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Neuropeptides 42 (2008) 435–440

Neuropeptides

Short communication

Central aminopeptidase and serotonin system activities:Possible relationship

L. Cicin-Sain a,*, S. Simaga b, A. Froebe c, M. Abramic b

a Department of Molecular Biology, ‘‘Rudjer Boskovic” Institute, Bijenicka 54, HR-10000 Zagreb, Croatiab Department of Organic Chemistry and Biochemistry, ‘‘Rudjer Boskovic” Institute, Zagreb, Croatia

c Department of Oncology and Nuclear Medicine, University hospital ‘‘Sestre milosrdnice”, Zagreb, Croatia

Received 24 January 2008; accepted 25 April 2008Available online 10 June 2008

Abstract

The co-localization of serotonin (5-hydroxytryptamine, 5HT) and neuroactive peptides in the same neuron points to the impor-tance of interactions between serotonergic and peptidergic systems in maintaining body homeostasis. In this work, we used anoriginal genetic rat model to search for possible interrelations between 5HT system functioning and the activities of aminopepti-dases, i.e. enzymes which are the key regulators of (neuro)peptides level/function.

The activities of three cytosolic exopeptidases: alanyl aminopeptidase (alanyl-AP), arginyl aminopeptidase (arginyl-AP) anddipeptidyl peptidase III (DPP III) were measured in brains and peripheral tissues of the sublines of rats with constitutionally upreg-ulated/downregulated 5HT transporter activity. These rat sublines, termed as high-5HT and low-5HT subline, have been obtainedpreviously by selective breeding for the extreme values of platelet 5HT level and velocity of 5HT uptake. Besides in the peripherythey show marked alterations also in brain 5HT function, indicating the differences in central 5HT transmission/homeostasis.

In this study, we have found that animals from the high-5HT subline have significantly lower activity of brain alanyl-AP(p < 0.05) and arginyl-AP (p < 0.01) as compared to control animals. No other differences were noticed regardless of the 5HT sub-line, investigated organ or analyzed aminopeptidase.

Results suggest that the constitutional upregulation of serotonergic activity may be related to a lowered brain cytosolic amino-peptidase activity which may have an influence on the cleavage of their physiological substrates.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Serotonin transporter; 5HTT; Aminopeptidase; Rat model

1. Introduction

Aminopeptidases (APs) are a large class of proteo-lytic enzymes that have an important role in fundamen-tal cell functions, such as signal transduction, cellgrowth, differentiation and apoptosis. The most inten-sively studied, with regard to their physiological role,are the APs in the central nervous system (neuroproteas-es, neuropeptidases). They are essential for the metabo-

0143-4179/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.npep.2008.04.007

* Corresponding author. Tel.: +385 1 4561 045; fax: +385 1 4561177.

E-mail address: cicinsai@irb.hr (L. Cicin-Sain).

lism of peptides acting as neurotransmitters andneuroendocrine hormones that control numerous sys-temic functions (see Hook, 2006), as well as specializedneural functions, such as pain, memory and cognition(Osada et al., 1999; Schott et al., 2000; Meighan et al.,2006). They are also thought to participate in the path-ogenesis of several neuropsychiatric (Hui et al., 1995;Osada et al., 1999) and neurodegenerative (Hook,2006; Hui, 2007) disorders. Neuroprotease inhibitorsrepresent promising analgesic and antidepressant drugs(Holsboer, 2003; Noble and Roques, 2007), and weresuggested as a therapeutic agents for Alzheimer’s disease(Hook, 2006). While the relationship between proteases

436 L. Cicin-Sain et al. / Neuropeptides 42 (2008) 435–440

and synaptic functioning is emerging, understanding ofthe mechanism behind this association is still incom-plete. Coexistence between neuropeptides and classicalneurotransmitters in the same neurons, described almost30 years ago (Hoekfelt et al., 1980), points to the impor-tance of interactions between different neurotransmittersystems.

Serotonin (5-hydroxytryptamine, 5HT), one of theclassical amine neurotransmitters, serves as a mastercontrol brain molecule which orchestrates the interac-tions of several other neurotransmitter systems, havingan important role in brain and peripheral (patho)physi-ological functions. Functional interactions between 5HTand peptide neurotransmitters have been shown bypharmacological, electrophysiological and molecular-genetic studies (Price et al., 1998; Santarelli et al.,2001; Pernar et al., 2004; Tan-No et al., 2007). Sincethe level of neuropeptides is regulated by enzyme activ-ities, neuropeptidases might be implicated in the men-tioned interactions. Indeed, the level of severalneuropeptidases was changed in the conditions wherealtered 5HT-ergic transmission is supposed (Banay-Sch-wartz et al., 1998; Prieto et al., 1998; van West et al.,2000; Banegas et al., 2006).

