Many neuropeptides and neurotransmitters/neuromodulators may be involved in the mechanisms of vasopressin and oxytocin release from the hypothalamo-neurohypophysial system (1-3).

Galanin (Gal) is a 29/30-amino acid peptide isolated originally from the porcine intestine (4). Many studies have revealed that Gal is widely distributed in the rat central nervous system (5-7). The highest densities of galanin-like immunoreactivity (Gal-LI) are found in brain structures such as the amygdaloid complex, the hypothalamus and the brainstem as well as posterior pituitary (5, 8, 9). In the hypothalamus Gal is particularly present in the preoptic area, the paraventricular (PVN) and supraoptic (SON) nuclei, and the arcuate nucleus/median eminence (7, 10, 11). Moreover, SON and PVN appear a high density of Gal binding sites (12, 13). In rat brain the presence of GalR1 mRNA in SON, magnocellular (m) and parvocellular (p) PVN as well as in dorsomedial, ventromedial and arcuate nuclei has been demonstrated (7). Yet, GalR2 mRNA is enriched in pPVN, but not SON (7). Several studies have revealed that Gal coexists with arginine vasopressin (AVP) in the same cell bodies of magnocellular neurons of the SON and PVN as well as in many parvocellular neurons in the PVN (14-16).

It has been shown earlier that the states of dehydration or salt loading reduce the Gal expression in the posterior lobe of the pituitary but raise (or had no effect) the hypothalamic Gal immunoreactivity; Gal mRNA levels increase in these conditions (17-19). Furthermore, some earlier reports have been showed that central injections of galanin inhibited osmotically stimulated arginine vasopressin release in the rat (20). Gal is also shown to inhibit oxytocin (OT) release in some circumstances (21).

The aim of our study was to investigate the effects of centrally injected Gal on AVP and OT hypothalamo-neurohypohysial content and their release into the blood of euhydrated or dehydrated rats.


MATERIALS AND METHODS

Animals.

Male Wistar rats, weighing 274 ± 17 g (mean ± S.D.), were used. All the experiments were performed with the acceptance of the Ethical Committee of Medical University of Lodz. The animals were fed normal pelleted laboratory diet and kept at room temperature of about + 20° C under 12 h light : 12 h dark cycle (artificial illumination from 6.00 a.m. to 6.00 p.m.). Four animals were hold per a cage. All animals had free access to tap water until the beginning of the experiment.

General experimental design.

Complete experimental protocol was followed in 40 animals divided into two groups: A - animals injected intracerebroventricularly, once daily at the same hour as the last injection before decapitation has been administered (see below), with artificial cerebrospinal fluid (aCSF) in a volume of 5 µl (the composition of 100 ml of aCSF was as follows: NaCl - 810 mg, KCl - 17 mg, CaCl2 - 14 mg, K2HPO4 - 42 mg, NaHCO3 - 176 mg, glucose - 61 mg, urea - 13 mg); B - animals injected i.c.v. once daily with solution of galanin (Gal; lot No 124H09511, SIGMA Chemical Co., St. Louis, USA) in a daily dose of 100 pM dissolved in 5 µl of aCSF. The pH was controlled for aCSF and aCSF enriched with Gal and was found within a range of 7.37-7.43 for both solutions. Osmolality of aCSF and aCSF with Gal solutions (Knauer's semimicroosmometer: Halbmikro-Osmometer, Knauer, Wissenchaftliche Geräte KG, Berlin) was within a range of 275-283 mOsm/kg H2O.

In each group two further subgroups were set up: I - controls not dehydrated; II - animals dehydrated for two days. The animals of subgroup B-I were decapitated 10 minutes after a single i.c.v. injection of Gal. The rats in subgroup B-II were killed after two days of water deprivation; in this subgroup the last injection of Gal was given 10 min before killing.

The animals of corresponding subgroups in group A were sacrificed in similar manner (subgroup A-I: controls not dehydrated; subgroup A-II: animals dehydrated, untreated).

Surgical preparation.

