In addition to its primary role in the regulation of pituitary thyrotropin hormone secretion, thyrotropin-releasing hormone (TRH), the hypothalamic neuropeptide, acts in the central nervous system (CNS) as a neurotransmitter or neuromodulator at a pre- or post-synaptic site (1, 2). TRH is supposed to be one of the neuropeptides/neuroregulators of the central nervous system engaged in the modulation of the hypothalamo-neurohypophysial system activity. Some evidence suggests TRH participation in vasopressin (VP) as well as oxytocin (OT) release under physiologic as well as pathologic states (3-5), however, in other experiments, no changes in OT and VP release after intracerebroventricularly (icv) injections of TRH were reported (6). Earlier studies from our laboratory have showed that TRH injected icv acts as an inhibitory neuromodulator of vasopressin and oxytocin release in 3 months old rat deprived of the access to tap water (7), in the conditions of the haemorrhage-provoked hypovolemia (8) as well as in suckling female rats during midlactation (9). TRH, injected intravenously (iv), diminished both neurohypophysial neurohormones release during the state of water deprivation (10). Similarly, TRH restricts VP and OT release in vitro from the hypothalamo-neurohypophysial system (11).

The process of vasopressin as well as oxytocin biosynthesis in the hypothalamus fluctuates during the animals life; the respective guide such as the neurohormones content in the hypothalamus and the neurohypophysis may be helpful to estimate the rate of this process. VP and OT content in the hypothalamo-neurohypophysial system is altered during different periods of the animal life. It has been demonstrated the rise, the decrease or no change VP blood level in old rats of Long-Evans, Wistar, Fischer or Spraque-Dawley strain (12-14). Hypothalamic vasopressin content diminished with advancing age of the rat while the neurohypophysial deposits similarly decreased or did not change (13, 14). As to oxytocin, the decline of its neurohypohysial content has been estimated without the changes in the hypothalamus and blood plasma (15).

Vasopressin and oxytocin, synthesized by magnocellular neurons of the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei, are released from the neurohypophysis into the blood in response to several stimuli, mainly hyperosmolality of extracellular fluid, hypovolemia, suckling and parturition (16, 17). The freshly synthesized amounts of both neurohormones are transported along the axons of vasopressinergic and oxytocinergic neurons towards the neurohypophysis (18, 19). The blockade of axonal transport caused by the use of colchicine induces the accumulation of VP and OT in the magnocellular neurons of the hypothalamus. Colchicine is an alkaloid usually used as the blocker of neurotransmitters/neuromodulators neuronal transport in different brain structures, among them, in the neurons of the hypothalamo-neurohypophysial system (18). We used then the colchicine procedure because it gave the possibility of the estimation of vasopressin and oxytocin biosynthesis rate over a constant time period by the comparison of colchicine-treated rats with untreated rats. Therefore, the present study was undertaken to examine the vasopressin and oxytocin biosynthesis rate in the hypothalamus as well as their release into the blood plasma in young not mature rats (1 month old) and young adult rats (3 or 7 months old) under treatment of intravenously administered TRH.


MATERIAL AND METHODS
Animals

One hundred and twenty three male Wistar rats weighing: 131 ± 7.7 g (±S.D.) (one month old) or 222 ± 6.1 g (±S.D.) (three months old) or 343 ± 9.7 g (±S.D.) (seven months old) were housed with free access to commercial food pellets as well as tap water ad libitum. All the animals were kept at room temperature in a controlled light (L) – dark (D) cycle L:D = 12:12; light was turned on at 07.00 a.m. There were four animals per a cage. All the experiments were performed with the acceptance (No. 37/LB339/2006) of the Local Ethical Committee, Lodz.

General experimental design

The animals were divided into three main series:
series I – rats at one month age,
series II – three months old rats
series III – seven months old rats.

In the each series the following groups of animals were selected:

group A (Veh-Salt) – rats injected iv, once daily for seven days, with vehicle solution (Veh; 0.9% NaCl; 100 µl/100 g b.w.) and received icv, twenty hours before the decapitation, 5 µl of 0.9% sodium chloride solution (Salt);

group B (TRH-Salt) – rats injected iv, once daily for seven days, with thyrotropin-releasing hormone (TRH; Sigma Chemical Co.; lot 4640815) at a dose of 100 ng/100 g b.w. and received icv, twenty hours before the decapitation, 5 µl of Salt;

group C (Veh-Colch) – rats similarly injected iv with Veh and received icv, twenty hours before the decapitation, injection of colchicine solution (Colchicine Crystalline; Sigma Chemical Co., 085K1290) in a dose of 5 µg/5 µl;

group D (TRH-Colch) – rats similarly injected iv with TRH and received icv, twenty hours before the decapitation, injection of 5 µg/5 µl Colch.

