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EFFECTS OF PSYCHOLOGICAL STRESS ON SERUM PROLYL ENDOPEPTIDASE AND DIPEPTIDYL PEPTIDASE IV ACTIVITY IN HUMANS: HIGHER SERUM PROLYL ENDOPEPTIDASE ACTIVITY IS RELATED TO STRESS-INDUCED ANXIETY
Psychoneuroendocrinology, Vol. 23, No. 5 pp. 485 – 495, 1998 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0306-4530/98 $19.00 + .00
PII: S0306-4530(98)00020-1
EFFECTS OF PSYCHOLOGICAL STRESS ON SERUM PROLYL ENDOPEPTIDASE AND DIPEPTIDYL PEPTIDASE IV ACTIVITY IN HUMANS: HIGHER SERUM PROLYL ENDOPEPTIDASE ACTIVITY IS RELATED TO STRESS-INDUCED ANXIETY Michael Maes1,2,3, Filip Goossens4, Ai-hun Lin1, Ingrid De Meester4, Ann Van Gastel1 and Simon Scharpe´4 1
Clinical Research Center for Mental Health (CRC-MH), 2060 Antwerp, Belgium 2 IRCCS, Istituto Fatebenefratelli, Brescia, Italy 3 Department of Psychiatry, Vanderbilt University, Nashville, TN, USA 4 Department of Medical Biochemistry, University of Antwerp, Wilrijk, Belgium
(Recei6ed 8 January 1998; in final form 24 March 1998)
SUMMARY There is now some evidence that psychiatric disorders, such as major depression, schizophrenia and post-traumatic stress disorder are associated with significant alterations in the serum activity of peptidases, such as prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV). The aims of the present study were to examine the effects of psychological stress on serum PEP and DPP IV activity in humans. Thirty-eight university students had repeated measurements of serum PEP and DPP IV activity a few weeks before and after (baseline conditions) as well as the day before a difficult academic examination (stress condition). Subjects were divided into anxiety responders and nonresponders to stress according to their stress-induced increase in the Spielberger State Anxiety Inventory. Serum PEP activity was somewhat lowered by stress in female, but not male, students. Serum PEP activity was significantly higher in the two baseline conditions and during the stress condition in anxiety responders than in anxiety nonresponders. There were no significant effects of stress on serum DPP IV activity and no significant differences between anxiety responders and nonresponders. Serum PEP and DPP IV activity were significantly higher in men than in women. The results suggest that increased baseline serum PEP activity is related to stress-induced anxiety. © 1998 Elsevier Science Ltd. All rights reserved. Keywords—Peptidases; Psychological stress; Prolyl endopeptidase; Dipeptidyl peptidase IV; Anxiety.
INTRODUCTION Recently, it has been shown that peptidases may play a role in the pathophysiology of various psychiatric disorders. Major depression is associated with lower serum activity of peptidases, such as prolyl endopeptidase (PEP, EC 3.4.21.26, post-proline cleaving enAddress correspondence and reprint requests to: M. Maes, University Department of Psychiatry, AZ Stuivenberg, 267 Lange Beeldekensstraat, 2060 Antwerp, Belgium (Fax: 32 3 4483265; E-mail: [email protected]). 485
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zyme, prolyl oligopeptidase) and dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) (Maes et al., 1991, 1994, 1995, 1996, 1997). Lower peptidase activity in major depression is in agreement with reports on low molecular weight hyperpeptiduria in that illness (Abassi et al., 1992; Saelid et al., 1985; Watanabe et al., 1993). Schizophrenia and post-traumatic stress disorder (PTSD), on the other hand, are accompanied by increased activity of serum PEP and/or DPP IV (Maes et al., 1995, 1999). PEP and DPP IV are two peptidases which are widely distributed among human tissues and body fluids, including the hypophysial portal system and the brain (Hopsu-Havu and Glenner, 1966; Kato et al., 1980a; Vanhoof et al., 1992; Goossens et al., 1996). PEP is a cytosolic endopeptidase which cleaves peptide bonds on the carboxyl side of proline in proteins of relatively small molecular mass (Moriyama et al., 1988; Walter et al., 1971). PEP plays a role in the regulation of intracellular protein turnover and in the degradation and processing of mature peptide hormones and neuropeptides (Welches et al., 1993). Many neuronally and behaviorally active peptides are substrates for PEP, such as arginine vasopressin (AVP), luteinizing hormone-releasing hormone (LH-RH), thyrotropin releasing hormone (TRH), substance P, oxytocin, bradykinin, neurotensin and angiotensin (Welches et al., 1993). DPP IV is a serine protease, which releases dipeptides from the aminoterminus when the penultimate residue of the peptide is a proline or alanine (De Meester, 1992; Hopsu-Havu and Glenner, 1966; Vanhoof et al., 1992). DPP IV has an important role in the processing of polypeptides and proteins, intestinal assimilation, renal handling of proline containing peptides, and adhesion and modulation of immune reactivity (Vanhoof et al., 1992; Yaron and Naider, 1993). DPP IV can process peptides, such as substance P, neuropeptide Y, and growth hormone RH (Frohman et al., 1989; Helander et al., 1996; Mentlein et al., 1993). On the T-lymphocyte cell surface, DPP IV is identical with the CD26 molecule and its expression is increased with that of other activation markers (Barton et al., 1990; Iwaki-Egawa et al., 1995; Scholz et al., 1985). Both membrane CD26 and the soluble CD26 molecule in plasma exhibit costimulatory functions for the response to recall antigens and play an important role in lymphocyte activation and production of cytokines such as interleukin-2 and interferon-g (Duke-Cohan et al., 1995; Reinhold et al., 1996; Schon et al., 1989; Tanaka et al., 1994). The observation that a number of cytokines, such as interleukin-1b (IL-1b) and IL-6, have a penultimate proline (Vanhoof et al., 1995) and that the costimulatory action of CD26 is linked to its DPP IV activity may suggest an immuneregulatory role for this molecule in cytokine synthesis (Gerli et al., 1996; Korom et al., 1997; Reinhold et al., 1997). It has remained elusive, however, whether the changes in serum PEP or DPP IV activity in major depression, schizophrenia or PTSD are related to these disorders per se or rather to the increased stress, stressor perception or anxiety, which accompany or characterize these disorders. In the present study, we have examined the effects of psychological stress on serum PEP and DPP IV, since psychological stress is accompanied by neuroendocrine and immune changes, which could be related to altered serum PEP and DPP IV activity. Thus, stress-induced changes in PEP and/or DPP IV activity could, in theory, be related to stress-induced hypothalamic–pituitary–adrenal (HPA)-axis hyperactivity (through changes in AVP degradation), stress-induced expression of the CD26 molecule (Maes et al., 1998c) and stress-induced changes in the secretion of pro-inflammatory cytokines (Maes et al., 1998a,b). Moreover, AVP, oxytocin, TRH, neurotensin and substance P profoundly modulate stress-related behaviors, such as stress-induced emotions, responsivity, social behavior, reward and positive reinforcement (Bluthe´ et al., 1990; Drago et al., 1996; Insel, 1992; Krappmann et al., 1994; Rinaman et al., 1995; Rompre, 1995).
