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JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2002, 53, 3, 423–437 www.jpp.krakow.pl

J. CIOSEK

VASOPRESSIN AND RELEASE AS INFLUENCED BY THYROTROPIN-RELEASING IN EUHYDRATED AND DEHYDRATED RATS

Department of Pathophysiology, Medical University of Lodz, Poland

Since the thyrotropin-releasing hormone (TRH) can modulate the processes of (AVP) and oxytocin (OT) biosynthesis and release mainly at the hypothalamo-neurohypophysial level, the present experiments were undertaken to estimate whether TRH, administered intravenously in different doses, modifies these mechanisms under conditions of osmotic stimulation, brought about by dehydration. AVP and OT contents in the and neurohypophysis as well as plasma

levels of AVP, OT, free thyroxine (FT4) and free (FT3) were studied after intravenously TRH treatment in euhydrated and dehydrated for two days male rats. Under conditions of equilibrated water metabolism TRH diminished significantly the hypothalamic and neurohypophysial AVP and OT content but was without the effect on plasma oxytocin level; however, TRH in a dose of 100 ng/100 g b.w. raised plasma AVP level. TRH, injected i.v. to dehydrated animals, resulted in a diminution of AVP content in the hypothalamus but did not affect the hypothalamic OT stores. After osmotic stimulation, neurohypophysial AVP and OT release was significantly restricted in TRH-treated rats. Under the same conditions, injections of TRH were followed by a significant decrease of plasma OT level. I.v. injected TRH

enhanced somewhat FT3 concentration in of euhydrated animals but

diminished FT4 plasma level during dehydration. Data from the present study suggest that TRH displays different character of action on vasopressin and oxytocin secretion in relation to the actual state of water metabolism.

Key words: thyrotropin-releasing hormone, intravenously administration, vasopressin, oxytocin, dehydration 424

INTRODUCTION

Many and may be involved in the control of the vasopressin and oxytocin secretion from the neurohypophysis (1-3). Thyrotropin-releasing hormone (TRH), the hypothalamic , is known to participate in the regulation of thyrotropin (TSH) secretion. Within the central nervous system (CNS) large amounts of TRH and of its receptors are distributed also over extrahypothalamic regions (4, 5). It is thought that, in the central nervous system, TRH may be involved in some regulatory events different from its TSH-releasing properties. TRH has been hypothesized to serve in CNS as a or neuromodulator at a pre- or post-synaptic site (6, 7). Outside the CNS, TRH immunoreactivity was observed in the gastrointestinal tract; the largest amounts of TRH were found to be localized within the (mainly in beta cells) (8). TRH inhibits gastric motility, acid secretion, and absorbtion of sugars from the gut (9, 10, 11). TRH has been implicated in the regulation of appetite and food intake (12, 13). An inhibitory effect of TRH on pancreatic enzyme secretion has been shown in rats (14, 15), dogs (16) and humans (17). There are some reports which indicate that TRH affects the neurohypophysis. When injected i.v., TRH was found to stimulate the release of vasopressin and oxytocin in the rabbit (18). Other authors noted that neither oxytocin nor vasopressin was released following i.v. TRH injection in euhydrated rats (19, 20). On the other hand, TRH injected intracerebroventricularly (i.c.v.) was shown to inhibit vasopressin release in euhydrated or dehydrated rats (1, 21, 22). The same effect of TRH has been observed in conditions in vitro (23). The present study has been performed, therefore, to investigate possible effects of intravenously (i.v.) injected thyrotropin-releasing hormone on the hypothalamo-neurohypophysial vasopressin and oxytocin content as well as their release in rats euhydrated or deprived of water.

MATERIALS AND METHODS

All experiments were designed in accordance with the generally accepted ethical standarts and the guidelines established by the Ethical Committee of Medical University of Lodz.

Animals Three-month old male Wistar rats were housed under a 12/12 hr light-dark schedule (lights on from 6 a.m.) and at a room temperature. The animals received standard pelleted food; before experimental use, they had free access to tap water.

