Neuropeptides 48 (2014) 387–397

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Neuropeptides j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / n p e p

Galanin and galanin-like peptide modulate vasopressin and oxytocin release in vitro: The role of galanin receptors Justyna Wodowska, Joanna Ciosek * Department of Neuropeptides Research, Faculty of Health Sciences, Medical University of Lodz, Narutowicza 60, 90-136 Lodz, Poland

A R T I C L E

I N F O

Article history: Received 26 March 2014 Accepted 22 October 2014 Available online 10 November 2014 Keywords: Galanin Galanin-like peptide Vasopressin Oxytocin Hypothalamus Neurohypophysis In vitro explant

A B S T R A C T

Galanin (Gal) and galanin-like peptide (GALP) may be involved in the mechanisms of the hypothalamoneurohypophysial system. The aim of the present in vitro study was to compare the influence of Gal and GALP on vasopressin (AVP) and oxytocin (OT) release from isolated rat neurohypophysis (NH) or hypothalamo-neurohypophysial explants (Hth–NH). The effect of Gal/GALP on AVP/OT secretion was also studied in the presence of galantide, the non-selective galanin receptors antagonist. Gal at concentrations of 10−10 M and 10−8 M distinctly inhibited basal and K+-stimulated AVP release from the NH and Hth–NH explants, whereas Gal exerted a similar action on OT release only during basal incubation. Gal added to the incubation medium in the presence of galantide did not exert any action on the secretion of either neurohormone from NH and Hth–NH explants. GALP (10−10 M and 10−9 M) induced intensified basal AVP release from the NH and Hth–NH complex as well as the release of potassium-evoked AVP from the Hth–NH. The same effect of GALP has been observed in the presence of galantide. GALP added to basal incubation medium was the reason for stimulated OT release from the NH as well as from the Hth–NH explants. However, under potassium-stimulated conditions, OT release from the NH and Hth–NH complexes has been observed to be distinctly impaired. Galantide did not block this inhibitory effect of GALP on OT secretion. It may be concluded that: (i) Gal as well as GALP modulate AVP and OT release at every level of the hypothalamo-neurohypophysial system; (ii) Gal acts in the rat central nervous system as the inhibitory neuromodulator for AVP and OT release via its galanin receptors; (iii) the stimulatory effect of GALP on AVP and OT release is likely to be mediated via an unidentified specific GALP receptor(s). © 2014 Elsevier Ltd. All rights reserved.

1. Introduction The neurohormones arginine vasopressin (AVP) and oxytocin (OT) perform homeostatic functions for water balance and reproduction. Structurally similar, differing by only two amino acids, AVP and OT are mainly derived from preprohormones synthesized by magnocellular neurons (MCNs) in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus (Hth), and are secreted from the posterior pituitary (the neurohypophysis; NH) into the general circulation, where they exert their multiple effects. It is generally believed that regulation of AVP and/or OT release occurs in both the somatodendritic regions in the hypothalamus and the nerve terminal regions in the neurohypophysis and involves both neuron– neuron and neuronal–glial cell interactions (Hussy, 2002; Landgraf and Neumann, 2004; Panatier, 2009; Son et al., 2013; Tobin et al., 2012). Numerous neuromodulators of the central nervous system

* Corresponding author. Department of Neuropeptides Research, Faculty of Health Sciences, Medical University of Lodz, Narutowicza 60, 90-136 Lodz, Poland. E-mail address: [email protected] (J. Ciosek). http://dx.doi.org/10.1016/j.npep.2014.10.005 0143-4179/© 2014 Elsevier Ltd. All rights reserved.

(CNS) can modify the mechanisms involved in AVP/OT synthesis and release (Bojanowska et al., 1999; Chu et al., 2009; Ciosek and Drobnik, 2013; Ciosek and Izdebska, 2009; Iovino et al., 2012; Juszczak et al., 2007; Sladek and Kapoor, 2001). Putative modulators include two members of the galanin neuropeptide family: “parental” galanin (Gal) and its cousin galanin-like peptide (GALP). Gal is a 29-amino acid residue peptide, comprising 30 amino acids in humans, isolated from the porcine intestine 30 years ago (Tatemoto et al., 1983). It has been shown to be involved in the regulation of numerous processes, including neuroendocrine control of systems such as the hypothalamic–pituitary–adrenal axis as well as feeding, intestine secretion, nerve regeneration, learning, memory or nociception (Butzkueven and Gundlach, 2010; Gundlach, 2002; Lang et al., 2007; Ogren et al., 2010; Shen and Gundlach, 2010; Tortorella et al., 2007). Gal was structurally unrelated to any known peptide in the mammalian brain. Today the galanin peptide family consists of the “parental” Gal, galanin-like peptide (GALP), galaninmessage-associated peptide (GMAP) and a recently-discovered peptide named “alarin” (Lang et al., 2007). Gal functions are mediated by at least three galanin receptor subtypes (GAL1, GAL2, and GAL3) which belong to G protein-coupled receptors (GPCRs) (Branchek et al., 2000). Galanin and galanin receptor-binding sites

