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Hydrolysis and protection from hydrolysis of circulating enkephalins
Camp. Biochem. Physiol. Printed in Great Britain
Vol. 8X,
No. 2, pp. 449-454,
0306-4492/86
1986
$3.00 + 0.00
PergamonJournals Ltd
MINI REVIEW HYDROLYSIS AND PROTECTION FROM HYDROLYSIS OF CIRCULATING ENKEPHALINS L. GIORGIO RODA*, FRANCEXA VENT~JRELLI and GIANNA RCBCETTI Dipartimento di Medicina Sperimentale e Scienze Bio~himiche, Universita’ degli Studi “Tor Vergata”, Rome, Italy (Received 24 February 1986)
1.
belonging to three classes: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases (e.g. Schwartz et al., 1981). (ii) The activities of these enzymes seem to be similar, but not identical, to the activities of the enkephalin-cleaving enzymes present in the nervous tissue (Roscetti et al., 1985). (iii) At least in man, but not in all the species investigated, the activity of the amino~ptidases prevails over that of the other enzymes. Thus, the first hydrolysis by-products are free tyrosine and the tetrapeptide Gly-Gly-Phe-(Leu or Met). Since all the tyrosine-lacking peptides are biologically inactive (e.g. Traficante et al., 1980), this step is the most physiologically relevant one. (iv) The half-lives reported in plasma range from a few seconds (DuPont et al., 1977) to several seconds in rat (Hambrook et al., 1976) and rabbit (HogueAngeletti and Roda, 1980), up to several minutes in human and murine plasma (Roscetti er al., 1985).
INTRODUCTION
Opioid peptides are secreted by and stored within the adrenal medulla chromaffin granule (~hultz~rg et al., 1978; Viveros et af., 1979). Thus, under physiological conditions they are released in the bloodstream, together with the whole soluble content of the granule (Stern et al., 1979; Clement-Jones et al., 1980; Yang et al., 1980). Once released, these peptides are hydrolyzed by several of the peptidases present in plasma. The enzyme degradation of the shorter opioids-at least in some laboratory animals-is fairly rapid (Hambrook et al., 1976; DuPont et al., 1977; Hogue-Angeletti and Roda, 1980). It has therefore been suggested that the considerable difficulties encountered to show the pharmacological activity of the opioid peptides when administered in vitro (eg. Terenius, 1978; Craves et al., 1978) may be caused by their rapid hydrolysis (e.g. Schwartz et al., 1981). In this respect, the activity of blood as a carrier seems to compete with its activity in degrading the carried substances. Yet, in the last few years there has been considerable evidence demonstrating the existence of endogenous substances capable of partially protecting the plasma-released enkephalins from enzyme hydrolysis (Possenti et al., 1983). The existence of these endogenous inhibitors, while making hydrolysis of the plasma-released opioids seem less puzzling, also makes it appear more complex. Nevertheless, it seems possible to start arranging the data we now have on this topic into a coherent pattern. Indeed, the purpose of the following pages is to review the existing data on enzyme hydrolysis of circulating enkephalins, and to try to ascertain the degree of logical consistency of the resulting pattern.
2.1. Hydrolysis pattern in human plasma The kinetics of formation and of degradation of the hydrolysis by-products in human plasma have been studied as follows: labelled leu-enkephalin was incubated with whole plasma and the incubation mixture was fractioned by steric exclusion chromatography to remove the high molecular weight material. The low molecular weight fractions containing enkephalin and hydrolysis by-products were then separated and quantified by reverse phase and ion exchange chromatography (Roscetti et al., 1985). Of the 13 fragments theoretically obtainable from a pentapeptide, eight are actually formed by the enzymes present in human plasma: (i) all the monomers; (ii) two dimers out of four Gly-Gly and PheLeu; (iii) one trimer out of three: Gly-Phe-Leu; (iv) one of the two possible tetramers, Gly-Gly-Phe Leu. Thus all the N-terminal peptides are absent. This indicates that the activity of the aminopeptidases is higher than the activity of the other enzyme groups. The aminopeptidases seem to be able to cleave the N-terminal glycine from the tetrapeptide Gly-GlyPhe-Leu, forming Gly-Phe-Leu, but to be unable to cleave this tripeptide further. Also absent are Gly-Phe
2. ENKEPHALINHYDROLYSISIN PLASMA The data available on enkephalin hydrolysis by plasma enzymes-less detailed than that existing on hydrolysis of opioid peptides by the nervous systemrelated enzymes-can be summarized as follows: (i) Enkephahns are hydrolyzed by several enzymes, _-..*Author to whom correspondence should be addressed. 449
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Fig. 1. Enkephalin-degrading enzyme activities in plasma as separated by steric exclusion chromatography. Top left: Homo; top right: Oryctolagus; middle left: Cauia; middle right: Rattus; bottom left: Mus; bottom right: Gallus. Dotted lines represent absorbance at 280nm; thick solid lines represent leu-enkephalin; lined areas: aminopeptidase; dotted areas: dipeptidylaminopeptidase; dark areas: dipeptidylcarboxypeptidase. Enkephalin and enzyme activities are expressed as moles per cent.
and Gly-Gly-Phe, is not cleaved.
