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Neuropeptide enzyme hydrolysis in human saliva
Archives of Oral Biology 45 (2000) 775±786
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Neuropeptide enzyme hydrolysis in human saliva Mario Marini, L. Giorgio Roda* Dipartimento di Neuroscienze, UniversitaÁ degli Studi di Roma ``Tor Vergata'', 00133 Roma, Italy Accepted 21 March 2000
Abstract The possible hydrolysis of neuropeptides by human saliva was studied using leucine enkephalin as a model. The data obtained indicate that in the presence of saliva this substrate is partially hydrolysed, and that its disappearance corresponds to the appearance of peptides whose composition is consistent with that of the substrate hydrolysis byproducts. The formation of these peptides indicates the presence of all three classes of enzymes known to hydrolyse enkephalins in other tissues: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. The activity of these enzymes appears to be altered by the presence of low molecular-weight substances, whose inhibitory activity is apparent on all three classes of enkephalin-degrading enzymes. Substrate degradation was higher in male than female saliva; these dierences appear to be caused by lower activity of the enzymes, and higher activity of the low molecular-weight inhibitors, measurable in female as compared to male saliva. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Neuropeptides; Enkephalins; Enzyme hydrolysis; Endogenous inhibitors
1. Introduction The human salivary glands synthesize and release several families of peptides (Barka, 1980), some of which, such as histatins (Xu et al., 1993), are described as saliva-speci®c. Many salivary peptides derive from in loco hydrolysis of precursor proteins (Perinpanayagam et al., 1995). Others [e.g. angiotensin II, vasoactive intestinal peptide, substance P, and gastrinreleasing peptide (Aalto et al., 1997)] belong to the family of brain±gut peptides, whose synthesis and release is controlled by the nervous system (Garrett, 1987). Not all of the physiological roles of salivary neuropeptides are easy to understand, however. Their
* Corresponding author. Tel.: +39-6-7259-6410; fax: +396-7259-6407. E-mail address: [email protected] (L.G. Roda).
most obvious function, to mediate the nervous control of saliva secretion, is supported by experimental data: according to EkstroÈm et al. (1988), tachykinins are transported by parasympathetic nerve ®bres to the salivary glands, where they regulate secretory functions (Holzer and Holzer-Petsche, 1997). A secretory role has also been described for adenylate cyclase-activating peptide by Tobin et al. (1995), and for vasoactive intestinal peptide by Tichen and Reid (1990). In addition, neuropeptides may have trophic eects on the salivary glands, such as those described by Mansson et al. (1990) for vasoactive intestinal peptide and substance P in the rat parotid. This kind of precise information is not available, unfortunately, for all of the neuropeptides present in saliva. It is possible, even obvious, to hypothesize that the presence of dierent mediators contributes to regulate ®nely the secretion of the salivary glands. Yet, some of the available data do not agree with the hy-
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pothesis that all salivary neuropeptides are directly released by nerve terminals. Aalto et al. (1997) report an increased concentration of bombesin-like material in the salivary ducts of rats in which bombesin immunoreactive nerve ®bres had been experimentally reduced. They interpreted this ®nding as indicating that bombesin can be released independently of axonal transport, and that this peptide pool is present in salivary ducts and is thus secreted in saliva. Similarly, Rougeot et al. (1994) suggest that peptides cleaved from submandibular rat 1 protein are selectively released in saliva (as well as in the bloodstream) following sympathetic stimulation. These data suggest that neuropeptides are not only released by, but also synthesized within, the salivary glands. Neuropeptides may also account for the control of environmental factors on saliva composition, as has been described by Backkelund Pedersen and Poulsen (1983) for the release of renin, and by Santavirta et al. (1997) for the release of vasoactive intestinal peptide. The role of environmental factors appears to be of particular relevance for the endocrine functions of the salivary glands: this was described as early as 1979 for renin (Bing and Poulsen, 1979; also Backkelund Pedersen and Poulsen, 1983) and, later on, for kallikrein (Berg et al., 1985, 1990), nerve growth factor (Aloe et al., 1986), and the peptides originated by the proteolytic cleavage of submandibular rat 1 protein (Rougeot et al., 1994). It seems possible therefore that saliva neuropeptides are transported into the bloodstream, contributing to the formation of the very large pool of plasma informational peptides. In such a context, the role of behavioural factors in controlling the release of these peptides by the salivary glands may be similar to that reported for other substances, such as monoamine oxidase in the central nervous system (Lemoineet al., 1990), secretion of proteases by mast cells (MacQueen et al., 1989), or enzyme degradation of opioid peptides in plasma (Young et al., 1989; Babst et al., 1999). As is known for other neuropeptides, some of the above-mentioned eects are regulated by enzyme degradation. Salivary proteolytic enzymes have been identi®ed mostly on the basis of their hydrolysis byproducts [e.g. kallikrein by Orstavik et al. (1982); prokallikrein by Kamada et al. (1990); the release of active peptides via the hydrolysis of Arg±Arg bonds by Rougeot et al. (1994)]. However, the proteases described by Xu et al. (1993) appear to be involved in the regulatory degradation of active peptides: according to them, the hydrolysis of histatins by salivary endopeptidases gives rise to by-products less active than the intact peptides. In addition, as in other tissues, notably plasma (e.g. Bolacchi et al., 1995), the presence of proteolytic enzymes is coupled with that of protease inhibitors: according to Nishikata et al. (1991), histatin 5 inhibits the trypsin-like protease released by Bacteroides gingi-
valis (and clostripain); Basak et al. (1997) showed that members of the histatin family have a (moderate) inhibitory activity on proprotein convertases; van't Hof et al. (1997) described the inhibition of cysteine proteinases by Von Ebner's gland lipocalin. These investigators suggest that this protein might have a role in controlling in¯ammatory processes via the inhibition of bacterial proteases. The co-presence in saliva of neuropeptides, proteases active on these peptides, and inhibitors of these proteases, suggests the existence of speci®c mechanisms capable of controlling the active concentration of neuropeptides. Similar processes are known to occur in various tissues, such as plasma (e.g. Roda et al., 1987), or in the respiratory tract (e.g. Johnson et al., 1985). Speci®cally, a regulatory role has been attributed to the enzyme proteolysis of active plasma peptides (e.g. Connelly et al., 1985; Pierart et al., 1988). The data that follow attempt to verify the possible existence of such mechanisms in human saliva using leucine enkephalin as a model substrate. The hydrolysis characteristics of this peptide in other tissues are known in detail (Hambrook et al., 1976; Marks et al., 1977; Roscetti et al., 1985). In addition, because of its small size, the pattern of its hydrolysis by-products is relatively simple. Therefore, even if the presence in saliva of leuenkephalin has not been described, it appears to be a suitable model for ascertaining the presence, distribution, and possible inhibition of enzymes capable of hydrolysing saliva neuropeptides. 2. Material and methods 2.1. Sample population The sample studied consisted of six individuals, three men and three women, mean age 32.2 years (SD 5.9). All donors were in good health, all were nonsmokers; the presence of in¯ammatory oral processes was speci®cally excluded. The administration of pharmacologically active substances was excluded for 5 days before taking the samples, as were oral contraceptives in the case of female donors. 2.2. Saliva collection Saliva samples were collected in 50-ml test-tubes, immediately transferred to an ice bath, dialysed at 48C against 10 mM HEPES, 100 mM NaCl, 2.5 10ÿ5 M ZnCl2 pH 7.2 (referred to henceforth as HEPES buffer), and centrifuged (10 min at 12,000 g). Samples to be fractionated by steric-exclusion chromatography were concentrated to 1/10 of the original volume on a PLCC membrane (Millipore Co., Bedford, MA, USA). All samples were used within 24 h of collection.