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The specific aims of the present study were to examine (i) whether psychological stress (academic examination stress in students) affects serum PEP and DPP IV activity and (ii) whether alterations in baseline serum PEP or DPP IV activity are related to stress-induced anxiety responses.
SUBJECTS AND METHODS Subjects The subjects were undergraduate students attending the second year of medical sciences at the Rijksuniversitair Centrum Antwerpen (RUCA), University of Antwerp, Wilrijk, Belgium. The study group consisted of 13 female students without use of contraceptive drugs, 13 females who had been taking contraceptive drugs for at least 3 consecutive months (monophasic oestroprogestativa) and 12 male students. The study protocol was approved by the institutional review board of the RUCA, Antwerp, Belgium. All subjects gave informed consent after the study design was fully explained. Exclusion criteria for the subjects were: (i) medical disorders, e.g. endocrine, immune and metabolic disorders; (ii) having suffered from an infectious, allergic or inflammatory response two weeks prior to this study; (iii) a past or present axis-I psychiatric disorder according to DSM-III-R criteria (American Psychiatric Association, 1987); (iv) ever having taken major psychotropic medications, including antidepressant and antipsychotic drugs; (v) a past or present history of substance abuse or substance use disorders (alcohol and any other drug of dependence); (vi) tobacco use of more than 15 cigarettes/day. There were six subjects with tobacco use and none of these changed their smoking behavior during the stress condition. We excluded subjects taking medical drugs during the study span, including over the counter drugs, and subjects who suffered from new illnesses during the study span, including influenza. All subjects denied use of psychostimulants during the study span. Accordingly, none of the subjects showed positive blood screenings, at any of the three conditions, for amphetamines and analogues (amphetamine, methamphetamine, MDMA, MDA and MDE, ephedrine, pseudoephedrine, norephedrine, diethylproprion+ metabolites, fenfluramine+ norfenfluramine, and phentermine). The latter were determined by means of gaschromatography with NPD detection after an extraction at alkaline pH (Demedts et al., 1996). Subjects were also excluded if they had abnormal baseline blood tests, such as liver tests (SGPT, SGOT, gGT), thyroid function tests (free thyroxine, free triiodothyronine, basal thyroid secreting hormone), BUN, WBC differentials and C-reactive protein concentrations. The subjects abstained from caffeine and nicotine for at least 8 h before each session. Since it is virtually impossible to examine all female students at the three conditions in a same phase of their menstrual cycle, we decided to adjust our results for possible effects of the sex-hormonal state by entering follicle stimulating hormone (FSH), luteinizing hormone (LH) and oestradiol as covariates in regression analyses. The number of alcoholic drinks during the last week before the three assessments was recorded (expressed in ml of beer daily). Methods Serum samples for the assay of PEP and DPP IV activity were collected at 0900h ( 9 45 min.) some weeks before (mean = 44.59 6.0 days; pre-stress condition) and after (mean= 36.09 5.9 days; post-stress condition) as well as 1 day before a difficult oral examination
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(the stress condition). Students completed, during the three sessions, the Spielberger State-Trait Anxiety Inventory (STAI) (Spielberger et al., 1987). Subjects were divided in two groups on the basis of the changes in the STAI scores from baseline to the stress condition: (i) anxiety-responders were defined as subjects with DSTAI values greater than eight, i.e. stress STAI minus pre-stress STAI; (ii) anxiety nonresponders, i.e. the remaining subjects. The Dutch translation of the Minnesota Multiphase Personality Inventory (MMPI-2™) (Derksen and de Mey, 1993) was used to assess personality characteristics, such as psychasthenia (Scale 7). The subjects were divided in two study groups, i.e. those with higher versus lower scores on the psychasthenia scale (cut-off value ] 10). In order to minimize effects of analytical variability, all serum specimens from patients and controls were assayed the same day, in a single run with a single lot number of reagents and consumables employed by a single operator. Serum PEP activity was determined by a fluorimetric method (Goossens et al., 1992) with the synthetic substrate Z-glycyl-prolyl-4-methylcoumarinyl-7-amide (Bachem Feinchemikalien AG, Bubendorf, Switserland). One unit (U) of PEP activity was defined as the enzyme catalytic activity which releases 1 mmol 7-amino-4-methylcoumarin in 1 min under the assay conditions. The assay is highly specific for PEP, since DPP IV and aminopeptidase P do not result in any breakdown of the substrate. Moreover, the possible interference from metalloproteases is prevented by the addition of EDTA to the incubation buffer. The intra-assay coefficient of variation (CV) in our laboratory was 4.8%. Serum DPP IV was determined by means of a colorimetric method (Nagatsu et al., 1976) using the chromogenic substrate glycyl-Lproline-p-nitroanilide tosylate (Sigma, Bornem, Belgium). Our laboratory has adapted the method for direct continuous measurement on the centrifugal analyzer Cobas Bio (Roche Diagnostics, Basel, Switzerland) (Vanhoof et al., 1992). One unit (U) of DPP IV activity is defined as the enzyme activity which produces 1 mmol of p-nitroaniline in 1 min under the above conditions. There is no interference from other dipeptidyl peptidases or aminopeptidases present in the serum. The intra-assay CV value of our DPP IV assay was 2.2%. LH and FSH were determined by means of chemoluminescent immunoassays (ACS, Ciba-Corning). The analytical intra-assay CV values for LH and FSH were 3.8 and 2.6%, respectively. Oestradiol was determined by means of an enzyme-linked fluorescent immunoassay (Vidas, Bio-Merieux). The intra-assay CV value was 7.3%. Statistics Group mean differences were assessed by means of analyses of variance (ANOVA). The independence of classification systems was ascertained by means of analysis of contingence (x 2-test). Relationships between variables were assessed by means of Pearson’s product moment correlation coefficients or through multiple regression analysis. Time-relationships (e.g. the changes from the pre-stress to the stress to the post-stress conditions) were checked by means of intra-class regression analyses, pooled over the repeated measurements in the 38 subjects. Repeated measures analyses of variance (RM ANOVAs) were used to examine: (i) the between-subject variability with effects of gender; anxiety responsivity (anxiety reactors vs. non-reactors); and high versus low psychasthenia scores; (ii) the within-subject variability with the pre/post-stress and stress conditions as time factor; (iii) two or three way interactions between time× gender; time× anxiety responsivity (or psychasthenia); and time× anxiety responsivity (or psychasthenia)× gender. A priori comparisons among treatment means were assessed with the Dunn test (Howell, 1982). Tests on simple effects were carried out in order to explore significant main effects
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or significant interaction patterns. A simple effect is defined as the effect of one variable at one level of the other variable (Howell, 1982). By means of tests on simple effects we tested, for example, the effects of time in anxiety responders and non-responders and differences in serum PEP or DPP IV between anxiety responders and nonresponders in the pre-stress, stress and post-stress conditions. RM analyses of covariance (ANCOVAs) were used to adjust for possible effects of alcohol use on serum PEP and DPP IV activity and the sex-hormonal state (by using FSH, LH and oestradiol as covariates).