indicating
that the Phe-Leu bond
2.2. Plasma enzymes The distribution of the enkephalin-degrading enzymes has been determined in the following species: Mus muscuius, Rattus rattus, Oryctolagus cuniculus, Cavia porcellus, Homo sapiens, and Gahs domesticus (Venturelli et al., 1985). Whole plasma was fractioned by steric exclusion chromatography, labelled leuenkephalin was incubated in the presence of the fractions thus separated, and the hydrolysis byproducts-identified by thin layer chromatographywere quantified by counting the cut thin layer sheets (Roscetti et al., 1985). Since Tyr, Tyr-Gly and TyrGly-Gly are very well separated, with this technique it is possible to identify and quantify the three enkephalin-cleaving enzyme groups present in
plasma. The distribution of these enzymes in the previously listed animals is shown in Fig. 1. In addition, when human plasma was further fractioned by ion exchange chromatography, six enzyme species were apparently separated by molecular weight and charge: three aminopeptidases, one dipeptidylcarboxypeptidase and two dipeptidylaminopeptidases (unpublished results). 3. ENKEPHALIN HYDROLYSIS IN DIFFERENT ANIMAL SPECIES
The kinetics of leu-enkephalin hydrolysis caused by plasma enzymes have been measured in several laboratory animals (genera Gallus, Mus, Rattus, Oryctolagus and Cavia) and in man (Venturelli et al.,
1985). Labelled leu-enkephalin was incubated in the presence of whole plasma obtained from the species
Enkephalin
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Fig. 2. Hydrolysis kinetics of leu-enkephalin in the presence of whole plasma. Dash-two dots line: Mus: dotted line:
Homo; dashdotted line: Oryctolagus: solid line: Gallus; dashed line: Raftus; dash-four dots line: Cauia.
indicated, and the hydrolysis by-products were separated from the intact pentapetide either by thin layer or reverse phase chromatography. The kinetics of hydrolysis determined as indicated are shown in Fig. 2. The relative half-lives are as follows: Mus, 13 min; Homo, 8 min; Oryctolagus, 1 min 54 set; Gallus, 1 min 28 set; Rat&s, 46 set; Cavia, 24 sec. These half-lives were measured close to the estimated plasma concentrations. It should be noted that the very low plasma levels of enkephalin (close to 1 x 10-l’ M, Clement-Jones et al., 1980; Ryder and Eng, 1982) make it rather difficult to obtain reliable quantitative measurements near these levels, even using very high-activity radiolabelled peptide. In addition, enkephalins have been reliably quantified only in man, while for the remaining species only preliminary data exist. Thus the figures shown must be interpreted with some caution. Nevertheless, these results seem to explain-at least partially-the very large differences (some two orders of magnitude) in the enkephalin half-lives reported by various authors (DuPont et al., 1977; Roscetti et al., 1985). While one conflict has thus been partially resolved, the large interspecific differences shown raise a further question, that is whether they indicate actual functional differences of these peptides in various species. Indeed, the 8 min half-life determined in man is consistent with a role of medium-term modulator. The same cannot be true for half-lives some 30 times shorter, like the ones measured in the genera Rattus and Oryctolagus, for which one is tempted to hypothesize a role in different, shorter-living phenomena. 4.
451
that these mechanisms must be of some relevance in the enkephalins’ metabolism. These data are described in some details in the following paragraphs.
*
g
hydrolysis
MECHANISMS
OF PROTECTION
As noted elsewhere (Roda et al., 1983), it is somehow difficult to fit the data available on enkephalin hydrolysis kinetics with the measured plasma levels (Clement-Jones et al., 1980; Ryder and Eng, 1982) and adrenal release (Clement-Jones et al., 1980). These discrepancies led us to specifically investigate the possible existence of physiological mechanisms able to protect the circulating enkephalins from enzyme proteolysis. The results obtained so far demonstrate the actual existence of such mechanisms. Moreover, enough data are by now available to show
mechanisms
within the cromafin
The adrenal medulla chromaffin granule storeswithin its seemingly uniform matrix-atecholamines (Blaschko and Welch, 1953; Hillarp et al., 1953), ions and proteins (Winkler, 1976; Njus and Radda, 1978; Hogue-Angeletti et al., 1980), together with enkephalins and other opioid and non-opioid peptides (Schultzberg et al., 1978; Viveros et al., 1979; Di Giulio et al., 1979; Lewis et al., 1979). Among the proteins, proteolytic enzymes are also present. These enzymes should be able to hydrolyze the polypeptides present within the granule, notably the precursors (Jones et al., 1979; Lewis et al., 1980) of the opioid peptides (Yang et al., 1980; Troy and Musacchio, 1982; Wallace et al., 1982; Fricker et al., 1982; Lindberg et al., 1982), as well as the opioid peptides themselves. Yet it is also possible that the smaller peptides-inherently scarcely resistant to the enzyme hydrolysis-are protected from the activity of these enzymes by some granule-contained mechanism. Indeed, it has been shown that enkephalins actually bind some chromaffin granule components, and that the bound material is partially protected from enzyme hydrolysis (Hogue-Angeletti and Roda, 1980). These results were obtained by incubating labelled leu-enkephalin in the presence of a chromaffin granule soluble lysate. The incubation mixture was subsequently fractioned by steric exclusion chromatography. With this approach, two chromaffin granule fractions of different molecular weight have been shown to bind to enkephalins. The higher molecular