JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2002, 53, 4, 823–834

www.jpp.krakow.pl

M. JUSZCZAK

NEUROKININ A AND THE NEUROHYPOPHYSIAL RESPONSE TO

MELATONIN: IN VITRO STUDIES.

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

The aim of this study was to investigate a possible role of (a member

of a family of known as tachykinins) in the pineal-neurohypophysial

interrelationship. The effect of neurokinin A (NKA) alone or in the presence of

+ pineal hormone - melatonin on basal and K -stimulated and

secretion from the hypothalamo-neurohypophysial system was studied in vitro. The

present results show that NKA stimulated basal vasopressin and oxytocin release

from the isolated hypothalamo-neurohypophysial system, when used at the

-7 concentration of 10 M/L. Melatonin diminished basal release of the

neurohypophysial hormones; it also significantly inhibited the NKA-stimulated

secretion of vasopressin and oxytocin. Lower concentrations of NKA did not affect

the neurohypophysial hormones basal release, however, when melatonin was added

-9 to the medium enriched with NKA at the concentration of 10 M/L, the vasopressin

secretion from the hypothalamo-neurohypophysial explants was decreased

+ significantly. The K -evoked release of neurohypophysial hormones was not further

modified by either NKA or melatonin. The present results confirm previous reports

as to the inhibitory effect of melatonin on both vasopressin and oxytocin secretion

from the hypothalamo-neurohypophysial complex in vitro. However, under present

experimental conditions, the contribution of NKA in the mechanisms of pineal-

neurohypophysial interrelationships has not been demonstrated.

Key words: neurokinin A, melatonin, oxytocin, vasopressin

INTRODUCTION

Vasopressin (AVP) and oxytocin (OT) are synthesized by magnocellular neurones of the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei and transported down axonal projections to terminals in the neurohypophysis (1).

The secretion of these neurohormones is known to be a component of 824

neuroendocrine response to several stimuli (2-8, also for references) and depends on the presence of numerous neuromediators and neuromodulators. A distinct and quite large group of neuromodulators, which modify the AVP and/or OT secretion from the neurohypophysis, is represented by . Indeed, Y, , glucagon-like -1, thyrotropin-releasing hormone, corticotropin-releasing hormone, and gonadotropin-releasing hormone were demonstrated to modify the AVP and OT secretion from rat neurohypophysis (9-13).

Tachykinins are protein products encoded by two genes: preprotachykinin A gene, which encodes for neurokinin A, , and neuropeptide γ as well as preprotachykinin B gene which encodes only for . The substance P acts preferentially on tachykinin NK-1 receptor, the neurokinin A, neuropeptide K and neuropeptide γ act on NK-2 receptor, whereas neurokinin B has preferential affinity for the tachykinin NK-3 receptor (14-15).

This family of peptides may regulate the secretion of anterior pituitary hormones, either by acting directly on the pituitary level or indirectly on the hypothalamic neurones. Namely, neurokinin A, substance P, neuropeptide K and neuropeptide γ were reported to control the secretion of growth hormone, prolactin, gonadotropins, thyrotropin or adrenocorticotropin (16-20). There are also findings which suggest that tachykinins may have a physiological role as regulators of the posterior pituitary endocrine function (10, 20-21). The hypothalamic SON and PVN neurones contain substance P, neuropeptide K, neurokinin A and B as well as they co-express the respective tachykinin receptors (21-23); the neurokinin A and substance P have also been found in the anterior and posterior pituitary (24-26).

It is well documented now that the pineal hormone, melatonin, modifies the

AVP and OT release from the hypothalamo-neurohypophysial system under physiological and/or pathological conditions both in vivo (2-7, 27) and in vitro

(28-32). The action of melatonin was found to depend on a dose applied (4, 27-

30) as well as on the time of day (31). However, the mechanisms responsible for modulation of the neurohypophysial hormones production and/or secretion by melatonin are less well understood.

Recently it was shown that neurokinin A inhibits OT release from the posterior pituitary (33), suggesting that this effect could be involved in the mechanisms of melatonin-dependent inhibition of AVP and OT secretion and play some role in the pineal-neurohypophysial interrelationship. The present study was, therefore, designed to investigate the effect of neurokinin A, alone or

+ in the presence of melatonin, on basal and K -stimulated AVP and OT secretion from the rat hypothalamo-neurohypophysial system in vitro.