The icv injections of colchicine vehicle (i.e., 0.9% sodium chloride) or colchicine solution (5 µg/5 µl) were made on the day before the decapitation. Animals were sacrificed 20 hr after colchicine or saline injection at the same time of a day (09.00-09.30 a.m.). The brain with the pituitary was removed from the skull, and the neurointermediate lobe was separated and homogenized in 0.25% acetic acid. A block of tissue containing the hypothalamus was dissected from the brain as described previously (20, 21) and homogenized in 0.5% acetic acid. The trunk blood was collected and VP and OT were extracted from the plasma using C18 Sep-Pak microcolumns (Waters Corp., Milford, Massachusetts, lot No W9224G1). The vasopressin and oxytocin content in the samples was determined by radioimmunoassay.

Radioimmunoassay (RIA)

The hypothalamic and neurohypophysial VP and OT content as well as plasma neurohormones concentration were determined by double-antibody specific RIA as previously described by Ciosek and Stempniak (22). Anti-VP and anti-OT antibodies were obtained in Department of Physiology and Biochemistry, Medical University of Lodz. The antibody titer was 1:24,000 for AVP and 1:80,000 for OT (both final dilutions) and the lower limit of detection for the assay was 1.25 pg VP/tube and 1.25 OT/tube. For standard curve preparation as well as iodination with 125I, using the chloramine-T method, the VP ([Arg8]-Vasopressin; lot 802958) and OT (OT-Oxytocin synth.; lot 027179) from Peninsula Laboratories Europe Ltd. were used. The intra-assay coefficients of variation for the VP was less than 3.5% and for OT 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

Vasopressin and oxytocin level was finally expressed in nanograms (ng) per mg of the hypothalamus tissue, in ng for whole neurohypophysis and in picograms (pg) per 1 ml of plasma. All results were reported as the mean ± standard error of the mean (S.E.M.). Statistical analysis of the experimental data was performed using “STATISTICA” (Version 6.0) software (StatSoft, Krakow, Poland). Data were calculated by use of the Kruskal-Wallis analysis of variance by ranks (ANOVA) test; if ANOVA revealed significant effects post hoc analyses were done using the U’Mann-Whitney test. P<0.05 was used as the minimal level of significance.

To compare the vasopressin and oxytocin biosynthesis rate in the hypothalamus of experimental animals we estimated the neurohormones biosynthesis rate over a 1-hr period on the base of the difference between the mean hypothalamic hormone content in the respective groups of rats. This difference was divided by 20 in view of the period of the time between the solutions injections and the decapitation (19, 23). Basing on these criteria, we compared VP and OT biosynthesis rate between: saline- or colchicine-treated rats (Veh-Colch vs Veh-Salt; Fig. 4) as well as vehicle- or TRH-injected ones (TRH-Salt vs Veh-Salt and TRH-Colch vs Veh-Colch; Fig. 5 and 6). Since the synthesis rate was estimated in such a way standards errors could not be calculated (19, 23). When the difference was negative (it has marked the reduction of neurohormonal biosynthesis rate by TRH treatment) the respective figures (Fig. 11-12) contented the co-ordinates system with the negative values in the Y axle.


RESULTS
TRH influence on the VP content in the hypothalamus

According to the obtained results we have observed the progressive raise of VP hypothalamic content during the maturation of control vehicle injected animals (Fig. 1). TRH injected iv into 1-month rats followed by the significant increase of VP hypothalamic content (Fig. 1); the respective differences seen in elderly rats were not statistically significant. The colchicine procedure was most effective in 1-month rats in which the augmentation VP content in the hypothalamus has been showed (Fig. 1). TRH distinctly increased vasopressin hypothalamic stores in colchicine-treated rats only in 3 months old rats (Fig. 1).

Fig. 1. Vasopressin (VP) content in the hypothalamus of different age rats (1, 3 or 7 months old) under influence of thyrotropin-releasing hormone (TRH) and/or colchicine (Colch) (mean+/-S.E.M.)

TRH influence on the VP content in the neurohypophysis

Intravenously injections of TRH were the reason of the distinct increase of neurohypophysial VP content in saline- and colchicine-treated 1-month old rats (Fig. 2). Similar effects of TRH in 7-months old saline-injected animals have been observed (Fig. 2). TRH did not changed VP content in the neurohypophysis of 3-months old rats (Fig. 2).

Fig. 2. Neurohypophysial (NH) vasopressin (VP) content of different age rats (1, 3 or 7 months old) under influence of thyrotropin-releasing hormone (TRH) and/or colchicine (Colch) (mean+/- S.E.M.)

TRH influence on the VP blood plasma concentration

Plasma vasopressin concentration did not change in 1-month as as well as 3-months old rats under TRH treatment, however, the tendency to some decrease of VP plasma level has been showed in this second case (Fig. 3). In 7-months old rats we have observed dramatic rise of VP plasma level in colchicine-treated animals; TRH distinctly restricted this effect (Fig. 3).