weight enkephalin-binding material corresponds to proteins: chromolipin, a granule-contained lipoprotein (Hogue-Angeletti and Sheetz, 1978) and chromogranin A (Helle, 1966). The lower molecular weight material was instead identified with a peptide family previously discovered in the chromaffin granule (Roda and Hogue-Angeletti, 1979). The enkephalins bound to the chromaffin granule components are partially protected from enzyme hydrolysis. This was demonstrated as follows: labelled leu-enkephalin was preincubated with a chromaffin granule crude lysate and then incubated with plasma. Under these conditions, the amount of intact enkephalin was found to be higher than in the controls; that is, the enkephalins not preincubated with the granule lysate. The inhibition of the hydrolysis measured under these conditions is not very high (the amount of intact enkephalin is increased by approximately 30%, Hogue-Angeletti and Roda, 1980). Yet the peculiar physico-chemical milieu existing in the chromaffin granule makes it difficult to reliably extrapolate to physiological conditions data obtained under experimental conditions so far removed from them. Two roles may tentatively be attributed to these systems. The first is to protext enkephalins-and presumably other peptides-from the activity of the granule-contained enzymes while they are still in the chromaffin granule, and/or to control the activity of these enzymes in the cleavage of the precursor mole-
L. GIORGIO RODA et
452
cules (e.g. Troy and Musacchio, 1982; Wallace et al., 1982). A second role may be to contribute to the stability of opioid peptides once they are released into the blood-stream, as will be detailed in the next paragraph. 4.2. Protection mechanisms in blood The existence of protecting substances in the chromaffin granule induced us to specifically investigate the possible existence of analogous mechanisms in plasma. Actually, these studies revealed the presence in plasma of two different systems protecting peripherally released enkephalins from enzyme hydrolysis (Possenti et al., 1983; Roscetti et al., 1985). These results were obtained as described below. Whole human plasma was incubated with labelled leu-enkephalin and the incubation mixture was fractioned by steric exclusion chromatography. Using this procedure, two groups of enkephalin-binding substances can be found: a protein (the serum albumin) and two groups of lower (400&5000 and 2OOG3000 Daltons) molecular weight substances, later identified as peptides. To ascertain whether the binding material could protect enkephalins from enzyme hydrolysis, labelled leu-enkephalin was incubated with whole plasma. To remove the interfering high-molecular weight substances, the incubation mixture was fractioned by steric exclusion chromatography. Intact enkephalin and its hydrolysis by-products were subsequently analyzed by reverse phase chromatography. Under the test conditions, over 90% of the albumin-bound peptide was eluted at the position of the intact enkephalin, while the unbound enkephalin was over 75% degraded. The same analyses were performed on the enkephalin bound to the peptidic fraction. In this case, an even higher amount of enkephalin (95%) was undegraded after the incubation. The aforementioned results have also been confirmed by direct measurements of the half-lives of enkephalins, performed in the presence and in the absence of the plasma enkephalin-protecting systems
al.
(serum albumin and peptides). Specifically, in the presence of the low molecular plasma material, and at enkephalin concentrations close to the physiological resting level, the enkephalin half-life is more than three times as long as the half-life measured in de-peptized plasma (that is, plasma from which the low molecular weight components are chromatographically removed and which is then re-concentrated to its original volume). The half-lives measured under these conditions (in the human species) are 8min in whole plasma and only 2.5min in depeptized plasma (Fig. 3). Moreover, after 60 min of incubation, the intact enkephalin is approximately 25% in whole plasma, but only 3% in de-peptized plasma (Roscetti et al., 1985). The same experiments have been repeated with mammals other than man, and the results show the existence of substances able to partially protect the enkephalin from enzyme hydrolysis in the low molecular weight region of the plasma of all the species examined (Venturelli et al., 1986). Moreover, the protection given by the low molecular weight plasma components-in terms of percent enhancement of the active lives and hence of the actual levels of leuenkephalin-is practically the same in all the species examined, even though the half-lives of the enkephalin differ by a factor of more than 30 (Venturelli et al., 1985). Finally, it should be noted that the physiological importance of the two systems described (serum albumin and peptides) seems to be totally different. Indeed, the low molecular weight system acts by inhibiting the enzymes’ activity (unpublished results). On the other hand, the enkephalin protected from the enzyme hydrolysis by the serum albumin is the enkephalin actually bound to the protein and, presumably, protected by sterical effects. Since this amount is low (approximately 5% of the total), the importance of this system seems to be mainly speculative. On the contrary-as shown above-the role of the low molecular weight system seems to be of considerable physiological relevance.
incubation time (minutes)
Fig. 3. Degradation kinetics of leu-enkephalin in the presence of whole (dashed line) and de-peptised (solid line) human plasma. Solid circles represent leu-enkephalin; stars represent tyrosine.