In this study, we examine the possibility that the con-stitutional differences in 5HT homeostasis are associatedwith alterations in central and/or peripheral neuropepti-dase activities. We used an original rat model: Wistar-Zagreb 5HT (WZ-5HT) rats, which has been developedpreviously by a selective breeding for the extreme valuesof platelet 5HT parameters: 5HT level and 5HT trans-porter activity (Jernej and Cicin-Sain, 1990; Cicin-Sainet al., 2005). Since 5HT transporter proteins on bothplatelets and neurons are encoded by the same gene,breeding procedure resulted in two rat sublines withconstitutional differences in peripheral and central5HT homeostasis. Thus, the animals from high-5HTsublines demonstrated a higher level of 5HT in bloodplatelets and several peripheral organs, higher platelet5HT uptake rate and 5HT transporter expression atthe level of both mRNA and protein (Jernej et al.,1999b; Hranilovic et al., 2001; Bordukalo-Niksic et al.,2004; Cicin-Sain et al., 2005), higher brain 5HT turnover(Bokulic et al., 2003), higher extracellular 5HT concen-tration basally and after pharmacological challenges(microdialysis study) and higher in vivo recovery of5HT (Romero et al., 1998). Behavioral studies showeddifferences in learning ability, anxiety- and depression-like behaviors and alcohol preference (Quevedo et al.,2002; Cicin-Sain et al., 2004; Hranilovic et al., 2005)between sublines. All these results suggest hyperactivityof serotonergic transmission in high-5HT rat subline incomparison with that of low-5HT subline.

There is an increasing body of evidence that cytosolicendo- and exopeptidases, in addition to membrane-bound proteases, are involved in the metabolism of bio-

logically active peptides (for review see Shrimpton et al.,2002; Hui, 2007). In this work, the activities of threecytosolic APs were measured in brains and body organsin WZ-5HT rats from both sublines. The assayed APactivities were those of alanyl aminopeptidase (alanyl-AP), arginyl aminopeptidase (arginyl-AP, aminopepti-dase B) and dipeptidyl peptidase III (DPP III, enkepha-linase B). Our previous results (Abramic et al., 1988;Abramic and Vitale, 1992) together with the contribu-tion of other research group indicated that these threecytosolic exopeptidases, being abundant in the rat braintissue and possessing a high affinity for a number of neu-ropeptides, could be good candidates to test a possiblerelationship with the 5HT system activity.

2. Material and methods

Females from the 23th breeding generation of WZ-5HT rats, aged 3.5 months were used. Their platelet5HT parameters were measured at the age of one monthand confirmed by the second determination performed aweek before taking the samples for enzyme analysis.Animals were housed in a colony room with 12:12 light-ing, 22 ± 2 �C, with food and water available ad libitum.Experiments were performed in accordance with theNIH Guide for the Care and Use of Laboratory Ani-mals, and research protocol was approved by a localethical committee. Sex- and age-matched unselected ani-mals were used as controls.

Methods for repetitive sampling of rat blood anddetermination of platelet 5HT parameters weredescribed previously (Jernej and Cicin-Sain, 1990; Jernejet al., 1999a). Organs to be assayed were rapidlyremoved from the rat after decapitation, rinsed withcold saline and blotted dry. The 10% (w/v) homogenateswere prepared in 50 mM Tris–HCl buffer, pH 7.6, con-taining 250 mM sucrose and 134 mM KCl, by the useof Ultra-Turrax homogenizer (three 5 s bursts) and werecentrifuged (17,000g, 45 min, 4 �C). The obtained super-natants were used for biochemical studies as ‘‘tissuecytosol”. Exopeptidase activities were measured spectro-photometrically by incubating samples with a saturatingconcentration of 2-naphthylamide (2NA) substrates aspreviously described (Abramic and Vitale, 1992; Abram-ic et al., 2004). Alanyl-AP and arginyl-AP were assayedin 25 mM Tris–HCl buffer, pH 7.5, with 0.07 mM Ala-2NA and Arg-2NA, respectively, while the DPP IIIactivity was determined with 0.04 mM Arg-Arg-2NAas a substrate in 50 mM Tris–HCl buffer, pH 8.6. Oneunit of aminopeptidase activity (U) corresponds tothe amount of enzyme which hydrolyzes 1 lmol of sub-strate per minute at 37 �C. Protein concentration wasdetermined following the Bradford method (Bradford,1976) and using the bovine serum albumin as astandard.