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2.3. Assay Enkephalin-degrading enzymes were identi®ed, and their activity was quanti®ed, on the basis of the labelled hydrolysis by-product formed. Portions (10 ml) of dialysed samples were transferred to 2-ml tapered test-tubes; tritiated leu-enkephalin (2 ml, corresponding to 3.4 10ÿ10 mol) was added to each sample. Reactions were carried out at 378C for the periods of time indicated in the ®gures (600 s for column eluent), and stopped with 3.5 ml of acetic acid. Intact leu-enkephalin and its labelled hydrolysis by-products were quanti®ed by thin-layer chromatography as described below. Proteins were determined by the micro-biuret method described by Itzhaki and Gill (1964). Carbohydrates were determined by the phenol±H2SO4 method described by Dubois et al. (1956) and modi®ed as described by Roda et al. (1977). 2.4. Thin-layer chromatography This was performed on aluminium-backed Kieselgel 60 sheets (Merck, Darmstadt, Germany) developed at 50 8C with 58:10:30:0.17 2-butyl alcohol:acetic acid:water:acetonitril. Sheets were cut according to internal standards, transferred to scintillation vials, and counted for tritium as described by Bolacchi et al. (1995). 2.5. Enzyme fractionation Enzymes were fractionated on a 5-mm 7.8 300 mm TSK G3000SW column (Toyo Soda Ltd, Tokyo, Japan) equilibrated in HEPES buer. Samples (350 ml) were eluted at 2.2 ml/cm2 per min with the same buffer. Absorbance at 280 nm was monitored, and 0.25ml fractions were collected and assayed as described above. 2.6. Low molecular weight inhibitors These were prepared with saliva obtained from a single donor (donor 6) as follows: low molecularweight substances were separated from enzymes on a TSK G3000SW column, equilibrated and eluted as described above, except that the buer used was 5 mM HEPES, pH 7.2, without the addition of NaCl [Fig. 2(a)]. The fractions (1 ml) eluted between K '=0 and K'=1.09 (referred to henceforth as K1' .09) were pooled, concentrated to their original volume in collodion bags (Sartorius AG, Gottingen, Germany), and used as the source of enzymes for subsequent assay. The fractions eluted from K '=1.09 up to K '=1.69 (pool 1) were concentrated by ultra®ltration on a PLCC membrane, the retentate was discarded and the
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eluent was concentrated in a rotating evaporator. The fractions eluted up to K'=2.28 (pool 2) and K'=2.73 (pool 3) were concentrated in a rotating evaporator only. 2.7. Data analysis One-way completely randomized ANOVA were performed with commercial software (Costat, Cohort Software, Minneapolis, MN, USA). Regression analyses were performed using a linear polynomial procedure of variable degree. Hydrolysis curves were interpolated with a non-linear iterative procedure (Marquardt±Levemberg), using the exponential equation y a e bx c e dx e: Parametric statistics were calculated according to standard procedures. 2.8. Material Leu-enkephalin, Tyr±Gly and Tyr±Gly±Gly were obtained from Bachem Feinchemikalien AG (Bubendorf, Switzerland), and Tyr from Serva Feinbiochemica GmbH (Heidelberg, Germany). Tritiated leuenkephalin (sp. act. 1.03 1011 Bq/mol), was prepared by isotope exchange; radiochemical purity of the tritiated peptide was checked by reverse-phase and thinlayer chromatography. All other material was obtained through local suppliers, and used without further puri®cation. 3. Results 3.1. Hydrolysis in whole saliva The possible hydrolysis of leu-enkephalin was measured by incubating the tritiated peptide in the presence of saliva. In samples obtained from all donors, the substrate concentration decreased timedependently, with the corresponding appearance of Tyr, Tyr±Gly, and Tyr±Gly±Gly (Fig. 1). The disappearance of the whole peptide, and the formation of the N-terminal leu-enkephalin hydrolysis by-products detected, can be assumed to indicate the presence of all three classes of enzymes known to hydrolyse enkephalins: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases (Hambrook et al., 1976; Craves et al., 1978). 3.1.1. Leu-enkephalin The curves describing the disappearance of leu-enkephalin over time [Fig. 1(a)] indicate that substrate hydrolysis was relatively fast in the ®rst 60±90 s, slowing down considerably afterwards. The average slope of the hydrolysis curves decreased from 11.21 between
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0 and 30 s to 0.30 between 240 and 1800 s, with a ¯ex point located between 40 and 65 s. Protein concentration was noticeably higher in male saliva (on average, 0.46 mg/ml) than in female saliva (0.20 mg/ml). The possible co-relation between total protein and substrate hydrolysis was, therefore, examined by performing linear regressions of variable degree between protein concentration and hydrolysis at 600 s. The results obtained (1st degree: r 2=0.60, F = 5.99, p = 0.07) were not statistically signi®cant ( p > 0.05); however, as they indicate a 93% probability of an associ-
ation between the two variables, these results strongly suggest the existence of such a co-relation. 3.1.2. Hydrolysis by-products Under our experimental conditions, the average substrate degraded at 600 s was 36% of the total in the reaction [Fig. 3(b)]. Of this, 61% was hydrolysed by aminopeptidases, 24% by dipeptidylaminopeptidases and 15% by dipeptidylcarboxypeptidases. As the hydrolysis curves were nearly parallel to each other (Fig. 1), this ratio was similar (within 210%) for the other reaction times. These data indicate a ratio
Fig. 1. Degradation of leu-enkephalin and formation of its labelled hydrolysis by-products in the presence of human saliva as a function of reaction time. (a) Leu-enkephalin; (b) Tyr; (c) Tyr±Gly; (d) Tyr±Gly±Gly. Dotted thick lines, female donors; dotted thin lines, male donors; solid thick lines, all donors (female and male). Symbols represent experimental points. Vertical bars represent statistical dispersion (21s). Curves were interpolated as described under Section 2.7.
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between the enzyme activities, and a predominance of aminopeptidases over the dipeptidylpeptidases (dipeptidylamino- and dipeptidylcarboxypeptidases), similar to that described in other tissues (Hambrook et al., 1976, Craves et al., 1978). 3.2. Sex-associated dierences The amount of substrate hydrolysed, and the ratio between the hydrolysis by-products formed, were dierent in salivas obtained from male and female donors. Speci®cally, as measured at 600 s, the amount
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of substrate hydrolysed, and the amount of Tyr, Tyr± Gly and Tyr±Gly±Gly formed in the presence of male saliva were 130%, 138%, 104% and 138% of the values measured in the presence of female saliva, respectively [Fig. 3(b)]. Similarly, the substrate half-life for female donors (1632 s, SD 167 s) was 128% of that for male donors [1277 s, SD 387 s; Fig. 1(a)]. The statistical signi®cance of these dierences was analysed by one-way ANOVA obtaining the following results: F 9:30 and p 0:053 for leu-enkephalin hydrolysis, F 17:60 and p 0:025 for the formation of Tyr, F 0:65 and p 0:478 for Tyr±Gly and F 3:61 and p 0:056
Fig. 2. Separation of enkephalin-degrading enzymes in human saliva by steric-exclusion chromatography. (a) Continuous line represents absorbance at 280 nm; dashed staircase represents protein as human serum albumin; dotted staircase represents carbohydrates as glucose. Solid horizontal bars represent pooled fractions; K identi®es K1' .09 pool; numerals 1, 2, and 3 identify the corresponding pool. (b) Aminopeptidases; (c) Dipeptidylaminopeptidases; (d) Dipeptidylcarboxypeptidases. Donors identi®ed in (b); thick lines represent female donors (f); thin lines represent male donors (m).
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for Tyr±Gly±Gly, respectively. These results indicate that there is a statistical probability of approx. 5% that the dierences observed between male and female donors in substrate degradation were only casual. They also indicate that these dierences were caused by highly signi®cant, sex-related, dierences in the activity of aminopeptidases, and by less signi®cant dierences in the activity of dipeptidylcarboxypeptidases. The dierences in the activity of dipeptidylaminopeptidases, and thus their contribution to the lowering of substrate hydrolysis in female saliva, were statistically non-signi®cant.
3.3. Hydrolysis in fractionated saliva To ascertain the approximate number and size distribution of the substrate-active enzymes, saliva samples were fractionated (Fig. 2); the activity of the separated fractions was then analysed [Fig. 2, panels (b)±(d)]. The substrate-active enzymes were eluted for the most part between approx. K'=0.25 and K'=0.76. This zone, unsurprisingly for a raw sample, did not correspond to speci®c features of the protein or carbohydrate elution pro®les [Fig. 2(a)]. However, the general shape of the elution envelopes of the column-
Fig. 3. Amount of leu-enkephalin degraded, and labelled hydrolysis by-products formed at 600 s in the presence of non-fractionated (a and b), and fractionated (c and d) human saliva, moles per cent. (a) and (c) represent individual donors, (b) and (d) represent averages. Open bars, leu-enkephalin; obliquely shaded bars, Tyr; vertically shaded bars, Tyr±Gly; solid bars, Tyr±Gly±Gly. Numerals identify individual donors; bold numerals, female donors; plain numerals, male donors.