Enkephalin hydrolysis 5. CONCLUSIONS
To summarize the data presented in the previous pages, the opioid peptides released in vertebrates’ plasma are degraded by several proteolytic enzymes. At the same time, they are protected from the activity of these enzymes by different groups of substances. The protecting substances are present both in plasma and in the chromaffin granule, whence they are co-released in plasma with the opioid peptides. The amount of protection given by the two groups of substances is considerably different. Expressed as the percent variation of the leu-enkephalin half-life, the protection given by the chromaffin granule components is close to 30%, while the protection given by the plasma substances is approximately 300%. These figures indicate that the activity of the plasma protecting systems can have a primary role in the control of the actual levels of circulating enkephalins. Moreover, the presence of the protecting substances implies that, in man, the enkephalin half-life is long enough to account for physiological activities of medium duration. This allows one to hypothesize a role as modulators for these molecules. This is not true in the case of other species. Indeed, differences in half-lives in the range determined so far (of approximately 30) seem to suggest that the opioid peptides may play different physiological roles in different species. The data reviewed seem also consistent with the hypothesis that the release, the hydrolysis and the protection from the hydrolysis of the opioid peptides form a set of closely related phenomena that must be considered as a whole. In this respect, the role of the degrading enzymes could be to actually end the action of the plasma-released opioids. This may be true if these peptides are active on structures devoid of the enzyme systems necessary for this function. This hypothesis contrasts with the alternative one, viz. that the role of the degrading enzymes is not functionally related to the metabolism of the opioid peptides. Indeed, the specificity of the proteolytic enzymes on low molecular weight peptides is in principle low. So, it is certainly possible that the smaller opioids are degraded non-specifically by the proteolytic enzymes present in plasma. Yet the simultaneous presence in plasma of two different systems with opposite roles favours the first hypothesis, viz. the existence of an homeostatic regulation of the activity of the peripheral opioids.
453
DuPont A., Cusan L., Garon M., Alvarado-Urbina G. and Labrie F. (1977) Extremely rapid degradation of ‘H methionine-enkephalin by various rat tissues in oiuo and in vitro. Life Sci. 21, 907-914. Fricker L. D., Supattapone S. and Snyder S. (1982) Enkephalin convertase: a specific enkephalin synthesizing carboxypeptidase in adrenal chromaffin granules, brain and pituitary gland. Life Sci. 31, 1841-1844. Hambrook J. M., Morgan B. A., Rance M. J. and Smith C. F. C. (1976) Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature 262, 782-783. Helle K. B. (1966) Antibody formation against soluble proteins from bovine adrenal medulla chromaffin granules. Biochem. biophys. Acfa 117, 107-110. Hillarp N. A., Lagenstedt S. and Nilson B. (1953) The isolation of a granular fraction from the suprarenal medulla, containing the sympatheticomimetic catecholamines. Acta physiol. stand. 29, 251-263. Hogue-Angeletti R. A. and Roda L. G. (1980) In vitro interaction of enkephalin with serum and chromaffin granule components. Experientia 36, 142G1421. Hogue-Angeletti R. A., Roda L. G., Nolan J. A. and Zaremba S. (1980) Catecholamine storage vesicles. In Proteins of the Nervous System (Edited by Bradshaw R. A. and Shneider D. M.), pp. 257-282. Raven Press, New York. Hogue-Angeletti R. A. and Sheetz P. B. (1978) A soluble lipid protein complex from bovine adrenal medulla chromaffin granules. J. biol. Chem. 253, 5613-5616. Jones B. N., Shively J. E., Kilpatrik D. L., Stern A. S., Lewis R. V., Kojima K. and Udenfriend S. (1979) Adrenal opioid proteins of 8,600 and 12,600 daltons: intermediates in proenkephalin processing. Proc. nafn. Acad. Sci. U.S.A. 76, 2096-2100. Lewis R. V., Stern A. S., Kimura S., Rossier J., Stein S. and Udenfriend S. (1980) An about 50,000-dalton protein in adrenal medulla: a common precursor of met- and leuenkephalin. Science 208, 1459-1461. Lewis R. V., Stern A. S., Rossier J., Stein S. and Udenfriend S. (1979) Putative enkephalin precursors in bovine adrenal medulla. Biochem. biophys. Res. Commun. 89, 822-829. Lindberg I., Yang H.-Y. T. and Costa E. (1982) Characterization of a partially-purified trypsin-like enkephalingenerating enzyme in bovine adrenal medulla. Life Sci. 31, 1713-1716. Njus D. and Radda G. K. (1978) Bioenergetic processes in chromaffin granules: a new perspective on some old problems. Biochim. biophys. Acta 463, 219-244. Possenti R., De Marco V. and Roda L. G. (1983) Enkephalin-binding systems in human plasma. Neurothem. Res. 81, 423432. Roda L. G., De Marco V. and Possenti R. (1983) Stability of peripheral enkephalins. In Dearadation of Endoaenous Opioids (Edited by Ehrenpreis, s. and Siciteri, Fy), pp. 2542. Raven Press. New York.
REFERENCES
Roda L. G. and Hague-Angeletti R. A. (1979) Peptides in the adrenal medulla chromaffin granule. FEBS Lett. 107,
Blaschko H. and Welch A. D. (1953) Localization of adrenaline in cytoplasmic particles of the bovine adrenal medulla. Naunyn-Schmiedeberg’s Arch. esp. Path. Pharmak. 219, 11-22. Clement-Jones V., Lowry P. J., Rees L. H. and Besser G. M. (1980) Met-enkephalin circulates in human plasma. Nature 283, 295-291. Craves F. B., Law P. Y., Hunt C. A. and Loh H. H. (1978) The metabolic disposition of radiolabelled enkephalins in vitro and in situ.. J. Pharmac. exp. Ther. 206, 442-506. Di Giulio A. M., Yang H.-Y. T., Fratta W. and Costa E. (1979) Decreased content of immunoreactive enkephalinlike peptide in peripheral tissue of spontaneously hypertensive rats. Nature 278, 64&647.