Fig. 3. Plasma vasopressin (VP) concentration in different age rats (1, 3 or 7 months old) under influence of thyrotropin-releasing hormone (TRH) and/or colchicine (Colch) (mean+/- S.E.M.)

Vasopressin synthesis rate in the hypothalamus under influence of colchicine or TRH

The influence of colchicine was most effective (i.e., most strongly blockade of VP transport) in younger 1-month rats in comparison with mature 3- and 7-months old rats (Fig. 4); the weakest colchicine effect in eldest animals has been demonstrated. TRH, chronically injected into saline-treated rats, was the reason of strongest and rapidest VP synthesis rate in the hypothalamus of youngest rats (Fig. 5); the slowest VP biosynthesis speed in 7-months rats has been showed. In animals injected icv with colchicine and simultaneously iv treated with TRH the VP biosynthesis rate in the hypothalamus was highest in 3-months old rats and lowest in 7-months old rats (Fig. 6).

Fig. 4. The effect of colchicine on vasopressin (VP) synthesis rate in different age rats

Fig. 5. The effect of TRH on the vasopressin (VP) synthesis rate in different age rats

Fig. 6. The effect of TRH on the vasopressin (VP) synthesis rate in different age and colchicine-treated rats

TRH influence on the OT content in the hypothalamus

Intravenously administered TRH significantly decreased OT hypothalamic content of 1-month old rats injected icv with saline or colchicine solution (Fig. 7). Similarly influence of TRH in 3-months old colchicine-treated rats and 7-months old saline-injected animals has been observed (Fig. 7). In all three series of animals icv injection of colchicine was the reason of distinct rise of OT content in the hypothalamus (Fig. 7).

Fig. 7. Oxytocin (OT) content in the hypothalamus of different age rats (1, 3 or 7 months old) under influence of thyrotropin-releasing hormone (TRH) and/or colchicine (Colch) (mean +/-S.E.M.)

TRH influence on the OT content in the neurohypophysis

Neurohypophysial OT content diminished significantly under influence of TRH in colchicine-treated rats in the age of 1- or 7-months (Fig. 8). On the contrary, OT neurohypophysial content increased following by TRH treatment in saline injected 7-months old rats (Fig. 8). It has been observed any significant TRH effect on OT release from the neurohypophysis of 3-months old rats.

Fig. 8. Neurohypophysial oxytocin (OT) content of different age rats (1, 3 or 7 months old) under influence of thyrotropin-releasing hormone (TRH) and/or colchicine (Colch) (mean +/- S.E.M.)

TRH influence on the OT blood plasma concentration

Our results showed the distinct diminution of OT blood plasma level in TRH-treated and injected icv with saline 3-months old rats (Fig. 9) or injected icv with colchicine 7-months old ones (Fig. 9).

Fig. 9. Oxytocin plasma concentration of different age rats (1, 3 or 7 months old) under influence of thyrotropin-releasing hormone TRH and/0r colchicine (Colch) (mean +/- S.E.M.)

Oxytocin synthesis rate in the hypothalamus under influence of colchicine or TRH

Similarly to vasopressin, oxytocin synthesis rate in animals injected icv with colchicine was highest in younger rats (1-month old) and decreased gradually to smallest level in rats in the age of 7 months (Fig. 10). Intravenous injections of TRH to salt-treated rats caused the decrease of OT synthesis rate – the strongest TRH effect in 7-months old rats has been showed (Fig. 11). Similarly, injections of TRH administered to colchicine-treated rats followed by the inhibition of oxytocin synthesis rate, however, the strongest TRH influence in 1-month old rats and the weekest in 7-months old rats have been noted (Fig. 12).

Fig. 10. The effect of colchicine on the oxytocin (OT) synthesis rate in different age rats

Fig. 11. The effect of TRH on the oxytocin (OT) synthesis rate in different age rats

Fig. 12. The effect of TRH on the oxytocin (OT) synthesis rate in different age and colchicine-treated ratsThe effect of TRH on the oxytocin (OT) synthesis rate in different age and colchicine-treated rats

DISCUSSION
In the discussion we intend to assume towards three main problems: (i) the modifications of vasopressin and oxytocin levels in the hypothalamus, neurohypophysis and blood plasma in different age rats; (ii) the estimation of both neurohormones hypothalamic biosynthesis rate by using the colchicine procedure (iii) thyrotropin-releasing hormone influence on vasopressin and oxytocin biosynthesis and release in saline-treated or colchicine-treated animals.