The animals were implanted with a permanent cannula for chronic injections inserted into the left cerebral ventricle under light hexobarbital anaesthesia (intraperitoneal (i.p.) injection of 7% Hexobarbital Natrium solution: 0.3 ml/100 g b.w.). The animals were immobilized in a simple stereotaxic apparatus as recommended by Noble et al. (22); a small hole was drilled in the skull (1.5-2.0 mm laterally and 1.5-2.0 mm posteriorly to the crossing of the sagittal and coronal sutures). A simple stainless stell cannula was inserted into the left lateral cerebral ventricle; its tip was 4.0 mm below the dorsal skull surface and its inner diameter was 0.5 mm. The cannula was fixed to the skull with dental cement (Duracryl; Spofa-Dental, Praha). After surgery the animals were allowed to recover for up to 7-10 days before starting the experimental protocol. The i.c.v infusions were made to previously trained conscious rats; a 50 µl Hamilton syringe (Hamilton Comp., Reno, NY) with plunger pushed by a microscrew was used. The syringe was connected through a polyethylene tube to the cannula and 5 µl of the appropriate solution were infused. The duration of the infusion was about 8-10 sec. The effectiveness of i.c.v. infusions was verified by injecting 10 µl of 0.25 per cent trypan blue solution to similarly operated separate control animals (one rat injected with the dye for every 10 animals injected with Gal solution or aCSF) and was found to be quite satisfactory, i.e., the dye was distributed in an uniform manner within all cerebral ventricles.

Experimental procedure.

The rats were weighed and killed by decapitation at 9.00-10.00 a.m. Mixed arterial-venous blood from the trunk was partly collected in heparinized capillaries for determination of the haematocrite index. The rest of the blood was collected in heparinized test tubes, centrifuged for 20 min (temperature + 4° C: rpm = 2500-3000), the plasma was removed and stored at - 70° C until radioimmunoassayed as well as for evaluation of the osmolality. Serum osmolality was estimated using a Knauer semimicroosmometer. The neurohormones were extracted from the plasma using C18 "Sep-Pak" microcolumns (Sep-Pak(R) C18, Lot No W9224G1; Waters Corp., Milford, Massachusetts, USA) as described by Forsling (23); the final extracts were preserved in frozen sealed vials until radioimmunoassay. The recoveries of hormones during extraction procedure were greater than 85% and therefore values were not corrected for procedural loses.

The brain with intact pituitary was quickly (i.e., not later than 2 min after decapitation) removed, infundibular stalk cut up and the neurointermediate lobe was separated. From the brain, hardened for a few minutes at - 70° C, the hypothalamic block was dissected as follows: rostral limit - frontal plane situated about 1 mm more rostral to the anterior margin of the optic chiasma; caudal limit - frontal plane just behind the mammillary bodies; lateral limits - sagittal planes passing on both sides through the hypothalamic fissures. The depth was about 2.0 mm from the base of the brain. The wet weight of such block of the tissue, containing the hypothalamus, was 28.3 ± 1.1 mg (mean ± S.D.).

The neurointermediate lobe (respectively, the hypothalamus) was homogenized in ultrasonic disruptor (MicrosonTM Ultrasonic Homogenizer; Labcaire, UK) in 2.0 ml of 0.25% (resp. 0.5%) acetic acid in 0.15 M sodium chloride solution. The tissue suspension was transferred into a centrifuge tube and then the sample was heated for 5 min on boiling water bath (in order to inactivate the proteolytic enzymes contained in the homogenized tissue) and next centrifuged for 30 min (temperature + 4° C; rpm = 4000). The supernatants were removed, frozen and stored at - 70° C until radioimmunoassay for AVP and OT.

Radioimmunoassay of vasopressin and oxytocin

Characteristic of antisera.
Anti-AVP (serum No 1228/1987-08-24) and anti-OT (serum No 1232/1988-02-03) antibodies were raised in rabbits in Department of Physiology, Institute of Physiology and Biochemistry, Medical University of Lodz, Poland. The antibody titer to be used in the radioimmunoassay was 1: 40 000 for anti-AVP and 1: 80 000 for anti-OT (both final dilutions). Cross reactivity with oxytocin for anti-AVP antibodies was 0,016%, with lysine vasopressin (LVP) - 2,7%, with luliberin (LH-RH), leucine enkephalin (Leu-Enk), angiotensin II (Ang II), substance P (SP), hexapeptide (PyrGlu6Tyr8)SP6-11 and hexaeptide (Tyr8)SP6-11 it was < 0,002%. Cross reactivity with AVP for anti-OT antibodies was 1,12%; with LH-RH, Leu-Enk and Ang II it was < 0,002%. The sensitivity of anti-AVP and anti-OT antisera was 0,78 pg/100 µl. Intra- and extra-assay coefficients of variation (cv) for the vasopressin assay were 2,5% and 6,3 %, respectively; for the oxytocin assay cv were 3,3 % and 8,3 %, respectively.