393-397. Roscetti G., Possenti R., Bassano R. and Roda L. G. (1985) Mechanisms of leu-enkephalin hydrolysis in human plasma. Neurochem. Res. 100, 1393-1404. Ryder S. W. and Eng J. (1982) Radioimmunoassay of leucine-enkephalin-like substance in human and canine plasma. J. clin. Endocr. Metab. 52, 367-369. Schultzberg M., Lundberg J. M., Hokfelt T., Terenius L., Brandt J., Elde R. P. and Goldstein M. (1978) Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla. Neurosci. 3, 1169-1186. Schwartz J. C., Malfroy B. and De La Baume S. (1981) Biological inactivation of enkephalins and the role
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of enkephalin dipeptidil-carboxypeptidase (“enkephalinase”) as neuropeptidase. Life Sci. 29, 1715-1740. Stern A. S., Lewis R. V., Kimura S., Rossier J., Stein S. and Udenfriend S. (1979) Isolation of the opioid heptapeptide met-enkephalin Arg6, Phe7 from bovine adrenal medullary granules and striatum. Proc. natn. Acad. Sci. U.S.A. 76, 668&6683. Terenius L. (1978) Endogenous peptides and analgesia. A. Rev. Pharmac. Toxic. 18, 1899204. Traticante L. J., Rotrosen J., Siekierski J., Tracer H. and Gershon S. (1980) Enkephalin inactivation by N-terminal tyrosine cleavage: purification and partial characterization of an highly specific enzyme from human brain. Life Sci. 261, 1697-1706. Troy C. M. and Musacchio J. M. (1982) Processing of enkephalin precursors by chromaffin granule enzymes. Life Sci. 31, 1717-1720. Venturelli F., Roscetti G., Vita F. and Roda L. G. (1985) Peripheral enkephalin hydrolysis in different animal speties: a comparative study. Neurochem. Res. 10, 1933202.
Venturelli F., Gilardi E., Roscetti G., Laugesen G., Barra P. F. A. and Roda L. G. (1986) Control mechanisms of peripheral enkephalin hydrolysis in mammalian plasma. Comp. Biochem. Physiol. 83C, 307-3 Il. Viveros 0. H., Diliberto R. J. J., Hasum E. and Chang K. J. (1979) Opiate-like material in the adrenal medulla: Evidence for storage and secretion with catecholamines. Molec. Pharmac. 16, 1101-l 108. Wallase E. F., Evans C. J., Jurik S. M., Mafford I. N. and Barchas J. D. (1982) Carboxypeptidase B activity from adrenal medulla. Is it involved in the processing of proenkephalin? Life Sci. 31, 1793-1796. Winkler H. (1976) The composition of adrenal chromaffin granules: an assessment of controversial results. Neurosci. 1, 65-80. Yang H.-T. T., Ci Giulio A. M., Fratta W., Hong J. S., Maiane E. A. and Costa E. (1980) Enkeuhalin in bovine adrenal gland: multiple molecular forms of Met5enkephalin immunoreactive peptides. Neuropharmacology 19, 209-215.
Vol. 8X,
No. 2, pp. 449-454,
0306-4492/86
1986
$3.00 + 0.00
PergamonJournals Ltd
MINI REVIEW HYDROLYSIS AND PROTECTION FROM HYDROLYSIS OF CIRCULATING ENKEPHALINS L. GIORGIO RODA*, FRANCEXA VENT~JRELLI and GIANNA RCBCETTI Dipartimento di Medicina Sperimentale e Scienze Bio~himiche, Universita’ degli Studi “Tor Vergata”, Rome, Italy (Received 24 February 1986)
1.
belonging to three classes: aminopeptidases, dypeptidylaminopeptidases and dypeptidylcarboxypeptidases (e.g. Schwartz et al., 1981). (ii) The activities of these enzymes seem to be similar, but not identical, to the activities of the enkephalin-cleaving enzymes present in the nervous tissue (Roscetti et al., 1985). (iii) At least in man, but not in all the species investigated, the activity of the amino~ptidases prevails over that of the other enzymes. Thus, the first hydrolysis by-products are free tyrosine and the tetrapeptide Gly-Gly-Phe-(Leu or Met). Since all the tyrosine-lacking peptides are biologically inactive (e.g. Traficante et al., 1980), this step is the most physiologically relevant one. (iv) The half-lives reported in plasma range from a few seconds (DuPont et al., 1977) to several seconds in rat (Hambrook et al., 1976) and rabbit (HogueAngeletti and Roda, 1980), up to several minutes in human and murine plasma (Roscetti er al., 1985).