The process of vasopressin and oxytocin synthesis in the hypothalamus and their release from the neurohypophysis into the blood of the experimental rats changes with the passage of the time. The alterations in the ability to regulate water excretion and urine production under influence of vasopressin with the ageing of animals or human have been noted (24). Some authors (13, 14) have observed the diminution of hypothalamic VP content in elder rats (30 months old) in comparison to young ones (1-2 months old) while the neurohypophysial VP deposits similarly decreased or did not change. It has been demonstrated the rise, the decrease or no change VP blood plasma level with ageing of different strains rats, i.e., Long Evans, Wistar, Fischer or Spraque-Dawley rats (12, 13, 25). Neurohypophysial OT content failed in aged rats without the changes in the hypothalamus and blood plasma (15).

In our study we have investigated the age-interval between 1 to 7 months of the life because of the frequent usage of such rats by the experimenters in numerous papers. Our data showed that in control vehicle-injected rats vasopressin synthesis increased gradually with maturation of animals; VP hypothalamic content was lowest in 1-month old and highest in 7-months old rats. Then, VP neurohypophysial content decreased to lowest values in 3-months old rats, then has risen in 7-months old animals to level similar this occurred in the youngest ones. It may be interpreted by transient declining axonal transport from the hypothalamus to the neurohypophysis. It could be consistent with the observation by Fotheringham et al. (26) indicating a significant reduction of up to 25% in the rate of axonal transport of the neurohormones but it was noted in animals with advancing age. On the other hand, some authors (27) hypothesized that the axonal transport of vasopressin is a process linked to synthesis rate rather than VP release from the neurohypophysis. In our paper lower VP values in the neurohypophysis correspond with higher plasma concentration of this neurohormone, so this observation could also testify to increasing VP release into the blood. However, above results are not quite consistent with the data of Zbuzek et al. (28) which showed that 3H-arginine incorporation into VP molecule as the index of neurohormonal biosynthesis was highest in the young, lower in the adult and lowest in the old rats as well as age-related decrease in the release of newly synthesized VP from the neurohypophysis. Furthermore, we showed that in contrast to VP oxytocin hypothalamic content in 1-month and 7-months old vehicle-treated rats were unaltered and at the similar level; the smallest values in 3-months old rats have been noted. In the same 3-months old animals the increase of neurohypohysial OT content attest to intensification of its axonal transport towards the neurohypophysis. Initial relatively high OT concentrations in blood plasma in Veh-Salt 1-month old rats undergo the debasement to lowest OT level in 7-months old rats. So, it seems that the process of OT plasma level normalization proceeds in opposite direction than in case of VP, i.e. from higher values in youngest rats to lower in adult rats.

Other aspect of our paper is the estimation the neurohormonal biosynthesis rate in the hypothalamus of different age animals with use of colchicine procedure. Colchicine was found to exert the strong inhibitory effect on the transport of the newly synthesized neurohormones from the perikarya of VP-ergic and OT-ergic neurons to the axonal terminals in the posterior lobe of the pituitary (18, 29). Colchicine administered into the third ventricle in doses in the range from 3.5 µg to 8 µg/rat blocked neurohormonal transport for 18-24 hours (19, 29). Earlier reports indicated that the colchicine method is useful in the detecting changes in the syntheses of VP and OT even in such small hypothalamic regions as supraoptic nucleus (29). In our paper we hope first describe that the hypothalamic biosynthesis rate of VP and OT is most effective in youngest rats and declines during the adolescence of animals to grow up.

Vasopressin and oxytocin act in the brain both as the neurotransmitters and neuromodulators of different biological processes. In particular, vasopressin is implicated in the central regulation of water-electrolyte homeostasis and cardiovascular system activity by binding with respective membrane receptors (V1a, V1b and V2). The participation of vasopressin in physiological regulation of these processes and its implication in the pathogenesis of brain edema and impairment of cerebral circulation have been described lately in detail by Kozniewska and Romaniuk (30).

In connection with biological central effects of AVP and OT the mechanisms related to both neurohormones biosynthesis and release require detailed and permanent studies. Thus, the biosynthesis as well as release of neurohypophysial neurohormones are the composed processes regulated by the neural and/or neurohormonal mechanisms. The numerous neurotransmitters, and/or neuromodulators, and/or neuropeptides of central nervous system have been reported to be involve in these processes as excitatory or inhibitory agents acting at the postsynaptic and/or presynaptic level. Such excitatory agents as glutamate, dopamine, histamine, acetylcholine, angiotensin II, and inhibitory agents such as GABA, galanin, adenosine, atrial natriuretic peptide, modulate vasopressinergic and oxytocinergic neurons activity. The respective information concerning these problems have been presented in interesting review of Dayanithi et al. (31). Among others, the following neuropeptide - thyrotropin-releasing hormone has been suggested to modify vasopressin and oxytocin release. In fact, some earlier data, also from this laboratory, suggest a modulatory role for TRH in the release of neurohypophysial neurohormones; the respective experiments were performed on 3-months old male or female rats (6, 10, 22, 32, 33).