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separated enzymes, a swell as many details, corresponded in the donors examined. This was particularly evident in the central region of the aminopeptidase elution envelope, where active species were eluted at K'=0.36, 0.45, 0.54 and 0.75 in nearly all donors [Fig. 2(b)]. The dipeptidylaminopeptidase elution envelopes for female donors were also similar to each other, with minor peaks matching below K '=0.40, and the main activity centred at K'=0.47±0.49. In contrast, in male saliva, the dipeptidylaminopeptidase elution envelopes appeared to be individual-speci®c. In this case, dipeptidylaminopeptidases were represented by two or three main peaks, located between K '=0.36 and K'=0.65, with few matching details, such as those located at K'=0.47, or the late activity at K '=0.80 [Fig. 2(c)]. Finally, the low and uniformly distributed activity of dipeptidylcarboxypeptidases did not permit the identi®cation of speci®c features [Fig. 2(d)]. The existence of corresponding features in the elution envelopes of dierent donors allows the hypothesis that these details may actually correspond to single enzyme species. The total amount of substrate hydrolysed, and the amounts of Tyr, Tyr±Gly and Tyr±Gly±Gly formed at 600 s by the fractionated enzymes, are shown in Fig. 3 [panels (c), (d)]. A comparison of these data with those in panels (a) and (b), measured under the same conditions but in the presence of non-fractionated saliva, indicated the existence of fractionation-associated variations of hydrolysis. As steric-exclusion techniques involve a considerable dilution of the original sample, the consequent lowering of enzyme concentration could account for the reduction of hydrolysis noticeable in fractionated as compared to whole samples. This was not, however, the sole measurable eect, as the hydrolysis ratio between the various donors was also altered, and the ratio between the activities of the three enzymes was modi®ed. Speci®cally, the relative amounts of substrate hydrolysed and hydrolysis byproducts formed in the presence of saliva obtained from each donor were modi®ed by fractionation, as indicated by the dierent order of the data in Fig. 3(a) and (c) (both arranged as a function of decreasing hydrolysis). The fractionation-associated dierences in the formation of hydrolysis by-products indicate that the dierences in the hydrolysis of the substrate can be attributed to variations in the relative activity of the three enzyme classes: measured at 600 s, the average per cent ratio of aminopeptidases to dipeptidylaminopeptidases to dipeptidylcarboxypeptidases changed from 61.2:23.9:14.9 in whole samples to 51.7:35.2:12.9 in fractionated samples. Therefore, in fractionated saliva the activity of dipeptidylaminopeptidases increased to 148% of the value measured in whole samples, while the activity of aminopeptidases and dipeptidylcarboxypeptidases decreased to 85% and
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87% of the value measured in non-fractionated samples, respectively. The above shifts between the relative amount of hydrolysis by-products formed in non-fractionated samples and that formed after fractionation also increased the sex-related dierences. Separately measured in male and female saliva, the per cent ratio between the activity of the three enzyme classes (aminopeptidase to dipeptidylaminopeptidases to dipeptidylcarboxypeptidases), which was 62.7:22.6:15.3 in male non-fractionated samples, became 59.3:26.0:14.7 after fractionation; the same ratio changed from 58.1:29.2:12.8 to 43.7:42.9:13.5 in female non-fractionated and fractionated samples, respectively [Fig. 3(b), (d)]. Therefore, the dierence between whole and fractionated samples was perceptibly higher in female than in male saliva. Speci®cally, fractionation induced a 5.4% and 24.8% decrease in the activity of aminopeptidases in male and female saliva, respectively; a 15.8% and 46.9% increase in the activity of dipeptidylaminopeptidases; a 3.9% and 5.5% decrease in the activity of dipeptidylcarboxypeptidases. The statistical signi®cance of the fractionation-induced variations was analysed separately for male and female saliva by one-way ANOVA, obtaining the following results: for male donors aminopeptidase F = 5.094, p = 0.087; dipeptidylaminopeptidases F = 11.555, p = 0.027; dipeptidylcarboxypeptidases F = 2.795, p = 0.331. For female donors: aminopeptidase F = 14.768, p = 0.018; dipeptidylaminopeptidases F = 26.900, p = 0.007; dipeptidylcarboxypeptidases F = 1.670, p = 0.266. ANOVA results indicate that the fractionation-induced variations in the relative activity of dipeptidylaminopeptidases were statistically signi®cant (i.e. p = 0.05) in both cases, and more signi®cant in females than in males, while variations in aminopeptidase activity were statistically signi®cant in female samples only. Variations in the activity of dipeptidylcarboxypeptidases were not signi®cant in either sex. 3.4. Proteolysis inhibitors The data reported above, indicating statistically signi®cant dierences between the hydrolysis pattern of whole and fractionated samples, might indicate the presence in saliva of low molecular-weight proteolysis inhibitors. As these substances, if present, must be separated from the enzymes by the fractionation procedure, their removal could account for the dierences noted above. To verify the possible presence of such inhibitors, enzyme-active fractions were separated from the low molecular-weight components; these were pooled and re-concentrated as described under ``Low molecular weight inhibitors'', and substrate hydrolysis was measured comparatively in the presence and absence of these substances.
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Enzyme activity can be modi®ed as a consequence of the steps necessary for separating enzymes from low molecular-weight substances. Substrate hydrolysis was, therefore, measured in the presence of K'1.09 saliva (i.e. the enzyme-active fractions) plus equal volumes either of HEPES buer, or of one of the three pools of high elution-volume substances, prepared as described under ``Low molecular weight inhibitors'' [Fig. 2(a)]. The results obtained (Fig. 4), although necessarily dierent from those obtained with unprocessed samples [Fig. 1(a)], indicate that hydrolysis measured in the presence of pool 1 or pool 3 was nearly identical to that measured in the presence of buer. On the contrary, in the presence of pool 2 (i.e. the substances eluted between K'=1.69 and K '=2.28), the amount of substrate hydrolysed, and of all three labelled hydrolysis by-products formed, was smaller. As measured at 600 s, the substrate hydrolysed decreased from 46% to 30% of the total peptide in reaction (i.e. to 65% of the
control); the amounts of Tyr, Tyr±Gly and Tyr±Gly± Gly released decreased from 25% to 19%, from 12% to 7% and from 7% to 3% of the total (i.e. to 76%, 58% and 43% of the control), respectively. These results appear to indicate the existence of substances capable of inhibiting the substrate-degrading enzymes within the high K' (i.e. low molecular weight) components of human saliva. In addition, although the high activity of aminopeptidases makes their inhibition more relevant in terms of substrate hydrolysis, the inhibitors were comparatively more active on the dipeptidylpeptidases. 4. Discussion Our data indicate that, in the presence of human saliva, leu-enkephalin was partially hydrolysed. Peptide degradation was paired with the formation of hydroly-
Fig. 4. Degradation of leu-enkephalin and formation of its labelled hydrolysis by-products as a function of reaction time in the presence of donor 6's K'1.09 saliva and: HEPES buer (thin solid line and open symbols); pool 1 (thin dashed lines and open symbols); pool 2 (thick solid lines and solid symbols); pool 3 (thin dotted lines and hollow symbols). Pools identi®ed in Fig. 2(a), assay conditions as described under ``Low molecular-weight inhibitors''. Symbols identify experimental points. Crosses, leu-enkephalin; circles, Tyr; lozenges, Tyr±Gly; squares, Tyr±Gly±Gly. Curves through data-points were interpolated as described under Section 2.
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sis by-products whose composition indicates the presence of all three classes of enzymes known to degrade this substrate in mammalian tissues: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. The activity of these enzymes was moderately, but signi®cantly, higher in male than in female saliva. These dierences are similar to, although not as evident as, those reported for other substances synthesized by the salivary glands. In this tissue, sexrelated dierences are directed towards higher activities (or concentrations), which are observed more often in males (e.g. Levi-Montalcini, 1958; Rougeot et al., 1994) and less frequently in females (BlaÈuer et al., 1996). The presence of low molecular-weight substances able to inhibit the substrate-degrading enzymes was also detected. Sex-related dierences were also observed for the inhibitors, which were more active in female than in male saliva. The sum of the data obtained can be interpreted as indicating that the sex-related dierences in the hydrolysis of the substrate, and in the pattern of the hydrolysis by-products, are induced by higher activity of the substrate-degrading enzymes in male saliva, together with higher activity of the low molecularweight inhibitors in female saliva. A comparative analysis of the data obtained with whole and fractionated saliva indicates that in male saliva chromatographic fractionation modi®ed only very modestly the hydrolysis ratio between the three enzyme classes (from 4.1:1.5:1.0 in whole samples to 4.0:1.8:1.0 in fractionated samples, aminopeptidases to dipeptidylaminopeptidases to dipeptidylcarboxypeptidases). In female saliva, the eect of fractionation was much more evident: the enzyme ratio changed from 4.5:2.3:1.0 in whole samples to 3.2:3.2:1.0 in fractionated samples. After steric exclusion fractionation, competition between the dierent enzyme species was reduced; in addition, the low molecular-weight inhibitors were separated from the enzymes, so their activity was at least drastically reduced, and possibly totally eliminated. Although it is quite dicult to distinguish the eects of the above two factors, the data obtained with fractionated saliva indicate that the sex-related dierences in enzyme activity increased with respect to those observed in whole samples. Speci®cally, in female saliva the activity of dipeptidylaminopeptidases was notably higher, and that of aminopeptidases lower, than in male saliva; this dierence was considerably increased by fractionation. As aminopeptidases are more active than the other enkephalin-degrading enzymes (Hambrook et al., 1976), this dierence may, at least in part, contribute to the lower overall hydrolysis observed in the samples obtained from female donors. According to this analysis, in female saliva the activity of the enzymes per se is dierent from that found in male saliva; however, the presence of inhibi-
