Copyright © 2004 by Institute of Pharmacology Polish Journal of Pharmacology Polish Academy of Sciences Pol. J. Pharmacol., 2004, 56, 421–425 ISSN 1230-6002

EFFECT OF DIFFERENT FORMS OF VASOACTIVE INTESTINAL PEPTIDE ON cAMP FORMATION IN THE MAMMALIAN CEREBRAL CORTEX

Agnieszka Dejda1, Hanna Gendek-Kubiak2, Jerzy Z. Nowak1,3,# Centre for Medical Biology, Polish Academy of Sciences, Lodowa 106, PL 93-232 £ódŸ, Department of Cytophysiology, Histology and Embryology, Medical University, Narutowicza 60, PL 90-136 £ódŸ, !Department of Pharmacology, Medical University, ¯eligowskiego 7/9, PL 90-752 £ódŸ, Poland

Effect of different forms of vasoactive intestinal peptide on cyclic AMP formation in the mammalian cerebral cortex. A. DEJDA, H. GENDEK- KUBIAK, J.Z. NOWAK. Pol. J. Pharmacol., 2004, 56, 421–425.

Chicken, guinea pig and mammalian (human/porcine/rat) vasoactive in- testinal peptides (VIP; 0.001–3 mM) were compared with respect to their ability to stimulate adenosine 3’, 5’-cyclic monophosphate (cAMP) forma- tion in the cerebral cortical slices of rat and guinea pig. Of the tested pep- tides, i.e. chicken VIP (cVIP), guinea pig VIP (gpVIP) and human/rat/por- cine (mammalian) VIP (mVIP), the strongest stimulator of cAMP synthesis was cVIP, and the weakest one – the gpVIP. Pituitary adenylate cyclase- activating polypeptide (PACAP) used as a reference drug at 0.1 mM concen- tration strongly stimulated cAMP formation in the cerebrum of both species, being, however, significantly more potent in the guinea pig model. The ob- tained data demonstrate significant differences in biological activity between cVIP and two distinct mammalian VIPs in the cerebral cortex of rat and guinea pig.

Key words: vasoactive intestinal peptide (VIP), chicken vasoactive intes- tinal peptide (cVIP), guinea pig vasoactive intestinal peptide (gpVIP), human, rat, porcine (mammalian) vasoactive intestinal peptide (mVIP), cyclic adeno- sine 3’, 5’-monophosphate (cAMP), cerebral cortex, guinea pig, rat

