Control mechanisms of peripheral enkephalin hydrolysis in mammalian plasma

Camp.

Biochem.Phvsiol. Vol.83C.

0306-4492/86 $3.00+ 0.00 '$“1986Per&um~n PressLtd

No. 2, pp. 307-31 I. 1986

Printed inGreatB&n

CONTROL MECHANISMS OF PERIPHERAL ENKEPHALIN HYDROLYSIS IN MAMMALIAN PLASMA FRANCESCA

*Cattedra di Fisiologia e Biochimica

ENZO GILARDI,t GIANNA ROSCETTI,* GITTE LAUGESEN,* PAOLO F. A. BARRA* and L. GIORGIO RODAQ

VENTURELLI,*

Patologica,

Umana,

Universita

degli Studi “Tor Vergata”, Rome, Italy, Metabolismo lstituto Superiore di Sanit$, Rome, Italy and SLaboratorio Unversiti degli Studi di Ancona, Ancona, Italy

TLaboratorio

di

di Farmacologia,

(Received 29 July 1985) Abstract-l. The protection of the adrenal-released enkephalins from enzyme hydrolysis by endogenous plasma components was studied in laboratory animals and in man. 2. The results indicate that mechanisms active in protecting leu-enkephalin from hydrolysis are present in the plasma of all species examined. 3. The protection seems to be due to two groups of substances, possibly of peptidic nature. 4. The amount of protection given by these substances seems to be sufficient to play a significant role in controlling the physiological levels of leu-enkephalin released into the bloodstream.

At least in man, the plasma-released enkephalins are partially protected from the activity of the cleaving enzymes by the presence of a group of substances which actually inhibit the enzymatic degradation of the enkephalins (Possenti et al., 1983). As already demonstrated (Roscetti et al., 1985; Roda et al., 1983), these mechanisms are physiologically significant in maintaining the proper levels of circulating enkephalins. Indeed, in the absence of these peptides the half-life of leu-enkephalin in human blood is reduced to almost exactly l/3 of the half-life measurable in the presence of the peptides (Roscetti et al., 1985). On the basis of these data, we thought it worthwhile to start a comparative study to ascertain the possible presence of protective systems in animals other than man. The final aim of this project is to better understand the physiological significance of the release, of the degradation and of the protection from hydrolysis of the opioid peptides.

INTRODUCTION Enkephalins, the opioid pentapeptides, are secreted by and stored within the chromaffin granule of the mammalian adrenal medulla (Schultzberg et al., 1978; Viveros et al., 1979). In the course of the physiological activity of the gland, these peptides are released into the bloodstream (Viveros et al., 1979; Clement Jones et al., 1980), presumably to be carried to their target organs. Nevertheless, the enkephalins thus released are inactivated by several groups of enzymes present in plasma (DuPont et al., 1977; Hambrook et al., 1976; Hogue-Angeletti and Roda, 1980). Although studied in considerably less detail than the enzymes present in the central nervous system (Schwartz et al., 1981; Craves et al., 1978), the distribution of these enzymes and the hydrolysis pattern of the enkephalins in human blood are relatively well known (Roscetti et al., 1985). As has been shown (Venturelli et al., 1985), the hydrolysis kinetics and the ratio between the activities of the different groups of enzymes vary considerably among the different species examined. Consequently, the pattern of hydrolysis of the enkephalins also differs in the various species. Briefly, the first-and the physiologically most relevant-step is the hydrolysis of the tyr-gly bond in all the species examined, except in the rabbit. In the latter species, the dipeptidylaminopeptidase activities are such that the first hydrolysis step is cleavage at the gly-gly bond. In several other animals, approximately 75% of the enkephalin is cleaved by the aminopeptidases and 25% by the dipeptidylaminopeptidases, while the role of the dipeptidylcarboxypeptidases seems to be a minor one in all the species investigated (Venturelli et al., 1985).

For the partial purification of the hydrolysis-protecting material the blood was collected in 10% of l50mM NH,COCH,, pH 4.0. For each 9ml blood, 1 ml NH,COCH, was measured in a test tube and the blood, collected with a syringe, was immediately transferred into the NH,COCH,-containing test tube. For the preparation of the proteolytic enzymes, the blood was collected with the same procedure, but using 1 mg/ml EDTA. In both cases, the blood was centrifuged for IO min at 25006 in a refrigerated centrifuge. Supernatant plasmas obtained from several (5 to 12) animals were pooled together to average the results. They were then divided into small aliquots and stored under liquid nitrogen until used.

$Author to whom correspondence should be addressed. Mailing address: c/o Cattedra di Fisiologia Umana, Universiti degli Studi “Tor Vergata”, Via 0. Raimondo, 1 00173 Rome, Italy.