MATERIALS AND METHODS

Three-months old male Wistar rats (weighing about 270-370 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. On the day of experiment, the animals were 825

decapitated between 11.00 and 12.00 a.m. The brain and the pituitary with intact pituitary stalk were carefully removed from the skull. Then, from the brain a block of hypothalamic tissue was quickly dissected as follows: rostral limit - frontal plane situated about 1.0 mm more anteriorily than the anterior margin of the optic chiasm; caudal limit - frontal plane just behind the mamillary bodies; lateral limits - sagittal planes passing, on both sides, just through the hypothalamic fissures (see: 3,

30-32). The depth of dissection was approximately 2.5-3.0 mm from the base of the brain; the total dissection time was about 3 min from decapitation. Such hypothalamo-neurohypophysial (HN) explant contained suprachiasmatic nucleus (SCN) as well as the SON and PVN hypothalamic nuclei with intact axonal projections to the neurohypophysis (13, 34). Each HN explant was immediately placed in one polypropylene tube with 1 ml 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 HN explants were equilibrated in KRF which was aspirated twice and replaced with 1 ml of fresh buffer. After 80 minutes of such preincubation, the KRF was discarded and the explants were incubated for 20 minutes in 1 ml of

KRF alone or containing the respective concentrations of neurokinin A (neurokinin α; Sigma-

Aldrich Chemie GmbH) and/or melatonin (N-Acetyl-5-methoxytryptamine; Sigma-Aldrich Chemie

GmbH).

The experimental procedures were done with the consent (No L/BD/82) of the Local Committee for the Animal Care.

+ In the first series of experiments the effect of neurokinin A (NKA) on basal and K -evoked AVP and OT release from the HN explants was studied. Explants were incubated successively in: 1 -

+ normal KRF {B1}; 2 - modified KRF containing the excess of K (56 mM KCl; the NaCl concentration was appropriately reduced to maintain medium osmolality) {S1}; 3 - the KRF as (1)

-11 -10 -9 -8 -7 alone or with NKA at the concentration of 10 , 10 , 10 , 10 or 10 M/L {B2}; 4 - the KRF as (2) alone or with NKA at the concentrations as (3) {S2}. In between incubation periods 2 and 3, the explants were washed for 20 minutes in normal KRF and these samples were discarded. After each incubation, the media were aspirated and samples immediately stored at -20°C.

In the second series of experiments the effect of both NKA and melatonin on basal as well as

+ K -evoked AVP and OT release from the isolated HN explants was studied. For these experiments two concentrations of NKA, which were the most effective in stimulation of the AVP and/or OT secretion in the first series, have been chosen. The experimental protocol was similar to that described for the series first except that the B2 and S2 buffers contained NKA (at the concentration

-9 -7 of 10 or 10 M/L) and additionally melatonin vehicle (0.1% ethanol) or melatonin solution at the

-9 concentration of 10 M/L.

The AVP and OT concentrations in the medium samples (from both series) were measured by radioimmunoassay.

Radioimmunoassay (RIA)

The concentrations of OT and AVP in the medium samples were determined by double-antibody specific RIAs. Anti-OT as well as anti-AVP 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: (12). For standard curve

125 preparation as well as for iodination with I, using the chloramine-T method, the OT (Oxytocin

8 synth.) and AVP ([Arg ]-Vasopressin) from Peninsula Laboratories Europe Ltd. were used. The intra- assay coefficients of variation for OT and AVP assay were less than 5% and 3.5%, respectively (all samples within the experiment were tested in the same RIA to avoid inter-assay variability). 826

Fig. 1. The effect of

different concentrations

-11 -10 -9 -8 (10 , 10 , 10 , 10 and

-7 10 M/L) of neurokinin A

(NKA) on basal vasopres-

sin (AVP; upper panel) and

oxytocin (OT; lower

panel) release from the

hypothalamo-neurohypo-

physial complex in vitro.

Each bar represents mean

+ S.E.M.; figures in bars

indicate the number of

animals in each group. All

the comparisons were

made with respect to the

control explants incubated

in Krebs-Ringer fluid with

no NKA; *p<0.05.

Statistical evaluation of the results

The neurohypophysial hormone secretion was estimated by using B2/B1 (basal release) and S2/S1

(stimulated release) ratios for each HN explant. All data are expressed as means ± S.E.M. Significance of the differences between means was evaluated by the analysis of variance (ANOVA) followed by

Student's "t" test (two means comparison); p<0.05 was considered as the minimal level of significance.

RESULTS

Series first. In agreement with previous in vitro studies (28-29, 35) high

+ concentration of K stimulated both AVP and OT release from the isolated hypothalamo-neurohypophysial explants (see: Tables 1-2).