INTRODUCTION
Opioid peptides are secreted by and stored within the adrenal medulla chromaffin granule (~hultz~rg et al., 1978; Viveros et af., 1979). Thus, under physiological conditions they are released in the bloodstream, together with the whole soluble content of the granule (Stern et al., 1979; Clement-Jones et al., 1980; Yang et al., 1980). Once released, these peptides are hydrolyzed by several of the peptidases present in plasma. The enzyme degradation of the shorter opioids-at least in some laboratory animals-is fairly rapid (Hambrook et al., 1976; DuPont et al., 1977; Hogue-Angeletti and Roda, 1980). It has therefore been suggested that the considerable difficulties encountered to show the pharmacological activity of the opioid peptides when administered in vitro (eg. Terenius, 1978; Craves et al., 1978) may be caused by their rapid hydrolysis (e.g. Schwartz et al., 1981). In this respect, the activity of blood as a carrier seems to compete with its activity in degrading the carried substances. Yet, in the last few years there has been considerable evidence demonstrating the existence of endogenous substances capable of partially protecting the plasma-released enkephalins from enzyme hydrolysis (Possenti et al., 1983). The existence of these endogenous inhibitors, while making hydrolysis of the plasma-released opioids seem less puzzling, also makes it appear more complex. Nevertheless, it seems possible to start arranging the data we now have on this topic into a coherent pattern. Indeed, the purpose of the following pages is to review the existing data on enzyme hydrolysis of circulating enkephalins, and to try to ascertain the degree of logical consistency of the resulting pattern.
2.1. Hydrolysis pattern in human plasma The kinetics of formation and of degradation of the hydrolysis by-products in human plasma have been studied as follows: labelled leu-enkephalin was incubated with whole plasma and the incubation mixture was fractioned by steric exclusion chromatography to remove the high molecular weight material. The low molecular weight fractions containing enkephalin and hydrolysis by-products were then separated and quantified by reverse phase and ion exchange chromatography (Roscetti et al., 1985). Of the 13 fragments theoretically obtainable from a pentapeptide, eight are actually formed by the enzymes present in human plasma: (i) all the monomers; (ii) two dimers out of four Gly-Gly and PheLeu; (iii) one trimer out of three: Gly-Phe-Leu; (iv) one of the two possible tetramers, Gly-Gly-Phe Leu. Thus all the N-terminal peptides are absent. This indicates that the activity of the aminopeptidases is higher than the activity of the other enzyme groups. The aminopeptidases seem to be able to cleave the N-terminal glycine from the tetrapeptide Gly-GlyPhe-Leu, forming Gly-Phe-Leu, but to be unable to cleave this tripeptide further. Also absent are Gly-Phe
2. ENKEPHALINHYDROLYSISIN PLASMA The data available on enkephalin hydrolysis by plasma enzymes-less detailed than that existing on hydrolysis of opioid peptides by the nervous systemrelated enzymes-can be summarized as follows: (i) Enkephahns are hydrolyzed by several enzymes, _-..*Author to whom correspondence should be addressed. 449
L.
450
100s
:
a
10.0
12.5
13.0
..:
GIORGIO RODA et al
:
17.5
20.0 ml
ml
ml
Fig. 1. Enkephalin-degrading enzyme activities in plasma as separated by steric exclusion chromatography. Top left: Homo; top right: Oryctolagus; middle left: Cauia; middle right: Rattus; bottom left: Mus; bottom right: Gallus. Dotted lines represent absorbance at 280nm; thick solid lines represent leu-enkephalin; lined areas: aminopeptidase; dotted areas: dipeptidylaminopeptidase; dark areas: dipeptidylcarboxypeptidase. Enkephalin and enzyme activities are expressed as moles per cent.
and Gly-Gly-Phe, is not cleaved.
indicating
that the Phe-Leu bond
2.2. Plasma enzymes The distribution of the enkephalin-degrading enzymes has been determined in the following species: Mus muscuius, Rattus rattus, Oryctolagus cuniculus, Cavia porcellus, Homo sapiens, and Gahs domesticus (Venturelli et al., 1985). Whole plasma was fractioned by steric exclusion chromatography, labelled leuenkephalin was incubated in the presence of the fractions thus separated, and the hydrolysis byproducts-identified by thin layer chromatographywere quantified by counting the cut thin layer sheets (Roscetti et al., 1985). Since Tyr, Tyr-Gly and TyrGly-Gly are very well separated, with this technique it is possible to identify and quantify the three enkephalin-cleaving enzyme groups present in
plasma. The distribution of these enzymes in the previously listed animals is shown in Fig. 1. In addition, when human plasma was further fractioned by ion exchange chromatography, six enzyme species were apparently separated by molecular weight and charge: three aminopeptidases, one dipeptidylcarboxypeptidase and two dipeptidylaminopeptidases (unpublished results). 3. ENKEPHALIN HYDROLYSIS IN DIFFERENT ANIMAL SPECIES
The kinetics of leu-enkephalin hydrolysis caused by plasma enzymes have been measured in several laboratory animals (genera Gallus, Mus, Rattus, Oryctolagus and Cavia) and in man (Venturelli et al.,
1985). Labelled leu-enkephalin was incubated in the presence of whole plasma obtained from the species
Enkephalin
* 1iz ‘L, Y
80
N
R \
SO
‘...:y..,..
‘\
40
.A.._., ..-..,
....
......._ ---. .-.. -..-..__, .-..... *... . .
1,i i\ \
‘\
I\
20
4.1. Protection granule
...;y
\
i ‘.
. . . . . . . . . . . ,,
‘\
12
12
20
24
. ... .