RESULTS The mean STAI scores in the anxiety nonresponders (n= 16) were: pre-stress 40.4 ( 99.3); stress 39.2 (9 8.3); and post-stress 32.9 (9 7.4); and in the anxiety responders (n =22): pre-stress 36.8 (99.8); stress 55.8 (9 10.3), and post-stress 30.8 (9 5.8). RM ANOVA, with the STAI score as dependent variable, showed a significant effect of time (F =24.1, df=2/68, p B10 − 4) and a significant time×anxiety responder status interaction (F =13.3, df=2/68, p B 10 − 4). There were no significant differences in the STAI score between male and female students (F=0.9, df= 1/34, p= .7) and no significant time ×gender interaction (F = 1.2, df=2/62, p= .3). Table I shows the mean serum PEP activities in females and male students in the pre-stress, stress and post-stress condition, as well as in anxiety responders and nonresponders. Since there were no significant differences in serum PEP activity between females with and without use of contraceptive drugs (t= 0.9, p= .6), we have carried out subsequent statistical analyses in the male versus female study groups. The between-subject analysis showed that: (i) serum PEP was significantly (F= 22.5, df = 1/34, p= .0001) higher in men (mean= 0.776 9 0.152 U/l) than in females (mean = 0.5639 0.156 U/l); (ii) serum PEP was significantly (F= 5.3, df =1/34, p=.02) higher in anxiety responders (mean =0.664 9 0.178 U/l) than in anxiety nonresponders (mean= 0.5899 0.182 U/l). The within-subject analysis showed a significant time× gender interaction (F= 4.6, df = 2/ 68, p =.01) and no significant time×anxiety responder status (F= 0.7, df= 2/68, p= .5) or time× gender ×anxiety responder status (F= 0.5, df= 2/68, p= .6) interactions. Analysis of simple effects showed: (i) significant differences in serum PEP between males and Table I. Measurements of serum prolyl endopeptidase (PEP) activity in female (F) and male (M) students some weeks before (pre) and after (post) as well as the day before a difficult examination (stress) F/M
AR/NR (n)
Pre-stress
Stress
Post-stress
F
NR (11) AR (15)
0.534 (0.117) 0.608 (0.129)
0.488 (0.135) 0.585 (0.148)
0.458 (0.105) 0.578 (0.136)
M
NR (5) AR (7)
0.687 (0.103) 0.815 (0.157)
0.704 (0.161) 0.795 (0.173)
0.722 (0.147) 0.857 (0.156)
All
(38)
0.635 (0.158)
0.611 (0.182)
0.614 (0.191)
The subjects were divided into anxiety responders (AR) and anxiety nonresponders (NR). All results are shown as mean (SD). PEP values are expressed in U/l. See statistics section for results of repeated measures ANOVAs and ANCOVAs.
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Table II. Measurements of dipeptidyl peptidase IV (DPP IV) activity in female (F) and male (M) students some weeks before (pre) and after (post) as well as the day before a difficult examination (stress) F/M
AR/NR (n)
Pre-stress
Stress
Post-stress
F
NR (11) AR (15)
38.1 (4.5) 35.5 (8.2)
35.8 (6.3) 36.0 (8.1)
34.5 (4.6) 34.6 (7.4)
M
NR (5) AR (7)
39.9 (6.1) 48.8 (8.8)
42.4 (8.6) 48.8 (6.3)
41.3 (7.7) 47.6 (8.3)
All
(38)
39.0 (7.7)
38.8 (8.4)
37.0 (7.5)
The subjects were divided into anxiety responders (AR) and nonresponders (NR). All results are shown as mean (SD). DPP IV values are expressed in U/l. See statistics section for results of repeated measures ANOVAs and ANCOVAs.