The presence of TRH, TRH receptors, mRNA for TRH and TRH receptors has been noted in the hypothalamus and neurohypophysis (34, 35). In hypothalamic neurons of supraoptic and paraventricular nuclei TRH is colocalized with vasopressin and oxytocin (35, 36). TRH is also present in VP-ergic and OT-ergic terminals in the neurohypophysis (37). The coexistence of TRH with vasopressin and/or oxytocin in the areas of the hypothalamus involved in the maintenance of the water-electrolyte balance indicates that this peptide can participate in the regulation of VP and OT biosynthesis and secretion.

In our experiments the route of TRH administration may be some of importance, however, it has been assumed that TRH applied intravenously penetrates to the brain through the blood-brain barrier quite well (38). It has been supposed that peripherally injected TRH has access to the central nervous system through cerebrospinal fluid (CSF) (39). Okuda et al. (40) has been noted quickly increase of TRH concentration in the cerebrospinal fluid after its iv administration to the conscious rats. So, exogenous TRH, applied icv or iv, exerts in CNS its influence on the hypothalamic areas containing VP-ergic and OT-ergic neurons and modifies the release of both neurohormones into the blood engaging the hypothalamic and/or pituitary TRH receptors.

This study demonstrates, we believe for the first time, thyrotropin-releasing hormone influence on vasopressin and oxytocin synthesis and release in rats in age period in the range from 1-month old to 7-months old rats. In present experiments TRH administered peripherally iv to saline- and colchicine-treated 1-month rats resulted in a significant rise in the hypothalamic and neurohypohysial VP content. Similar TRH influence has been noted in the hypothalamus of colchicine-treated 3-months rats as well in neurohypohysial VP content of saline-injected 7-months animals. However, VP concentration in blood plasma diminished only in the colchicine-injected 7-months rats. These observations could be the ground for the supposition that TRH modulates vasopressin biosynthesis and release at the different levels. On the one hand, in the young animals TRH stimulates neurohormonal biosynthesis and the axonal transport to the neurohypophysis. On the other hand, in elder animals TRH rather inhibits vasopressin release from the neurohypophysis into the blood. In fact, stimulatory TRH influence on VP biosynthesis rate is strongly marked in 1 month saline-injected rats and declines with passage of time. When TRH was administered to colchicine-treated rats the distinct increase of VP biosynthesis rate in 3-months old rats has been noted which suggests immediately influence of this peptide on the VP-ergic neurons at the hypothalamus level. These results are consistent with our earlier experiments in vitro in which TRH inhibited vasopressin and oxytocin release from the rat hypothalamo-neurohypophysial explants (11). Similarly, the present results are in agreement with former data from this laboratory, which showed that TRH injected icv inhibited vasopressin release in euhydrated as well as dehydrated, haemorrhaged or salt-loaded rats (7, 41-44). However, the present study do not confirm other our previous findings (10) which showed that TRH injected iv to animals of equilibrated water metabolism resulted in a decrease of hypothalamic and neurohypohysial VP contents. This difference difficult for the interpretation at this moment remains to subsequent elucidation.

This study shows that TRH exerts an opposite effect on the hypothalamic OT synthesis in comparison to VP. We have found that chronically administered TRH was able to inhibit OT biosynthesis in 1 month old Veh-Salt rats as well as 3 months old Veh-Colch and 7 months old Veh-Salt rats. The OT biosynthesis rate is inhibited following by TRH administration and this effect is most strong marked in 7-months old saline-injected animals and 1-month old colchicine-treated ones. What is more, neurohypophysial OT deposits decreased under influence of TRH in 1- and 7-months old colchicine-treated rats; however, the increase of OT content in the neurohypophysis of 7-months saline-treated rats after TRH injections has been noted. These results are in agreement with some earlier our observation (10) but do not confirm other data from this laboratory (7, 41- 44).

It cannot be excluded that TRH modifies VP and OT release indirectly, i.e., by stimulation of the pituitary – thyroid system and/or by participation of the anterior pituitary hormone prolactin.

The results of the studies concerning possible role of the thyroid in the regulation of VP and OT secretion are not quite consistent. For example, Ali et al. (45) has found the diminution of plasma vasopressin level in the hypothyroid rats. However, in other study there were no differences in plasma or pituitary VP levels as well as hypothalamic VP mRNA content during aminotriazole-induced hypothyroidism in rats (46). On the other hand, treatment of rats with triiodothyronine increased OT mRNA expression, the neurohypophysial OT content as well as OT level in blood (47). In patients with hyperthyroidism the increase of VP plasma level has been observed (48). Thyroxine injected into neonatal rats did not change the concentration of VP mRNA in the neurons of the hypothalamic PVN (49).