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tors combines with competition to increase the activity of aminopeptidases and reduce that of dipeptidylaminopeptidases, inducing a hydrolysis pattern that is similar to that observed in male saliva. This phenomenon resembles that already described in plasma, where the hydrolysis pattern is controlled to a notable extent by the low molecular-weight inhibitors (Bolacchi et al., 1995). The above data have been discussed under the assumption that all proteolyses found in saliva are actually released by the salivary glands. It is, however, necessary to observe that proteolytic enzymes released by oral micro-organisms (e.g. Nishikata et al., 1991) might contribute to the hydrolysis of the model substrate. The technique used for drawing samples, as well as the lack of characterization of the bacterial content of the samples analysed, does not exclude that a part of the enzyme activities described might actually derive from bacterial sources. However, it seems at least unlikely that proteinases of bacterial origin could induce a pattern of enkephalin-degrading enzymes so similar within the dierent donors as that found here and, particularly, similar to that found in other human tissues (Hambrook et al., 1976; Marks et al., 1977; Roscetti et al., 1985). Even more unlikely is the notion that bacteria-released enzymes could induce the sex-related dierences shown here. The dierences, once again, are similar to those found in other active proteins secreted by the salivary glands (Rougeot et al., 1994; BlaÈuer et al., 1996) under endocrine control (Kurachi and Oka, 1985; Black et al., 1992; BlaÈuer et al., 1996). Therefore, whereas it seems possible that proteolyses from oral bacteria could modify the base enzyme pattern, it seems unlikely that they determine it. A dierent source of variation can be found in the sample population itself. Indeed, no attempt was made to synchronize the taking of female saliva samples with the menstrual cycle. As synthesis and release of sexspeci®c substances by the salivary glands are controlled by hormones, including steroid hormones (Kurachi and Oka, 1985; Black et al., 1992), the pattern of enkephalin-degrading enzymes, and possibly the inhibition pattern as well, might also depend on the menstrual cycle (and, presumably, on age). Therefore, the sex-related dierences reported here could be quantitatively dierent from those that might be observed if the menstrual cycle was taken into account. From a functional point of view, it appears that, like other proteins, enkephalin-degrading enzymes must be transported across the plasma membrane by dedicated mechanisms. Consequently, their presence in saliva can be assumed to indicate the existence of speci®c mechanisms of regulative degradation of salivary neuropeptides. The simultaneous presence of enzymes able to hydrolyse leu-enkephalin, together
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with their inhibitors, suggests that active levels of saliva neuropeptides can be controlled by homeostatic mechanisms similar to those that have been described in other tissues, such as plasma (Roda et al., 1987), or in immunocompetent cells (e.g. Amoscato et al., 1989). The sex-related dierences reported here also appear to support, although indirectly, this hypothesis. Indeed, the dierences observed are similar to, although less evident than, those referred to in Section 1 concerning several active proteins synthesized in the salivary glands (Barka et al., 1980; Rougeot et al., 1994; BlaÈuer et al., 1996), whose activity in regulating mucosal morphology and functions has been ascertained (Sarosiek et al., 1991). Finally, the presence of multiple enzymes may, again indirectly, support the hypothesis of an active role of saliva neuropeptides. A multiplicity of neuropeptide-active enzymes is known in which similar regulative mechanisms have been described, such as for plasma, both membrane (Amoscato et al., 1989; Mari et al., 1992) and soluble (Marini et al., 1992) immunocompetent-cell proteolyses, the gastrointestinal tract (Cohen et al., 1983) and the respiratory tract (e.g. Sekizawa et al., 1987; De Gouw et al., 1996). It is, ®nally, worth noting that the very limited three-dimensional structure in polar solvent that is characteristic of small peptides, notably enkephalins (e.g. Temussi et al., 1987), implies a low substrate speci®city. This has actually been observed, as in the case of the hydrolysis of neurotensin (Checler et al., 1983), gastrin (Deschodt-Lanckman et al., 1988), substance P and enkephalins (Matsas et al., 1983) by endopeptidase 24.11, or the hydrolysis of several peptides by endopeptidase 24.16 and 24.15 (Mentlein and Dahms, 1994). Therefore, at least in principle, the data reported here, obtained using leu-enkephalin as a model, may be extended to other neuropeptides and small proteins whose presence in saliva is ascertained, and that are involved in the transmission of information (Connelly et al., 1985; Pierart et al., 1988; Drapeau et al., 1991), mediate secretory eects (e.g. Tobin et al., 1995; Holzer et al., 1997) or trophic eects (Mansson et al., 1990), or ful®l any of the many other roles of saliva neuropeptides, not all of which have yet been ascertained.
References Aalto, Y., Kjorell, U., Henriksson, R., Franzen, L., Forsgren, S., 1997. Bombesin-like peptide is present in duct cells in salivary glands: studies on normal and irradiated animals. Neuropeptides 31, 167±173. Aloe, L., Alleva, E., BoÈhm, A., Levi-Montalcini, R., 1986. Aggressive behavior induces release of nerve growth factor from mouse salivary gland into the bloodstream. Proc. Natl. Acad. Sci. USA 83, 6184±6187.
Amoscato, A.A., Alexander, J.W., Babcock, G.F., 1989. Surface aminopeptidase activity of human lymphocytes. J. Immunol. 142, 1245±1252. Babst, R., Bongiorno, L., Marini, M., Roda, L.G., Spagnoli, G., Urbani, A., 1999. Trauma decreases leucine enkephalin hydrolysis in human plasma. J. Pharmacol. Exp. Ther. 288, 766±773. Backkelund Pedersen, E., Poulsen, K., 1983. Aggression-provoked huge release of submaxillary mouse renin to saliva. Acta Endocrinol. 104, 510±512. Barka, T., 1980. Biologically active polypeptides in the submandibular glands. J. Histochem. Cytochem. 28, 836±859. Basak, A., Ernst, B., Brewer, D., Seidah, N.G., Munzer, J.S., Lazure, C., Lajoie, G.A., 1997. Histidine-rich human salivary peptides are inhibitors of proprotein convertase furin and PC7 but act as substrate for PC1. J. Pept. Res. 49, 596±603. Berg, T., Johansen, L., Nustad, K., 1985. Enzymatic activity of rat submandibular gland kallikrein released into blood. Am. J. Physiol. 249, H1134±H1142. Berg, T., Johansen, L., Poulsen, K., 1990. Exocrine and endocrine release of kallikrein after re¯ex-induced salivary secretion. Acta Physiol. Scand. 139, 29±37. Bing, J., Poulsen, K., 1979. Dierences in renal and submaxillary renin release after stimulation with isoprenaline and noradrenaline. Acta Physiol. Scand. 105, 58±63. Black, M.A., Lefebvre, F.-A., Pope, L., Lefebvre, Y.A., Walker, P., 1992. Thyroid hormone and androgen regulation of nerve growth factor gene expression in the mouse submandibular gland. Mol. Cell. Endocrinol. 84, 145±154. BlaÈuer, M., Wichmann, L., Punnonen, R., Tuohimaa, P., 1996. Measurement of activin B in human saliva and localization of activin subunits in rat salivary gland. Biochem. Biophys. Res. Comm. 222, 230±235. Bolacchi, F., Marini, M., Urbani, A., Roda, L.G., 1995. Enzymes and inhibitors of leu-enkephalin metabolism in human plasma. Neurochem. Res. 20, 991±999. Checler, F., Vincent, J.P., Kitabgi, P., 1983. Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-convertin enzyme, and other unidenti®ed peptididases. J. Neurochem. 41, 375±384. Cohen, M.L., Geary, L.E., Wiley, K.S., 1983. Enkephalin degradation in the guinea-pig ileum: eect of aminopeptidase inhibitors, puromycin and bestatin. J. Pharmacol. Exp. Ther. 224, 379±385. Connelly, J.C., Skidgel, R.A., Schultz, W.W., Johnson, A.R., ErdoÈs, E.G., 1985. Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. Proc. Natl. Acad. Sci. USA 82, 8737±8741. Craves, F.B., Law, P.Y., Hunt, C.A., Loh, H.H., 1978. The metabolic disposition of radiolabeled enkephalins in vitro and in situ. J. Pharmacol. Exp. Ther. 206, 492±506. De Gouw, H.W.F.M., Diamant, Z., Kuijpers, E.A.P., Sont, J.K., Sterk, P.J., 1996. Role of neutral endopeptidase in exercise-induced bronchoconstriction in asthmatic subjects. J. Appl. Physiol. 81, 673±678. Deschodt-Lanckman, M., Pauwels, S., Najdovsky, T., Dimaline, R., Dockray, J., 1988. In vitro and in vivo degradation of human gastrin by endopeptidase 24.11. Gastroenterology 94, 712±721.