females in the pre-stress (F =14.8, p= .0004), stress (F= 19.7, p= .0001) and post-stress (F= 32.2, p B10 − 4; all df= 1/102) conditions; (ii) a significant effect of time in female (F= 4.1, df=2/68, p =.02), but not male (F= 1.8, df= 2/68, p= .2) subjects; (iii) significant differences in serum PEP between anxiety responders and nonresponders in the pre-stress (F =4.1, p =.04), stress (F= 4.5, p= .03) and post-stress (F= 7.7, p =.006; all df = 1/102) conditions; (iv) no significant effects of time on serum PEP in anxiety responders (F =1.8, df =2/68, p =.2) or nonresponders (F= 0.7, df = 2/68, p = 0.5). The index of spherificity was 0.964 and not one of the above results changed after correction for spherificity. There were no significant time-relationships between the changes in serum PEP activity and the changes in the STAI (r= 0.02, p= .8) (results of regression analysis pooled over the subjects). RM ANOVA showed (i) a significant time effect on use of alcohol between the pre-stress (mean=195 9190 ml), stress (mean= 32.5972.5 ml) and post-stress (mean= 205 9315 ml) conditions (F =14.6, df =2/68, p=.00004); (ii) a significant greater use of alcohol in men than in women (F= 4.3, df=1/34, p= .04). By means of RM ANCOVA with use of alcohol as covariate, the differences in serum PEP activity between anxiety responders versus nonresponders (F= 5.2, df= 1/34, p= .03) and the time×gender interaction (F= 4.8, df=2/68, p =.01) remained significant. By means of RM ANCOVA with the serum concentrations of FSH, LH and oestradiol as covariates, the differences in serum PEP activity between anxiety responders versus nonresponders (F= 5.7, df= 1/34, p=.02) and the time × gender interaction (F= 4.6, df = 2/68, p = .01) remained significant. RM ANOVA with gender and psychasthenia as between-subject factors showed that there were no significant differences in serum PEP between subjects with low and high psychasthenia scores (F =0.2, df= 1/34, p= .6); and no significant time× psychasthenia (F = 0.6, df=2/68, p =.5) or time×psychasthenia×gender (F =0.4, df= 2/68, p= .7) interactions. Analyses on simple effects showed no significant differences in serum PEP between subjects with low and high psychasthenia scores in the pre-stress (F= 0.00, p= .9), stress (F =0.07, p = .8) and post-stress (F= 0.1, p= .7) conditions (all df= 1/102). Table II shows the mean serum DPP IV activity in female and male subjects in the three conditions. Since there were no significant differences in serum DPP IV activity between females with and without use of contraceptive drugs (t= 0.9, p = .7), we have carried out
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subsequent statistical analyses in the male versus female study groups. The between-subject analysis showed a significant difference between men and women (F=12.8, df= 1/34, p= .001), but not between anxiety responders and nonresponders (F= 1.1, df= 1/34, p= .3). There was no significant interaction between gender × anxiety responder status (F = 2.5, df= 1/34, p =.1). The within-subject analysis showed no significant effects of time (F =1.7, df= 2/68, p =.2), and no significant time× gender (F= 1.7, df= 2/68, p=.2), time×anxiety responder status (F= 0.1, df= 2/68, p= .9) or time× gender× anxiety responder status (F =1.9, df= 2/68, p= .1) interactions. Tests on simple effects showed (i) significant differences between men and women in the pre-stress (F= 10.4, df=1/102, p = .002), stress (F =15.6, p= 1/102, p= .0003) and post-stress (F= 16.6, df= 1/102, p =.0002) conditions. The index of spherificity was 0.964 and not one of the above results changed after correction for spherificity. There were no significant time-relationships between the changes in serum DPP IV activity and the changes in the STAI (r= 0.19, p = .08) (results of regression analysis pooled over the subjects). RM ANOVA with gender and psychasthenia as between-subject factors showed that there were no significant differences in serum DPP IV between subjects with low and high psychasthenia scores (F = 1.7, df=1/34, p= .2); and no significant time×psychasthenia (F =0.0, df =2/68, p = .99) or time ×psychasthenia× gender (F= 2.4, df = 2/68, p= .1) interactions. There were significant and positive time-relationships between serum PEP and DPP IV activity (r = 0.35, p=.002; results of intra-class regression analysis pooled over the subjects).
DISCUSSION To the best of our knowledge, this is a first study reporting that increased baseline serum PEP activity is significantly related to stress-induced anxiety. These findings extent our findings that PTSD, an anxiety disorder, is accompanied by significantly increased serum PEP activity (Maes et al., 1998a). Although our findings do not prove a causal relationship between increased PEP activity and stress-induced anxiety, such a relationship could be explained by the hypothesis that increased PEP activity may be accompanied by an increased inactivation of behaviorally active peptides, which play an important role in emotion, stress responsivity, avoidance behavior and social interactions. TRH reduces the total immobility time of rats tested in the despair test and TRH-analogues may ameliorate memory and cognitive deficits, as assessed by means of facilitation of the acquisition of active avoidance behavior (Drago et al., 1990, 1996). Substance P has positive reinforcing capacities (Huston and Oitzl, 1989; Krappmann et al., 1994). Neurotensin is involved in the activity of dopaminergic neurons that mediate behaviors motivated by positive reinforcers (Rompre and Bauco, 1992; Rompre, 1995). Oxytocin has anxiolytic properties, facilitates social encounters by reducing associated anxiety (McCarthy, 1995), promotes social bonds, stimulates social behavior, decreases fear-induced freezing behavior, and hastens the extinction of passive and active avoidance responses (Insel, 1992; Rinaman et al., 1995; Stoehr et al., 1992; Uvnas Moberg et al., 1994; Witt et al., 1992). Finally, AVP facilitates positive conditioning, improves social recognition, and increases social interactions (Bluthe´ et al., 1990; Engelmann et al., 1994; Metzger et al., 1994; Popik and Vetulani, 1993). There are, however, reports showing that blockade of AVP neurotransmission in the septum may have anxiolytic-like effects (Liebsch et al., 1996).
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There is now evidence that peripheral serum PEP activity may be relevant for the brain. Smaller neuropeptides, such as AVP, oxytocin and neurotensin, can penetrate the brain from the peripheral blood to exert their central activity, and, consequently, peripheral degradation of these peptides may play a role in the central activity of these neuropeptides. In sheep, PEP is released from the vascular bed of the head and the brain might be a major source of circulating PEP (Lawrence et al., 1992). Moreover, high PEP activity is observed in the central cortex (Wilk 1983; Kato et al., 1980b) and in the hypophysial-portal plasma and median eminence (Lawrence et al., 1992). Another hypothesis is that the gender differences in serum PEP activity (greater in men) and the significant gender differences in stress-induced changes in serum PEP (decreased in women) may be of relevance for the gender-differences in neuroendocrine responses to stress. For example, in depression, serum PEP activity may be related to HPA-axis hyperactivity through changes in AVP secretion. Depression is characterized by increased serum AVP (van London et al., 1997) and by a significant inverse correlation between lower serum PEP activity and increased post-dexamethasone cortisol values (Maes et al., 1994). These results suggest that diminished inactivation of AVP by lower PEP activity may play a role in HPA-axis hyperactivity in depression (van London et al., 1997). It has repeatedly been shown that female rats have greater stress-induced corticosterone responses than male rats (Curzon, 1989; Haleem et al., 1988; Pericic and Pivac, 1995). The results of this study suggest that lower serum PEP activity in females may contribute to greater stress-induced HPA-axis responses in females through decreased inactivation of AVP. In conclusion, the results show that subjects, who had a significant stress-induced anxiety response, have significantly increased serum PEP activity during the baseline conditions as well as during the stress condition; and that psychological stress has a significant suppressant effect on serum PEP activity in females only. It is hypothesized that increased serum PEP activity is related to stress-induced anxiety through increased degradation of behaviorally active neuropeptides, such as oxytocin, AVP,TRH, neurotensin and substance P. Acknowledgements: The research reported was supported in part by the Clinical Research Center for Mental Health (CRC-MH), Antwerp, Belgium; the Funds for Scientific Research, Vlaanderen, Belgium; and the Staglin Investigator Award to Dr M. Maes (NARSAD); F. Goossens is a senior research assistant of the funds for Scientific Research, Vlaanderen, Belgium.
