidase activities. The observed increment (about 6-17-fold) of the soluble basic aminopeptidase and prolyl endopeptidase and soluble and particulate neutral and prolyl dipeptidyl aminopeptidase IV activities in elicited macrophages was particularly relevant, as these might contribute to an increased ability of this cell to inactivate several susceptible substrates known to be inflammatory and/or immunological mediators.     

In vitro and in vivo effects of kelatorphan on enkephalin metabolism in rodent brain

Biologically relevant assays were used to compare the potency of kelatorphan (N-[3(R)-[(hydroxyamino)carbonyl]-2-benzyl-1-oxopropyl]-L-alanine) as inhibitor of the peptidase-induced metabolism of enkephalins to that of bestatin, a non-specific inhibitor of aminopeptidase and thiorphan, a highly potent blocker of the neutral endopeptidase (EC 3.4.24.11) designated as enkephalinase. Kelatorphan almost completely inhibited the formation of the three metabolites [3H]Tyr, [3H]Tyr-Gly and [3H]Tyr-Gly-Gly produced by incubation of [3H][Tyr1,Met5]enkephalin with rat striatal slices. Co-administered with [Met5]enkephalin in mouse brain, kelatorphan was able to prevent by 80% the degradation of the exogenous peptide. Moreover, a mixture of thiorphan (1 microM) and bestatin (20 microM) or kelatorphan alone (20 microM) induced a 2.2 to 2.5-fold increase in endogenous [Met5]enkephalin overflow after evoked depolarization of superfused rat striatal slices. In this assay, kelatorphan was the only compound to increase by 63% the basal level of released [Met5]enkephalin. Kelatorphan was about 100 times less potent than bestatin to inhibit the total rat striatal aminopeptidases, but as efficient (IC50 = 4 X 10(-7) M) as bestatin to inhibit a minor aminopeptidase activity resembling aminopeptidase M. Therefore the reported enhanced analgesic potency of kelatorphan with regard to the association of bestatin and thiorphan is very likely related to its ability to almost completely inhibit enkephalin-degrading enzymes (including the Tyr-Gly releasing peptidase) and to its better selectivity for the biologically relevant aminopeptidase M. Kelatorphan would be a valuable probe, preferable to the association of bestatin and thiorphan, to investigate the physiological functions regulated by a phasic enkephalinergic activity.