M. Marini, L.G. Roda / Archives of Oral Biology 45 (2000) 775±786 Drapeau, G., Chow, A., Ward, P.E., 1991. Metabolism of bradykinin analogs by angiotensin I converting enzyme and carboxypeptidase. N. Peptides 12, 631±638. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28, 350±356. EkstroÈm, J., Ekman, R., HaÊkanson, R., SjoÈgren, S., Sundler, F., 1988. Calcitonin gene-related peptide in the salivary glands: neuronal localization, depletion upon nerve stimulation, and eects on salivation in response to substance P. Neuroscience 26, 933±949. Garrett, J.R., 1987. The proper role of nerves in salivary secretion: a review. J. Dent. Res. 66, 387±397. Hambrook, J.M., Morgan, B.A., Rance, M.J., Smith, C.F., 1976. Mode of deactivation of the enkephalins by rat and human plasma and rat brain homogenates. Nature 262, 782±783. Holzer, P., Holzer-Petsche, U., 1997. Tachykinins in the gut. Part II. Roles in neural excitation, secretion and in¯ammation. Pharmacol. Ther. 73, 219±263. Itzhaki, R.F., Gill, D.M., 1964. A micro-biuret method for estimating proteins. Anal. Biochem. 9, 401±410. Johnson, A.R., Ashton, J., Schulz, W.W., ErdoÈs, E.G., 1985. Neutral metalloendopeptidase in human lung tissue and cultured cells. Am. J. Respir. Dis. 132, 564±568. Kamada, M., Furuhata, N., Yamaguchi, T., Ikekita, K., Kizuki, K., Moriya, H., 1990. Observation of tissue prokallikrein activation by some serine proteases, arginine esterases in rat submandibular gland. Biochem. Biophys. Res. Comm. 166, 231±237. Kurachi, H., Oka, T., 1985. Regulation of the level of epidermal growth factor by oestrogen in the submandibular gland of female mice. J. Endocr. 109, 221±225. Lemoine, A.P., Armando, I., Brun, J.C., Sgura, E.T., Barontini, M., 1990. Footshock aects heart and brain MAO and MAO inhibitory activity and open ®eld behavior in rats. Pharmacol. Biochem. Behav. 36, 85±88. Levi-Montalcini, R., 1958. Chemical stimulation of nerve growth factor. In: McElroy, W.D., Glass, B. (Eds.), Chemical Basis of Development. Johns Hopkins Press, Baltimore, pp. 646±664. MacQueen, G., Marshall, J., Perdue, M., Siegel, S., Bienenstock, J., 1989. Pavlovian conditioning of rat mucosal mast cells to secrete rat mast cell protease II. Science 243, 83±85. Mansson, B., Nilsson, B.O., EkstroÈm, J., 1990. Eects of repeated infusion of substance P and vasoactive intestinal peptide on the weights of salivary glands subjected to atrophying in¯uences in rats. Br. J. Pharmacol. 101, 853±858. Mari, B., Checler, F., Ponzio, G., Peyron, J.-F., Manie, S., Farahifar, D., Rossi, B., Auberger, P., 1992. Jurkat T cells express a functional neutral endopeptidase activity (CALLA) involved in T cell activation. EMBO J. 11, 3875±3885. Marini, M., Urbani, A., Bongiorno, L., Roda, L.G., 1992. Hydrolysis and association of leucine enkephalin to lymphomic and erythroleukaemic cell lines, III: separation of enzymes released by cells. Int. J. Immunopharmac. 14, 995±1002. Marks, N., Grynbaum, A., Neidle, A., 1977. On the degradation of enkephalins and endorphins by rat and mouse
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brain extracts. Biochem. Biophys. Res. Comm. 74, 1552± 1559. Matsas, R., Fulcher, I.S., Kenny, A.J., Turner, A.J., 1983. Substance P and [Leu] enkephalin are hydrolyzed by an enzyme in pig caudate synaptic membranes identical with the endopeptidase of kidney microvilli. Proc. Natl. Acad. Sci. USA 80, 3111±3115. Mentlein, R., Dahms, P., 1994. Endopeptidase 24.16 and 24.15 are responsible for the degradation of somatostatin, neurotensin, and other neuropeptides by cultivated rat cortical astrocytes. J. Neurochem. 62, 27±36. Nishikata, M., Kanehira, T., Oh, H., Tani, H., Tazaki, M., Kuboki, Y., 1991. Salivary histatin as an inhibitor of a protease produced by the oral bacterium Bacteroides gingivalis. Biochem. Biophys. Res. Comm. 174, 625±630. Orstavik, T.B., Carretero, O.A., Scicli, A.G., 1982. Kallikrein±renin system in regulation of submaxillary gland blood ¯ow. Am. J. Physiol. 242, H1010±H1014. Perinpanayagam, H.E., Van Wuickhuse, B.C., Ji, Z.S., Tabak, L.A., 1995. Characterization of low-molecularweight peptides in human parotid saliva. J. Dent. Res. 74, 345±350. Pierart, M.E., Najdovski, T., Appelboom, T.E., DeschodtLanckman, M.M., 1988. Eect of human endopeptidase 24.11 (``enkephalinase'') on IL-1-induced thymocyte proliferation activity. J. Immunol. 140, 3808±3811. Roda, L.G., Tomasi, M., Battistini, A., Luzzi, I., Mastrantonio, P., Zampieri, A., D'Agnolo, G., 1977. Heterogeneity of puri®ed cholera toxin. Biochem. Biophys. Acta 492, 303±315. Roda, L.G., Venturelli, F., Roscetti, G., 1987. Hydrolysis and protection from the hydrolysis of circulating enkephalins. Comp. Biochem. Physiol. 85 (C), 449±454. Roscetti, G., Possenti, R., Bassano, E., Roda, L.G., 1985. Mechanisms of leu-enkephalin hydrolysis in human blood. Neurochem. Res. 10, 1393±1404. Rougeot, C., Rosinski-Chupin, I., Njamkepo, E., Rougeon, F., 1994. Selective processing of submandibular rat 1 protein at dibasic cleavage sites. Salivary and bloodstream secretion products. Eur. J. Biochem. 219, 765±773. Santavirta, N., Konttinen, Y.T., ToÈrnwall, J., Sogerberg, M., Santavirta, S., Matucci-Cerinic, M., BjoÈrwell, I., 1997. Neuropeptides of the autonomic nervous system in SjoÈgren's syndrome. Ann. Rheum. Dis. 56, 737±740. Sarosiek, J., Feng, T., McCallum, R.W., 1991. The interrelationship between salivary epidermal growth factor and the functional integrity of the esophageal mucosal barrier in the rat. Am. J. Med. Sci. 302, 359±363. Sekizawa, K., Tamaoki, J., Graf, P.D., Basbaum, C.B., Borson, D.B., Nadel, J.A., 1987. Enkephalinase inhibitor potentiates mammalian tachykinin-induced contraction in ferret trachea. J. Pharmacol. Exp. Ther. 243, 1211±1217. Temussi, P.A., Tancredi, T., Pastore, A., Castiglione-Morelli, M.A., 1987. Experimental attempt to simulate receptor site environment. A 500-MHz 1H nuclear magnetic resonance study of enkephalin amides. Biochemistry 26, 7856±7863. Tichen, D., Reid, A.M., 1990. Non-adrenergic, non-cholinergic control of salivary gland function. In: Young, J.A., Wong, P.Y.D. (Eds.), Epithelial Secretion of Water and Electrolytes. Springer, Berlin, pp. 217±228. Tobin, G., AszteÂly, A., Edwards, A.V., EkstroÈm, J.,
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HaÊkanson, R., Sundler, F., 1995. Presence and eects of pituitary adenylate cyclase activating peptide in the submandibular gland of the ferret. Neuroscience 66, 227±235. van't Hof, W., Blankenvoorde, M.F.J., Veerman, E.C.I., Nieuw Amerongen, A.V., 1997. The salivary lipocalin von Ebner's gland protein is a cysteine proteinase inhibitor. J. Biol. Chem. 272, 1837±1841.
Young, E.A., Houghten, R.A., Akil, H., 1989. Degradation of [3H]b-endorphin in rat plasma is increased with chronic stress. Eur. J. Pharmacol. 167, 229±236. Xu, L., Lal, K., Santarpia, R.P., Pollock, J.J., 1993. Salivary proteolysis of histidine-rich polypeptides and the antifungal activity of peptide degradation products. Arch. Oral. Biol. 38, 277±283.
www.elsevier.com/locate/archoralbio
Neuropeptide enzyme hydrolysis in human saliva Mario Marini, L. Giorgio Roda* Dipartimento di Neuroscienze, UniversitaÁ degli Studi di Roma ``Tor Vergata'', 00133 Roma, Italy Accepted 21 March 2000
Abstract The possible hydrolysis of neuropeptides by human saliva was studied using leucine enkephalin as a model. The data obtained indicate that in the presence of saliva this substrate is partially hydrolysed, and that its disappearance corresponds to the appearance of peptides whose composition is consistent with that of the substrate hydrolysis byproducts. The formation of these peptides indicates the presence of all three classes of enzymes known to hydrolyse enkephalins in other tissues: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. The activity of these enzymes appears to be altered by the presence of low molecular-weight substances, whose inhibitory activity is apparent on all three classes of enkephalin-degrading enzymes. Substrate degradation was higher in male than female saliva; these dierences appear to be caused by lower activity of the enzymes, and higher activity of the low molecular-weight inhibitors, measurable in female as compared to male saliva. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Neuropeptides; Enkephalins; Enzyme hydrolysis; Endogenous inhibitors
1. Introduction The human salivary glands synthesize and release several families of peptides (Barka, 1980), some of which, such as histatins (Xu et al., 1993), are described as saliva-speci®c. Many salivary peptides derive from in loco hydrolysis of precursor proteins (Perinpanayagam et al., 1995). Others [e.g. angiotensin II, vasoactive intestinal peptide, substance P, and gastrinreleasing peptide (Aalto et al., 1997)] belong to the family of brain±gut peptides, whose synthesis and release is controlled by the nervous system (Garrett, 1987). Not all of the physiological roles of salivary neuropeptides are easy to understand, however. Their
* Corresponding author. Tel.: +39-6-7259-6410; fax: +396-7259-6407. E-mail address: [email protected] (L.G. Roda).