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PII: S0306-4530(98)00020-1
EFFECTS OF PSYCHOLOGICAL STRESS ON SERUM PROLYL ENDOPEPTIDASE AND DIPEPTIDYL PEPTIDASE IV ACTIVITY IN HUMANS: HIGHER SERUM PROLYL ENDOPEPTIDASE ACTIVITY IS RELATED TO STRESS-INDUCED ANXIETY Michael Maes1,2,3, Filip Goossens4, Ai-hun Lin1, Ingrid De Meester4, Ann Van Gastel1 and Simon Scharpe´4 1
Clinical Research Center for Mental Health (CRC-MH), 2060 Antwerp, Belgium 2 IRCCS, Istituto Fatebenefratelli, Brescia, Italy 3 Department of Psychiatry, Vanderbilt University, Nashville, TN, USA 4 Department of Medical Biochemistry, University of Antwerp, Wilrijk, Belgium
(Recei6ed 8 January 1998; in final form 24 March 1998)
SUMMARY There is now some evidence that psychiatric disorders, such as major depression, schizophrenia and post-traumatic stress disorder are associated with significant alterations in the serum activity of peptidases, such as prolyl endopeptidase (PEP) and dipeptidyl peptidase IV (DPP IV). The aims of the present study were to examine the effects of psychological stress on serum PEP and DPP IV activity in humans. Thirty-eight university students had repeated measurements of serum PEP and DPP IV activity a few weeks before and after (baseline conditions) as well as the day before a difficult academic examination (stress condition). Subjects were divided into anxiety responders and nonresponders to stress according to their stress-induced increase in the Spielberger State Anxiety Inventory. Serum PEP activity was somewhat lowered by stress in female, but not male, students. Serum PEP activity was significantly higher in the two baseline conditions and during the stress condition in anxiety responders than in anxiety nonresponders. There were no significant effects of stress on serum DPP IV activity and no significant differences between anxiety responders and nonresponders. Serum PEP and DPP IV activity were significantly higher in men than in women. The results suggest that increased baseline serum PEP activity is related to stress-induced anxiety. © 1998 Elsevier Science Ltd. All rights reserved. Keywords—Peptidases; Psychological stress; Prolyl endopeptidase; Dipeptidyl peptidase IV; Anxiety.
INTRODUCTION Recently, it has been shown that peptidases may play a role in the pathophysiology of various psychiatric disorders. Major depression is associated with lower serum activity of peptidases, such as prolyl endopeptidase (PEP, EC 3.4.21.26, post-proline cleaving enAddress correspondence and reprint requests to: M. Maes, University Department of Psychiatry, AZ Stuivenberg, 267 Lange Beeldekensstraat, 2060 Antwerp, Belgium (Fax: 32 3 4483265; E-mail: [email protected]). 485
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zyme, prolyl oligopeptidase) and dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) (Maes et al., 1991, 1994, 1995, 1996, 1997). Lower peptidase activity in major depression is in agreement with reports on low molecular weight hyperpeptiduria in that illness (Abassi et al., 1992; Saelid et al., 1985; Watanabe et al., 1993). Schizophrenia and post-traumatic stress disorder (PTSD), on the other hand, are accompanied by increased activity of serum PEP and/or DPP IV (Maes et al., 1995, 1999). PEP and DPP IV are two peptidases which are widely distributed among human tissues and body fluids, including the hypophysial portal system and the brain (Hopsu-Havu and Glenner, 1966; Kato et al., 1980a; Vanhoof et al., 1992; Goossens et al., 1996). PEP is a cytosolic endopeptidase which cleaves peptide bonds on the carboxyl side of proline in proteins of relatively small molecular mass (Moriyama et al., 1988; Walter et al., 1971). PEP plays a role in the regulation of intracellular protein turnover and in the degradation and processing of mature peptide hormones and neuropeptides (Welches et al., 1993). Many neuronally and behaviorally active peptides are substrates for PEP, such as arginine vasopressin (AVP), luteinizing hormone-releasing hormone (LH-RH), thyrotropin releasing hormone (TRH), substance P, oxytocin, bradykinin, neurotensin and angiotensin (Welches et al., 1993). DPP IV is a serine protease, which releases dipeptides from the aminoterminus when the penultimate residue of the peptide is a proline or alanine (De Meester, 1992; Hopsu-Havu and Glenner, 1966; Vanhoof et al., 1992). DPP IV has an important role in the processing of polypeptides and proteins, intestinal assimilation, renal handling of proline containing peptides, and adhesion and modulation of immune reactivity (Vanhoof et al., 1992; Yaron and Naider, 1993). DPP IV can process peptides, such as substance P, neuropeptide Y, and growth hormone RH (Frohman et al., 1989; Helander et al., 1996; Mentlein et al., 1993). On the T-lymphocyte cell surface, DPP IV is identical with the CD26 molecule and its expression is increased with that of other activation markers (Barton et al., 1990; Iwaki-Egawa et al., 1995; Scholz et al., 1985). Both membrane CD26 and the soluble CD26 molecule in plasma exhibit costimulatory functions for the response to recall antigens and play an important role in lymphocyte activation and production of cytokines such as interleukin-2 and interferon-g (Duke-Cohan et al., 1995; Reinhold et al., 1996; Schon et al., 1989; Tanaka et al., 1994). The observation that a number of cytokines, such as interleukin-1b (IL-1b) and IL-6, have a penultimate proline (Vanhoof et al., 1995) and that the costimulatory action of CD26 is linked to its DPP IV activity may suggest an immuneregulatory role for this molecule in cytokine synthesis (Gerli et al., 1996; Korom et al., 1997; Reinhold et al., 1997). It has remained elusive, however, whether the changes in serum PEP or DPP IV activity in major depression, schizophrenia or PTSD are related to these disorders per se or rather to the increased stress, stressor perception or anxiety, which accompany or characterize these disorders. In the present study, we have examined the effects of psychological stress on serum PEP and DPP IV, since psychological stress is accompanied by neuroendocrine and immune changes, which could be related to altered serum PEP and DPP IV activity. Thus, stress-induced changes in PEP and/or DPP IV activity could, in theory, be related to stress-induced hypothalamic–pituitary–adrenal (HPA)-axis hyperactivity (through changes in AVP degradation), stress-induced expression of the CD26 molecule (Maes et al., 1998c) and stress-induced changes in the secretion of pro-inflammatory cytokines (Maes et al., 1998a,b). Moreover, AVP, oxytocin, TRH, neurotensin and substance P profoundly modulate stress-related behaviors, such as stress-induced emotions, responsivity, social behavior, reward and positive reinforcement (Bluthe´ et al., 1990; Drago et al., 1996; Insel, 1992; Krappmann et al., 1994; Rinaman et al., 1995; Rompre, 1995).