-9 -7 Under basal conditions, the two concentrations of NKA (i.e., 10 and 10

M/L) were effective in stimulation of OT secretion from the isolated HN explants,

-7 while AVP release was increased by NKA only at the concentration of 10 M/L; 827

+ + Table 1: The effect of high concentration of K (S1), when compared with normal level of K (B1), on the vasopressin and oxytocin secretion into the incubation medium (first series). NKA –

-11 -10 -9 -8 -7 neurokinin A at the concentration of 10 , 10 , 10 , 10 or 10 M/L; KRF - Krebs-Ringer fluid; values represent mean ± S.E.M.; n – number of animals per group.

Vasopressin (pg/ml) Oxytocin (pg/ml)

Groups B1 S1 B1 S1

KRF with 397.5 ± 60.8 3065.1 ± 537.8 464.5 ± 73.8 1350.4 ± 132.5

no NKA n = 19 n = 17 n = 20 n = 20

216.0 ± 26.0 3879.1 ± 571.0 463.6 ± 66.0 1539.9 ± 149.7

-11 NKA(10 ) n = 8 n = 7 n = 8 n = 8

304.5 ± 63.8 5508. ± 900.3 309.4 ± 48.2 1384.6 ± 155.6

-10 NKA(10 ) n = 8 n = 8 n = 8 n = 8

257.9 ± 45.2 3808.7 ± 831.7 353.5 ± 101.6 970.1 ± 80.6

-9 NKA(10 ) n = 7 n = 7 n = 7 n = 7

262.5 ± 36.2 1586.7 ± 107.6 244.3 ± 42.1 974.0 ± 74.1

-8 NKA(10 ) n = 8 n = 6 n = 8 n = 7

430.2 ± 99.5 3731.7 ± 653.2 238.3 ± 28.5 1417.8 ± 203.0

-7 NKA(10 ) n = 8 n = 8 n = 8 n = 8

+ + Table 2: The effect of high concentration of K (S1), when compared with normal level of K (B1), on vasopressin and oxytocin secretion into the incubation medium (second series). MLT – melatonin; VEH – melatonin vehicle; KRF-NKA-0 - Krebs-Ringer fluid with no neurokinin A;

-9 -7 NKA - neurokinin A at the concentration of 0, 10 or 10 M/L; values represent mean ± S.E.M.; n

– number of animals per group.

Vasopressin (pg/ml) Oxytocin (pg/ml)

Groups B1 S1 B1 S1

KRF- 461.9 ± 114.2 2112.9 ± 424.8 379.2 ± 61.1 2377.2 ± 353.4

NKA-0 n = 9 n = 9 n = 10 n = 10

VEH- 420.2 ± 79.7 1389.1 ± 285.1 436.9 ± 60.2 2594.8 ± 505.4

NKA-0 n = 10 n = 10 n = 10 n = 10

MLT- 196.9 ± 34.8 861.7 ± 244.3 401.3 ± 93.9 2694.0 ± 594.5

NKA-0 n = 9 n = 9 n = 8 n = 9

VEH 395.4 ± 54.7 1053.4 ± 139.4 216.3 ± 37.2 1701.6 ± 143.1

-9 NKA-10 n = 9 n = 9 n = 9 n = 9

MLT- 424.4 ± 112.4 880.6 ± 187.1 269.7 ± 50.3 1337.3 ± 275.8

-9 NKA-10 n = 10 n = 10 n = 10 n = 10

VEH- 323.1 ± 48.3 1151.1 ± 248.3 317.3 ± 78.7 2452.4 ± 474.3

-7 NKA-10 n = 10 n = 10 n = 10 n = 10

MLT- 171.4 ± 23.3 814.6 ± 183.5 276.4 ± 86.4 1912.6 ± 323.0

-7 NKA-10 n = 10 n = 10 n = 10 n = 10

the other concentrations of NKA were ineffective in the process in question (Fig.

+ 1). The NKA did not substantially alter the K -stimulated release of either AVP or OT from the isolated hypothalamo-neurohypophysial explants (Fig. 2). 828

Fig. 2. The effect of

different concentrations

-11 -10 -9 -8 (10 , 10 , 10 , 10 and

-7 10 M/L) of neurokinin A

+ (NKA) on K -stimulated

vasopressin (AVP; upper

panel) and oxytocin (OT;

lower panel) release from

the hypothalamo-neuro-

hypophysial complex in

vitro. Each bar represents

mean + S.E.M.; figures in

bars indicate the number

of animals in each group.

All the comparisons were

made with respect to the

control explants incubated

in Krebs-Ringer fluid with

no NKA.