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are widely distributed within CNS, but historically much research has been focused on hypothalamic galanin systems including those in the preoptic area, paraventricular nucleus, supraoptic nucleus, and arcuate nucleus (ARC) (Gundlach et al., 2001; Mennicken et al., 2002; Waters and Krause, 2000). Most galanin-immunoreactive neurons (Gal-ir) in SON and PVN project directly to the posterior lobe of the pituitary, indicating that galanin may play a role in the modulation of hypophyseal secretion (Arai et al., 1990). Immunohistochemical and in vitro studies have confirmed the coexistence of Gal with AVP in the rat hypothalamic magnocellular neurons in the SON and PVN (mPVN) and/ or pPVN (Bartfai, 1995; Gai et al., 1990; Gundlach and Burazin, 1998; Meister et al., 1990; Sanchez et al., 2001). While studying the role of galanin in the modulation of the release of neurohypophysial hormones, Ciosek and co-workers reported that galanin affects vasopressin and oxytocin release from the hypothalamoneurohypophysial system in hemorrhaged, dehydrated or saltloaded rats. This increase in plasma vasopressin and oxytocin during hemorrhaging was inhibited in rats previously treated intracerebroventricularly (icv) with galanin (Ciosek et al., 2003). Gal acts as an inhibitory neuromodulator of AVP and OT secretion in rats experiencing a hemorrhage or those which are dehydrated or salt loaded (Ciosek and Cisowska, 2003; Ciosek et al., 2003; Cisowska-Maciejewska and Ciosek, 2005). An in vitro study by Izdebska and Ciosek (2010) showed that a range of Gal concentrations exerted an inhibitory effect on the AVP secretion of all incubated tissues as well as on OT release from the neurohypophysis and hypothalamo-neurohypophysial explants. Recently, Ciosek and Drobnik (2013) demonstrated that Gal acts as a stimulatory neuromodulator of OT release in vitro in response to prolonged osmotic stimulus and that, conversely, acute osmotic stimulus blocks OT-ergic neurons susceptible to Gal. Galanin-like peptide, the second member of the galanin peptide family, was discovered while searching for additional ligands capable of activating galanin receptors (Ohtaki et al., 1999). GALP was identified as a 60-amino-acid peptide, which, unlike Gal, has a nonamidated C-terminus. GALP shares sequence homology to galanin (1–13) in positions 9–21 and can activate three galanin receptor subtypes (GAL1-3) with a preference for GAL2 and GAL3 over GAL1 (Lang et al., 2005). A second region, highly conserved between different species, is unique to the GALP peptide and lies between residues 38–54. It is hypothesized to be a binding region for a putative GALPspecific receptor (Man and Lawrence, 2008; Robinson et al., 2006), which is as yet unknown. In contrast to the broad distribution of Gal in the central nervous system, GALP mRNA was not detected in magnocellular neurons of the supraoptic or paraventricular nucleus (Shen et al., 2001). GALP mRNA and protein are expressed only in the neurons of the hypothalamic ARC, the median eminence, and in the pituicytes of the posterior pituitary, specialized astrocytes that are thought to modulate posterior pituitary hormone release by changing the amount of contacts between axon terminals and fenestrated capillaries (Kerr et al., 2000). GALP neurons in the ARC receive neuropeptide-Y (NPY) projections and more than 85% of these neurons express leptin receptor (Takatsu et al., 2001). There is lack of GALP mRNA in the magnocellular neurons of the paraventricular and supraoptic nuclei, which supply the majority of neuronal projections into the neurohypophysis (Shen et al., 2001). However, dense staining of GALP-containing fibers was found in the anterior parvicellular part of the PVN (pPVN) (Takatsu et al., 2001). Some previous findings indicate that GALP has an influence on AVP and OT secretion from the posterior pituitary gland. Cunningham et al. (2004) noted that the expression of GALP mRNA was increased in the neurohypophysis of lactating rats compared to nonlactating rats, whereas GALP mRNA expression in the ARC was unaffected by lactation. Onaka et al. (2005) demonstrated that icv administration of GALP caused significant increases of plasma