28
MINUTES
Fig. 2. Hydrolysis kinetics of leu-enkephalin in the presence of whole plasma. Dash-two dots line: Mus: dotted line:
Homo; dashdotted line: Oryctolagus: solid line: Gallus; dashed line: Raftus; dash-four dots line: Cauia.
indicated, and the hydrolysis by-products were separated from the intact pentapetide either by thin layer or reverse phase chromatography. The kinetics of hydrolysis determined as indicated are shown in Fig. 2. The relative half-lives are as follows: Mus, 13 min; Homo, 8 min; Oryctolagus, 1 min 54 set; Gallus, 1 min 28 set; Rat&s, 46 set; Cavia, 24 sec. These half-lives were measured close to the estimated plasma concentrations. It should be noted that the very low plasma levels of enkephalin (close to 1 x 10-l’ M, Clement-Jones et al., 1980; Ryder and Eng, 1982) make it rather difficult to obtain reliable quantitative measurements near these levels, even using very high-activity radiolabelled peptide. In addition, enkephalins have been reliably quantified only in man, while for the remaining species only preliminary data exist. Thus the figures shown must be interpreted with some caution. Nevertheless, these results seem to explain-at least partially-the very large differences (some two orders of magnitude) in the enkephalin half-lives reported by various authors (DuPont et al., 1977; Roscetti et al., 1985). While one conflict has thus been partially resolved, the large interspecific differences shown raise a further question, that is whether they indicate actual functional differences of these peptides in various species. Indeed, the 8 min half-life determined in man is consistent with a role of medium-term modulator. The same cannot be true for half-lives some 30 times shorter, like the ones measured in the genera Rattus and Oryctolagus, for which one is tempted to hypothesize a role in different, shorter-living phenomena. 4.
451
that these mechanisms must be of some relevance in the enkephalins’ metabolism. These data are described in some details in the following paragraphs.
*
g
hydrolysis
MECHANISMS
OF PROTECTION
As noted elsewhere (Roda et al., 1983), it is somehow difficult to fit the data available on enkephalin hydrolysis kinetics with the measured plasma levels (Clement-Jones et al., 1980; Ryder and Eng, 1982) and adrenal release (Clement-Jones et al., 1980). These discrepancies led us to specifically investigate the possible existence of physiological mechanisms able to protect the circulating enkephalins from enzyme proteolysis. The results obtained so far demonstrate the actual existence of such mechanisms. Moreover, enough data are by now available to show
mechanisms
within the cromafin
The adrenal medulla chromaffin granule storeswithin its seemingly uniform matrix-atecholamines (Blaschko and Welch, 1953; Hillarp et al., 1953), ions and proteins (Winkler, 1976; Njus and Radda, 1978; Hogue-Angeletti et al., 1980), together with enkephalins and other opioid and non-opioid peptides (Schultzberg et al., 1978; Viveros et al., 1979; Di Giulio et al., 1979; Lewis et al., 1979). Among the proteins, proteolytic enzymes are also present. These enzymes should be able to hydrolyze the polypeptides present within the granule, notably the precursors (Jones et al., 1979; Lewis et al., 1980) of the opioid peptides (Yang et al., 1980; Troy and Musacchio, 1982; Wallace et al., 1982; Fricker et al., 1982; Lindberg et al., 1982), as well as the opioid peptides themselves. Yet it is also possible that the smaller peptides-inherently scarcely resistant to the enzyme hydrolysis-are protected from the activity of these enzymes by some granule-contained mechanism. Indeed, it has been shown that enkephalins actually bind some chromaffin granule components, and that the bound material is partially protected from enzyme hydrolysis (Hogue-Angeletti and Roda, 1980). These results were obtained by incubating labelled leu-enkephalin in the presence of a chromaffin granule soluble lysate. The incubation mixture was subsequently fractioned by steric exclusion chromatography. With this approach, two chromaffin granule fractions of different molecular weight have been shown to bind to enkephalins. The higher molecular weight enkephalin-binding material corresponds to proteins: chromolipin, a granule-contained lipoprotein (Hogue-Angeletti and Sheetz, 1978) and chromogranin A (Helle, 1966). The lower molecular weight material was instead identified with a peptide family previously discovered in the chromaffin granule (Roda and Hogue-Angeletti, 1979). The enkephalins bound to the chromaffin granule components are partially protected from enzyme hydrolysis. This was demonstrated as follows: labelled leu-enkephalin was preincubated with a chromaffin granule crude lysate and then incubated with plasma. Under these conditions, the amount of intact enkephalin was found to be higher than in the controls; that is, the enkephalins not preincubated with the granule lysate. The inhibition of the hydrolysis measured under these conditions is not very high (the amount of intact enkephalin is increased by approximately 30%, Hogue-Angeletti and Roda, 1980). Yet the peculiar physico-chemical milieu existing in the chromaffin granule makes it difficult to reliably extrapolate to physiological conditions data obtained under experimental conditions so far removed from them. Two roles may tentatively be attributed to these systems. The first is to protext enkephalins-and presumably other peptides-from the activity of the granule-contained enzymes while they are still in the chromaffin granule, and/or to control the activity of these enzymes in the cleavage of the precursor mole-
L. GIORGIO RODA et
452
cules (e.g. Troy and Musacchio, 1982; Wallace et al., 1982). A second role may be to contribute to the stability of opioid peptides once they are released into the blood-stream, as will be detailed in the next paragraph. 4.2. Protection mechanisms in blood The existence of protecting substances in the chromaffin granule induced us to specifically investigate the possible existence of analogous mechanisms in plasma. Actually, these studies revealed the presence in plasma of two different systems protecting peripherally released enkephalins from enzyme hydrolysis (Possenti et al., 1983; Roscetti et al., 1985). These results were obtained as described below. Whole human plasma was incubated with labelled leu-enkephalin and the incubation mixture was fractioned by steric exclusion chromatography. Using this procedure, two groups of enkephalin-binding substances can be found: a protein (the serum albumin) and two groups of lower (400&5000 and 2OOG3000 Daltons) molecular weight substances, later identified as peptides. To ascertain whether the binding material could protect enkephalins from enzyme hydrolysis, labelled leu-enkephalin was incubated with whole plasma. To remove the interfering high-molecular weight substances, the incubation mixture was fractioned by steric exclusion chromatography. Intact enkephalin and its hydrolysis by-products were subsequently analyzed by reverse phase chromatography. Under the test conditions, over 90% of the albumin-bound peptide was eluted at the position of the intact enkephalin, while the unbound enkephalin was over 75% degraded. The same analyses were performed on the enkephalin bound to the peptidic fraction. In this case, an even higher amount of enkephalin (95%) was undegraded after the incubation. The aforementioned results have also been confirmed by direct measurements of the half-lives of enkephalins, performed in the presence and in the absence of the plasma enkephalin-protecting systems
al.
