JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2004, 55, 4, 739749 www.jpp.krakow.pl M. JUSZCZAK, K. FURYKIEWICZ-NYKI, B. STEMPNIAK ROLE OF TACHYKININ RECEPTORS AND MELATONIN IN OXYTOCIN SECRETION FROM ISOLATED RAT HYPOTHALMO- NEUROHYPOPHYSIAL SYSTEM Department of Pathophysiology, Medical University of £ód, £ód, Poland Present investigations were undertaken to study the influence of peptide NK-1 and NK-2 receptor agonists and antagonists as well as substance P and neurokinin A (the natural ligands for these tachykinin receptors) on oxytocin (OT) release from isolated rat hypothalamo-neurohypophysial (H-N) system as well as to determine whether the tachykinin NK-1 and/or NK-2 receptors contribute to the response of oxytocinergic neurons to melatonin. The results show, for the first time, that highly selective NK- 9 11 1 receptor agonist, i.e., [Sar ,Met(O2) ]-Substance P, enhances while the NK-1 6 7 9 receptor antagonist (Tyr ,D-Phe ,D-His )-Substance P (6-11) - sendide - diminishes significantly OT secretion; the latter peptide was also found to antagonize the substance P-induced hormone release from isolated rat H-N system, when used at the -7 concentration of 10 M/L. Melatonin significantly inhibited basal and substance P- stimulated OT secretion. Neurokinin A and the NK-2 receptor selective agonist (ß- 8 5 6,8,9 Ala )-Neurokinin A (4-10) as well as the NK-2 receptor antagonist (Tyr ,D-Trp , 10 Lys-NH2 )-Neurokinin A (4-10) were essentially inactive in modifying OT release from the rat H-N system in vitro. The present data indicate a distinct role for tachykinin NK-1 (rather than NK-2) receptor in tachykinin-mediated regulation of OT secretion from the rat H-N system. Under present experimental conditions, however, a role of respective tachykinin receptors in the response of oxytocinergic neurons to melatonin has not been found. Key words: NK-1/NK-2 receptors, substance P, neurokinin A, melatonin, oxytocin INTRODUCTION Oxytocin (a neurohormone synthesized by magnocellular neurons of the hypothalamic paraventricular {PVN} and supraoptic {SON} nuclei) is secreted 740 from the neurohypophysis into general circulation in response to several stimuli, e.g., parturition, suckling, hypovolaemia, plasma hypertonicity or stress (1-6). The release of this hormone depends on the presence of numerous neuromediators and neuromodulators. Abundant evidence supports the importance of neuropeptides as neuromodulators in oxytocin (OT) secretion process (3, 4, 7-9). The hypothalamic PVN and SON neurons and the posterior pituitary contain substance P (SP) and neurokinin A (NKA), members of a family of peptides known as tachykinins (10-14). Numerous SP-containing afferent pathways project to the magnocellular hypothalamic nuclei (11); the SP-immunorective nerve fibres are also present in moderate densities in the rat median eminence and neurohypophysis (15-16). Also a high density of SP binding sites was observed in the PVN and SON nuclei (12, 17) and recently, morphological evidence show that oxytocin-containing neurons co-express SP receptor (18). After intracerebroventricular (icv) injection, SP was found to increase the firing rate of oxytocinergic neurons of the SON (19), while after intravenous (iv) administration it failed to change the OT plasma level in normal men (20). On the other hand, NKA has been found to inhibit the OT secretion from the rat posterior pituitary (21). The above data suggest, therefore, that SP and NKA are differentially involve in the control of the hypothalamic magnocellular neurons function, which could result from stimulation of different classes of tachykinin receptors, for which these two peptides are natural ligands. SP and NKA are protein products encoded by the same preprotachykinin A (PPT-A) gene and they are mostly colocalized and cosynthesized in SP/NKA- ergic neurons (22, 23). They exert their biological activity via tachykinin receptors. Namely, SP acts preferentially on NK-1 receptor, whereas NKA has preferential affinity for the tachykinin NK-2 receptor. As a matter of fact, NKA is also efficient substitute of SP as endogenous agonist at NK-1 receptor and, in turn, SP could act as an agonist at NK-2 receptor (24, 25). These two classes of tachykinin receptors are widely distributed in both central and peripheral nervous system (25-27) and they regulate the function of cardiovascular, respiratory, gastrointestinal and genitourinary systems as well as they are involve in autonomic reflexes and pain transmission (25, 28-32). However, functional importance of tachykinin NK-1 and/or NK-2 receptors for OT secretion has not been studied. The first goal of the present experiments was to determine whether these two classes of neurokinin receptors