 correspondence; e-mail: [email protected] A. Dejda, H. Gendek-Kubiak, J.Z. Nowak

INTRODUCTION vasodilatation and smooth muscles relaxation [4, 5, 16, 22, 31]. Vasoactive intestinal peptide (VIP) is a 28- Various biological effects of VIP are mediated amino acid polypeptide widely distributed in the through interaction with two types, named tissues of various vertebrates. VIP was originally VPAC and VPAC , which recognize with a similar isolated from porcine [33]. Yet, later on, high affinity both VIP and PACAP. Yet, PACAP its presence has been detected in the gastrointesti- can also bind to another receptor type, called PAC , nal, respiratory, cardiovascular, genitourinary, and which expresses modest or weak affinity for VIP immune systems, endocrine glands and brain [1, 2, [17]. These three receptors are members of a large 5, 11, 19–21, 29, 32, 33, 35]. VIP belongs to a su- family of G-protein-coupled receptors, and are perfamily of structurally related peptides, embracing preferentially linked to stimulation of adenylyl cy- pituitary adenylate cyclase-activating polypeptide clase activity [17]. (PACAP), , , glucagon-like peptide-1 The aim of this paper was to analyze in a paral- (GLP-1), glucagon-like peptide-2 (GLP-2), gluco- lel study the effects of three different VIPs, i.e. the se-dependent insulinotropic polypeptide (GIP), human/rat/porcine (mammalian), guinea pig and growth -releasing hormone (GHRH), pep- chicken one, on receptor-linked cAMP formation in tide histidine-isoleucine (PHI) and its human coun- the cerebral cortex of two mammals, guinea pig terpart – peptide histidine-methionine (PHM) [36]. and rat. The primary structure of VIP is highly conserved among lower and higher vertebrates [13, 18], being MATERIALS and METHODS identical in ten out of twelve mammals studied thus far, i.e. human, monkey, dog, cow, goat, sheep, pig, Animals rabbit, rat, mouse. Exceptions are guinea pig and opossum, whose peptides have four and five substi- Experiments were carried out on male guinea tutions, respectively [9, 10]. VIP present in non- pigs (Dankin Hartley; 250–300 g) and rats (Wistar; mammalian vertebrates shares 82–86% amino acid 180–250 g). The animals were maintained under sequence identity with the human/rat/porcine pep- a 12 h light : 12 h dark lighting schedule (lights on tide, often referred to as mammalian VIP (mVIP); between 07.00 and 19.00) with standard food and chicken VIP (cVIP) differs from its mammalian tap water available ad libitum. On the day of ex- counterpart in four positions, although the total periment, the animals were killed by decapitation, number of amino acid residues is the same [24]: brains were quickly removed, and cerebral cortex (without white matter) was isolated and processed Human, monkey, dog, HSDAVFTDNYTRLRKQMAVKKY for biochemical measurements. The experiments cow, goat, pig, sheep, LNSILN were carried out in strict accordance with the Pol- rabbit, mouse, rat ish governmental regulations concerning experi- Guinea pig HSDALFTDTYTRLRKQMAMKKY ments on animals (Dz.U.97.111.724). LNSVLN Opossum HSDAVFTDSYTRLLKQMAMRKY Assay of cAMP formation LDSILN Each experiment was carried out on the cerebral S F Chicken HSDAVFTDNY R RQMAVKKYL cortex isolated from one guinea pig or rat and re- NSVLT peated two-three times (each sample in triplicate). Cross-chopped slices (250 mm; prepared with VIP exerts pleiotropic biological activity. In ad- McIlwain tissue chopper) of the tissue were sus- dition to its role as a /neuromodu- pended in cold, O /CO (95:5) gassed, glucose- lator, VIP has been demonstrated to function as a supplemented modified Krebs-Henseleit medium hormone/neurohormone, a neurotrophic, growth, (containing in mM: 118 NaCl, 5 KCl, 1.3 CaCl , and neuroprotective factor, and an anti-inflammatory 1.2 MgSO", 1.2 KH PO", 25 NaHCO! and 11.7 D- agent [8, 13–15, 36]. Furthermore, VIP modulates the glucose; pH 7.4). The formation of [!H]cAMP in secretory activity of the pituitary gland, adrenal [!H]adenine prelabeled preparation of the cerebral gland and , as well as ion transport in the cortex was assayed according to Shimizu et al. intestine [3, 28, 30, 38]. This peptide also leads to [37]. The formed [!H]cAMP was isolated by a se-

422 Pol. J. Pharmacol., 2004, 56, 421–425 VIP AND cAMP IN MAMMALIAN BRAIN quential Dowex-alumina chromatography accord- gpVIP, produced net increases in the nucleotide ing to Salomon et al. [34]. The results were indi- production (in per cent conversion) by, respec- vidually corrected for a percentage recovery with tively, 8.88, 9.08 and 7.86 in the guinea pig cere- the aid of [ "C]cAMP added to each column system bral cortex, and by 3.66, 3.84 and 2.98 in the rat prior to the nucleotide extraction. The accumula- cerebral cortex. In both species, the most potent tion of cAMP during a 15-min stimulation period stimulator of cAMP synthesis was cVIP, whereas was assessed as a per cent of the conversion of the weakest one was gpVIP. A high potency of [!H]adenine to [!H]cAMP. Details of the whole cVIP in comparison with mVIP in stimulating procedure were described by us earlier [26]. cAMP production we have already observed in our earlier study carried out on the rat brain [25], as Chemicals well as in several fowls, such as chick, goose and The following were used: VIP turkey [6, 27; Zawilska et al., unpublished observa- (chicken – SP022212) from Neosystem (Stras- tion]. A comparatively low potency of the gpVIP to bourg, France), VIP (guinea pig – 1182) from increase cAMP formation in guinea pig cerebral Tocris Cookson Ltd. (Bristol, UK), VIP (human, cortex seems rather surprising, as this peptide is a porcine, rat – V 6130) and PACAP38 (mammalian native one to this mammal. In fact, the gpVIP was – A 1439) from Sigma (St. Louis, MO, USA). Ra- even less potent than the human/rat/porcine VIP diolabeled compounds: [!H]adenine (specific activ- which is an observation that we cannot explain at ity 24.2 Ci/mmol) and [ "C]cAMP (specific activity the moment. It can be only hypothesized that of the 56 mCi/mmol) were purchased from PerkinElmer tested VIPs, whose amino acid sequences vary in 4 Life Sciences Inc. (Boston, MA, USA) and Mo- positions (different in each species), the chemical ravek Biochemical Inc. (Brea, CA, USA), respec- structure of the gpVIP may be the least favorable tively. Other chemicals were of analytical purity for its interaction with adenylyl cyclase-linked and were obtained mainly from Sigma (St. Louis, VPAC type receptor, resulting in a comparatively MO, USA). small cAMP response. This hypothesis is supported by the recently published findings showing that Statistical analysis modification of only one amino acid residue in