concentrations of neurohypophyseal hormones in male rats. Shen et al. (2001) showed that the expression of galanin-like peptide mRNA in the rat posterior pituitary gland was markedly upregulated by chronic osmotic stimuli such as dehydration, salt-loading and intraperitoneal administration of lipopolysaccharide: challenges that stimulate the secretion of vasopressin and oxytocin. Kawasaki et al. (2007) examined whether the expression of the GALP gene in the NH and ARC would be induced after intraperitoneal (ip) administration of hypertonic saline, that is, acute osmotic stimulus, in rats. Previous findings indicate the ability of Gal and GALP to alter AVP and OT secretion from the posterior pituitary gland, but several questions remain unclear. An important question is why Gal and GALP sometimes share such similar features as stimulation of gonadotropin secretion and orexigenic effects in rats but differ in their effects, such as regulation of neurohypophysial hormones. As it might be the case that these effects are based on interactions with different receptors, the aim of the present in vitro study was to investigate the effect of various concentrations of Gal and GALP on basal and K+-stimulated AVP and OT secretion from isolated rat neurohypophysis and hypothalamo-neurohypophysial explants. To investigate whether the presence of galanin receptors is required for Gal and GALP to exert an influence on AVP/OT secretion, the role of the non-selective galanin receptor antagonist, galantide (M15; galanin(1–12)-Pro-substanceP(5–11)-amide) was also examined. 2. Materials and methods 2.1. Animals Male adult (3-months) Wistar rats weighing 280 ± 30 g (the mean ± SD) were housed under controlled temperature (21– 23 °C) and in a controlled 12 h light:12 h dark cycle with lights on at 0600 h. All animals had free access to commercial food pellets (LSM, Bacutil, Poland) as well as tap water ad libitum. Four animals were kept per cage. The study was approved by the Local Ethics Commission, Lodz. 2.2. Reagents The following reagents were used in the study: galanin [Gal] [galanin (rat), Bachem, lot 0560209], galanin-like peptide [trifluoroacetate salt (rat), Bachem, lot 1017885] and galantide [(M15), galanin (1–13)-substance P (5–11) amide, Bachem, lot 0560022]. The [Arg8]-vasopressin and oxytocin, used for standard curve preparation as well for iodination with 125I, were obtained from Peninsula Laboratories Europe Ltd. 2.3. Procedures On the day of the experiment, the animals were decapitated between 9:00 and 10:00 a.m. Euthanasia was performed very quickly with the use of a guillotine for experimental animals. Each rat was decapitated in the laboratory room separately from the other animals. The brain and the pituitary with intact pituitary stalk were carefully removed from the skull and then two types of neuronal tissue were prepared: (i) the neurohypophysis, or (ii) intact hypothalamoneurohypophysial system. The total dissection time was about 3 min from decapitation. The neurohypophysis and the hypothalamoneurohypophysial systems were taken from the brains of different animals. A block of hypothalamic tissue was dissected as follows: the rostral border as the frontal plane situated about 1.0 mm anterior to the anterior margin of the optic chiasm; the caudal limit being the frontal plane just behind the mammillary bodies, and the lateral limits being the sagittal planes, passing on both sides, just through the hypothalamic fissures (Cisowska-Maciejewska and Ciosek, 2005).

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The depth of dissection was approximately 2.5–3.0 mm from the base of the brain. This hypothalamo-neurohypophysial explant contained the suprachiasmatic nucleus as well as the SON and PVN hypothalamic nuclei with intact axonal projections to the neurohypophysis: the anterior lobe of the pituitary was excised (Bojanowska et al., 1999; Cisowska-Maciejewska and Ciosek, 2005). In other group of animals, the posterior lobe of the pituitary was prepared under the microscope. Each type of the isolated neural tissue was placed immediately in a polypropylene tube with 1 ml of Krebs–Ringer fluid [termed as normal KRF (nKRF)] 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 = 7.37–7.46; osmolality 280–290 mOsm/kg H2O). Tubes were placed in a water bath at 37 °C and constantly gassed with carbogen (a mixture of 95% O2 and 5% CO2). In the first series of the experiments, the effect of Gal on the basal and K+-stimulated AVP and OT release was studied. In the second series of the experiments, GALP influence on the release of both neurohormones was studied. Each series contained six subgroups. At the beginning of the experiment, the appropriately prepared tissue was equilibrated in nKRF [aspirated twice (2 × 1 ml)] for two 40-min periods. After 80 min of such preincubation, the nKRF was discarded. Next, the incubation (stage 1) was still carried out in nKRF for 20 min to estimate basal neurohormone secretion into incubation medium (B1 fraction). In the next stage (stage 2), nKRF was replaced for 20 min by modified KRF containing excess potassium ions (S1 fraction): the K+ concentration being 56 mM, with the NaCl concentration in the medium reduced to maintain the osmolality. The next period of the incubation in nKRF (stage 3) was performed to wash out the excess of K+ ions and this sample was discarded. Depending on the experimental series and subgroups, further steps of the incubation (stages 4 and 5) were different. In the first subgroup (control: Gal 0 or GALP 0), the isolated tissues were again incubated in nKRF (B2 fraction; stage 4) but the last stage of the incubation (stage 5) was carried out in KRF with the excess of K+ ions (S2 fraction). The detailed plan of the incubation protocol is presented as follows: Preincubation Stage 1