L. Cicin-Sain et al. / Neuropeptides 42 (2008) 435–440 437

Data were analyzed by using Statistica (StatSoft Inc.,Version 5.0) software with the level of significance set atp < 0.05. The normality assumption was examined byKolmogorov and Smirnov test. Analyses were per-formed by the use of Student’s two-tailed unpaired t-test(platelet 5HT parameters) or two-tailed Mann–WhitneyU-test (APs activities).

3. Results

Platelet 5HT measures in 5HT sublines, determined aweek before tissue sampling, are shown in Fig. 1. Thedifference in the mean values of both platelet serotoninlevel and velocity of platelet serotonin uptake was highlysignificant between high-5HT and low-5HT sublines(p < 0.001, t-test), with the mean values of control ani-mals falling in between.

Aminopeptidase activities assayed against alanyl-(Ala-AP), arginyl-(Arg-AP) and arginyl-arginyl-2NA(DPP-III) in the brain and body organs cytosols of therat sublines are shown in Fig. 2. Comparison across dif-ferent tissues revealed heterogeneous regional distribu-tion of AP activities: 3-fold and 5-fold difference wereobserved between the tissue with the highest and lowestactivities. None of measured AP activities was homoge-nously distributed among the different tissues, regardlessof the 5HT subline. In general, the highest levels of APactivity were found in the brain, kidney and intestine.The distribution of the DPP III activity was clearly dif-ferent from the other two, since a high level of activitywas determined in the liver as well, and the DPP IIIactivity in lung extracts was relatively higher than theactivity towards Ala-2NA and Arg-2NA.

Fig. 1. Platelet serotonin level (lg 5HT/mg platelet protein) andvelocity of platelet serotonin uptake (nmol 5HT/mg platelet protein/min) in animals from high-5HT and low-5HT sublines and in controlanimals, expressed as mean ± standard deviations. N = 5 per group.Mean values of platelet 5HT uptake in the group of 15 animals fromwhich 5 animals were chosen at random for this study was 1.52 ± 0.17.Differences by t-test, **p < 0.01 high-5HT subline vs. control;***p < 0.001 high-5HT vs. low-5HT subline.

There was no difference in DPP III activity betweenthe groups differing in 5HT parameters in any rat organ.However, animals from the high-5HT subline demon-strated significantly lower activity of both enzymes, ala-nyl-AP (p < 0.05, U-test) and arginyl-AP (p < 0.01, U-test), in their brains as compared to control animals.

4. Discussion

This study indicates that the activity of brain cyto-solic aminopeptidases may be related to the activity ofserotonergic system. Results were achieved in an origi-nal rat model consisting of sublines with constitutivealterations in 5HT transporter activity, and conse-quently, whole 5HT homeostasis. Differences betweenhigh-5HT and low-5HT sublines were found in brains,and not in peripheral tissues.

Differences in the brain activity of Arg-AP and Ala-AP observed between the sublines of rats with hyperac-tivity/hypoactivity of 5HT system may suggest the dif-ferent controls of functions in which these enzymes areinvolved. Our further studies on the relation between5HT system and APs activity will be focused on discretebrain regions of rats from 5HT sublines.

In support of the inverse relationship between 5HTand AP activity levels are the reports that mice withgenetically induced decrease of (puromycin-sensitive)AP activity showed an increased anxiety and impairedpain response (Osada et al., 1999), both thought to beassociated with an increased 5HT activity. Also, thememory enhancing effects of protease inhibition (Gar-cia-Horsman et al., 2007) are in line with a better mem-ory performance, which we have observed in animalsfrom the 5HT-high subline (Quevedo et al., 2002), andhere we demonstrated their lower AP activities(Fig. 2). Measuring 5HT activity in a recently developedAPN-null mice (Rangel et al., 2007) and/or in a mouseline deficient in the gene for puromycin-sensitive AP(PSA, cytosol alanyl-AP) (Osada et al., 1999) mightprobably help to elucidate a link between central APsand 5HT system activities.