Iodination of arginine vasopressin and oxytocin. Arginine vasopressin

(Arg8)-Vasopressin, (Bachem AG, lot 511731) and oxytocin (Peninsula Lab. Ltd., lot 027179) were iodinated with 125I using the chloramine-T method (24). Unreacted iodide was removed by mixing the reaction mixture with Amberlite (Aldrich Chemical Company, USA). Further purification was carried out on a column of Sephadex G-25 (Aldrich Chemical Company, USA) fine pre-equilibrated and eluated with 0,05 mol/l acetic acid. Labelled vasopressin and oxytocin were identified in the third peak by their ability to bind to the corresponding antibodies (25). The effectiveness of the iodination procedure was 80 % - 90 %. The top or the 1th descending portion of this peak was used as the tracer in RIA. Labelled hormones retained their antibody bindability for up to four weeks. All the specimens from particular experiments were measured in duplicate in the same assay. For a single estimation 100 µl of the respective extract were used.

Statistical evaluation of the results.

The vasopressin or oxytocin content was finally expressed in nanograms per mg of the hypothalamic tissue, in nanograms per whole neurointermediate lobe and in picograms per millilitre of blood plasma. All findings are reported as mean ± standard error of the mean (±S.E.M.). Data were calculated by the analysis of variance (ANOVA); if ANOVA revealed significant effects, post hoc analyses were done using U Mann-Whitney test (p< 0,05 was considered to be statistically significant). For statistical assessment of the data the programme "STATISTICA" (Version 5.0), copyright StatSoft Inc., licensed to Department of Pathophysiology, Medical University of Lodz, was used.


RESULTS

The results are summarized on Tables 1 and 2 as well as on Figures 1-6.

Validation of the dehydration degree (the changes of haematocrite index and osmolality values) (Tables 1 and 2).

Under conditions of water deprivation, progressive increase of both haematocrite index and serum osmolality was noted (Table 1 and 2: subgroup A-II vs A-I). Gal administered i.c.v. to euhydrated animals did not result in any significant change of both parameters in question. Following two days of dehydration, however, serum osmolality and haematocrite index were more marked in animals treated with Gal (Table 1 and 2: subgroup B-II vs A-II).

Table 1. Serum osmolality in euhydrated and dehydrated rats (in mOsm/kg H2O; mean ± S.E.M.)

Table 2. Haematocrite index in euhydrated and dehydrated rats (mean ± S.E.M.)

NS - not significant

The effect of galanin on the vasopressin and oxytocin content in the hypothalamus and neurohypophysis (Fig. 1, 2, 4 and 5).

Both the hypothalamic and neurohypophysial vasopressin and oxytocin content decreased progressively under conditions of dehydration (Fig. 1, 2, 4 and 5: subgroup A-II vs A-I).

Gal injected i.c.v. to euhydrated rats (subgroup B-I) had no distinct influence on the hypothalamic storage of oxytocin as well as neurohypophysial OT and AVP content, however, resulted in a decrease of hypothalamic AVP resources when compared with rats injected with aCSF (Fig. 1: subgroup B-I vs A-I). Instead, Gal distinctly restrained AVP and OT depletion in the neurointermediate lobe of animals dehydrated over two days (Fig. 2 and 5: subgroup B-II vs A-II). Similarly, the diminution of the hypothalamic oxytocin stores (as brought about by stimulation of osmoreceptors) was distinctly less marked under Gal treatment (Fig. 4: subgroup B-II vs A-II). No significant differences were found, however, in the hypothalamic AVP content in the rats deprived of water and simultaneously injected with Gal (Fig. 1: subgroup B-II vs A-II).

Fig. 1. The hypothalamic (Hth) vasopressin (AVP) content in euhydrated and dehydrated rats as influenced by Gal injected i.c.v. (mean +/- S.E.M.)