Stage 2

Stage 3

Stage 4

Stage 5

nKRF B1 fraction

KRF + K+ S1 fraction

nKRF

nKRF B2 fraction

KRF + K+ S2 fraction

In the subgroups estimating the influence of tested peptides (Gal or GALP) on AVP and OT release, the incubation outline was the following:

nKRF B1 fraction

After each incubation, the media (fractions B1, S1, B2 and S2) were aspirated and samples were immediately frozen and stored at −25 °C until AVP and OT determination by radioimmunoassay. 2.4. Radioimmunoassay (RIA) The concentrations of AVP and OT in the medium samples were determined by double-antibody specific RIA. Anti-AVP antibodies and anti-OT antibodies were raised in the Department of Experimental Physiology, Chair of Experimental and Clinical Physiology, Medical University of Lodz. A more detailed description of antibodies has been given earlier by Cisowska-Maciejewska and Ciosek (2005). For iodination with 125I and for standard curve preparation, the chloramine-T method using standard VP ([Arg 8 ] − vasopressin) and OT (oxytocin synth.) was applied. The lower limit of detection for the assay was 1.25 pg AVP/100 μl and 1.25 pg OT/ 100 μl; the intra-assay coefficient of variation was less than 3.5% for AVP and less than 5% for OT. All samples within each experimental series were tested in the same RIA to avoid inter-assay variability. 2.5. Statistical evaluation of the results The received absolute values of AVP and OT contents in incubate media quite often showed great differentiations within the group. So, AVP and OT release into incubation media was estimated by calculating the ratios between two incubation periods B2/ B1 (basal release) and S2/S1 (stimulated release) for the data obtained from the incubation of the NH or Hth–NH system. In each experimental group, the results were calculated and expressed as means ± SEM. To analyze the data (comparison between two groups) we used the Mann–Whitney “U” test, a non-parametric test that does not assume a Gaussian distribution in the data being analyzed. P < 0.05 was considered as the minimal level of significance. 3. Results 3.1. K+-evoked AVP and OT release from the isolated rat NH or Hth–NH explants in each series The potassium-stimulated AVP and OT release from the NH or Hth–NH complex as calculated from the experiments of the first and second series (period S1; incubation in Krebs–Ringer fluid enriched with potassium ions) were markedly higher than the basal secretion of both hormones (period B1; incubation in normal Krebs– Ringer fluid), thus suggesting that the incubation procedure did not disturb the viability of the explants. There is a significant (p < 0.0001 or p < 0.00001) K+-dependent effect on the AVP and OT release (Fig. 1.). 3.2. Galanin influence on vasopressin and oxytocin release in vitro; the role of galanin receptor antagonist

Preincubation Stage 1

389

Stage 2 +

KRF + K S1 fraction

Stage 3

Stage 4

Stage 5

nKRF

KRF + X* B2 fraction

KRF + K+ + X* S2 fraction

X* in series I:Subgroup 2 – Gal at the concentration 10−10 M; subgroup 3 – Gal at the concentration 10−8 M; subgroup 4 – M15 at the concentration 10−10 M; subgroup 5 – Gal at the concentration 10−10 M and M15 at the concentration 10−10 M; subgroup 6 – Gal at the concentration 10−8 M and M15 at the concentration 10−10 M. X* in series II:Subgroup 2 – GALP at the concentration 10−10 M; subgroup 3 – GALP at the concentration 10−9 M; subgroup 4 – M15 at the concentration 10−10 M; subgroup 5 – GALP at the concentration 10−10 M and M15 at the concentration 10−10 M; subgroup 6 – GALP at the concentration 10−9 M and M15 at the concentration 10−10 M.