Experimental design Sixty rats were used for the experiments. The animals were divided into three groups: A – animals injected intravenously (i.v.), once daily over 2 days, with 0.9% chloride 425 solution in a daily dose of 0,1 ml/100 g b.w.; B – animals similarly injected i.v. with thyrotropin- releasing hormone [TRH; SIGMA Chemical Co.; lot 4640815] in a daily dose of 10 ng/100 g b.w.; C – animals similarly injected i.v. with TRH in a daily dose of 100 ng/100 g b.w. In each group two further experimental subgroups were set up: I – controls not dehydrated; II – animals dehydrated for two days. The animals of subgroups A-I - C-I were decapitated 20 min after last i.v. injection of TRH or saline. The rats in subgroups A-II, B-II and C-II were killed after two days of water deprivation; the last i.v. injection of TRH or vehicle was given 20 min before killing.

Experimental procedure The experiments were carried out between 9.00 and 10.00 a.m. On the day of the experiments after the decapitation of rats mixed arterial-venous blood from the trunk was collected in heparinized tubes for AVP, OT as well as FT3 and FT4 estimation. Moreover, the blood was preserved for evaluation serum osmolality or collected in heparinized capillaries for determination of the haematocrite index. Serum osmolality was estimated using a Knauer semimicroosmometer (Halbmikro-Osmometer, Knauer, Weissenschaftliche Geräte KG, Berlin). The were extracted from the plasma using C18 Sep-pak columns (Sep-Pak(R) C18 Cartridges, lot No W9224G1; Waters Corp., Milford, Massachusetts) as described by Forsling (24); the final extracts were preserved in frozen sealed vials until radioimmunoassay. The recoveries of during extraction procedure were greater than 80% and therefore values were not corrected for procedural loses. The with intact pituitary was quickly (i.e., not later than 2 min after decapitation) removed, the infundibular stalk cut up and the neurointermediate lobe was separated from the anterior lobe. From the brain, rapidly frozen for a few minutes at –70° C, the hypothalamic block was dissected as previously described (25). The wet weight of such a block of the brain was 38.5 ± 1.7 mg (mean ± S.D.). We did not recount the neurohormones content per milligram of hypothalamic because of fact that the biosynthesis of vasopressin and oxytocin exists only in some areas of the hypothalamus (, paraventricular nucleus, ). Hence it appears that the presence of the neurohormones in the isolated block of tissue (containing the hypothalamus and a part of ) is not uniformly and the results could be falsely diminished. The neurointermediate lobe (respectively, the hypothalamus) was homogenized at + 4° C by sonication in MicrosonTM Ultrasonic Homogenizer (Labcaire, UK) in 2.0 ml of 0.25% (resp. 0.5%) acetic acid dissolved in 0.9% saline. 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 at 2000 rpm at +4° C for 20 min. The supernatants were removed, frozen, and stored at –70° C until radioimmunoassay.

Radioimmunoassays The AVP and OT content of the neurohypophysial and hypothalamic extracts as well as plasma AVP and OT levels were determined by double-antibody specific radioimmunoassay as previously described by Ciosek et al. (25). Anti-AVP and anti-OT antibodies were raised by Dr. M. Or³owska- Majdak (Department of Physiology and Biochemistry, Medical University of Lodz). The antibody titer was 1 : 24000 for anti-AVP and 1 : 80000 for anti-OT (both final dilutions). Cross reactivity for anti-AVP antibodies was with oxytocin 0.016%, with vasopressin (LVP) 2.7%, with -releasing hormone (Gn-RH), TRH, leucine (Leu-Enk), II (Ang II) and (SP) less than 0.002%. Cross reactivity for anti-OT antibodies was with vasopressin 1.12%, with Gn-RH, TRH, Leu-Enk, Ang II and SP less than 0.002%. The sensitivity 426 of anti-AVP and anti-OT antisera was 1.25 pg AVP or OT per tube. vasopressin (Peninsula Lab. Ltd.; catalog No. RAS 8103, lot 032179) as well as oxytocin (Peninsula Lab. Ltd.; catalog No. RAS 8152, lot 027179) were used for standard curves preparation as well as for iodination with 125I using the chloramine-T method. Intra-assay coefficient of variation (cv) for the AVP and OT was 2.0% and 3.7%, respectively.