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The specific aims of the present study were to examine (i) whether psychological stress (academic examination stress in students) affects serum PEP and DPP IV activity and (ii) whether alterations in baseline serum PEP or DPP IV activity are related to stress-induced anxiety responses.
SUBJECTS AND METHODS Subjects The subjects were undergraduate students attending the second year of medical sciences at the Rijksuniversitair Centrum Antwerpen (RUCA), University of Antwerp, Wilrijk, Belgium. The study group consisted of 13 female students without use of contraceptive drugs, 13 females who had been taking contraceptive drugs for at least 3 consecutive months (monophasic oestroprogestativa) and 12 male students. The study protocol was approved by the institutional review board of the RUCA, Antwerp, Belgium. All subjects gave informed consent after the study design was fully explained. Exclusion criteria for the subjects were: (i) medical disorders, e.g. endocrine, immune and metabolic disorders; (ii) having suffered from an infectious, allergic or inflammatory response two weeks prior to this study; (iii) a past or present axis-I psychiatric disorder according to DSM-III-R criteria (American Psychiatric Association, 1987); (iv) ever having taken major psychotropic medications, including antidepressant and antipsychotic drugs; (v) a past or present history of substance abuse or substance use disorders (alcohol and any other drug of dependence); (vi) tobacco use of more than 15 cigarettes/day. There were six subjects with tobacco use and none of these changed their smoking behavior during the stress condition. We excluded subjects taking medical drugs during the study span, including over the counter drugs, and subjects who suffered from new illnesses during the study span, including influenza. All subjects denied use of psychostimulants during the study span. Accordingly, none of the subjects showed positive blood screenings, at any of the three conditions, for amphetamines and analogues (amphetamine, methamphetamine, MDMA, MDA and MDE, ephedrine, pseudoephedrine, norephedrine, diethylproprion+ metabolites, fenfluramine+ norfenfluramine, and phentermine). The latter were determined by means of gaschromatography with NPD detection after an extraction at alkaline pH (Demedts et al., 1996). Subjects were also excluded if they had abnormal baseline blood tests, such as liver tests (SGPT, SGOT, gGT), thyroid function tests (free thyroxine, free triiodothyronine, basal thyroid secreting hormone), BUN, WBC differentials and C-reactive protein concentrations. The subjects abstained from caffeine and nicotine for at least 8 h before each session. Since it is virtually impossible to examine all female students at the three conditions in a same phase of their menstrual cycle, we decided to adjust our results for possible effects of the sex-hormonal state by entering follicle stimulating hormone (FSH), luteinizing hormone (LH) and oestradiol as covariates in regression analyses. The number of alcoholic drinks during the last week before the three assessments was recorded (expressed in ml of beer daily). Methods Serum samples for the assay of PEP and DPP IV activity were collected at 0900h ( 9 45 min.) some weeks before (mean = 44.59 6.0 days; pre-stress condition) and after (mean= 36.09 5.9 days; post-stress condition) as well as 1 day before a difficult oral examination
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(the stress condition). Students completed, during the three sessions, the Spielberger State-Trait Anxiety Inventory (STAI) (Spielberger et al., 1987). Subjects were divided in two groups on the basis of the changes in the STAI scores from baseline to the stress condition: (i) anxiety-responders were defined as subjects with DSTAI values greater than eight, i.e. stress STAI minus pre-stress STAI; (ii) anxiety nonresponders, i.e. the remaining subjects. The Dutch translation of the Minnesota Multiphase Personality Inventory (MMPI-2™) (Derksen and de Mey, 1993) was used to assess personality characteristics, such as psychasthenia (Scale 7). The subjects were divided in two study groups, i.e. those with higher versus lower scores on the psychasthenia scale (cut-off value ] 10). In order to minimize effects of analytical variability, all serum specimens from patients and controls were assayed the same day, in a single run with a single lot number of reagents and consumables employed by a single operator. Serum PEP activity was determined by a fluorimetric method (Goossens et al., 1992) with the synthetic substrate Z-glycyl-prolyl-4-methylcoumarinyl-7-amide (Bachem Feinchemikalien AG, Bubendorf, Switserland). One unit (U) of PEP activity was defined as the enzyme catalytic activity which releases 1 mmol 7-amino-4-methylcoumarin in 1 min under the assay conditions. The assay is highly specific for PEP, since DPP IV and aminopeptidase P do not result in any breakdown of the substrate. Moreover, the possible interference from metalloproteases is prevented by the addition of EDTA to the incubation buffer. The intra-assay coefficient of variation (CV) in our laboratory was 4.8%. Serum DPP IV was determined by means of a colorimetric method (Nagatsu et al., 1976) using the chromogenic substrate glycyl-Lproline-p-nitroanilide tosylate (Sigma, Bornem, Belgium). Our laboratory has adapted the method for direct continuous measurement on the centrifugal analyzer Cobas Bio (Roche Diagnostics, Basel, Switzerland) (Vanhoof et al., 1992). One unit (U) of DPP IV activity is defined as the enzyme activity which produces 1 mmol of p-nitroaniline in 1 min under the above conditions. There is no interference from other dipeptidyl peptidases or aminopeptidases present in the serum. The intra-assay CV value of our DPP IV assay was 2.2%. LH and FSH were determined by means of chemoluminescent immunoassays (ACS, Ciba-Corning). The analytical intra-assay CV values for LH and FSH were 3.8 and 2.6%, respectively. Oestradiol was determined by means of an enzyme-linked fluorescent immunoassay (Vidas, Bio-Merieux). The intra-assay CV value was 7.3%. Statistics Group mean differences were assessed by means of analyses of variance (ANOVA). The independence of classification systems was ascertained by means of analysis of contingence (x 2-test). Relationships between variables were assessed by means of Pearson’s product moment correlation coefficients or through multiple regression analysis. Time-relationships (e.g. the changes from the pre-stress to the stress to the post-stress conditions) were checked by means of intra-class regression analyses, pooled over the repeated measurements in the 38 subjects. Repeated measures analyses of variance (RM ANOVAs) were used to examine: (i) the between-subject variability with effects of gender; anxiety responsivity (anxiety reactors vs. non-reactors); and high versus low psychasthenia scores; (ii) the within-subject variability with the pre/post-stress and stress conditions as time factor; (iii) two or three way interactions between time× gender; time× anxiety responsivity (or psychasthenia); and time× anxiety responsivity (or psychasthenia)× gender. A priori comparisons among treatment means were assessed with the Dunn test (Howell, 1982). Tests on simple effects were carried out in order to explore significant main effects
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or significant interaction patterns. A simple effect is defined as the effect of one variable at one level of the other variable (Howell, 1982). By means of tests on simple effects we tested, for example, the effects of time in anxiety responders and non-responders and differences in serum PEP or DPP IV between anxiety responders and nonresponders in the pre-stress, stress and post-stress conditions. RM analyses of covariance (ANCOVAs) were used to adjust for possible effects of alcohol use on serum PEP and DPP IV activity and the sex-hormonal state (by using FSH, LH and oestradiol as covariates).