Gal was added to the media at concentrations of 10−10 or 10−8 M to examine its direct influence on AVP and OT release in vitro. Both concentrations were found to inhibit the basic release of both neurohormones separately from the NH [Fig. 2; B2/B1: Gal 10 vs Gal 0: p < 0.01, Gal 8 vs Gal 0: p < 0.01; Fig. 4; B2/B1: Gal 10 vs Gal 0: p < 0.05] or Hth–NH explant (Fig. 3; B2/B1: Gal 10 vs Gal 0: p < 0.02; Gal 8 vs Gal 0: p < 0.02; Fig. 5; B2/B1: Gal 10 vs Gal 0: p < 0.01; Gal 8 vs Gal 0: p < 0.01). Also, while 10−10 M Gal reduced K+-stimulated AVP secretion from the NH explant (Fig. 2; S2/S1: Gal 10 vs Gal 0: p < 0.01), 10−8 M Gal demonstrated a similar action on the Hth–NH explant (Fig. 3; S2/S1: Gal 8 vs Gal 0: p < 0.05). In the case of the NH, the addition of Gal in 10−10 M or 10−8 M concentration reduced the B2/B1 ratio to about half (46% and 42%, respectively), which

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Fig. 1. The comparison of the basal (period B1; incubation in normal KRF) and stimulated (period S1; incubation in medium enriched with excess potassium ions) AVP and OT release from the isolated rat NH or Hth–NH explants as estimated from experiments of each series (mean ± SEM). Numbers above bars indicate group size.

Fig. 2. The effect of galanin (Gal) or galantide (M15 at the concentration 10−10 M) or Gal together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) AVP release from the neurohypophysis in vitro (series 1). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no Gal.

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Fig. 3. The effect of galanin (Gal) or galantide (M15 at the concentration 10−10 M) or Gal together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) AVP release from the hypothalamo-neurohypophysial explant in vitro (series 1). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no Gal.

Fig. 4. The effect of galanin (Gal) or galantide (M15 at the concentration 10−10 M) or Gal together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) OT release from the neurohypophysis in vitro (series 1). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no Gal.

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Fig. 5. The effect of galanin (Gal) or galantide (M15 at the concentration 10−10 M) or Gal together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) OT release from the hypothalamo-neurohypophysial explant in vitro (series 1). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no Gal.

represents relatively large differences. The effect of Gal in the concentration of 10−10 M on the S2/S1 ratio was much smaller (about 26%). For the Hth–NH complex incubated in KRF fluid containing Gal in the same concentrations, we could observe the similar dependences: the decrease of the B2/B1 ratio was in a range of 40% and 34%, while the effect of Gal 10−8 M on the S2/S1 ratio was about 20%. Similarly, both Gal concentrations tested during the incubation of the NH and Hth–NH explants reduced distinctly the B2/B1 ratios as compared to control incubation (about 44% and 31% for the NH as well as about 70% and 43% for the Hth–NH). However, Gal was not found to have any such influence on potassiumstimulated OT release from either explant. Galantide, an antagonist of galanin receptors, added to the incubation fluid at a concentration of 10−10 M did not change AVP and OT release from the NH or Hth–NH complex under either basal or potassium-stimulated conditions. Neither concentration of Gal was found to exert any influence on AVP or OT secretion from the NH and Hth–NH explants in the presence of galantide in the medium. 3.3. Galanin-like peptide influence on AVP and OT secretion in vitro; the relation to galanin receptors The presence of GALP at concentrations of 10−10 and 10−9 M in the basal incubation media was the cause of intensified AVP release from both the NH (Fig. 6; B2/B1: GALP 10 vs GALP 0: p < 0.05, GALP 9 vs GALP 0: p < 0.01) and Hth–NH complexes (Fig. 7; B2/B1: GALP 10 vs GALP 0: p < 0.05, GALP 9 vs GALP 0: p < 0.001). For the NH, the B2/B1 ratios increased about 75% or 84%. This effect was strongest demonstrated (the sevenfold increase) when the Hth–NH complex was incubated with addition of 10−9 M GALP. Although the AVP concentration of the K+-enhanced medium was also increased