most obvious function, to mediate the nervous control of saliva secretion, is supported by experimental data: according to EkstroÈm et al. (1988), tachykinins are transported by parasympathetic nerve ®bres to the salivary glands, where they regulate secretory functions (Holzer and Holzer-Petsche, 1997). A secretory role has also been described for adenylate cyclase-activating peptide by Tobin et al. (1995), and for vasoactive intestinal peptide by Tichen and Reid (1990). In addition, neuropeptides may have trophic eects on the salivary glands, such as those described by Mansson et al. (1990) for vasoactive intestinal peptide and substance P in the rat parotid. This kind of precise information is not available, unfortunately, for all of the neuropeptides present in saliva. It is possible, even obvious, to hypothesize that the presence of dierent mediators contributes to regulate ®nely the secretion of the salivary glands. Yet, some of the available data do not agree with the hy-
0003-9969/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 0 ) 0 0 0 4 0 - 6
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pothesis that all salivary neuropeptides are directly released by nerve terminals. Aalto et al. (1997) report an increased concentration of bombesin-like material in the salivary ducts of rats in which bombesin immunoreactive nerve ®bres had been experimentally reduced. They interpreted this ®nding as indicating that bombesin can be released independently of axonal transport, and that this peptide pool is present in salivary ducts and is thus secreted in saliva. Similarly, Rougeot et al. (1994) suggest that peptides cleaved from submandibular rat 1 protein are selectively released in saliva (as well as in the bloodstream) following sympathetic stimulation. These data suggest that neuropeptides are not only released by, but also synthesized within, the salivary glands. Neuropeptides may also account for the control of environmental factors on saliva composition, as has been described by Backkelund Pedersen and Poulsen (1983) for the release of renin, and by Santavirta et al. (1997) for the release of vasoactive intestinal peptide. The role of environmental factors appears to be of particular relevance for the endocrine functions of the salivary glands: this was described as early as 1979 for renin (Bing and Poulsen, 1979; also Backkelund Pedersen and Poulsen, 1983) and, later on, for kallikrein (Berg et al., 1985, 1990), nerve growth factor (Aloe et al., 1986), and the peptides originated by the proteolytic cleavage of submandibular rat 1 protein (Rougeot et al., 1994). It seems possible therefore that saliva neuropeptides are transported into the bloodstream, contributing to the formation of the very large pool of plasma informational peptides. In such a context, the role of behavioural factors in controlling the release of these peptides by the salivary glands may be similar to that reported for other substances, such as monoamine oxidase in the central nervous system (Lemoineet al., 1990), secretion of proteases by mast cells (MacQueen et al., 1989), or enzyme degradation of opioid peptides in plasma (Young et al., 1989; Babst et al., 1999). As is known for other neuropeptides, some of the above-mentioned eects are regulated by enzyme degradation. Salivary proteolytic enzymes have been identi®ed mostly on the basis of their hydrolysis byproducts [e.g. kallikrein by Orstavik et al. (1982); prokallikrein by Kamada et al. (1990); the release of active peptides via the hydrolysis of Arg±Arg bonds by Rougeot et al. (1994)]. However, the proteases described by Xu et al. (1993) appear to be involved in the regulatory degradation of active peptides: according to them, the hydrolysis of histatins by salivary endopeptidases gives rise to by-products less active than the intact peptides. In addition, as in other tissues, notably plasma (e.g. Bolacchi et al., 1995), the presence of proteolytic enzymes is coupled with that of protease inhibitors: according to Nishikata et al. (1991), histatin 5 inhibits the trypsin-like protease released by Bacteroides gingi-
valis (and clostripain); Basak et al. (1997) showed that members of the histatin family have a (moderate) inhibitory activity on proprotein convertases; van't Hof et al. (1997) described the inhibition of cysteine proteinases by Von Ebner's gland lipocalin. These investigators suggest that this protein might have a role in controlling in¯ammatory processes via the inhibition of bacterial proteases. The co-presence in saliva of neuropeptides, proteases active on these peptides, and inhibitors of these proteases, suggests the existence of speci®c mechanisms capable of controlling the active concentration of neuropeptides. Similar processes are known to occur in various tissues, such as plasma (e.g. Roda et al., 1987), or in the respiratory tract (e.g. Johnson et al., 1985). Speci®cally, a regulatory role has been attributed to the enzyme proteolysis of active plasma peptides (e.g. Connelly et al., 1985; Pierart et al., 1988). The data that follow attempt to verify the possible existence of such mechanisms in human saliva using leucine enkephalin as a model substrate. The hydrolysis characteristics of this peptide in other tissues are known in detail (Hambrook et al., 1976; Marks et al., 1977; Roscetti et al., 1985). In addition, because of its small size, the pattern of its hydrolysis by-products is relatively simple. Therefore, even if the presence in saliva of leuenkephalin has not been described, it appears to be a suitable model for ascertaining the presence, distribution, and possible inhibition of enzymes capable of hydrolysing saliva neuropeptides. 2. Material and methods 2.1. Sample population The sample studied consisted of six individuals, three men and three women, mean age 32.2 years (SD 5.9). All donors were in good health, all were nonsmokers; the presence of in¯ammatory oral processes was speci®cally excluded. The administration of pharmacologically active substances was excluded for 5 days before taking the samples, as were oral contraceptives in the case of female donors. 2.2. Saliva collection Saliva samples were collected in 50-ml test-tubes, immediately transferred to an ice bath, dialysed at 48C against 10 mM HEPES, 100 mM NaCl, 2.5 10ÿ5 M ZnCl2 pH 7.2 (referred to henceforth as HEPES buffer), and centrifuged (10 min at 12,000 g). Samples to be fractionated by steric-exclusion chromatography were concentrated to 1/10 of the original volume on a PLCC membrane (Millipore Co., Bedford, MA, USA). All samples were used within 24 h of collection.
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2.3. Assay Enkephalin-degrading enzymes were identi®ed, and their activity was quanti®ed, on the basis of the labelled hydrolysis by-product formed. Portions (10 ml) of dialysed samples were transferred to 2-ml tapered test-tubes; tritiated leu-enkephalin (2 ml, corresponding to 3.4 10ÿ10 mol) was added to each sample. Reactions were carried out at 378C for the periods of time indicated in the ®gures (600 s for column eluent), and stopped with 3.5 ml of acetic acid. Intact leu-enkephalin and its labelled hydrolysis by-products were quanti®ed by thin-layer chromatography as described below. Proteins were determined by the micro-biuret method described by Itzhaki and Gill (1964). Carbohydrates were determined by the phenol±H2SO4 method described by Dubois et al. (1956) and modi®ed as described by Roda et al. (1977). 2.4. Thin-layer chromatography This was performed on aluminium-backed Kieselgel 60 sheets (Merck, Darmstadt, Germany) developed at 50 8C with 58:10:30:0.17 2-butyl alcohol:acetic acid:water:acetonitril. Sheets were cut according to internal standards, transferred to scintillation vials, and counted for tritium as described by Bolacchi et al. (1995). 2.5. Enzyme fractionation Enzymes were fractionated on a 5-mm 7.8 300 mm TSK G3000SW column (Toyo Soda Ltd, Tokyo, Japan) equilibrated in HEPES buer. Samples (350 ml) were eluted at 2.2 ml/cm2 per min with the same buffer. Absorbance at 280 nm was monitored, and 0.25ml fractions were collected and assayed as described above. 2.6. Low molecular weight inhibitors These were prepared with saliva obtained from a single donor (donor 6) as follows: low molecularweight substances were separated from enzymes on a TSK G3000SW column, equilibrated and eluted as described above, except that the buer used was 5 mM HEPES, pH 7.2, without the addition of NaCl [Fig. 2(a)]. The fractions (1 ml) eluted between K '=0 and K'=1.09 (referred to henceforth as K1' .09) were pooled, concentrated to their original volume in collodion bags (Sartorius AG, Gottingen, Germany), and used as the source of enzymes for subsequent assay. The fractions eluted from K '=1.09 up to K '=1.69 (pool 1) were concentrated by ultra®ltration on a PLCC membrane, the retentate was discarded and the
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eluent was concentrated in a rotating evaporator. The fractions eluted up to K'=2.28 (pool 2) and K'=2.73 (pool 3) were concentrated in a rotating evaporator only. 2.7. Data analysis One-way completely randomized ANOVA were performed with commercial software (Costat, Cohort Software, Minneapolis, MN, USA). Regression analyses were performed using a linear polynomial procedure of variable degree. Hydrolysis curves were interpolated with a non-linear iterative procedure (Marquardt±Levemberg), using the exponential equation y a e bx c e dx e: Parametric statistics were calculated according to standard procedures. 2.8. Material Leu-enkephalin, Tyr±Gly and Tyr±Gly±Gly were obtained from Bachem Feinchemikalien AG (Bubendorf, Switzerland), and Tyr from Serva Feinbiochemica GmbH (Heidelberg, Germany). Tritiated leuenkephalin (sp. act. 1.03 1011 Bq/mol), was prepared by isotope exchange; radiochemical purity of the tritiated peptide was checked by reverse-phase and thinlayer chromatography. All other material was obtained through local suppliers, and used without further puri®cation. 3. Results 3.1. Hydrolysis in whole saliva The possible hydrolysis of leu-enkephalin was measured by incubating the tritiated peptide in the presence of saliva. In samples obtained from all donors, the substrate concentration decreased timedependently, with the corresponding appearance of Tyr, Tyr±Gly, and Tyr±Gly±Gly (Fig. 1). The disappearance of the whole peptide, and the formation of the N-terminal leu-enkephalin hydrolysis by-products detected, can be assumed to indicate the presence of all three classes of enzymes known to hydrolyse enkephalins: aminopeptidases, dipeptidylaminopeptidases, and dipeptidylcarboxypeptidases (Hambrook et al., 1976; Craves et al., 1978). 3.1.1. Leu-enkephalin The curves describing the disappearance of leu-enkephalin over time [Fig. 1(a)] indicate that substrate hydrolysis was relatively fast in the ®rst 60±90 s, slowing down considerably afterwards. The average slope of the hydrolysis curves decreased from 11.21 between
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0 and 30 s to 0.30 between 240 and 1800 s, with a ¯ex point located between 40 and 65 s. Protein concentration was noticeably higher in male saliva (on average, 0.46 mg/ml) than in female saliva (0.20 mg/ml). The possible co-relation between total protein and substrate hydrolysis was, therefore, examined by performing linear regressions of variable degree between protein concentration and hydrolysis at 600 s. The results obtained (1st degree: r 2=0.60, F = 5.99, p = 0.07) were not statistically signi®cant ( p > 0.05); however, as they indicate a 93% probability of an associ-
ation between the two variables, these results strongly suggest the existence of such a co-relation. 3.1.2. Hydrolysis by-products Under our experimental conditions, the average substrate degraded at 600 s was 36% of the total in the reaction [Fig. 3(b)]. Of this, 61% was hydrolysed by aminopeptidases, 24% by dipeptidylaminopeptidases and 15% by dipeptidylcarboxypeptidases. As the hydrolysis curves were nearly parallel to each other (Fig. 1), this ratio was similar (within 210%) for the other reaction times. These data indicate a ratio
Fig. 1. Degradation of leu-enkephalin and formation of its labelled hydrolysis by-products in the presence of human saliva as a function of reaction time. (a) Leu-enkephalin; (b) Tyr; (c) Tyr±Gly; (d) Tyr±Gly±Gly. Dotted thick lines, female donors; dotted thin lines, male donors; solid thick lines, all donors (female and male). Symbols represent experimental points. Vertical bars represent statistical dispersion (21s). Curves were interpolated as described under Section 2.7.