Fig. 2. The neurohypophysial (NH) vasopressin (AVP) content in euhydrated and dehydrated rats as influenced by Gal injected i.c.v. (mean +/- S.E.M.)

The effect of galanin on the vasopressin and oxytocin concentration in the blood plasma (Fig. 3 and 6).

In the animals injected with aCSF and simultaneously deprived of water for two days, plasma concentrations of AVP and OT were significantly elevated (Fig. 3 and 6: subgroup A-II vs A-I).

Fig. 3. The blood plasma vasopressin (AVP) concentration in euhydrated and dehydrated rats as influenced by Gal injected i.c.v. (mean +/- S.E.M.)

Fig. 4. The hypothalamic (Hth) oxytocin (OT) content in euhydrated and dehydrated rats as influenced by Gal injected i.c.v. (mean +/- S.E.M.)

Fig. 5. The neurohypophysial (NH) oxytocin (OT) content in euhydrated and dehydrated rats as influenced by Gal injected i.c.v. (mean+/- S.E.M.)

Fig. 6. The blood plasma oxytocin (OT) concentration in euhydrated and dehydrated rats as influenced by Gal injected i.c.v. (mean+/- S.E.M.)

Under conditions of equilibrated water metabolism i.c.v. treatment with Gal resulted in an increase of both vasopressin and oxytocin blood plasma concentrations (Fig. 3 and 6: subgroup B-I vs A-I). In rats deprived of tap water for 2 days Gal distinctly decreased AVP and OT blood plasma level (Fig. 3 and 6: subgroup B-II vs A-II).


DISCUSSION

Vasopressin and oxytocin release during dehydration.

In animals deprived of access to tap water occur the symptomps of hypertonic dehydration in which the water deficiency prevails over the electrolyte losses. In these conditions the diminution of both extracellular and intracellular fluid volume as well as the hyperosmolality of these spaces have been observed (26, 27). The dehydration persisting 48 hours resulted in about 18% decrease of blood volume and about 5-6% increase of serum osmolality; simultaneously, vasopressin and oxytocin concentrations in blood plasma significantly raised (28). Sugahara et al. (29) have observed in rats deprived of water 8-10 time higher AVP level in the blood. In the conditions of the dehydration intensified secretion of vasopressin and oxytocin depends on the rise of the blood plasma osmolality as well as the decrease of blood volume. However, it is difficult to estimate univocally which parameter change has a crucial importance for the stimulation of the hypothalamo-neurohypophysial function (30).

Dehydration as the state of intensified osmoreceptors activity is known to be a major factor affecting the release of vasopressin as well as oxytocin from the hypothalamo-neurohypophysial system. Several studies have shown an increase in the neurohypophysial hormones biosynthesis, axonal transport and their release into the blood (for references see: 31, 32) as well as an increase in the bioelectrical discharge of vasopressinergic and oxytocinergic neurons of magnocellular hypothalamic nuclei during progressive dehydration (33, 34). In these conditions the decrease of vasopressin and oxytocin content in the neurohypophysis as well as the elevation of their plasma concentrations has been observed (2, 28, 35, 36). It is known, that in the conditions of the extracellular fluid hyperosmolality the vasopressin and oxytocin release into the blood (34, 37-39) as well as into the cerebrospinal fluid (39) is then intensified. It is important to note that OT-ergic neurons are more sensitive for the changes of the extracellular fluid osmolality than AVP-ergic one (40). The diminution of the neurohypophysial oxytocin storages during the dehydration occurs more quickly than the decrease of AVP content (41).

Moreover, AVP and OT mRNAs levels are enhanced in magnocellular neurons in the rat hypothalamus under influence of osmotic stimulation (42). Similarly, c-fos mRNA and Fos protein are expressed in vasopressinergic and oxytocinergic neurons of the hypothalamus in these conditions (29, 43-45). The degree of the c-Fos expression is correlated with long-term duration of the dehydration; it depends also on the day-time when the dehydration started (46). Summy-Long et al. (47) have observed that protein Fos content in the magnocellular neurons of SON increased parallel to the blood osmolality rise.

The neurohypophysial vasopressin and oxytocin contents associated with their plasma concentrations reflect the actual degree of the neurohormones release into the blood. Next, the hypothalamic stores of both vasopressin and oxytocin depend on the degree of their biosynthesis and the rate of axonal transport towards the neurohypophysis. Under conditions of dehydration these processes are greatly modified by the impulsation of osmotic origin (36, 48, 49).