by both GALP concentrations, this difference was only statistically significant for the Hth–NH explant (Fig. 7; S2/S1: GALP 10 vs GALP 0: p < 0.01, GALP 9 vs GALP 0: p < 0.05; the S2/S1 ratios significantly increased about 122% or 89%). The addition of M15 at the concentration of 10−10 M into the incubation medium did not change AVP secretion from the incubated tissues, either during the basal or the potassium-stimulated periods. On the other hand, GALP added into Krebs–Ringer fluid in the presence of galantide (10−10 M) exerted a significant impact on AVP secretion. Hence, 10−10 and 10−9 M GALP stimulated basal release of AVP from NH (Fig. 6; B2/B1: GALP 10 + M15 vs GALP 0: p < 0.05, GALP 9 + M15 vs GALP 0: p < 0.01), with 10−9 M GALP exerting the greatest effect (Fig. 6; B2/B1: GALP 10 + M15 vs GALP 9 + M15: p < 0.001). Similarly, both GALP concentrations intensified basal AVP secretion from the Hth–NH system incubated in the presence of M15 (Fig. 7; B2/B1: GALP 10 + M15 vs GALP 0: GALP 9 + M15 vs GALP 0: p < 0.001). When the neurointermediate lobe was incubated in the K+enriched medium with galantide and 10−9 M GALP, increased AVP secretion was observed (Fig. 6; S2/S1: GALP9 + M15 vs M15: p < 0.05). Both GALP concentrations significantly stimulated K+-evoked AVP release from the Hth–NH explants, irrespective of the presence of M15 (Fig. 7; S2/S1: GALP 10 vs GALP 0: p < 0.01; GALP 9 vs GALP 0: p < 0.05; GALP10 + M15 vs M15: p < 0.01; GALP9 + M15 vs M15: p < 0.001). It is important to note that 10−9 M GALP exerted a greater impact. GALP at concentrations of 10−10 and 10−9 M added to normal KRF enhanced OT release from both the NH (Fig. 8; B2/B1: GALP 10 vs GALP 0: p < 0.01, GALP 9 vs GALP 0: p < 0.05) and Hth–NH explants (Fig. 9; B2/B1: GALP 10 vs GALP 0: p < 0.001, GALP 9 vs GALP 0: p < 0.001). Similarly to AVP, the B2/B1 ratios representing OT release

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393

Fig. 6. The effect of galanin-like peptide (GALP) or galantide (M15 at the concentration 10−10 M) or GALP together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) AVP release from the neurohypophysis in vitro (series 2). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no GALP.

from the incubated NH increased about 111% and 178%. In the case of the Hth–NH explant, the increases of the B2/B1 ratios were dramatically high and received the values of 591% and 523% for both tested GALP concentrations. However, under potassium-stimulated conditions, the presence of 10−10 or 10−9 M GALP has been observed to significantly impair OT release from the NH and Hth– NH complexes (Fig. 8; S2/S1: GALP 10 vs GALP 0: p < 0.01, GALP 9 vs GALP 0: p < 0.001; Fig. 9; S2/S1: GALP 10 vs GALP 0: p < 0.01, GALP 9 vs GALP 0: p < 0.01). Galantide did not modify the OT secretion from the NH or the Hth–NH systems when added into K+-enriched media. However, 10−10 and 10−9 M GALP markedly increased basal OT release from the isolated NH or Hth–NH systems incubated in the presence of M15 (Fig. 8; B2/B1: GALP 9 + M15 vs M15: p < 0.01; Fig. 9; B2/B1: GALP 10 + M15 vs M15: p < 0.001; GALP 9 + M15 vs M15: p < 0.001). GALP added into incubation media together with M15 significantly reduced K+-evoked OT release from the NH and Hth–NH explant into the medium (Fig. 8; S2/S1: GALP 10 + M15 vs M15: p < 0.01; GALP 9 + M15 vs M15: p < 0.001; Fig. 9; S2/S1: GALP 10 + M15 vs M15: p < 0.01). 4. Discussion The results of the present in vitro study clearly demonstrate the roles played by both Gal and GALP in the processes of AVP and OT release from the rat neurohypophysis or hypothalamoneurohypophysial explants incubated in normal or modified Krebs– Ringer fluid. The use of galantide, an antagonist of galanin receptors, indicates that Gal modifies the release of both neurohormones via its binding sites but GALP action is exerted by another unidentified specific receptor.