Plasma free thyroxine (FT4) and free triiodothyronine (FT3) concentrations were determined in duplicate by RIA kits provided by POLATOM (Oœrodek Badawczo-Rozwojowy Izotopów,

Otwock-Œwierk, Poland; lot No 112028 and 11027, respectively). The intra-assay cv for FT4 and for

FT3 were 5.03% and 4.88%, respectively.

Statistical evaluation of the results The vasopressin and oxytocin content was finally expressed in nanograms for whole hypothalamus or neurointermediate lobe and in picograms per millilitre of plasma. The plasma FT4 and FT3 concentrations were expressed in picomols per liter. All results were reported as the mean ± standard error of the mean (S.E.M.). Statistical analysis of the experimental data was performed using a Pentium I computer and “STATISTICA” software. Significance of the differences between the means was assessed using non-parametric analysis of variance (ANOVA) followed by two-way Wilcoxon test. P<0.05 was used as the minimal level of significance.

RESULTS

Validation of the actual state of water metabolism In untreated, euhydrated animals the serum osmolality and haematocrite index were 250.5 ± 2.98 mOsm/Kg H2O and 42.9 ± 0.87, respectively (Tabl. 1 and 2: subgroup A-I).Under conditions of water deprivation a progressive

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

S E R U M O S M O L A L I T Y

Group A: Group B: Group C: Statistical significance animals animals injected i.v. animals injected i.v. Subgroups untreated with 10 ng TRH with 100 ng TRH A versus B A versus C of animals I – animals not dehydrated 250.5±2.98 278±13.25 275.5±12.17 p<0.05 NS

II – two days of dehydration 323±3.43 319.5±5.24 343.3±5.83 NS p<0.05

Statistical significance:

I versus II p<0.01 p<0.05 p<0.01

NS - not significant 427

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

H A E M A T O C R I T E I N D E X

Group A: Group B: Group C: Statistical significance animals animals injected i.v. animals injected i.v. Subgroups untreated with 10 ng TRH with 100 ng TRH A versus B A versus C of animals I – animals not dehydrated 42.9±0.87 41±0.75 42.4±1.00 NS NS

II – two days of dehydration 50.5±0.69 51.8±1.15 53.2±1.16 NS NS

Statistical significance:

I versus II p<0.01 p<0.01 p<0.01

NS – not significant increase of haematocrite index and of serum osmolality were noted (Tabl. 1 and 2: A-I vs A-II). TRH administered i.v. to euhydrated animals did not change the haematocrite index; it increased serum osmolality (Tabl. 1: A-I vs B-I), when was applied in a dose of 10 ng. Following two days of water deprivation only serum osmolality was more marked under treatment with TRH in a dose of 100 ng (Tabl. 1: A-II vs C-II).

TRH influence on the vasopressin content in the hypothalamus, neurohypohysis and blood plasma level The neurohypophysial vasopressin content decreased significantly in rats deprived of water for 48 hours (Fig. 2: subgroup A-II), down to about 41% of the respective control value in subgroup A-I. TRH in both doses (i.e., 10 ng/100 g b.w. or 100 ng/100 g b.w.) administered to euhydrated animals was followed by a distinct decrease of hypothalamic and neurohypophysial vasopressin content (Fig. 1 and 2: A-I vs B-I and A-I vs C-I). TRH significantly stimulated vasopressin depletion in the hypothalamus of dehydrated animals (Fig. 1: A-II vs C-II). On the contrary, i.v. injections of TRH restrained the progressive decrease of neurohypophysial vasopresin content under conditions of dehydration (Fig. 2: A-II vs B-II and A-II vs C-II). Plasma vasopressin concentration was distinctly raised in vehicle-treated rats during dehydration (Fig. 3: subgroup A-II) as compared with the respective control values (subgroup A-I). After i.v. injections of higher TRH dose (i.e., 100 ng) to the control euhydrated animals AVP plasma concentration distinctly raised (Fig. 3: A-I vs C-I). On the other hand, vasopressin plasma level in dehydrated 428