Series second. Basal release of neurohypophysial hormones was not different from the control (i.e., normal KRF) when melatonin vehicle was added to the medium (Tab. 3). Melatonin itself diminished basal release of AVP and OT when compared to vehicle; it also significantly inhibited the NKA-evoked (used at the

-7 concentration of 10 M/L) secretion of AVP and OT. Basal release of OT and

-9 AVP was not stimulated by NKA at the concentration of 10 M/L, however, when melatonin was added to KRF enriched with NKA at such concentration, the AVP

+ secretion from the HN explants was decreased significantly (Tab. 3). The K - evoked release of OT and AVP was not further modified by either NKA or melatonin (Tab. 3).

DISCUSSION

-7 In the present study, NKA (when used at the concentration of 10 M/L) was found to stimulate basal release of both AVP and OT from isolated hypothalamo- 829

+ Table 3: The effect of neurokinin A and melatonin on basal (B2/B1) and K -stimulated (S2/S1) vasopressin and oxytocin release into the incubation medium (KRF). MLT – melatonin; VEH – melatonin vehicle; KRF-NKA-0 - Krebs-Ringer fluid with no neurokinin A; NKA - neurokinin A

-9 -7 at the concentration of 0, 10 or 10 M/L; values represent mean ± S.E.M.; n – number of animals per group.

Vasopressin release Oxytocin release

Groups B2/B1 S2/S1 B2/B1 S2/S1

1) KRF- 1.56 ± 0.27 0.85 ± 0.16 1.92 ± 0.37 0.72 ± 0.1

NKA-0 n = 9 n = 9 n = 10 n = 10

2) VEH- 1.81 ± 0.32 0.86 ± 0.15 2.26 ± 0.29 0.63 ± 0.12

NKA-0 n = 10 n = 10 n = 10 n = 10

3) MLT- 0.77 ± 0.16 0.99 ± 0.22 1.01 ± 0.2 0.6 ± 0.09

NKA-0 n = 9 n = 9 n = 8 n = 9

4) VEH- 1.87 ± 0.46 0.77 ± 0.09 2.33 ± 0.51 0.68 ± 0.04

-9 NKA-10 n = 9 n = 9 n = 9 n = 9

5) MLT- 0.77 ± 0.19 0.69 ± 0.11 1.16 ± 0.27 0.78 ± 0.12

-9 NKA-10 n = 10 n = 10 n = 10 n = 10

6) VEH- 2.6 ± 0.29 1.28 ± 0.17 3.53 ± 0.64 0.71 ± 0.14

-7 NKA-10 n = 10 n = 10 n = 10 n = 10

7) MLT- 1.19 ± 0.14 1.12 ± 0.31 2.07 ± 0.34 0.65 ± 0.09

-7 NKA-10 n = 10 n = 10 n = 10 n = 10

Significance

of the difference:

1 versus 2 NS NS NS NS

2 versus 4 NS NS NS NS

2 versus 6 P<0.05 NS P<0.05 NS

2 versus 3 P<0.05 NS P<0.05 NS

4 versus 5 P<0.05 NS NS NS

6 versus 7 P<0.05 NS P<0.05 NS

NS – not significant neurohypophysial system. This observation confirms those of other studies, both in vivo and in vitro, in which tachykinins were described to influence the secretory activity of the magnocellular hypothalamic neurones and modify the secretion of posterior pituitary hormones. Namely, neurokinin B was reported to induce the secretion of AVP from the hypothalamic PVN and SON neurones (21).

Centrally injected substance P was found to inhibit water and salt intake (36) as well as to increase the secretion of AVP and OT into the general circulation (10,

20). However, our present data are not consistent with those described by De

Laurentiis et al. (33), who have reported that NKA reduced OT release from the posterior pituitary but not from the hypothalamus of male rats. The authors also postulate that the inhibitory action of NKA on OT release from the posterior pituitary is mediated by nitric oxide (33). There is probably a number of reasons for dissimilar effects of NKA on AVP and/or OT secretion in our present experiments and the studies of De Laurentiis et al. In our experimental paradigm, 830

we have incubated the whole hypothalamo-neurohypophysial complex with intact axons of the vasopressinergic and oxytocinergic neurones, while in the other studies (33) the hypothalamic explants or posterior pituitaries were incubated separately. In the present study NKA could, therefore, stimulate the AVP/OT secretion acting directly on axon endings located in the posterior pituitary and/or axonal transport of the hormones to terminals in the neurohypophysis; the NKA could also modify the rate of hormone synthesis in cell bodies of the vasopressinergic/oxytocinergic neurones located in the hypothalamic SON and