As TRH is well-known prolactin liberator the distinct relationships between PRL and neurohypophysial neurohormones release may be taken under consideration. The prolactin gene expression and immunoreactive PRL forms as well as PRL receptor mRNA in magnocellular neurons of the supraoptic and paraventricular nuclei have been detected (50-52). Prolactin and its 16 kDa N-terminal fragment stimulate release of VP in vitro by a direct effect on the hypothalamo-neurohypophysial neurons (53). Prolactin intensifies OT mRNA expression (54) and oxytocin release in vitro from the hypothalamic explants (55). What is more, intracerebroventricular injection of PRL induces of c-Fos in the SON (56). On the other hand, electrophysiological studies have been showed that prolactin induced a significant decrease in firing rates of OT-ergic neurons without the effect on the VP-ergic neurons activity in nonpregnant rats (57).

Taken together these findings suggest that:

1. during the maturation of male rats vasopressin synthesis and release increase whereas these processes in relation to oxytocin diminish;
2. VP and OT biosynthesis rate in the hypothalamus is most effective in youngest rats and declines with the passage of the time of animals life;
3. thyrotropin-releasing hormone affects VP-ergic and OT-ergic hypothalamic neurons activity and both neurohormones biosynthesis process. This effect, however, is opposed: TRH acts as a stimulator of vasopressin biosynthesis most of all in young male rats and as an inhibitor for oxytocin biosynthesis especially in adult animals.
   

This work has been supported by the grant No. 502-16-654 of Medical University of Lodz.

Acknowledgements: The authors wish to thank to Dr. Jacek Drobnik (Department of Connective Tissue Metabolism) for his helpful discussion of the results and to Mrs. Lucyna Grzywna (Department of General and Experimental Pathology) for her technical assistance with the Sep-pak method for VP and OT extraction.

Conflict of interests: None declared.