play a role in the process in question, by studying the effect of tachykinin NK-1 and NK-2 receptor peptide agonists and antagonists on basal OT release from the rat hypothalamo-neurohypophysial system in vitro. Pineal hormone, melatonin, is known to modify the OT secretion under different experimental conditions, both in vivo (2, 5, 6) and in vitro (33-36). Recently, we have demonstrated that melatonin inhibits the NKA- and SP- stimulated OT output from isolated rat hypothalamo-neurohypophysial system 741 [37-38]. To date, however, the effect of coexposure to melatonin and tachykinin NK-1 and/or NK-2 receptor agonists or antagonists on OT release from the hypothalamo-neurohypophysial system has not been evaluated. The second aim of the present investigation was, therefore, to assess the modulatory effect of peptide NK-1 and/or NK-2 receptor agonists (or the tachykinin receptors natural ligands, i.e., SP and NKA) and antagonists on the response of OT to melatonin. MATERIALS AND METHODS Animals Three-months old male Wistar rats (weighing about 250-350 g) were housed under a 12/12 hr light/dark schedule (lights on from 6 a.m.) and at room temperature. The animals received standard pelleted food and had free access to tap water. Drugs 9 11 All peptides, i.e.: substance P, tachykinin NK-1 receptor agonist [(Sar ,Met(O2) )-Substance P] 6 7 9 and antagonist [(Tyr ,D-Phe ,D-His )-Substance P (6-11)] (Sendide) as well as neurokinin A, 8 5 6,8,9 tachykinin NK-2 receptor agonist [(ß-Ala )-Neurokinin A (4-10)] and antagonist [(Tyr ,D-Trp , 10 Lys-NH2 )-Neurokinin A (4-10)], were purchased from BACHEM AG, Bubendorf, Switzerland. Melatonin (N-acetyl-5-methoxytryptamine) come from Sigma-Aldrich Chemie GmbH. Experimental protocol On the day of experiment, animals were decapitated between 10.30 and 11.30 a.m. The brain and the pituitary with intact pituitary stalk were carefully removed from the skull and a block of hypothalamic tissue was dissected as previously described (37). Such hypothalamo- neurohypophysial (H-N) explant was placed immediately in one polypropylene tube with 1ml of Krebs-Ringer fluid (KRF) containing: 120 mM NaCl, 5 mM KCl, 2.6 mM CaCl2, 1.2 mM KH2PO4, 0.7 mM MgSO4, 22.5 mM NaHCO3, 10 mM glucose, 1.0 g/l bovine serum albumin and 0.1 g/l ascorbic acid (pH = 7.4-7.5, osmolality = 285-295 mOsm/Kg). Tubes were placed in a water bath at 37°C and constantly gassed with carbogen (a mixture of 95% O2 and 5% CO2). At the beginning of experiment, the H-N explants were equilibrated in KRF which was aspirated twice and replaced with 1 ml of fresh buffer. After 80 minutes of such preincubation, the media were discarded and explants were incubated for 20 minutes in 1 ml of KRF alone or containing the respective peptide. Series I. In the first series of experiments, the effect of specific NK-1 or NK-2 receptor agonists on basal OT secretion from the H-N explants was tested in vitro. Explants were therefore incubated successively in: (1) normal KRF {B1}; (2) incubation fluid as B1 alone (control; n - number of samples per subgroup, n = 8) or containing either NK-1 (n = 8) or NK-2 (n = 6) receptor agonist at -7 -9 the concentration of 10 or 10 M/L {B2}. The B2 buffer was enriched with either melatonin -9 vehicle (0.1% ethanol) or melatonin solution at the concentration of 10 M/L. After each incubation period, the media were aspirated and samples immediately frozen and stored at -20°C until OT estimation by the radioimmunoassay (RIA). Series II. In the second series of experiments, the effect of specific NK-1 (or NK-2) receptor antagonist and SP (or NKA) on OT secretion from the H-N explants was tested in vitro. After incubation in normal KRF {B1}, explants were consequently incubated in one of the following 742 media: a - incubation fluid as B1 (control; n = 10), b - KRF containing either NK-1 (n = 8) or NK- -9 2 (n = 9) receptor antagonist at the concentration of 10 M/L, c - KRF containing either NK-1 (n = -7 8) or NK-2 (n = 9) receptor antagonist at the concentration of 10 M/L, d - KRF containing either SP (n = 10) or NKA (n = 10) alone, e - KRF containing either SP together with the NK-1 receptor antagonist (n = 9) or NKA together with the NK-2 receptor antagonist (n = 10) at the concentration -7 of 10 M/L {B2}. The B2 buffer additionally comprised either vehicle (0.1% ethanol) or melatonin -9 solution at the concentration of 10 M/L. After each incubation period, the media were aspirated and samples immediately frozen and stored at -20°C until OT estimation by the RIA. To determine the OT secretion, the B2/B1 ratio was calculated for each H-N explant in both series. The experimental procedures were done with the consent (No L/BD/82) of the Local Committee for the Animal Care.