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Vasopressin and Oxytocin Release As Influenced by Thyrotropin-Releasing Hormone in Euhydrated and Dehydrated Rats

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Vasopressin and Oxytocin Release As Influenced by Thyrotropin-Releasing Hormone in Euhydrated and Dehydrated Rats JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2002, 53, 3, 423437 www.jpp.krakow.pl J. CIOSEK VASOPRESSIN AND OXYTOCIN RELEASE AS INFLUENCED BY THYROTROPIN-RELEASING HORMONE IN EUHYDRATED AND DEHYDRATED RATS Department of Pathophysiology, Medical University of Lodz, Poland Since the thyrotropin-releasing hormone (TRH) can modulate the processes of vasopressin (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 hypothalamus and neurohypophysis as well as plasma levels of AVP, OT, free thyroxine (FT4) and free triiodothyronine (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 blood plasma 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 neuropeptides and neurotransmitters may be involved in the control of the vasopressin and oxytocin secretion from the neurohypophysis (1-3). Thyrotropin-releasing hormone (TRH), the hypothalamic neuropeptide, 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 neurotransmitter 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 pancreas (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% sodium 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 neurohormones 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 hormones during extraction procedure were greater than 80% 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, 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 tissue because of fact that the biosynthesis of vasopressin and oxytocin exists only in some areas of the hypothalamus (supraoptic nucleus, paraventricular nucleus, suprachiasmatic nucleus). Hence it appears that the presence of the neurohormones in the isolated block of tissue (containing the hypothalamus and a part of thalamus) 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 lysine vasopressin (LVP) 2.7%, with gonadotropin-releasing hormone (Gn-RH), TRH, leucine enkephalin (Leu-Enk), angiotensin II (Ang II) and substance P (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. Arginine 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 (Orodek Badawczo-Rozwojowy Izotopów, Otwock-wierk, 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.
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