PVN. On the other hand, when posterior pituitaries are incubated separately, the

NKA may influence only the secretion of AVP and/or OT from axon endings in the neurohypophysis, but neither axonal transport of the hormones or their biosynthesis in the hypothalamus. Therefore, it could be hypothesized that the opposite effects of NKA on the secretion of OT in our present experiments and the studies of De Laurentiis et al. (33) may result from: 1 - possible inhibitory effect of NKA on the OT/AVP release, through mechanisms involving among others nitric oxide (NO), on the level of neurohypophysis (an inhibitory role of

NO in AVP and OT secretion is now well documented; 37) and 2 - stimulatory effect of NKA, on the level of hypothalamus and/or neurohypophysis, by an NO- independent mechanisms. Such a mode of NKA action is consistent with the evidence that hypothalamic SON and PVN neurones contain NKA and they co- express the respective receptors (23-24, 26, 36), while the presence of tachykinin

NK-2 receptor (the receptor subtype to which NKA binds with preferential affinity) possibly located in the posterior pituitary, remains to be demonstrated.

The NKA is present in rat pineal gland and may have a role in regulation of the pineal function (18). Since pineal gland is known to mediate the effects of light on rhythms of pituitary hormone secretion, and a circadian rhythm of NKA in plasma, hypothalamus and posterior pituitary has been observed in the male rat

(26), it was reasonable to suppose that this peptide could be, in some way, involved in the pineal-neurohypophysial interrelationships. However, under present experimental conditions, the NKA and melatonin were found to produce opposite effects on the AVP and OT secretion from isolated rat hypothalamo- neurohypophysial system. Therefore, the contribution of NKA in melatonin- mediated inhibition of AVP and OT secretion has not been demonstrated and the hypothesis as to the role of NKA in pineal-neurohypophysial interrelationship could not be confirmed.

The present results showed that melatonin inhibited the OT and AVP release from isolated hypothalamo-neurohypophysial explants. This observation is consistent with the results obtained in hamster neurointermediate lobe (28) and rat hypothalamus (30) which, in general, show that melatonin is effective in inhibiting the AVP and OT release from the hypothalamo-neurohypophysial system in vitro at the concentration which is relatively close to its physiological level in the blood; the concentration of melatonin employed in the present

-9 experiment (i.e., 10 M/L) was at the range of physiological level of the hormone. 831

However, the mechanisms by which melatonin affects the release of neurohypophysial hormones are still not fully answered question in neuroendocrinology. In rats and hamsters, melatonin receptors have been detected in different parts of the brain, especially in the median eminence/pars tuberalis region, SCN and other hypothalamic nuclei (38). Thanks to direct neuronal projection from the SCN to PVN (39) or SON (40) melatonin may influence, at least in part, the activity of vasopressinergic and oxytocinergic neurones acting via melatonin receptors located in the SCN; the hypothalamo-neurohypophysial explant, we employed in the present studies, contained SCN as well as the SON and PVN hypothalamic nuclei with intact axonal projections to the neurohypophysis. Apart from direct effect of the hormone, via melatonin receptors in the hypothalamus, melatonin could also inhibit the AVP and OT secretion acting indirectly via modification of metabolism of some neuromediators or neuromodulators in the hypothalamus and/or in the neurointermediate lobe (41-42). Indeed, acetylcholine, dopamine and prostaglandins were shown to be involved in melatonin-mediated inhibition of the neurohypophysial hormone secretion (27, 32).

Studies as to the intracellular mechanisms of melatonin action show that the intracellular calcium as well as the cAMP may participate in these mechanisms, some of them being similar in the pituitary and SCN (43). Interestingly, the inhibition of AVP release by melatonin in the SCN-slice culture depends partly

on melatonin MT2 (Mel 1b) receptor (44). There is also a possibility that the pineal hormone modifies neuronal activity by a direct action on the genome; due to its high ability to pass through cell membranes, melatonin modulates some cellular functions without interaction with a specific membrane receptor (45).

In summary, this paper confirms previous evidence that melatonin inhibits

AVP and OT secretion from the hypothalamo-neurohypophysial complex in vitro.

However, under present experimental conditions, the contribution of NKA in mechanisms of the pineal-neurohypophysial interrelationships has not been demonstrated.

Acknowledgements: I wish to thank Dr Bo¿ena Stempniak (Department of Pathophysiology

Medical University of £ódŸ) for her help in hormonal RIA. This work has been supported by

Medical University of £ódŸ, contract No. 502-11-632.

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Received: May 13, 2002

Accepted: October 29, 2002

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

University of £ódŸ, Narutowicza 60,90-136 £ódŸ, Poland, Tel: +42 630 61 87, Fax: +42 631 97 23

E-mail: [email protected]