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between the enzyme activities, and a predominance of aminopeptidases over the dipeptidylpeptidases (dipeptidylamino- and dipeptidylcarboxypeptidases), similar to that described in other tissues (Hambrook et al., 1976, Craves et al., 1978). 3.2. Sex-associated dierences The amount of substrate hydrolysed, and the ratio between the hydrolysis by-products formed, were dierent in salivas obtained from male and female donors. Speci®cally, as measured at 600 s, the amount
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of substrate hydrolysed, and the amount of Tyr, Tyr± Gly and Tyr±Gly±Gly formed in the presence of male saliva were 130%, 138%, 104% and 138% of the values measured in the presence of female saliva, respectively [Fig. 3(b)]. Similarly, the substrate half-life for female donors (1632 s, SD 167 s) was 128% of that for male donors [1277 s, SD 387 s; Fig. 1(a)]. The statistical signi®cance of these dierences was analysed by one-way ANOVA obtaining the following results: F 9:30 and p 0:053 for leu-enkephalin hydrolysis, F 17:60 and p 0:025 for the formation of Tyr, F 0:65 and p 0:478 for Tyr±Gly and F 3:61 and p 0:056
Fig. 2. Separation of enkephalin-degrading enzymes in human saliva by steric-exclusion chromatography. (a) Continuous line represents absorbance at 280 nm; dashed staircase represents protein as human serum albumin; dotted staircase represents carbohydrates as glucose. Solid horizontal bars represent pooled fractions; K identi®es K1' .09 pool; numerals 1, 2, and 3 identify the corresponding pool. (b) Aminopeptidases; (c) Dipeptidylaminopeptidases; (d) Dipeptidylcarboxypeptidases. Donors identi®ed in (b); thick lines represent female donors (f); thin lines represent male donors (m).
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for Tyr±Gly±Gly, respectively. These results indicate that there is a statistical probability of approx. 5% that the dierences observed between male and female donors in substrate degradation were only casual. They also indicate that these dierences were caused by highly signi®cant, sex-related, dierences in the activity of aminopeptidases, and by less signi®cant dierences in the activity of dipeptidylcarboxypeptidases. The dierences in the activity of dipeptidylaminopeptidases, and thus their contribution to the lowering of substrate hydrolysis in female saliva, were statistically non-signi®cant.
3.3. Hydrolysis in fractionated saliva To ascertain the approximate number and size distribution of the substrate-active enzymes, saliva samples were fractionated (Fig. 2); the activity of the separated fractions was then analysed [Fig. 2, panels (b)±(d)]. The substrate-active enzymes were eluted for the most part between approx. K'=0.25 and K'=0.76. This zone, unsurprisingly for a raw sample, did not correspond to speci®c features of the protein or carbohydrate elution pro®les [Fig. 2(a)]. However, the general shape of the elution envelopes of the column-
Fig. 3. Amount of leu-enkephalin degraded, and labelled hydrolysis by-products formed at 600 s in the presence of non-fractionated (a and b), and fractionated (c and d) human saliva, moles per cent. (a) and (c) represent individual donors, (b) and (d) represent averages. Open bars, leu-enkephalin; obliquely shaded bars, Tyr; vertically shaded bars, Tyr±Gly; solid bars, Tyr±Gly±Gly. Numerals identify individual donors; bold numerals, female donors; plain numerals, male donors.
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separated enzymes, a swell as many details, corresponded in the donors examined. This was particularly evident in the central region of the aminopeptidase elution envelope, where active species were eluted at K'=0.36, 0.45, 0.54 and 0.75 in nearly all donors [Fig. 2(b)]. The dipeptidylaminopeptidase elution envelopes for female donors were also similar to each other, with minor peaks matching below K '=0.40, and the main activity centred at K'=0.47±0.49. In contrast, in male saliva, the dipeptidylaminopeptidase elution envelopes appeared to be individual-speci®c. In this case, dipeptidylaminopeptidases were represented by two or three main peaks, located between K '=0.36 and K'=0.65, with few matching details, such as those located at K'=0.47, or the late activity at K '=0.80 [Fig. 2(c)]. Finally, the low and uniformly distributed activity of dipeptidylcarboxypeptidases did not permit the identi®cation of speci®c features [Fig. 2(d)]. The existence of corresponding features in the elution envelopes of dierent donors allows the hypothesis that these details may actually correspond to single enzyme species. The total amount of substrate hydrolysed, and the amounts of Tyr, Tyr±Gly and Tyr±Gly±Gly formed at 600 s by the fractionated enzymes, are shown in Fig. 3 [panels (c), (d)]. A comparison of these data with those in panels (a) and (b), measured under the same conditions but in the presence of non-fractionated saliva, indicated the existence of fractionation-associated variations of hydrolysis. As steric-exclusion techniques involve a considerable dilution of the original sample, the consequent lowering of enzyme concentration could account for the reduction of hydrolysis noticeable in fractionated as compared to whole samples. This was not, however, the sole measurable eect, as the hydrolysis ratio between the various donors was also altered, and the ratio between the activities of the three enzymes was modi®ed. Speci®cally, the relative amounts of substrate hydrolysed and hydrolysis byproducts formed in the presence of saliva obtained from each donor were modi®ed by fractionation, as indicated by the dierent order of the data in Fig. 3(a) and (c) (both arranged as a function of decreasing hydrolysis). The fractionation-associated dierences in the formation of hydrolysis by-products indicate that the dierences in the hydrolysis of the substrate can be attributed to variations in the relative activity of the three enzyme classes: measured at 600 s, the average per cent ratio of aminopeptidases to dipeptidylaminopeptidases to dipeptidylcarboxypeptidases changed from 61.2:23.9:14.9 in whole samples to 51.7:35.2:12.9 in fractionated samples. Therefore, in fractionated saliva the activity of dipeptidylaminopeptidases increased to 148% of the value measured in whole samples, while the activity of aminopeptidases and dipeptidylcarboxypeptidases decreased to 85% and
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87% of the value measured in non-fractionated samples, respectively. The above shifts between the relative amount of hydrolysis by-products formed in non-fractionated samples and that formed after fractionation also increased the sex-related dierences. Separately measured in male and female saliva, the per cent ratio between the activity of the three enzyme classes (aminopeptidase to dipeptidylaminopeptidases to dipeptidylcarboxypeptidases), which was 62.7:22.6:15.3 in male non-fractionated samples, became 59.3:26.0:14.7 after fractionation; the same ratio changed from 58.1:29.2:12.8 to 43.7:42.9:13.5 in female non-fractionated and fractionated samples, respectively [Fig. 3(b), (d)]. Therefore, the dierence between whole and fractionated samples was perceptibly higher in female than in male saliva. Speci®cally, fractionation induced a 5.4% and 24.8% decrease in the activity of aminopeptidases in male and female saliva, respectively; a 15.8% and 46.9% increase in the activity of dipeptidylaminopeptidases; a 3.9% and 5.5% decrease in the activity of dipeptidylcarboxypeptidases. The statistical signi®cance of the fractionation-induced variations was analysed separately for male and female saliva by one-way ANOVA, obtaining the following results: for male donors aminopeptidase F = 5.094, p = 0.087; dipeptidylaminopeptidases F = 11.555, p = 0.027; dipeptidylcarboxypeptidases F = 2.795, p = 0.331. For female donors: aminopeptidase F = 14.768, p = 0.018; dipeptidylaminopeptidases F = 26.900, p = 0.007; dipeptidylcarboxypeptidases F = 1.670, p = 0.266. ANOVA results indicate that the fractionation-induced variations in the relative activity of dipeptidylaminopeptidases were statistically signi®cant (i.e. p = 0.05) in both cases, and more signi®cant in females than in males, while variations in aminopeptidase activity were statistically signi®cant in female samples only. Variations in the activity of dipeptidylcarboxypeptidases were not signi®cant in either sex. 3.4. Proteolysis inhibitors The data reported above, indicating statistically signi®cant dierences between the hydrolysis pattern of whole and fractionated samples, might indicate the presence in saliva of low molecular-weight proteolysis inhibitors. As these substances, if present, must be separated from the enzymes by the fractionation procedure, their removal could account for the dierences noted above. To verify the possible presence of such inhibitors, enzyme-active fractions were separated from the low molecular-weight components; these were pooled and re-concentrated as described under ``Low molecular weight inhibitors'', and substrate hydrolysis was measured comparatively in the presence and absence of these substances.