The neurohypophysis incubated in vitro provides an excellent model for studying the regulation of secretion at the terminal level, free from the complex synaptic effects present throughout the rest of the CNS (Ciosek, 2007; Juszczak, 2002). The axons of these neurons in the posterior lobe of the pituitary maintain the ability to secrete AVP and OT in response to appropriate stimuli for at least a few hours (Ciosek and Drobnik, 2013; Ciosek and Guzek, 1992). In this incubation model, the differences in AVP and OT secretion are due to the direct influence of various biologically active compounds, Gal or GALP in the present study, on AVP-ergic and OT-ergic neuron endings creating the neurohypophysis. The whole isolated hypothalamo-neurohypophysial system may be also used for studying the in vitro release of AVP and OT (Ciosek and Gałecka, 2011; Izdebska and Ciosek, 2010). In such a prepared specimen, the permanence of neural hypothalamo-neurohypophysial tracts is maintained, as are most processes related to the biosynthesis of these neurohormones, their transport in the infundibular stalk and the secretion into the medium. It is also assumed that most neuron fibers secreting different neuromodulators/neuromediators and impinging on the AVP-ergic and OT-ergic cell bodies within the paraventricular and supraoptic nuclei of the hypothalamus are intact (Gregg and Sladek, 1984). Some data from earlier in vivo and in vitro experiments confirm that Gal plays a modulatory role in the release of AVP and OT from the neurohypophysis. The results of the in vivo studies are ambiguous. Kondo et al. (1991) showed Gal to have an inhibitory effect on AVP release in hypertonic saline-treated rats. Balment and al Barazanji (1992) observed a transitory diuresis after central Gal infusion, which may be the result of impaired AVP secretion. Similarly, a significant reduction of OT plasma level has been observed in rats after icv injection of Gal (Björkstrand et al., 1993). However, Gayman

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Fig. 7. The effect of galanin-like peptide (GALP) or galantide (M15 at the concentration 10−10 M) or GALP together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) AVP release from the hypothalamo-neurohypophysial explant in vitro (series 2). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no GALP.

and Falke (1990) reported Gal to have no influence on OT release from posterior lobe of the pituitary in rats. Landry et al. (1995) demonstrated that the expression of AVP mRNA (but not OT mRNA) in PVN and SON of the rat hypothalamus decreased following an icv injection of Gal into dehydrated rats. Moreover, the increase of AVP mRNA level was observed after administration of the galanin receptor antagonist M15 (Landry et al., 1995, 2000). However, Molnár et al. (2005) did not report any significant influence of Gal on AVP release, i.e. an intravenous (iv) injection of 1.0 nmol of Gal did not modify the plasma basal AVP level and AVP release evoked by osmotic factor. An icv injection of 100 pmol of Gal did not influence the basal AVP plasma level. Ciosek and Cisowska (2003) reported that Gal injected icv did not affect OT content in the Hth and NH but diminished the hypothalamic AVP content without any change of neurohypophysial storage in rats which were not dehydrated. However, the same treatment distinctly inhibited AVP and OT release from the Hth–NH system in dehydrated rats (Ciosek and Cisowska, 2003). Furthermore, icv injection of Gal to salt-loaded rats caused a marked increase of both AVP and OT in the Hth and NH and a decrease in blood plasma (Cisowska-Maciejewska and Ciosek, 2005). Gal was found to have a similar impact on AVP and OT release from the Hth–NH system into the circulation in hemorrhaged rats (Ciosek et al., 2003). The present in vitro experiments confirm previous suppositions (Ciosek and Gałecka, 2011; Izdebska and Ciosek, 2010) that Gal modulates AVP and OT release by acting at every level of the hypothalamo-neurohypophysial system. In fact, both 10−10 M and 10−8 M Gal diminished basal release of AVP and OT from the isolated NH and Hth–NH explants. In the case of OT release from the Hth–NH explant, Gal exerts the strongest inhibitory impact in the concentration of 10−10 M. This effect seems to be incidental as our

earlier reports (Ciosek and Drobnik, 2013; Izdebska and Ciosek, 2010) did not show the differences in the activity of both Gal concentrations. The present results confirm those of in vitro studies by Gálfi et al. (2002;2003) and Nagyéri et al. (2009) who observed that Gal inhibits AVP release from the incubated neurohypophysial tissue. Moreover, the present results show that Gal has the same inhibitory effect on AVP release when added into the medium enriched with excess potassium ions. However, this effect of Gal on OT release was not marked during the incubation of the NH or Hth–NH complex in the K+-enriched medium. On the basis of these results it may be supposed that Gal acts as an inhibitory neuromodulator of AVP and OT secretion affecting the axon terminals in the neurohypophysis and modifying the processes related to transport and release of both neurohormones at the level of the hypothalamo-neurohypophysial system. It is possible that the influence of Gal did not depend on the tested concentration during the incubation, and that Gal is an endogenous factor disturbing the release of AVP and OT. The second objective of our study was to evaluate the potential influence of GALP on AVP and OT release from the hypothalamoneurohypophysial system in vitro. An in vivo experiment conducted by Onaka et al. (2005) indicates that GALP may play an important role in the release of AVP and OT from the neurohypophysis. After intracerebroventricular administration of GALP (2 nmol), plasma concentrations of both hormones were significantly increased as compared with values in the saline-injected control rats. Plasma hormone concentrations after GALP returned to the level of salineinjected control rats within 30–60 min (Onaka et al., 2005). In turn, the expression of GALP mRNA in the posterior pituitary was markedly increased in rats after dehydration or salt loading, manipulations that stimulate the secretion of AVP and OT (Suzuki et al., 2010). In streptozotocin-induced diabetic rats, which are in a hyperosmotic