The plasma enkephalin-degrading enzymes were partially purified by steric exclusion chromatography on a 7.6 x 600 mm TSK G3000 SW column as described below, The enzyme-containing fractions were concentrated to l/2 of the

MATERIALS AND METHODS Blood collection

307

FRANCESCA VENTUKELLI (‘1 01

30x

original plasma volume using a Millipore PCAC membrane (Milliporc Co. Bedford, MA, USA) in a Millipore stirred cell.

High enzyme G3000 Hepes.

pressure steric exclusion chromatography used for preparation was performed on a 7.6 x 600 mm TSK SW column &ted at 2.2 m!/cm2/min with 25 mM l25mM NaCl, 2.5 x 10m5M Zn(COCH),, pH 7.0 (henceforth called Hepes buffer). Steric exclusion chromatography for the partial purification of the enkephalinprotecting material was performed on a 1.6 x 90 cm column of Fractogel40 TSK HW 2540 pm (E. Merck, Darmstadt, FRG) eluted at 0.33 ml/cm’/min with 50 mM NH,COCH,, pH 4.0. For the determination of molecular weights, a 7.6 x 600 mm TSK G2000 column~quilibrated in Hepes buffer -was calibrated with myoglobin, cytochrome c, lysozyme. insulin. insulin b chain and cyanocobalamin, and the calibration curve was interpolated with a linear least-squares procedure. Fractions were collected by means of a LKB Ultrorak fraction collector (LKB instruments, Bromma, Sweden). Thin layer chromatography for the enzyme assays was performed on Kiesegel 60 plates (Merck) developed with rt-buty! a!cohol:acetic acid: water 3.5: 1: 1.5.

Labelled leu-enkephaiin was quantified by scintillation counting of sample aliyuots. Protein concentration was measured by the Lowry assay (Lowry et al., 1951) and carbohydrates were measured with a slight modification of the method of Dubois et ul. (1956). The radiochemica1 purity of the labelled material was assayed by high pressure chromatography as described elsewhere (Possenti et al., 1983).

Amino acid analyses were performed on acid-hydrolysed material (24 hr at I IO’C under reduced pressure) with a Carlo Erba 3A29 amino acid analyser (Carlo Erba Strumentazione, Corsica, Italy).

Tritiated leu-enkephalin (tyrosyl-3,5[‘H](N)-H-leucine enkephalin, specific activity 6 x IO” Ci/mo!e) was obtained from New England Nuclear (Boston, MA, USA). HC! for hydrolyses was Suprapur grade and Na citrate for the amino acid analyser buffers was special grade from E. Merck. All other substances used were of reagent grade and used without further purification. RESUllFS

This was tested as follows: the samples under test--dried under reduced pressure and washed twice with water-were resuspended in I/Z of their original plasma volume of hepes buffer. Twenty-five microlitres of the resuspended sample were added to an equal volume of reconcentrated enzymes plus Spl labelled leu-enkephalin (5 x 105 DPM, viz. 1 x 10. ‘moles) and incubated for three times the half-life of the species under study (Venturelli et al., 1985). except where otherwise indicated. The reaction was terminated by the addition of 3 icl of CH,COOH and the reaction mixture was stored at -30 C until tested by thin layer chromatography. The thin layer plates, once developed, were stained with ninhydrin (0.1% in acetone) to show the standards. then cut to separate the spots relative to the intact leu-enkephalin. to tyr. to tyr-gly and to tyr-gly-gly (Roscctti e/ ul.. 1985). The fragments thus separated were counted for tritium in a LKB 1211 scintillation counter and the results were expressed as the percentage of the total DPM. lnhibil~on activity was measured against a blank containing only the enzymes and labelled ieu-enkephalin.

ia)

I

AND DISCUSSION

To study the possible existence of hydrolysisinhibiting substances in the plasma of the species examined, the low molecular weight material was separated from the enkephalin-hydrolysing enzymes by steric exclusion chromatography on a G3000 TSK column. The two groups of substances were obtained from separate runs, eluting the same column with different buffers. as described under Materials and Methods. Figure 1 shows the chromatograms obtained with human plasma. The shaded area in panel a shows the pool made to separate the enzymes. The shaded area in panel b indicates the pool made to separate the low molecular weight material. Similar runs (not shown) were repeated using the plasma of the other species examined, and the resulting material was reconcentrated as described under Materials and Methods. The material obtained as described above was used to check the possible existence of substances active