Data (showing net increases in the cAMP re- mVIP, i.e. [Leu ]VIP or [Ala ]VIP, can lead to sponse, i.e. differences between peptide-stimulated and basal values) were expressed as means ± SEM, and were analyzed for statistical significance by one-way analysis of variance followed by post-hoc Student-Newman-Keuls test using GraphPad soft- ware.

RESULTS and DISCUSSION

The following peptides were tested for their ability to stimulate cAMP formation in the guinea pig and rat cerebral cortex: chicken VIP (cVIP; 0.001–3 mM), guinea pig VIP (gpVIP; 0.001–3 mM), and mammalian (human/rat/porcine) VIP (mVIP; 0.01–3 mM). All the peptides stimulated the synthesis of cAMP in a concentration-depen- Fig. 1. Effect of chicken VIP (cVIP), guinea pig VIP (gpVIP) and mammalian VIP (mVIP) on cAMP formation in dent manner, with maximal effects being decisively [!H]adenine-prelabeled slices of the rat cerebral cortex. Basal larger in guinea pig than in rat tissue (Fig. 1 and 2). activities (in per cent of conversion) were: 1.040 ± 0.076 (14), The relative rank order of potency of the tested 0.878 ± 0.063 (5) and 0.876 ± 0.056 (13) for the above peptides, VIPs to stimulate cAMP formation was: cVIP >> respectively. Results are expressed as net increases and ³ represent means ± SEM from n = 3–15. Values statistically mVIP gpVIP in the guinea pig cerebrum, and different from respective controls: *p < 0.05, **p < 0.01, cVIP > mVIP > gpVIP in the rat tissue. At the high- ***p < 0.001. PACAP38 serves as a positive control of cAMP est tested concentration, i.e. 3 mM, cVIP, mVIP and formation

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stimulatory PACAP-driven signals from three re- ceptors, i.e. VPAC ,VPAC ,PAC , likely contrib- ute to the overall biological effect of this peptide. In conclusion, the present work demonstrates the existence of differences between the chicken, guinea pig and mammalian (human/rat/porcine) VIPs in their ability to stimulate cAMP production in the cerebral cortex of the guinea pig and rat, with the guinea pig peptide (gpVIP) being the least po- tent in evoking the cAMP response in both species.