RESULTS The mean STAI scores in the anxiety nonresponders (n= 16) were: pre-stress 40.4 ( 99.3); stress 39.2 (9 8.3); and post-stress 32.9 (9 7.4); and in the anxiety responders (n =22): pre-stress 36.8 (99.8); stress 55.8 (9 10.3), and post-stress 30.8 (9 5.8). RM ANOVA, with the STAI score as dependent variable, showed a significant effect of time (F =24.1, df=2/68, p B10 − 4) and a significant time×anxiety responder status interaction (F =13.3, df=2/68, p B 10 − 4). There were no significant differences in the STAI score between male and female students (F=0.9, df= 1/34, p= .7) and no significant time ×gender interaction (F = 1.2, df=2/62, p= .3). Table I shows the mean serum PEP activities in females and male students in the pre-stress, stress and post-stress condition, as well as in anxiety responders and nonresponders. Since there were no significant differences in serum PEP activity between females with and without use of contraceptive drugs (t= 0.9, p= .6), we have carried out subsequent statistical analyses in the male versus female study groups. The between-subject analysis showed that: (i) serum PEP was significantly (F= 22.5, df = 1/34, p= .0001) higher in men (mean= 0.776 9 0.152 U/l) than in females (mean = 0.5639 0.156 U/l); (ii) serum PEP was significantly (F= 5.3, df =1/34, p=.02) higher in anxiety responders (mean =0.664 9 0.178 U/l) than in anxiety nonresponders (mean= 0.5899 0.182 U/l). The within-subject analysis showed a significant time× gender interaction (F= 4.6, df = 2/ 68, p =.01) and no significant time×anxiety responder status (F= 0.7, df= 2/68, p= .5) or time× gender ×anxiety responder status (F= 0.5, df= 2/68, p= .6) interactions. Analysis of simple effects showed: (i) significant differences in serum PEP between males and Table I. Measurements of serum prolyl endopeptidase (PEP) activity in female (F) and male (M) students some weeks before (pre) and after (post) as well as the day before a difficult examination (stress) F/M
AR/NR (n)
Pre-stress
Stress
Post-stress
F
NR (11) AR (15)
0.534 (0.117) 0.608 (0.129)
0.488 (0.135) 0.585 (0.148)
0.458 (0.105) 0.578 (0.136)
M
NR (5) AR (7)
0.687 (0.103) 0.815 (0.157)
0.704 (0.161) 0.795 (0.173)
0.722 (0.147) 0.857 (0.156)
All
(38)
0.635 (0.158)
0.611 (0.182)
0.614 (0.191)
The subjects were divided into anxiety responders (AR) and anxiety nonresponders (NR). All results are shown as mean (SD). PEP values are expressed in U/l. See statistics section for results of repeated measures ANOVAs and ANCOVAs.
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Table II. Measurements of dipeptidyl peptidase IV (DPP IV) activity in female (F) and male (M) students some weeks before (pre) and after (post) as well as the day before a difficult examination (stress) F/M
AR/NR (n)
Pre-stress
Stress
Post-stress
F
NR (11) AR (15)
38.1 (4.5) 35.5 (8.2)
35.8 (6.3) 36.0 (8.1)
34.5 (4.6) 34.6 (7.4)
M
NR (5) AR (7)
39.9 (6.1) 48.8 (8.8)
42.4 (8.6) 48.8 (6.3)
41.3 (7.7) 47.6 (8.3)
All
(38)
39.0 (7.7)
38.8 (8.4)
37.0 (7.5)
The subjects were divided into anxiety responders (AR) and nonresponders (NR). All results are shown as mean (SD). DPP IV values are expressed in U/l. See statistics section for results of repeated measures ANOVAs and ANCOVAs.