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Enzyme activity can be modi®ed as a consequence of the steps necessary for separating enzymes from low molecular-weight substances. Substrate hydrolysis was, therefore, measured in the presence of K'1.09 saliva (i.e. the enzyme-active fractions) plus equal volumes either of HEPES buer, or of one of the three pools of high elution-volume substances, prepared as described under ``Low molecular weight inhibitors'' [Fig. 2(a)]. The results obtained (Fig. 4), although necessarily dierent from those obtained with unprocessed samples [Fig. 1(a)], indicate that hydrolysis measured in the presence of pool 1 or pool 3 was nearly identical to that measured in the presence of buer. On the contrary, in the presence of pool 2 (i.e. the substances eluted between K'=1.69 and K '=2.28), the amount of substrate hydrolysed, and of all three labelled hydrolysis by-products formed, was smaller. As measured at 600 s, the substrate hydrolysed decreased from 46% to 30% of the total peptide in reaction (i.e. to 65% of the
control); the amounts of Tyr, Tyr±Gly and Tyr±Gly± Gly released decreased from 25% to 19%, from 12% to 7% and from 7% to 3% of the total (i.e. to 76%, 58% and 43% of the control), respectively. These results appear to indicate the existence of substances capable of inhibiting the substrate-degrading enzymes within the high K' (i.e. low molecular weight) components of human saliva. In addition, although the high activity of aminopeptidases makes their inhibition more relevant in terms of substrate hydrolysis, the inhibitors were comparatively more active on the dipeptidylpeptidases. 4. Discussion Our data indicate that, in the presence of human saliva, leu-enkephalin was partially hydrolysed. Peptide degradation was paired with the formation of hydroly-
Fig. 4. Degradation of leu-enkephalin and formation of its labelled hydrolysis by-products as a function of reaction time in the presence of donor 6's K'1.09 saliva and: HEPES buer (thin solid line and open symbols); pool 1 (thin dashed lines and open symbols); pool 2 (thick solid lines and solid symbols); pool 3 (thin dotted lines and hollow symbols). Pools identi®ed in Fig. 2(a), assay conditions as described under ``Low molecular-weight inhibitors''. Symbols identify experimental points. Crosses, leu-enkephalin; circles, Tyr; lozenges, Tyr±Gly; squares, Tyr±Gly±Gly. Curves through data-points were interpolated as described under Section 2.
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sis by-products whose composition indicates the presence of all three classes of enzymes known to degrade this substrate in mammalian tissues: aminopeptidases, dipeptidylaminopeptidases and dipeptidylcarboxypeptidases. The activity of these enzymes was moderately, but signi®cantly, higher in male than in female saliva. These dierences are similar to, although not as evident as, those reported for other substances synthesized by the salivary glands. In this tissue, sexrelated dierences are directed towards higher activities (or concentrations), which are observed more often in males (e.g. Levi-Montalcini, 1958; Rougeot et al., 1994) and less frequently in females (BlaÈuer et al., 1996). The presence of low molecular-weight substances able to inhibit the substrate-degrading enzymes was also detected. Sex-related dierences were also observed for the inhibitors, which were more active in female than in male saliva. The sum of the data obtained can be interpreted as indicating that the sex-related dierences in the hydrolysis of the substrate, and in the pattern of the hydrolysis by-products, are induced by higher activity of the substrate-degrading enzymes in male saliva, together with higher activity of the low molecularweight inhibitors in female saliva. A comparative analysis of the data obtained with whole and fractionated saliva indicates that in male saliva chromatographic fractionation modi®ed only very modestly the hydrolysis ratio between the three enzyme classes (from 4.1:1.5:1.0 in whole samples to 4.0:1.8:1.0 in fractionated samples, aminopeptidases to dipeptidylaminopeptidases to dipeptidylcarboxypeptidases). In female saliva, the eect of fractionation was much more evident: the enzyme ratio changed from 4.5:2.3:1.0 in whole samples to 3.2:3.2:1.0 in fractionated samples. After steric exclusion fractionation, competition between the dierent enzyme species was reduced; in addition, the low molecular-weight inhibitors were separated from the enzymes, so their activity was at least drastically reduced, and possibly totally eliminated. Although it is quite dicult to distinguish the eects of the above two factors, the data obtained with fractionated saliva indicate that the sex-related dierences in enzyme activity increased with respect to those observed in whole samples. Speci®cally, in female saliva the activity of dipeptidylaminopeptidases was notably higher, and that of aminopeptidases lower, than in male saliva; this dierence was considerably increased by fractionation. As aminopeptidases are more active than the other enkephalin-degrading enzymes (Hambrook et al., 1976), this dierence may, at least in part, contribute to the lower overall hydrolysis observed in the samples obtained from female donors. According to this analysis, in female saliva the activity of the enzymes per se is dierent from that found in male saliva; however, the presence of inhibi-
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tors combines with competition to increase the activity of aminopeptidases and reduce that of dipeptidylaminopeptidases, inducing a hydrolysis pattern that is similar to that observed in male saliva. This phenomenon resembles that already described in plasma, where the hydrolysis pattern is controlled to a notable extent by the low molecular-weight inhibitors (Bolacchi et al., 1995). The above data have been discussed under the assumption that all proteolyses found in saliva are actually released by the salivary glands. It is, however, necessary to observe that proteolytic enzymes released by oral micro-organisms (e.g. Nishikata et al., 1991) might contribute to the hydrolysis of the model substrate. The technique used for drawing samples, as well as the lack of characterization of the bacterial content of the samples analysed, does not exclude that a part of the enzyme activities described might actually derive from bacterial sources. However, it seems at least unlikely that proteinases of bacterial origin could induce a pattern of enkephalin-degrading enzymes so similar within the dierent donors as that found here and, particularly, similar to that found in other human tissues (Hambrook et al., 1976; Marks et al., 1977; Roscetti et al., 1985). Even more unlikely is the notion that bacteria-released enzymes could induce the sex-related dierences shown here. The dierences, once again, are similar to those found in other active proteins secreted by the salivary glands (Rougeot et al., 1994; BlaÈuer et al., 1996) under endocrine control (Kurachi and Oka, 1985; Black et al., 1992; BlaÈuer et al., 1996). Therefore, whereas it seems possible that proteolyses from oral bacteria could modify the base enzyme pattern, it seems unlikely that they determine it. A dierent source of variation can be found in the sample population itself. Indeed, no attempt was made to synchronize the taking of female saliva samples with the menstrual cycle. As synthesis and release of sexspeci®c substances by the salivary glands are controlled by hormones, including steroid hormones (Kurachi and Oka, 1985; Black et al., 1992), the pattern of enkephalin-degrading enzymes, and possibly the inhibition pattern as well, might also depend on the menstrual cycle (and, presumably, on age). Therefore, the sex-related dierences reported here could be quantitatively dierent from those that might be observed if the menstrual cycle was taken into account. From a functional point of view, it appears that, like other proteins, enkephalin-degrading enzymes must be transported across the plasma membrane by dedicated mechanisms. Consequently, their presence in saliva can be assumed to indicate the existence of speci®c mechanisms of regulative degradation of salivary neuropeptides. The simultaneous presence of enzymes able to hydrolyse leu-enkephalin, together
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with their inhibitors, suggests that active levels of saliva neuropeptides can be controlled by homeostatic mechanisms similar to those that have been described in other tissues, such as plasma (Roda et al., 1987), or in immunocompetent cells (e.g. Amoscato et al., 1989). The sex-related dierences reported here also appear to support, although indirectly, this hypothesis. Indeed, the dierences observed are similar to, although less evident than, those referred to in Section 1 concerning several active proteins synthesized in the salivary glands (Barka et al., 1980; Rougeot et al., 1994; BlaÈuer et al., 1996), whose activity in regulating mucosal morphology and functions has been ascertained (Sarosiek et al., 1991). Finally, the presence of multiple enzymes may, again indirectly, support the hypothesis of an active role of saliva neuropeptides. A multiplicity of neuropeptide-active enzymes is known in which similar regulative mechanisms have been described, such as for plasma, both membrane (Amoscato et al., 1989; Mari et al., 1992) and soluble (Marini et al., 1992) immunocompetent-cell proteolyses, the gastrointestinal tract (Cohen et al., 1983) and the respiratory tract (e.g. Sekizawa et al., 1987; De Gouw et al., 1996). It is, ®nally, worth noting that the very limited three-dimensional structure in polar solvent that is characteristic of small peptides, notably enkephalins (e.g. Temussi et al., 1987), implies a low substrate speci®city. This has actually been observed, as in the case of the hydrolysis of neurotensin (Checler et al., 1983), gastrin (Deschodt-Lanckman et al., 1988), substance P and enkephalins (Matsas et al., 1983) by endopeptidase 24.11, or the hydrolysis of several peptides by endopeptidase 24.16 and 24.15 (Mentlein and Dahms, 1994). Therefore, at least in principle, the data reported here, obtained using leu-enkephalin as a model, may be extended to other neuropeptides and small proteins whose presence in saliva is ascertained, and that are involved in the transmission of information (Connelly et al., 1985; Pierart et al., 1988; Drapeau et al., 1991), mediate secretory eects (e.g. Tobin et al., 1995; Holzer et al., 1997) or trophic eects (Mansson et al., 1990), or ful®l any of the many other roles of saliva neuropeptides, not all of which have yet been ascertained.
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