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Fig. 1. The effect of i.v. administered TRH on the hypothalamic (Hth) vasopressin (AVP) content in euhydrated as well as dehydrated male rats (mean +/- S.E.M.; number of animals: n = 7-10)

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Fig. 2. The effect of i.v. administered TRH on the neurohypophysial (NH) vasopressin content in euhydrated as well as dehydrated male rats (mean +/- S.E.M.; number of animals: n = 6-10)

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Fig. 3. The effect of i.v. administered TRH on the vasopressin (AVP) concentration in the blood plasma in euhydrated as well as dehydrated male rats (mean +/- S.E.M.; number of animals: n = 6-10) 429 and simultaneously TRH-treated animals (Fig. 3: subgroups B-II and C-II) was lower than in rats similarly dehydrated but injected with vehicle; however, the respective differences were not statistically significant.

TRH influence on the oxytocin content in the hypothalamus, neurohypohysis and blood plasma level Under conditions of dehydration the hypothalamic as well as neurohypophysial oxytocin content distinctly diminished, down to about 55% and 45% of the respective control values (Fig. 4 and 5: A-I vs A-II). TRH significantly reduced hypothalamic and neurohypophysial oxytocin content in rats drinking tap water ad libitum (Fig. 4 and 5: A-I vs B-I and A-I vs C-I). In dehydrated and TRH-treated rats (Fig. 4: subgroups B-II and C-II) hypothalamic oxytocin stores were not altered when compared to respective control (subgroup A-II). TRH injected i.v. in a daily dose of 10 ng/100 g b.w. significantly inhibited

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Fig. 4. The effect of i.v. administered TRH on the hypothalamic (Hth) oxytocin (OT) content in euhydrated as well as dehydrated male rats (mean +/- S.E.M.); number of animals: n = 8-10)

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Fig. 5. The effect of i.v. administered TRH on the neurohypophysial (NH) oxytocin (OT) content in euhydrated as well as dehydrated male rats (mean +/- S.E.M.; number of animals: n = 6-10) 430 the oxytocin release from the neurohypophysis as brought about by stimulation of (Fig. 5: A-II vs B-II). Plasma oxytocin concentration was distinctly raised in dehydrated and vehicle-treated rats (Fig. 6: A-I vs A-II). TRH, injected i.v., did not modify OT plasma level in animals under conditions of equilibrated water metabolism (Fig. 6: subgroup B-I and C-I). TRH, administered in both doses, was the reason

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Fig. 6. The effect of i.v. administered TRH on the oxytocin (OT) concentration in the blood plasma in euhydrated as well as dehydrated male rats (mean +/- S.E.M.; number of animals: 6-10) of OT plasma level diminution in animals dehydrated for two days (Fig. 6: A-II vs B-II and A-II vs C-II).

TRH influence on free thyroxine (FT4) and free triiodothyronine (FT3) concentrations in blood plasma

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Fig. 7. The effect of i.v. administered TRH on free thyroxine (FT4) concentration in the blood plasma in euhydrated as well as dehydrated male rats (mean +/- S.E.M.); number of animals: n = 9-10) 431

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Fig. 8. The effect of i.v. administered TRH on free triiodothyronine (FT3) concentration in the blood plasma in euhydrated as well as dehydrated male rats (mean +/- S.E.M.; number of animals: n = 9-10)

The plasma FT4 and FT3 concentrations of animals treated i.v. with 0.9% NaCl significantly increased during dehydration (Fig. 7 and 8: subgroup A-II as compared with subgroup A-I). In euhydrated animals i.v. injections of TRH in a dose of 10 ng elevated the concentration of FT3 in blood plasma (Fig. 8: A-I vs B-I). On the other hand, i.v. injection of 100 ng TRH was followed by decreased FT4 plasma level in rats deprived of water (Fig. 7: subgroup C-II) when compared with the respective control value in subgroup A-II.