REFERENCES

1. Reichlin S. Neural functions of TRH. Acta Endocrinol 1986; 276(Suppl): 21-33.
2. Ciosek, J. Tyreoliberyna (TRH): biosynteza, wystepowanie, receptory, metabolizm. Endokrynol Pol 2004; 55: 608-615.
3. Horita A, Carino MA. Centrally administered TRH produces a vasopressor response in rabbits. Proc West Pharmacol Soc 1977; 20: 303-304.
4. Weitzman RE, Firemark NM, Glatz TH, Fisher DA. Thyrotropin releasing hormone stimulates release of arginine vasopressin and oxytocin in vivo. Endocrinology 1979; 104: 2154-2160.
5. Ciosek J. Wplyw tyreoliberyny na uwalnianie wazopresyny i oksytocyny z ukladu podwzgorzowo-przysadkowego w warunkach in vivo oraz in vitro. Post Hig Med Dosw 2007; 61: 429-437.
6. Kasting NW. Simultaneous and independent release of vasopressin and oxytocin in the rat. Can J Physiol Pharmacol 1988; 66: 22-26.
7. Ciosek J, Guzek JW, Orlowska-Majdak M. Thyrotropin-releasing hormone (TRH) modulates vasopressin and oxytocin release from the hypothalamo-neurohypohysial system in dehydrated rats. J Physiol Pharmacol 1993; 44: 293-302.
8. Ciosek J, Orlowska-Majdak M. Thyrotropin-releasing hormone (TRH) inhibits the release of vasopressin but not that of oxytocin from the hypothalamo-neurohypophysial system in haemorrhaged rats. Endocr Regul 1995; 29: 47-55.
9. Ciosek J, Guzek JW. Thyrotropin-releasing hormone affects the oxytocin, vasopressin and prolactin release in female rats during midlactation: relation to suckling. J Physiol Pharmacol 1998; 49: 135-150.
10. Ciosek J. Vasopressin and oxytocin release as influenced by thyrotropin-releasing hormone in euhydrated and dehydrated rats. J Physiol Pharmacol 2002; 53: 423-437.
11. Ciosek J, Stempniak B. Thyrotropin-releasing hormone (TRH) inhibits vasopressin and oxytocin release from the rat hypothalamo-neurohypohysial explants in vitro. Acta Neurobiol Exp 1996; 56: 35-40.
12. Silverman WF, Aravich PA, Sladek JR, Sladek CD. Physiological and biochemical indices of neurohypophyseal function in the aging Fischer rat. Neuroendocrinology 1990; 52: 181-190.
13. Terwel D, Markering M, Jolles J. Age-related changes in concentrations of vasopressin in the central nervous system and plasma of the male Wistar rat. Mech Ageing and Develop 1992; 65: 127-136.
14. Zbuzek VK, Zbuzek V, Wu W. The effect of aging on vasopressin system in Fischer 344 rats. Exp Gerontol 1983; 18: 305-311.
15. Zbuzek V, Fuchs AR, Zbuzek VK, Wu W. Neurohypophyseal aging: differential changes in oxytocin and vasopressin release, studied in Fischer 344 and Spraque-Dawley rats. Neuroendocrinology 1988; 48: 619-626.
16. Bourque CW, Oliet SHR, Richard D. Osmoreceptors, osmoreception, and osmoregulation. Front Neuroendocrinol 1994; 15: 231-274.
17. Ciosek J. Uwalnianie wazopresyny i oksytocyny z czesci nerwowej przysadki - obecny stan wiedzy. Endokrynol Pol 2000, 51: 113-123.
18. Alonso G. Effects of colchicine on the intraneuronal transport of secretory material priori to the axon: A morphofunctional study in hypothalamic neurosecretory neurons of the rat. Brain Res 1988; 453: 191-203.
19. Robinson AG, Roberts MM, Wayne AE, Janocko LE, Hoffman GE. Total translation of vasopressin and oxytocin in neurohypophysis of rats. Am J Physiol 1989; 257: R109-R117.
20. Ciosek J, Cisowska A. Centrally administered galanin modifies vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of euhydrated and dehydrated rats. J Physiol Pharmacol 2003; 54: 625-641.
21. Cisowska-Maciejewska A, Ciosek J. Galanin influences vasopressin and oxytocin release from the hypothalamo-neurohypophysial system of salt-loaded rats. J Physiol Pharmacol 2005; 56: 673-688.
22. Ciosek J, Stempniak B. Thyroliberin and the daily rhythm of vasopressin and oxytocin release from the hypothalamo-neurohypophysial system. Pathophysiology 1998; 5: 131-139.
23. Juszczak M, Bojanowska E, Dabrowski R. Melatonin and the synthesis of vasopressin in pinealectomized male rats. PSEBM 2000; 225: 207-210.
24. Pavo I, Varga C, Szucsa M, Laszlo F. Effects of testosterone on the rat medullary receptor concentration and the antidiuretic response. Life Sci 1995; 56: 1215-1222.
25. Frolkis VV, Golovchenko SF, Medved VI, Frolkis RA. Vasopressin and cardiovascular system in aging. Gerontology 1982; 28: 290-302.
26. Fotheringham AP, Davidson YS, Davies I, Morris JA. Age-associated changes in neuroaxonal transport in the hypothalamo-neurohypophysial system of the mouse. Mech Ageing Dev 1991; 60: 113-121.
27. Roberts MM, Robinson AG, Hoffman GE, Fitzsimmons MD. Vasopressin transport regulation is coupled to the synthesis rate. Neuroendocrinology 1991; 53: 416-422.
28. Zbuzek VK, Zbuzek V, Wu WT. Age-related differences in the incorporation of 3H-arginine into vasopressin in Fischer 344 rats. Exp Gerontol 1987; 22: 113-125.
29. Liu B, Kwok RP, Fernstrom JD. Colchicine-induced increases in immunoreactive neuropeptide levels in hypothalamus: use as an index of biosynthesis. Life Sci 1991; 49: 345-352.
30. Kozniewska E, Romaniuk K. Vasopressin in vascular regulation and water homeostasis in the brain. J Physiol Pharmacol 2008; 59: 109-116.
31. Dayanithi G, Viero C, Shibuya I. The role of calcium in the action and release of vasopressin and oxytocin from CNS neurones/terminals to the heart. J Physiol Pharmacol 2008; 59: 7-26.