females in the pre-stress (F =14.8, p= .0004), stress (F= 19.7, p= .0001) and post-stress (F= 32.2, p B10 − 4; all df= 1/102) conditions; (ii) a significant effect of time in female (F= 4.1, df=2/68, p =.02), but not male (F= 1.8, df= 2/68, p= .2) subjects; (iii) significant differences in serum PEP between anxiety responders and nonresponders in the pre-stress (F =4.1, p =.04), stress (F= 4.5, p= .03) and post-stress (F= 7.7, p =.006; all df = 1/102) conditions; (iv) no significant effects of time on serum PEP in anxiety responders (F =1.8, df =2/68, p =.2) or nonresponders (F= 0.7, df = 2/68, p = 0.5). The index of spherificity was 0.964 and not one of the above results changed after correction for spherificity. There were no significant time-relationships between the changes in serum PEP activity and the changes in the STAI (r= 0.02, p= .8) (results of regression analysis pooled over the subjects). RM ANOVA showed (i) a significant time effect on use of alcohol between the pre-stress (mean=195 9190 ml), stress (mean= 32.5972.5 ml) and post-stress (mean= 205 9315 ml) conditions (F =14.6, df =2/68, p=.00004); (ii) a significant greater use of alcohol in men than in women (F= 4.3, df=1/34, p= .04). By means of RM ANCOVA with use of alcohol as covariate, the differences in serum PEP activity between anxiety responders versus nonresponders (F= 5.2, df= 1/34, p= .03) and the time×gender interaction (F= 4.8, df=2/68, p =.01) remained significant. By means of RM ANCOVA with the serum concentrations of FSH, LH and oestradiol as covariates, the differences in serum PEP activity between anxiety responders versus nonresponders (F= 5.7, df= 1/34, p=.02) and the time × gender interaction (F= 4.6, df = 2/68, p = .01) remained significant. RM ANOVA with gender and psychasthenia as between-subject factors showed that there were no significant differences in serum PEP between subjects with low and high psychasthenia scores (F =0.2, df= 1/34, p= .6); and no significant time× psychasthenia (F = 0.6, df=2/68, p =.5) or time×psychasthenia×gender (F =0.4, df= 2/68, p= .7) interactions. Analyses on simple effects showed no significant differences in serum PEP between subjects with low and high psychasthenia scores in the pre-stress (F= 0.00, p= .9), stress (F =0.07, p = .8) and post-stress (F= 0.1, p= .7) conditions (all df= 1/102). Table II shows the mean serum DPP IV activity in female and male subjects in the three conditions. Since there were no significant differences in serum DPP IV activity between females with and without use of contraceptive drugs (t= 0.9, p = .7), we have carried out
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subsequent statistical analyses in the male versus female study groups. The between-subject analysis showed a significant difference between men and women (F=12.8, df= 1/34, p= .001), but not between anxiety responders and nonresponders (F= 1.1, df= 1/34, p= .3). There was no significant interaction between gender × anxiety responder status (F = 2.5, df= 1/34, p =.1). The within-subject analysis showed no significant effects of time (F =1.7, df= 2/68, p =.2), and no significant time× gender (F= 1.7, df= 2/68, p=.2), time×anxiety responder status (F= 0.1, df= 2/68, p= .9) or time× gender× anxiety responder status (F =1.9, df= 2/68, p= .1) interactions. Tests on simple effects showed (i) significant differences between men and women in the pre-stress (F= 10.4, df=1/102, p = .002), stress (F =15.6, p= 1/102, p= .0003) and post-stress (F= 16.6, df= 1/102, p =.0002) conditions. The index of spherificity was 0.964 and not one of the above results changed after correction for spherificity. There were no significant time-relationships between the changes in serum DPP IV activity and the changes in the STAI (r= 0.19, p = .08) (results of regression analysis pooled over the subjects). RM ANOVA with gender and psychasthenia as between-subject factors showed that there were no significant differences in serum DPP IV between subjects with low and high psychasthenia scores (F = 1.7, df=1/34, p= .2); and no significant time×psychasthenia (F =0.0, df =2/68, p = .99) or time ×psychasthenia× gender (F= 2.4, df = 2/68, p= .1) interactions. There were significant and positive time-relationships between serum PEP and DPP IV activity (r = 0.35, p=.002; results of intra-class regression analysis pooled over the subjects).
DISCUSSION To the best of our knowledge, this is a first study reporting that increased baseline serum PEP activity is significantly related to stress-induced anxiety. These findings extent our findings that PTSD, an anxiety disorder, is accompanied by significantly increased serum PEP activity (Maes et al., 1998a). Although our findings do not prove a causal relationship between increased PEP activity and stress-induced anxiety, such a relationship could be explained by the hypothesis that increased PEP activity may be accompanied by an increased inactivation of behaviorally active peptides, which play an important role in emotion, stress responsivity, avoidance behavior and social interactions. TRH reduces the total immobility time of rats tested in the despair test and TRH-analogues may ameliorate memory and cognitive deficits, as assessed by means of facilitation of the acquisition of active avoidance behavior (Drago et al., 1990, 1996). Substance P has positive reinforcing capacities (Huston and Oitzl, 1989; Krappmann et al., 1994). Neurotensin is involved in the activity of dopaminergic neurons that mediate behaviors motivated by positive reinforcers (Rompre and Bauco, 1992; Rompre, 1995). Oxytocin has anxiolytic properties, facilitates social encounters by reducing associated anxiety (McCarthy, 1995), promotes social bonds, stimulates social behavior, decreases fear-induced freezing behavior, and hastens the extinction of passive and active avoidance responses (Insel, 1992; Rinaman et al., 1995; Stoehr et al., 1992; Uvnas Moberg et al., 1994; Witt et al., 1992). Finally, AVP facilitates positive conditioning, improves social recognition, and increases social interactions (Bluthe´ et al., 1990; Engelmann et al., 1994; Metzger et al., 1994; Popik and Vetulani, 1993). There are, however, reports showing that blockade of AVP neurotransmission in the septum may have anxiolytic-like effects (Liebsch et al., 1996).
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There is now evidence that peripheral serum PEP activity may be relevant for the brain. Smaller neuropeptides, such as AVP, oxytocin and neurotensin, can penetrate the brain from the peripheral blood to exert their central activity, and, consequently, peripheral degradation of these peptides may play a role in the central activity of these neuropeptides. In sheep, PEP is released from the vascular bed of the head and the brain might be a major source of circulating PEP (Lawrence et al., 1992). Moreover, high PEP activity is observed in the central cortex (Wilk 1983; Kato et al., 1980b) and in the hypophysial-portal plasma and median eminence (Lawrence et al., 1992). Another hypothesis is that the gender differences in serum PEP activity (greater in men) and the significant gender differences in stress-induced changes in serum PEP (decreased in women) may be of relevance for the gender-differences in neuroendocrine responses to stress. For example, in depression, serum PEP activity may be related to HPA-axis hyperactivity through changes in AVP secretion. Depression is characterized by increased serum AVP (van London et al., 1997) and by a significant inverse correlation between lower serum PEP activity and increased post-dexamethasone cortisol values (Maes et al., 1994). These results suggest that diminished inactivation of AVP by lower PEP activity may play a role in HPA-axis hyperactivity in depression (van London et al., 1997). It has repeatedly been shown that female rats have greater stress-induced corticosterone responses than male rats (Curzon, 1989; Haleem et al., 1988; Pericic and Pivac, 1995). The results of this study suggest that lower serum PEP activity in females may contribute to greater stress-induced HPA-axis responses in females through decreased inactivation of AVP. In conclusion, the results show that subjects, who had a significant stress-induced anxiety response, have significantly increased serum PEP activity during the baseline conditions as well as during the stress condition; and that psychological stress has a significant suppressant effect on serum PEP activity in females only. It is hypothesized that increased serum PEP activity is related to stress-induced anxiety through increased degradation of behaviorally active neuropeptides, such as oxytocin, AVP,TRH, neurotensin and substance P. Acknowledgements: The research reported was supported in part by the Clinical Research Center for Mental Health (CRC-MH), Antwerp, Belgium; the Funds for Scientific Research, Vlaanderen, Belgium; and the Staglin Investigator Award to Dr M. Maes (NARSAD); F. Goossens is a senior research assistant of the funds for Scientific Research, Vlaanderen, Belgium.
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