In agreement with former data, also from this laboratory (2, 35, 50), the present results show that, under conditions of hyperosmotic dehydration, the stores of both vasopressin and oxytocin are depleted in the hypothalamus and the neurohypophysis. Simultaneously, the vasopressin and oxytocin plasma levels distinctly raised. It seems that the infundibular trasport of both neurohormones - although intensified in the conditions of water deprivation - is probably insufficient to compensate the hormonal quantities released into the circulation. Moreover, the blood plasma osmolality and the haematocrite index as the indirect factors of the progressive dehydration were statistically higher in comparison with these parameters values noted in euhydrated rats.

Galanin influence on vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of dehydrated rats.

Some data suggest a modulating role for Gal in the release of the hypothalamo-pituitary hormones (51, 52). For example, galanin peptide is implicated in neuroendocrine regulation of prolactin, growth hormone, adrenocorticotropic hormone or thyrotropic hormone release in the rat (51, 53-58). Lactotrophs, somatotrophs, corticotrophs and thyrotrophs have been shown to contain immunoreactive galanin. Moreover, Gal acts presynaptically to modulate the secretion of GnRH or GHRH from the hypothalamus (59, 60). The hypothalamus is thought to be one of the major sites of Gal action in the central nervous system. The high affinity binding receptors for galanin have been detected in the brain, mainly in the medial preoptic area, paraventricular and supraoptic nuclei (52, 61). In situ hybridization studies have demonstrated the high density of GalR1 mRNA in SON, mPVN and pPVN. In contrast, GalR2 mRNA prevails in pPVN but is not detected in SON (7).

A great number of galanin-like neurons are found in the magnocellular hypothalamo-neurohypophysial system as well as in the parvocellular hypothalamo-anterior pituitary system (8). Cell bodies of galanin-immunoreactive neurons are present mainly in the supraoptic and paraventricular nuclei; their axons terminate in the posterior lobe of the pituitary (8, 9, 62). Immunohistochemical and in vitro studies have shown that Gal coexists with AVP in the magnocellular neurons in the SON and PVN and/or parvocellular neurons in the PVN of the human and rat hypothalamus (14, 17, 51, 62, 63). Gal appears also in OT-ergic neurons particularly in the experimental conditions such as colchicin treatment (64, 65) or hypophysectomy (17, 66).

Some findings seem to confirm the hypothesis that Gal may be engaged in the water-electrolyte homeostasis regulation. Earlier reports have been showed that Gal mRNA content raised in the supraoptic and paraventricular nuclei of rats deprived of tap water as well as Brattleboro rats with hereditary diabetes insipidus; Gal-like immunoreactivity in the neurohypophysis was then reduced (12, 18, 19). The level of GalR1 mRNA in the SON and magnocellular PVN was increased in salt-loaded or dehydrated rats (67).

Hence, Gal seems to have a modulatory role in the release of vasopressin and oxytocin. Kondo et al. (20) demonstrated that centrally administered Gal inhibits vasopressin release in hypertonic saline-treated rats. Landry et al. (68) showed in dehydrated animals that osmotically stimulated AVP mRNA content in PVN and SON decreases after i.c.v. injection of galanin; any influence of Gal on OT mRNA content after stimulation by dehydration has not been found. On the other hand, Björkstrand et al. (21) reported a significant decrease of OT plasma level in anaesthetized rats 20 min after i.c.v. injection of 0.1 or 1 µg of galanin. In the study of Gayman and Falke (69) Gal had no effect on oxytocin release from the rat neurohypophysis. Intravenous infusion of human galanin resulted in any significant change in plasma antidiuretic hormone (70). Moreover, Skofitsch et al. (19) observed that Gal injected intravenously resulted in mild diuresis, however, had no effect on AVP-induced anti-diuresis process. Similarly, Balment and al Barazanji (71) observed a transistory diuresis after central galanin infusion. In our latest study (72) we have demonstrated for the first time the consequences of i.c.v. injection of Gal on vasopressin and oxytocin release in the rats in the state of hypovolemia induced by haemorrhage. We have showed that Gal distinctly supressed AVP as well as OT release (intensified in the state of hypovolemia) from the neurohypophysis into the blood. Accordingly, the hypothalamic and neurohypophysial stores of AVP and OT significantly raised in these animals.