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of leu-enkephalin in the presence (solid line) and in the absence (dashed c: rat; d: rabbit. weight plasma material. Panel a: man: b: guinea-pig;

in inhibiting the enkephalin-degrading enzymes. The enzymes were incubated with tritiated leu-enkephalin for 10, 20, 40 and 80sec and for 4, 8 and 30min both in the presence and in the absence of the low molecular weight material, and the enkephalin hydrolysis was followed by thin layer chromatography as described under Materials and Methods. Figure 2 shows the curves representing intact leu-enkephalin as a function of the incubation time for each one of the species examined, both in the presence (solid line) and in the absence (dashed line) of the low molecular weight material. As shown, in the presence of the low molecular weight material the leu-enkephalin hydrolysis is considerably reduced, thus indicating the existence of material able to protect leu-enkephalin from the enzyme hydrolysis in the low molecular weight region of the plasma of all the species examined. The curves shown in Fig. 2 seem to suggest that the protection from enzyme hydrolysis is quantitatively different in the various species under test. However, the data shown in Table l-which shows the inhibitory activities calculated at the plasma half-lives of the leu-enkephalin for each speciesshow that the protection-as measured at rhe halflife-is quite similar in all the species tested. This Table I. Percentage inhibition of leu-enkephalin hydrolysts as calculated at the plasma half-life determined for each species Species Man (Homo .sapirnsf Rabbit (Og~rolagus) Guinea-pig (Cmicr porcellus) Rat (Rarrus rams)

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result means that-in terms of percentage enhancement of the active lives, and hence of the actual levels of leu-enkephalin--the protection given by the low molecular weight plasma components is practically the same in all the species examined, even though the half-lives of the enkephalin differ by a factor of more than 20 (Venturelli et al., 1985). These results indicate the existence of substancespresent in the plasma of the four species examinedwhich are able to partially protect the peripherally released enkephalins from enzyme hydrolysis. To obtain some information on the nature of these substances, the following experiments were performed. The plasma of each species was fractioned by steric exclusion chromatography on Fractogel 4OSW. and the eluled fractions were tested for inhibition of enzyme activities as described under Materials and Methods. The chromatograms thus obtained are shown in Fig. 3. The dark-shaded histogram bars, which represent the percentage of inhibition in each fraction, indicate the presence of inhibitory material in two different zones of the chromatograms. The first-eluted active fractions represent on average approximately 60% of the total enkephalin-protecting activity, which is centred at an elution volume of about 30ml; a second group of active fractions, accounting for about 20% of the total protection, is eluted between 70 and 85 ml, except in man. As indicated in Fig. 3, carbohydrates are associated with the first-eluted active material, which is also positive to the Lowry assay. On the other hand, the last-eluted material is under the detection limit of the carbohydrate assay (approximately 5 x 10 ‘g/ml),

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and close to this limit for the Lowry assay. Moreover, the carbohydrates/activity ratio in the first-eluted fractions does not seem to be consistent with the hypothesis that the protecting activity can be actually due to carbohydrates. On the contrary, the amino acid analyses shown in Table 2 seem to indicate that the amino acids are associated with all the active fractions. Therefore, these results-albeit partialfavour the hypothesis that the inhibition is due to peptidic material. Finally, to obtain an indication of its molecular weight, the active material eluted from the Fractogel Table 2. Amino acid analyses of the inhibitory plasma components as eked from the Fractogel 40. A indicates the first-eluted and B indicates the last-eluted group of active fractions Amino acid

Man

Species Rabbit Guinea-pig A B A B

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16.6 10.3 t 5.6 t 5.6 I I.2 t 6.4 6.1 5.1 2.3

40 column was pooled, concentrated and chromatographed again on a TSK G3000 column, calibrated as described under Materials and Methods. The elution volume of the first-eluted active fractions is consistent with an apparent molecular weight of approximately 9000 for all the species examined. The apparent molecular weight of the last-eluted active fractions is approximately 2000 for the human species and a few hundred for the other species examined. These data indicate that the leu-enkephalin released into the bloodstream is partially protected from the enzyme hydrolysis and that the inhibition of the enzyme hydrolysis measured in the presence of the enkephalin-protecting substances is nearly the same in all the species. Specifically, under the experimental conditions described above, the active life of the leu-enkephahn is increased by 55575% in the presence of the protective substances. Thus, the existence of mechanisms active in controlling the enzyme hydrolysis of the adrenal-released enkephalinsalready identified in man-is certainly confirmed in other mammal species. Nevertheless, certain points should be noted. First, in man, at concentrations close to the physiological levels of leu-enkephalin (from our data. approximately 1 x lo-l4 moles/ml), the half-life of this peptide is almost exactly three times (8 min instead of 150 set) the half-life determined in the absence of the protecting substances (Roscetti et ul., 1985). This contrasts with the 76% of inhibition measured for leu-enkephalin at the peptide concentrations used in this study. Therefore, the protecting substances seem to be more active at the very low enkephalin concentrations existing in the human species. This is probably due to the inhibitory effect