Regarding substrate specificity, two cytosolic APs areable to cleave L-Arg-2NA: Arg-AP (aminopeptidase B)and PSA. However, towards amino acid 2-naphthyla-mides, PSA has a broad specificity: Ala-2NA andArg-2NA are hydrolyzed at approximately equal rates.We have chosen Ala-2NA as a second AP substratebecause Arg-AP does not hydrolyze it and the PSA isa major cytosolic protease which cleaves this syntheticsubstrate. Based on the hydrolytic activity towards thesetwo substrates, we can conclude that significantdifferences in AP activity between 5HT sublines mayreflect predominantly the differences in PSA level. It isknown that PSA is most abundant in rodent brain(Dando and Barrett, 2004). Recently, important role

Fig. 2. Specific activities of arginyl aminopeptidase (Arg-AP), alanyl aminopeptidase (Ala-AP) and dipeptidyl peptidase (DPP III) in brains andbody organs of high-5HT and low-5HT sublines and in control animals, expressed as nmol of aminoacyl-2-naphthylamide hydrolyzed per min permg protein. Values represent mean ± standard error of the means. N = 5 per group. Differences for high-5HT subline vs. control by Mann–WhitneyU-test, *p < 0.05, **p < 0.01; B-brain (whole brain without cerebellum), H – heart, L – lungs, Lv – liver, K – kidney, I – intestine (distal third of thesmall intestine).

438 L. Cicin-Sain et al. / Neuropeptides 42 (2008) 435–440

of this aminopeptidase has been recognized in protec-tion against tau-induced neurodegeneration in vivo

(Karsten et al., 2006).It has been shown previously that brain AP activities

might fluctuate during the estrous cycle in the female rat(de Gandarias et al., 1993, 1996), but these changes seemto be specific to the brain areas which are related toreproduction and are dependent on the type and

localization of the enzyme. In our study, the enzymeactivities were measured in homogenates of the wholebrains, and animals were grouped at random (i.e. stagesof estrous cycle are expected to be distributed similarlybetween groups), so we believe that the observeddecrease in soluble APs in brains of high-5HT animalswas not influenced by the hormonal status of theanimals.

L. Cicin-Sain et al. / Neuropeptides 42 (2008) 435–440 439

The possibility that individual differences in 5HTtransmission may be related to neuropeptidase activitiesmight have clinical implications. For example, it hasbeen suggested that any factor that leads to brain APdownregulation would increase pathological aggrega-tion of tau proteins, which, in turn, could contributeto susceptibility to neurodegenerative diseases (Leeet al., 2001; Karsten et al., 2006). In this sense our resultsindicate that 5HT system may be a modifier of diseasesusceptibility and thus may represent a potential targetfor therapeutics in neurodegenerative diseases. A possi-ble relevance for the neurobiology of autism should alsobe considered. Namely, one of the hypotheses regardingetiology of autism postulates that excessive amounts ofexogenous/endogenous peptides (Shattock and White-ley, 2002) caused probably by defective (peripheral) pep-tidase activity may be important in pathophysiology ofthis disorder. The most consistent, but unexplained,neurochemical finding in autistic patients is their ele-vated blood 5HT levels (hyperserotoninemia) (Janusoniset al., 2006). Study on our high-5HT rats having consti-tutive hyperserotoninemia (Fig. 1), and low peptidaseactivity in brain, but not in periphery (Fig. 2) may pos-sibly help to understand how genetically based 5HTalteration may result in peptidase deficiency supposedin autism.

In contrast to the brain where differences in APsactivities between 5HT sublines were evident, no analo-gous changes were detected outside the CNS (Fig. 2),which is in agreement with differential APs regulation/role depending on their location. Regarding the distribu-tion among body organs in rats, our data (Fig. 2.) aremostly in line with the literature (Lee and Snyder,1982; Agirregoitia et al., 2005).

In conclusion, we have found that constitutionallyupregulated 5HT activity is related to lowered braincytosolic aminopeptidase activity which may have influ-ence on cleavage of their physiological substrates.

Acknowledgements

We thank Branimir Jernej, MD PhD, for the criticalreading of the manuscript and Mrs. Katarina Karlo forskillful technical assistance. This work was supported byGrants from the Croatian Ministry of Science, Educa-tion and Sport (No. 098-1081870-2397 and 098-1191344-2938).

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