Acknowledgment. This study was supported by grants from the Medical University in £ódŸ, No. 502-11-93 and No. 502-11-168 and by funds from the Centre for Medical Fig. 2. Effect of chicken VIP (cVIP), guinea pig VIP (gpVIP) Biology, Polish Academy of Sciences in £ódŸ, Poland. and mammalian VIP (mVIP) on cAMP formation in [!H]adenine-prelabeled slices of the guinea pig cerebral cortex. REFERENCES Basal activities (in per cent of conversion) were: 0.587 ± 0.088 (7), 1.376 ± 0.161 (7) and 0.98 ± 0.140 (14), for the above 1. Ahren B, Alumets J, Ericson M, Fahrenkrug J, Fahrenk- peptides, respectively. Results are expressed as net increases rug L, Hakanson R, Hedner P et al.: VIP occurs in intra- and represent means ± SEM from n = 3–9. Values statistically thyroidal nerves and stimulates hormone se- different from respective controls: *p < 0.05, ***p < 0.001. cretion. Nature, 1980, 287, 343–345. PACAP38 serves as a positive control of cAMP formation 2. Aliakbari J, Sreedharan SP, Turck CW, Goetzl EJ: Se- lective localization of vasoactive intestinal peptide dramatic changes in the peptide affinity for VPAC and substance P in human eosinophils. Biochem Bio- or VPAC receptor [12]. In addition, our recent ob- phys Res Commun, 1987, 148, 1440–1445. servation showed that substitution of Gly for D-Phe 3. Bailey CJ, Wilkes LC, Conlon JM, Armstrong PH, Bu- in the fourth position of PHI, a VIP-like peptide, chanan KD: Effects of gastric inhibitory polypeptide, va- resulted in a minimal effect of the modified peptide soactive intestinal polypeptide and peptide histidine iso- leucine on the secretion of by isolated mouse on cAMP formation in the rat cerebral cortex and pancreatic islets. J Endocrinol, 1990, 125, 375–379. in comparison with the effect of un- 4. Blank MA, Brown JR, Hunter JC, Bloom SR, Tyers changed PHI in the studied models [6, 7]. On the MB: Effects of VIP and related peptides and Gila other hand, regarding the structure of receptors for monster venom on genitourinary smooth muscle. Eur VIP, it has been demonstrated that even small J Pharmacol, 1986, 132, 155–161. changes in the amino acid sequence of the human 5. Dalsgaard T, Hannibal J, Fahrenkrug J, Larsen CR, VPAC receptor led to significant changes in both Ottesen B: VIP and PACAP display different vasodila- # tory effects in rabbit coronary and cerebral arteries. radioligand ([ I]VIP) binding and the ability of Regul Pept, 2003, 110, 179–188. VIP to stimulate cAMP production [23]. 6. Dejda A, Matczak I, Nowak JZ: Peptide histidine- In addition to VIP, in each experiment, we used isoleucine (PHI) – from receptor to function (Polish). PACAP38 (0.1 mM) as a positive control, which, Dzia³alnoœæ Naukowa PAN, 2003, 15, 126–128. according to our earlier findings, is a highly potent 7. Dejda A, Matczak I, Wiktorowska-Owczarek A, Nowak stimulator of the nucleotide production in the verte- JZ: Cyclic AMP formation in chicken brain: effect of va- brate brain [25, 26]. In different experiments, PA- soactive intestinal peptide, peptide histidine-isoleucine (PHI), and some PHI-related peptides. Pol J Pharma- CAP38 evoked net increases in cAMP production col, 2003, 55, 747–751. of 10.93 ± 0.47 in the guinea pig cerebral cortex, 8. Delgado M, Abad C, Martinez C, Juarranz MG, Ar- and of 2.62 ± 0.18 in the rat cerebral cortex (the ranz A, Gomariz RP, Leceta J: Vasoactive intestinal data represent the mean values ± SEM from n = 20 peptide in the immune system: potential therapeutic and n = 23, respectively), showing a clearly greater role in inflammatory and autoimmune diseases. J Mol sensitivity of the receptor-coupled cAMP generat- Med, 2002, 80, 16–24. 9. Du BH, Eng J, Hulmes JD, Chang M, Pan YCE, Yalow ing system of the guinea pig than that of the rat RS: Guinea pig has a unique mammalian VIP. Biochem brain (Fig. 1 and 2). It has to be stressed that, in con- Biophys Res Commun, 1985, 128, 1093–1098. trast to VIP which interacts only with VPAC type re- 10. Eng J, Yu J, Rattan S, Yalow RS: Isolation and amino ceptors, in the PACAP-evoked cAMP response, acid sequences of opossum vasoactive intestinal

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