DISCUSSION

Stimulation of the hypothalamo-neurohypophysial system function as the aspect of neuroendocrine regulation during the dehydration. Dehydration is known to be 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 release (for references see: 26, 27) as well as an increase in the bioelectrical discharge of vasopressinergic and oxytocinergic of magnocellular hypothalamic nuclei during progressive dehydration (28). 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 (21, 29, 30). It is known, that in the conditions of the 432 hyperosmolality the vasopressin and oxytocin release into the blood (31, 32, 33) as well as into the (33) is then intensified. Moreover, AVP and OT mRNAs levels are enhanced in magnocellular neurons in the rat hypothalamus under influence of osmotic stimulation (34). Similarly, c-fos mRNA and Fos protein are expressed in vasopressinergic and oxytocinergic neurons of the hypothalamus in these conditions (35, 36, 37). 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 (30, 38, 39). In agreement with former data, also from this laboratory (1, 21), 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 transport of both neurohormones – although increased in the conditions of water deprivation – is probably insufficient to compensate the hormonal quantities released into the circulation.

Thyrotropin-releasing hormone and the function of the hypothalamo- neurohypophysial system. Some data suggest a modulating role for TRH in the release of neurohypophysial hormones. Studies of the effects of TRH on vasopressin or oxytocin release in the rat and other species suggest the existence of both stimulating and inhibiting actions, depending on different dosages and the routes of TRH administration. TRH has been noted to stimulate the relase of oxytocin and vasopresin in the rabbit (18, 40), when applied i.c.v. or i.v. in the doses of 3 nmol or 30 nmol. Similarly, i.c.v. TRH, in the doses of 10 ng – 5 µg, was followed by an increase of vasopressin release in the rat (41). In other experiments, no changes in oxytocin and vasopressin release after i.v. injections of TRH in the doses of 0.05 – 5 ng were reported (19). What is more, no changes in oxytocin and vasopressin release, following i.v. administration of 500 ng TRH, were noted in the rat (20). Similarly, no change in oxytocin and vasopressin release has been noted following intravenous injections of TRH in humans (42). Skowsky and Swan (43) and also Ciosek and Guzek (1) reported that TRH increased the vasopressin release from the neurointermediate lobes in vitro but inhibited that of oxytocin. On the other hand, TRH inhibited the vasopressin and oxytocin release from the rat hypothalamo-neurohypophysial explants in vitro (23). As shown in the study of Ciosek and Stempniak (22), administration of i.c.v. TRH in doses of 50 ng or 100 ng to euhydrated rats resulted in a decrease of the neurohypophysial vasopressin content, but an opposite effect (i.e., significant increase of neurohypophysial 433 vasopressin content) under influence of comparatively higher (200 ng i.c.v.) dose of TRH was noted. Moreover, this dose of TRH inhibited vasopressin release in dehydrated, haemorrhaged or salt-loaded rats but stimulated that of oxytocin under similar experimental conditions (1, 21, 44, 45). In present experiments TRH injected i.v. to animals in the state of equilibrated water metabolism resulted in a decrease of hypothalamic and neurohypophysial vasopressin and oxytocin contents; it may be assumed that the axonal transport as well as release of both neurohormones were then dramatically intensified. However, vasopressin concentration in blood plasma raised only in the rats treated i.v. with TRH in a dose of 100 ng/100 g b.w. Nevertheless, we can conclude that TRH, injected i.v., may facilitate vasopressin and oxytocin release into the blood from the neurohypophysis in rats having free access to tap water. In this respect, our results seem to be consistent with earlier work of Weitzman et al. (18) and of Horita and Carino (40), both carried out on rabbits as well as with study of Ciosek and Stempniak (22) in euhydrated rats. The present study confirm previous findings from this laboratory (1, 21) that TRH, when administered to dehydrated animals, increased the vasopressin storage in the neurohypophysis but diminished that in the hypothalamus. The oxytocinergic neurons respond to TRH in the same way as the vasopressinergic do. In fact, under conditions of dehydration the depletion of neurohypophysial vasopressin and oxytocin content was distinctly less marked in animals treated i.v. with TRH. Most probably, this event is to be commented in terms of diminished AVP and OT release into the blood. Indeed, the level of oxytocin decreased in the blood plasma under conditions of dehydration. It may be, therefore, suggested that activation of the vasopressinergic and oxytocinergic systems after osmotic stimulation is less pronounced after i.v. TRH administration. It is important to note that TRH injected i.v. penetrates to the brain through the blood-brain barrier quite well (46). For example, Okuda et al. (47) has been observed that after i.v. administration of TRH to the conscious rats, the concentration in the cerebrospinal fluid (CSF) collected from the fourth ventricle increased quickly during the first 20 min. It has been supposed that peripherally administered TRH has access to the central nervous system through CSF (48). Other authors suggested, however, that intravenous TRH administration produces effects through different mechanisms due to lower permeability of the blood brain-barrier for TRH (49). Nevertheless, TRH, given i.c.v. or i.v., exerts its effects in the central nervous sytem mainly at the level of the hypothalamus which is thought to be one of most important sites of TRH action. TRH receptors with high levels of binding have been demonstrated in several brain areas, especially in the hypothalamic paraventricular (PVN) and supraoptical (SON) nuclei (50, 51, 52) as well as in the (51, 52). Exogenous TRH may, therefore, modify the release of neurohypophysial hormones into the blood acting via hypothalamic and 434 pituitary TRH receptors or by direct influence on vasopressinergic and oxytocinergic neurons. Indeed, TRH was shown to inhibited the vasopressin and oxytocin release from the rat hypothalamo-neurohypophysial explants in vitro (23). However, this possible action of TRH may be disturbed by its rapid metabolism and (53, 54). The studies with i.v. or i.c.v. administration of TRH in rats and humans have shown that the plasma half-life of this peptide is about 6-9 min (55, 56) but its half-life in the brain tissues was determined to be about 9-11 min (57). What is more, TRH is known to rapidly processed in the blood and within the brain to some derivatives of increased neuropharmacological potency (57).