(serum albumin and peptides). Specifically, in the presence of the low molecular plasma material, and at enkephalin concentrations close to the physiological resting level, the enkephalin half-life is more than three times as long as the half-life measured in de-peptized plasma (that is, plasma from which the low molecular weight components are chromatographically removed and which is then re-concentrated to its original volume). The half-lives measured under these conditions (in the human species) are 8min in whole plasma and only 2.5min in depeptized plasma (Fig. 3). Moreover, after 60 min of incubation, the intact enkephalin is approximately 25% in whole plasma, but only 3% in de-peptized plasma (Roscetti et al., 1985). The same experiments have been repeated with mammals other than man, and the results show the existence of substances able to partially protect the enkephalin from enzyme hydrolysis in the low molecular weight region of the plasma of all the species examined (Venturelli et al., 1986). Moreover, the protection given by the low molecular weight plasma components-in terms of percent enhancement of the active lives and hence of the actual levels of leuenkephalin-is practically the same in all the species examined, even though the half-lives of the enkephalin differ by a factor of more than 30 (Venturelli et al., 1985). Finally, it should be noted that the physiological importance of the two systems described (serum albumin and peptides) seems to be totally different. Indeed, the low molecular weight system acts by inhibiting the enzymes’ activity (unpublished results). On the other hand, the enkephalin protected from the enzyme hydrolysis by the serum albumin is the enkephalin actually bound to the protein and, presumably, protected by sterical effects. Since this amount is low (approximately 5% of the total), the importance of this system seems to be mainly speculative. On the contrary-as shown above-the role of the low molecular weight system seems to be of considerable physiological relevance.
incubation time (minutes)
Fig. 3. Degradation kinetics of leu-enkephalin in the presence of whole (dashed line) and de-peptised (solid line) human plasma. Solid circles represent leu-enkephalin; stars represent tyrosine.
Enkephalin hydrolysis 5. CONCLUSIONS
To summarize the data presented in the previous pages, the opioid peptides released in vertebrates’ plasma are degraded by several proteolytic enzymes. At the same time, they are protected from the activity of these enzymes by different groups of substances. The protecting substances are present both in plasma and in the chromaffin granule, whence they are co-released in plasma with the opioid peptides. The amount of protection given by the two groups of substances is considerably different. Expressed as the percent variation of the leu-enkephalin half-life, the protection given by the chromaffin granule components is close to 30%, while the protection given by the plasma substances is approximately 300%. These figures indicate that the activity of the plasma protecting systems can have a primary role in the control of the actual levels of circulating enkephalins. Moreover, the presence of the protecting substances implies that, in man, the enkephalin half-life is long enough to account for physiological activities of medium duration. This allows one to hypothesize a role as modulators for these molecules. This is not true in the case of other species. Indeed, differences in half-lives in the range determined so far (of approximately 30) seem to suggest that the opioid peptides may play different physiological roles in different species. The data reviewed seem also consistent with the hypothesis that the release, the hydrolysis and the protection from the hydrolysis of the opioid peptides form a set of closely related phenomena that must be considered as a whole. In this respect, the role of the degrading enzymes could be to actually end the action of the plasma-released opioids. This may be true if these peptides are active on structures devoid of the enzyme systems necessary for this function. This hypothesis contrasts with the alternative one, viz. that the role of the degrading enzymes is not functionally related to the metabolism of the opioid peptides. Indeed, the specificity of the proteolytic enzymes on low molecular weight peptides is in principle low. So, it is certainly possible that the smaller opioids are degraded non-specifically by the proteolytic enzymes present in plasma. Yet the simultaneous presence in plasma of two different systems with opposite roles favours the first hypothesis, viz. the existence of an homeostatic regulation of the activity of the peripheral opioids.
453
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