JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2004, 55, 4, 739–749

www.jpp.krakow.pl

M. JUSZCZAK, K. FURYKIEWICZ-NYKIŒ, B. STEMPNIAK

ROLE OF TACHYKININ RECEPTORS AND MELATONIN IN

SECRETION FROM ISOLATED RAT HYPOTHALMO-

NEUROHYPOPHYSIAL SYSTEM

Department of Pathophysiology, Medical University of £ódŸ, £ódŸ, Poland

Present investigations were undertaken to study the influence of NK-1 and

NK-2 receptor agonists and antagonists as well as 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 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 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.

Radioimmunoassay

The OT concentration in all samples was determined by double-antibody specific RIA. Anti-OT antibodies were raised by Dr. Monika Or³owska-Majdak (Department of Experimental and Clinical

Physiology, Institute of Physiology and Biochemistry, Medical University of £ódŸ); for the characteristic of antibodies see: Juszczak (6). The lower limit of detection for the assay was 1.25 pg

125 OT per tube. For standard curve preparation as well as for iodination with I, using the chloramine-

T method, the OT (Oxytocin synth.) from Peninsula Laboratories Europe Ltd. was used. The intra- assay coefficient of variation for the OT assay was less than 5% (all samples within the experiment were tested in the same RIA to avoid inter-assay variability).

Statistical evaluation of the results

All data are expressed as means ± S.E.M. Significance of the differences between means was evaluated by use of the Kruskal-Wallis analysis of variance (ANOVA) by ranks for each set of data

(all subgroups). Thereafter, the statistical significance of differences between means of two subgroups compared was determined by Mann-Whitney "U" test, using p<0.05 as the minimal level of significance.

Fig. 1. Effect of melatonin

(MLT) and the NK-1 receptor

9 11 agonist [Sar ,Met(O ) ]- VEH MLT 2 Substance P or the NK-2

2,5 b 8 receptor agonist (ß-Ala )- 2 Neurokinin A (4-10), both at -9 -7 the concentration of 10 or 10 a a 1,5 M/L, on oxytocin (OT) release 1 from the rat hypothalamo- neurohypophysial complex in

0,5 vitro. Each bar represents mean

OT release (B2/B1) + S.E.M.; number of samples

0 a per subgroup (n) = 6-8; p<0.05

control NK1(-9) NK1(-7) NK2(-9) NK2(-7) - significantly different versus

agonists explants incubated in medium

containing melatonin vehicle

b (VEH); p<0.05 - significantly

different versus control. 743

RESULTS

9 11

Series I. The NK-1 receptor agonist (Sar ,Met(O2) )-Substance P, at the

-7 concentration of 10 M/L, enhanced markedly OT secretion from isolated rat H-N

-9 explants, but it failed to affect the hormone release at the concentration of 10

M/L; the latter effect was observed when melatonin vehicle or melatonin solution was present in the medium. Melatonin itself diminished basal release of OT when compared to the vehicle (control subgroup); it also significantly inhibited the NK-

1 receptor agonist-stimulated release of the hormone (Fig. 1).

Basal OT secretion into the medium was not different from the control when

8 NK-2 receptor agonist (ß-Ala )-Neurokinin A (4-10) was added to the buffer;

-9 -7 both concentrations (i.e., 10 and 10 M/L) of the peptide were ineffective in modifying the OT output from isolated H-N explants independently of the presence of melatonin or its vehicle in the medium (Fig. 1).

Series II. Basal release of OT was inhibited significantly when the NK-1

6 7 9 receptor antagonist (Tyr ,D-Phe ,D-His )-Substance P (6-11) - sendide - was added to the medium; sendide also completely blocked the stimulatory effect of SP on the

OT output from isolated H-N system (Fig. 2A). Melatonin itself diminished basal release of the hormone when compared to the vehicle (control group); it also significantly inhibited the SP-induced OT secretion into the medium (Fig. 2A).