Our present results seem to be consistent with these reports. It is noteworthy that in euhydrated rats Gal distinctly increased plasma AVP and OT concentration; however, Gal did not affect the hypothalamic OT (AVP level was diminished) and neurohypophysial OT and AVP content. On the contrary, Gal administered i.c.v. to rats deprived of water distinctly inhibited AVP and OT release from the hypothalamo-neurohypophysial system. Simultaneously, plasma AVP and OT level was significantly diminished after Gal treatment in dehydrated rats.

The mechanisms of Gal actions on the vasopressinergic and oxytocinergic neurons are still not quite clear. It is possible that exogenous Gal injected i.c.v. may influence the synthesis and/or release of vasopressin and oxytocin by a direct effect on the hypothalamic neurons or by modified neurotransmission in the brain. This direct influence Gal exerts through the binding to the respective galanin receptors at the level of the hypothalamo-neurohypophysial system. Galanin binding with GalR1 or GalR2 can modulate bioelectrical and neurosecretory function of AVP- and/or OT-ergic neurons in the autocrine/paracrine mechanism (73). According to the report of Pieribone et al. (74) Gal has been shown to provoke intracellular K+ current in the magnocellular hypothalamic neurons which is followed by long-term cell membrane hyperpolarization and the inhibition of neuronal bioelectrical discharge. This mechanism can also handicap the process of egzocytosis of the neurosecretory vesicles containing AVP and OT from the endings of AVP- and OT-ergic neurons. It could reduce AVP and OT biosynthesis and release into the blood. Therefore, Gal seem to be the inhibitory neuromodulator of the magnocellular neurons activity (75).

Moreover, it cannot be excluded the indirect influence of Gal on the mechanisms of AVP and OT release via participation of specific innervation of AVP-ergic and OT-ergic neurons. It has been shown that Gal may be involved in the modulation of activity of monoamine system with widespread projections in the central nervous system (76). For example, galanin-positive nerve endings could be seen in the synaptic contact with dendrites and soma of noradrenergic neurons in the locus coeruleus (77, 78). On the other hand, galanin immunoreactive neurons from the locus coeruleus project to the anterior and periventricular parts of the PVN (79). Electron microscope studies of Landry et al. (80) demonstrated synaptic contacts between galanin-containing fibers and magnocellular hypothalamic neurons. What is more, the adrenergic afferentation of SON and PVN neurons modify the neurohormones biosynthesis and release (81-83). It could be therefore possible that functional interactions between galaninergic and monoaminergic neurons could modify magnocellular neurons activity under exogenous Gal influence. It is also of interest that exogenous Gal is able to regulate galanin expression itself in hypothalamic magnocellular neurons. In the study of Landry et al. (80), after galanin injection, Gal mRNA level decreased in salt-loaded rats whereas the expression of galanin immunoreactivity increased.

To sum up, in present study we would like to accentuate on two directions of Gal action. OT content (but not AVP) in the hypothalamus of dehydrated rats increased significantly following galanin injection. Similarly, AVP and OT neurohypophysial content in the same rats remarkably raised after Gal treatment; both plasma neurohormones level was then diminished. So, we suggest that under osmotic stimulation Gal influences as inhibitory neuromodulator on the biosynthesis and release of AVP and OT. Under Gal influence restrained axonal transport of AVP and OT as well as their release into the blood could be the reason of the accumulation and the increase of the neurohormones content in the hypothalamus and neurohypophysis. Secondly, the significant raise in AVP and OT plasma level observed in not dehydrated rats after Gal treatment could be recommended for the hypothesis that Gal may act as a stimulatory neuromodulator of both neurohormones secretion under conditions of equilibrated water balance.

The present results suggest that modulatory efect of galanin on vasopressin and oxytocin release depends on the actual state of water metabolism. Gal acts as an inhibitory neuromodulator of AVP and OT secretion under conditions of the dehydration but stimulates this process in the state of equilibrated water metabolism.

Acknowledgements: Conducted under contract No 6PO5A 13721 with the State Committee for Scientific Research, Warsaw.


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