32. Siren AL, Lake CR, Feuerstein G. Hemodynamic and neural mechanisms of action of thyrotropin-releasing hormone in the rat. Circ Res 1988; 62: 139-154.
33. Ciosek J, Stempniak B. Thyrotropin-releasing hormone (TRH) modifies oxytocin release from the hypothalamo-neurohypophysial system in salt-loaded rats. J Physiol Pharmacol 1995; 46: 169-178.
34. Kanaka S, Yamada M, Satoh T et al. Expression of thyrotropin-releasing hormone (TRH) receptor mRNA in somatotrophs in the rat anterior pituitary. Endocrinology1997; 138: 827-830.
35. Mitsuma T, Rhue N, Sobue G et al. Distribution of thyrotropin releasing hormone receptor in rats: an immunohistochemical study. Endocr Regul 1995; 29: 129-134.
36. Sharif NA. Quantitavive autoradiography of TRH receptor in discrete brain regions of different mammalian species. Ann NY Acad Sci 1985; 553: 147-175.
37. Rondeel JM, Klootwijk W, Linkels E, van Haasteren GA, de Greef WJ, Visser TJ. Regulation of thyrotropin-releasing hormone in the posterior pituitary. Neuroendocrinology 1995; 61: 421-429.
38. Koskinen L-OD. Thyrotropin-releasing hormone and cerebral blood flow. In Vasodilatation, PM Vanhoutte (ed). New York, Raven Press, 1988, pp 75-80.
39. Zlokovic BV, Segal MB, Begley DJ, Davson H, Rakic L. Permeability of the blood-cerebrospinal fluid and blood-brain barriers to thyrotropin-releasing hormone. Brain Res 1985; 358: 191-199.
40. Okuda C, Tanaka H, Miyazaki M. Cardiovascular effect of intravenously administered thyrotropin-releasing hormone and its concentration in push-pull perfusion of the fourth ventricle in conscious and pentobarbital-anesthetized rats. Life Sci 1988; 42: 1181-1188.
41. Ciosek J, Guzek JW. Thyrotropin-releasing hormone (TRH) and vasopressin and oxytocin release: in vitro as well as in vivo studies. Exp Clin Endocrinol 1992; 100: 152-159.
42. Ciosek J, Stempniak B, Orlowska-Majdak M. Thyrotropin-releasing hormone (TRH) inhibits vasopressin release from the hypothalamo-neurohypophysial system of rats drinking hypertonic saline. Endocr Regul 1993; 27: 29-34.
43. Ciosek J, Orlowska-Majdak M. Thyrotropin-releasing hormone (TRH) inhibits release of vasopressin but not that of oxytocin from the hypothalamo-neurohypophysial system in haemorrhaged rats. Endocr Regul 1995; 29: 47-55.
44. Ciosek J, Stempniak. Thyrotropin-releasing hormone (TRH) and release of neurohypophysial hormones in the rat. Pol J Endocrinol 1997; 48: 23-34.
45. Ali M, Guillon G, Cantan B et al. A comparative study of plasma vasopressin levels and V1 and V2 vasopressin receptor properties in congenital hypothyroid rat under thyroxine or vasopressin therapy. Horm Metab Res 1987; 19: 624-628.
46. Howard RL, Summer S, Rossi N, Kim JK, Schrier RW. Short-term hypothyroidism and vasopressin gene expression in the rat. Am J Kidn Dis 1992; 19: 573-577.
47. Adan RA, Cox JJ, van Kats JP, Burbach JP. Thyroid hormones regulates the oxytocin gene. J Biol Chem 1992; 267: 3771-3777.
48. Vargas F, Baz MJ, Luna JD et al. Urinary excretion of digoxin-like immunoreactive factor and arginine-vasopressin in hyper- and hypo-thyroid rats. Clin Sci 1991; 81: 471-476.
49. Dakine N, Oliver C, Grino M. Thyroxine modulates corticotropin-releasing factor but not arginine vasopressin gene expression in the hypothalamic paraventricular nucleus of the developing rat. J Neuroendocrinol 2000; 12: 774-783.
50. Mejia S, Morales MA, Zetina ME, Martinez de la Escalera G, Clapp C. Immunoreactive prolactin forms colocalize with vasopressin in neurons of the hypothalamic paraventricular and supraoptic nuclei. Neuroendocrinology 1997; 66: 151-159.
51. Bakowska JC, Morrell JI. The distribution of mRNA for the short form of the prolactin receptor in the forebrain of the female rat. Mol Brain Res 2003; 116: 50-58.
52. Pi XJ, Grattan DR. Differential expression of the two forms of prolactin receptor mRNA within microdissected hypothalamic nuclei of the rat. Mol Brain Res 1998; 59: 1-12.
53. Mejia S, Torner LM, Jeziorski MC et al. Prolactin and 16K prolactin stimulate release of vasopressin by a direct effect on hypothalamo-neurohypohyseal system. Endocrine 2003; 20: 155-162.
54. Popeski N, Amir S, Woodside B. Prolactin and oxytocin in the paraventricular and supraoptic nuclei: effects on oxytocin mRNA and nitric oxide synthase. J Neuroendocrinol 2003; 15: 687-696.
55. Parker SL, Armstrong WE, Sladek CD, Grosvenor CE, Crowley WR. Prolactin stimulates the release of oxytocin in lactating rats: evidence for a physiological role via an action at the neural lobe. Neuroendocrinology 1991; 53: 503-510.
56. Cave BJ, Wakerley JB, Luckman SM, Tortonese DJ. Hypothalamic targets for prolactin: assessment of c-Fos induction in tyrosine hydroxylase- and proopiomelanocortin-containing neurons in the rat arcuate nucleus following acute central prolactin administration. Neuroendocrinology 2001; 74: 386-395.
57. Kokay IC, Bull PM, Davis RL, Ludwig M, Grattan DR. Expression of the long form of the prolactin receptor in magnocellular oxytocin neurons is associated with specific prolactin regulation of oxytocin neurons. Am J Physiol Regul Integr Comp Physiol 2006; 290: R1216-R1225.