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Fig. 8. The effect of galanin-like peptide (GALP) or galantide (M15 at the concentration 10−10 M) or GALP together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) OT release from the neurohypophysis in vitro (series 2). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no GALP.

state with elevated plasma AVP levels, GALP mRNA levels were increased approximately 20-fold in the neural lobe as compared to control (Shen and Gundlach, 2004). GALP expression was unaffected in the ARC during these states. The expression of GALP mRNA in the neural lobe of the pituitary is also induced by another condition associated with activation of magnocellular neurones, lactation (Cunningham et al., 2004). On the other hand, Dungan-Lemko et al. (2008) reported that congenital deficiency of GALP does not interfere with normal lactation in mice. The precise nature of the actions of GALP on vasopressinergic and oxytocinergic neurons is still not quite clear. Our results are consistent with those of previous in vivo studies. They demonstrate that both 10 −9 and 10 −10 M GALP distinctly stimulate basal and K+ ion-stimulated AVP release from the neurohypophysis as well as the hypothalamo-neurohypophysial explant. An important role in this neurosecretory control may be played by GALP-containing neurohypophysial astrocytes (pituicytes). Under normal physiological conditions, astrocytes act as a physical and chemical barrier, limiting neuron–neuron interactions, as well as the diffusion of neurotransmitters in the extracellular space (Rosso and Mienville, 2009; Stern and Filosa, 2013). These changes can be also produced in vitro in neurohypophysial explants (Hatton et al., 1984). The likely synthesis/release of GALP by these specialized astrocytes and its transcriptional regulation by osmotic challenges strongly suggests a role for this novel peptide in the regulation of pituicyte morphology. The results for OT achieved in the present study were not so clear. GALP at concentrations of 10−9 M and 10−10 M stimulated basal release of OT from the isolated NH and Hth–NH explants. GALP-stimulated OT release was not abolished by the galanin receptor antagonist, galantide. However, GALP was also found to exert an inhibitory effect on OT release when added into a medium

enriched with potassium ions. It is hard to comment on this significant difference between the reactions of OT-ergic neurons to GALP. The possibility that GALP exerts an indirect influence on the function of the hypothalamo-neurohypophysial system via activation of NPY neurons should be considered (Larsen et al., 1994; Seth et al., 2003). Furthermore, our results are the first to demonstrate that the stimulating effect of GALP on AVP release in rats is independent of galanin receptors. The same stimulatory GALP influence on AVP secretion has been also observed in the presence of galantide, an antagonist of Gal receptors in the incubation media. One possible explanation is that GALP has its own receptor(s), distinct from the known galanin receptors, an idea confirmed by the results of other studies which also suggest that GALP generally acts via a novel receptor. Centrally administered GALP was found to produce different physiological effects on food intake, LH release and c-Fos induction than Gal, a mature agonist of the galanin receptor (Dong et al., 2006; Man and Lawrence, 2008). Moreover, central administration of a GAL2/3 agonist in rats did not induce c-Fos in any of the brain regions that expressed this protein after GALP injection (Man and Lawrence, 2008). Boughton et al. (2010) proposed that alarin, a splice variant of the GALP gene, may act as a ligand for the same unidentified receptor or receptors. The concept of an unidentified GALP-specific receptor also has a potential basis in the structure of GALP. Although galanin and GALP share a partial sequence identity, there is a highly conserved and unique amino acid sequence in GALP not shared by galanin (GAL 38–54) that may dictate its interaction with a unique GALP receptor (Ohtaki et al., 1999; Robinson et al., 2006). In summary, Gal and GALP, while having some structural overlap, shared receptors, also have some very important differences. Here,

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Fig. 9. The effect of galanin-like peptide (GALP) or galantide (M15 at the concentration 10−10 M) or GALP together with M15 on the basal (B2/B1) and K+-stimulated (S2/S1) OT release from the hypothalamo-neurohypophysial explant in vitro (series 2). Each bar represents mean ± SEM; numbers in bars indicate group size. All comparisons were made with respect to the control tissue incubated in KRF with no GALP.

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