Fig. 2. Effect of melatonin

(MLT) and the NK-1 receptor

6 7 3 Aantagonist (Tyr ,D-Phe ,D- 9 His )-Substance P (6-11) 2,5 b {anNK1} and/or substance P 2 {SP} [A] as well as the NK-2

VEH 5 1,5 receptor antagonist (Tyr ,D-

MLT 6,8,9 10

Trp ,Lys-NH2 )-Neurokinin A 1 a bb a c (4-10) {anNK2} and/or a 0,5 OT release (B2/B1) neurokinin A {NKA} [B], at the

-9 -7 0 concentration of 10 or 10

control anNK1(-9) anNK1(-7) SP(-7) SP/an(-7) M/L, on oxytocin (OT) release

from the rat hypothalamo- 2,5 B neurohypophysial complex in

2 vitro. Each bar represents mean

a + S.E.M.; n = 8-10; p<0.05 - 1,5 VEH significantly different versus MLT explants incubated in medium 1 aa a containing melatonin vehicle a 0,5 (VEH) and the respective OT release (B2/B1) b peptide; p<0.05 - significantly

0 c different versus control; p<0.05 control anNK2(-9) anNK2(-7) NKA(-7) NKA/an(-7) - significantly different versus

explants incubated in medium

containing SP and VEH. 744

5 6,8,9 10

Neither NKA nor NK-2 receptor antagonist (Tyr ,D-Trp ,Lys-NH2 )-

Neurokinin A (4-10) affected significantly the release of OT from isolated H-N system (Fig. 2B). Melatonin diminished basal secretion of the hormone (control group); it also inhibited the OT release in the presence of NKA and/or NK-2 receptor antagonist (Fig. 2B).

DISCUSSION

There are several lines of evidence that point to neuropeptides participating in the regulation of OT secretion in the rat. However, very little is known about the influence of SP and/or NKA on OT secretion and, what is more, the previous data suggest existence of both stimulatory (19, 37-39) and inhibitory (21) actions.

Results from the present study, showing the stimulatory effect of SP on OT output from isolated H-N complex, are in agreement with previous in vitro observations

(38, 39). Additionally, we have demonstrated, for the first time, that highly

9 11 selective NK-1 receptor agonist, i.e., [Sar ,Met(O2) ]-Substance P, enhances significantly the release of OT from the rat H-N complex in vitro.

When potency of sendide (highly selective antagonist of tachykinin NK-1 receptor) in antagonizing the effects of SP in central and peripheral nervous system was studied previously, it was found to antagonize several SP-induced behavioural responses in the rat (40). In our present studies, we have found that this NK-1 receptor antagonist not only blocked the stimulatory effect of exogenous SP on OT secretion into the medium, but also inhibited by itself the OT release from the rat

H-N complex in vitro. The latter effect could hypothetically result from the fact that probably present in isolated H-N explant endogenous SP could not participate in stimulation of OT secretion into the medium because of the NK-1 receptor blockade by sendide. The above results strongly suggest that SP influences the OT secretion acting via NK-1 .

When a role in central and peripheral nervous system of the very potent antagonist with high affinity and selectivity for tachykinin NK-2 receptor, i.e.,

5 6,8,9 10

(Tyr ,D-Trp ,Lys-NH2 )-Neurokinin A (4-10) was studied in rats and guinea- pig, it has been found to antagonize the effects of NKA (a physiological ligand

8 for NK-2 receptor) or the NK-2 receptor agonist (ß-Ala )-Neurokinin A (4-10), without affecting the response to an NK-1 receptor agonist (28-32). Under present experimental conditions, however, the above mentioned tachykinin NK-2 receptor selective agonist and antagonist were essentially inactive in modifying the OT release from isolated rat H-N system. Moreover, the response under study was increased when NKA was added into the medium, although this change did not reach significance in the present experiment. Taking together, present findings implicate a major role for tachykinin NK-1, rather than for NK-2, receptors in stimulatory effect of endogenous tachykinins on OT secretion from the rat H-N system. Such a suggestion is consistent with the results of binding studies, which 745

have given conclusive evidence for the occurrence of central NK-1 binding sites and mRNA for the SP receptor (17, 41), while only low levels of NK-2 receptors have been detected in some specific nuclei of the brain (26, 27). Since NKA has been described as an efficient agonist also at NK-1 receptor (24, 25) it could be, therefore, suggested that NKA, like SP, may be acting via NK-1 receptor to affect the oxytocinergic neurones function.

Several studies have demonstrated the coexistence of a variety of putative and/or neuropeptides in SP- and/or SP/NKA-ergic neurons.

Identified substances within such neurons include serotonin (23), acetylcholine (11), catecholamines (11, 39) as well as some neuropeptides. Moreover, several neuroactive agents such as γ-aminobutyric acid - GABA, nitric oxide or ATP (21, 39) were reported to be of some importance for the mechanisms by which oxytocinergic neurones are influenced by tachykinins. All the above mentioned neurotransmitters and/or neuromodulators are involve in modifying OT release from the H-N system

(1, 7) and certain combination of these agents elicits a potent resultant effect.