It is also of interest that TRH exerts on FT4 and FT3 plasma levels in a different manner. TRH intravenous injections to euhydrated rats has been observed to result in an increase of FT3 plasma level. This stimulatory effect is in an agreement with previous data (58). In contrast to this observation, the concentration of FT4 in blood plasma diminished in rats treated with TRH and simultaneously deprived of water. On the other hand, in the study of Iversen and

Laurberg (59) there was no effect of TRH on secretion of T4 and T3 from perfused dog thyroid lobes in vitro. The mechanisms of TRH action on the vasopressinergic and oxytocinergic neurons are still not clear. As mentioned above, it is possible that exogenous TRH may influence on the synthesis and/or release of vasopressin and oxytocin by a direct effect on the hypothalamic neurons or by modified neurotransmission in the brain (for example, via mechanisms involving noradrenergic neurotransmission in PVN and/or SON (60)). Peripherally administration of TRH does not exclude the similar mechanisms of this peptide action. Moreover, it is possible that the character of TRH action could change in accordance with the actual state of the activity of vasopressinergic and oxytocinergic neurons (excited in conditions of the dehydration, hypovolemia or hypotonia). In summary, the present findings show that TRH appears to be potential stimulating factor for vasopressin and oxytocin release during equilibrated water metabolism but an inhibiting factor under conditions of osmotic stimulation.

Acknowledgements: I wish to thank Dr. B. Stempniak (Department of Pathophysiology, Medical University of £ódŸ) for her kindly assistance with radioimmunoassay technique.

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Received: November 12, 2001 Accepted: July 11, 2002

Author’s address: Joanna Ciosek, Ph.D., D.Sc. Department of Pathophysiology, Medical University of Lodz, ul. Narutowicza 60, 90-136 Lodz, Poland. Tel. (4842) 630-61-87, fax. (4842) 631-97-23, E-mail: [email protected]