311

Peripheral enkephalin hydrolysis of the hydrolysis by-products on the degrading enzymes (Roscetti et al., 1985). Since no definitive data are available on the physiological concentrations of the opioid peptides in species other than the human species, it is as yet impossible to determine the exact amount of protection under physiological conditions. Nevertheless, at the half-life the level of protection is very much the same in all the species examined. Since preliminary results tend to indicate that the leuenkephalin levels in the mammals examined are not far from the levels existing in man, the data relative to the human species may tentatively be extrapolated to other species. Second, the experiments reported above were performed under the conditions necessary to study the possible presence of protecting substances, coexisting in the same tissue with the substances on which the former are active. This made it mandatory to separate the two groups of substances, viz. peptides and enzymes. In turn, this procedure alters the hydrolysis kinetics with respect to the kynetics measured with unfractioned plasma (Roscetti et al., 1985; Possenti et al., 1983). Thus, the protection can only be estimated, but not directly measured. under physiological conditions. A last point worth mentioning is that, unlike the protective activities, the enkephalin-degrading enzymes present in the plasma of the various species show a considerable degree of intraspecific variation (unpublished). This results in the considerable variations in the plasma half-lives of the enkephalins already detected. Very different activities of the degrading systems may cause large variations in the plasma levels of the enkephalins at nearly constant release. On the other hand, the release of the opioid peptides could show a large degree of intraspecific variation, and the enkephalin levels could thus be similar in the various species. Preliminary results, indicating that the enkephalin resting levels in mammals are of the same order of magnitude, tend to confirm the latter hypothesis. The two separate systems present in mammalian plasma, one able to degrade, the other able to inhibit the degradation of the opioid peptides, can be viewed as one single system, acting as a functional unit capable of regulating the levels of the circulating enkephalins even in the presence of a steady release. Any attempt to confirm or disprove this hypothesis would contribute significantly to our understanding of the role of the peripherally released opioid peptides. We hope that the determination of the enkephalins’ plasma levels in various animals, and the purification of the hydrolysis-inhibiting material already in progress in our laboratory will help to improve our understanding of these phenomena.

Acknowledgement-The present work was undertaken within the framework of and with the partial support of the “Progetto Finalizzato Chimica Fine e Secondaria” of the CNR (National Council for Scientific Research).

REFERENCES

Clement Jones V., Lowry P. J., Rees L. H. and Besser G. M. (1980) Met-enkephalin circulates in human plasma. Nature, Lond. 283, 295-297. Craves F. B., Law P. Y. and Loh H. H. (1978) The metabolic disposition of radiolabeled enkephalins in vitro and in situ. J. Pharmac. exp. Ther. 206, 492-506. Dubois M., Gilles K. A., Hamilton J. K., Rebers P. A. and Smith F. (1956) Calorimetric method for the determination of sugars and related substances. Analyf. Chem. 28, 356. DuPont A., Cusan L., Garon M., Alvarado-Urbina G. and Labrie F. (1977) Extremely rapid degradation of [‘HImethionine-enkephalin by various rat tissues in viva and in rritro. Life Sci. 21, 907-914. Hambrook J. M., Morgan B. A., Rance M. J. and Smith C. F. (1976) Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature, Lond. 262, 782-783. Hogue-Angeletti R. A. and Roda L. G. (1980) In vitro interaction of enkephalin with serum and chromaffin granule components. Experientia 36, 142G-1421. Lowry 0. H., Rosenbrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin uhenol reagent. J.‘biol. Chem. 193, 265-275. Possenti R., De Marco V., Cherubini 0. and Roda L. G. (1983) Enkephalin-binding systems in human plasma. Neurochem. Res. 8, 423432. Roda L. G., De Marco V. and Possenti R. (1983) Stability of peripheral enkephalins. In Degradution of Endogenous Opioids (Edited by Ehrenpreis S. and Sicuteri F.), pp. 2542. Raven Press, New York. Roscetti G.. Possenti R.. Bassano E. and Roda L. G. (1985) Peripheral enkephalin hydrolysis in human plasma: Neuiochem. Re.7.. 10, 1393-1404. Schultzbere M.. Lundberg J. M.. Hokfelt T.. Terenius L.. Brandt ‘J., Elde R. P. and’ Goldenstein M. (1978) Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla. Neuroscience 3, 1169-l 186. Schwartz J.-C., Malfroy B. and De La Baume S. (1981) Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptidase (“enkephalinase”) as neuropeptidase. Life Sri. 29, 1715-1740. Venturelli F., Roscetti G., Possenti R., Vita F. and Roda L. G. (1985) Peripheral enkephalin hydrolysis in different animal species: a comparative study. Neurochem. Res. 10, 333-342. Viveros 0. H., Diliberto E. J., Hazum E. and Chang K.-J. (1979) Opiate-like material in the adrenal medulla. Evidence for storage and secretion with catecholamines. Molec. Pharmac. 16, 1101-I 108.