Studies of the effect of melatonin on OT release in the rat suggested existence of both stimulatory and inhibitory actions, depending on a dose of the hormone (2,

5, 6, 33-35). The concentration of melatonin employed in the present experiment,

-9 i.e., 10 M/L is at the range of physiological level of the hormone, and was found to inhibit the OT release from isolated H-N explants. Action of melatonin on neurohypophysial hormone release from the rat hypothalamic explants in vitro was found to depend on the time of day (36). Namely, melatonin inhibited OT secretion when the hypothalamic tissue was obtained from animals at light conditions (i.e.,

2-3 h after lights on), but no effect of the hormone could be seen when tissue samples were obtained during the night, i.e., 4-5 h after lights off [36]. Present experiments were, therefore, performed during light period of the light/dark cycle, i.e., about 4 h after lights on, and the inhibitory effect of melatonin on basal release of OT is in concordance with previous in vitro (34-37) and in vivo (2) data.

In the present in vitro experiment, the NK-1 receptor antagonist, similarly to melatonin, blocked the stimulating effect of SP on OT release. However, similar influence of the selective NK-2 receptor antagonist on the OT output from isolated H-N complex could not be seen. The data here reported show, therefore, that melatonin and SP (and tachykinin NK-1 receptor) are differentially involve in the process under study.

It is postulated that melatonin modifies OT secretion acting via specific melatonin membrane receptors localized in the hypothalamus, especially those located in the suprachiasmatic (SCN) nucleus (42). Direct neuronal projection from the SCN to the PVN (43) or SON (44) may, at least in part, participate in the process under discussion; the hypothalamo-neurohypophysial explant, we employed in the present experiment in vitro, contained the hypothalamic SCN,

SON and PVN with intact axonal projections to the neurohypophysis. It has been found that the SCN neurons can influence function of the PVN and/or SON cells by releasing from their axonal endings either excitatory (glutamate) or inhibitory 746

(GABA) amino acids (43); L-glutamate has been shown to increase plasma OT level, whereas GABA exerts an inhibitory influence on this hormone secretion

(7). Additionally, the SCN neurons contain several peptides including SP, the specific tachykinin receptors (25, 45) as well as the SP-immunoreactive fibres and nerve terminals (45). Presence of preprotachykinin-A mRNA in same parts of the SCN indicates that synthesis of SP and/or NKA also takes place in the SCN neurons (45). Some of the SCN neurons could, therefore, integrate the afferent signals (also derived from melatonin via its membrane receptors) and thereafter transmit them to oxytocinergic neurons in the PVN and/or SON, which could potentially be mediated by projection of the SP-ergic cells from the SCN.

However, present results, indicating the opposite effects of SP and melatonin on

OT secretion, exclude such a hypothesis.

The pineal hormone can, therefore, modify neuronal activity by interaction with specific melatonin membrane receptors or via direct action on the genome

(46), without interaction with membrane receptors; the involvement of brain- specific nuclear RZR receptors in melatonin action on target tissues has been described (47). Melatonin may also affect the release of OT by acting directly on oxytocinergic axons localized in the neurohypophysis or indirectly via modification of the metabolism of some neuromediators and/or neuromodulators in the hypothalamus and/or neurointermediate lobe (48, 49). In fact, acetylcholine, dopamine and prostaglandins were found to be involved in melatonin-mediated inhibition of OT secretion (50). The intracellular calcium as well as the cAMP and cGMP are postulated to participate in the intracellular mechanisms of melatonin action (51). However, well understanding of the mechanisms underlying the modulation of oxytocinergic neurons function by melatonin, still requires further studies.

In summary: the present study confirms previous data as to the inhibitory effect of melatonin on OT secretion from the rat H-N complex in vitro. It also provides an evidence for tachykinin-induced modulation of OT secretion as mediated by tachykinin NK-1 receptor. However, under present experimental conditions, the contribution of tachykinin NK-1 or NK-2 receptors to the response of oxytocinergic neurons to melatonin has not been demonstrated.

Acknowledgements: This work has been supported by Medical University of £ódŸ, contract No.

502-16-140.

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Received: May 18, 2004

Accepted: November 16, 2004

Author’s address: Marlena Juszczak, Ph.D., D.Sc., Department of Pathophysiology, Medical

University of £ódŸ, ul. Narutowicza 60, 90-136 £ódŸ, Poland, Tel/Fax: (+4842) 630 61 87.