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Upregulation of the expression of vasopressin gene in the paraventricular and supraoptic nuclei of the lithium-induced diabetes insipidus rat
Brain Research 772 Ž1997. 161–166
Research report
Upregulation of the expression of vasopressin gene in the paraventricular and supraoptic nuclei of the lithium-induced diabetes insipidus rat Hirofumi Anai a, ) , Yoichi Ueta b , Ryota Serino b , Masayoshi Nomura b , Narutoshi Kabashima b , Izumi Shibuya b , Masayuki Takasugi a , Yasuhide Nakashima a , Hiroshi Yamashita b a
b
Second Department of Internal Medicine, School of Medicine, UniÕersity of Occupational and EnÕironmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan Department of Physiology, School of Medicine, UniÕersity of Occupational and EnÕironmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan Accepted 8 July 1997
Abstract The expression of arginine vasopressin ŽAVP. gene in the paraventricular ŽPVN. and supraoptic nuclei ŽSON. was investigated in rats with lithium ŽLi.-induced polyuria, using in situ hybridization histochemistry and radioimmunoassay. The male Wistar rats consuming a diet that contained LiCl Ž60 mmolrkg. for 4 weeks developed marked polyuria. The Li-treated rats produced a large volume of hypotonic urine with low ionic concentrations. Plasma sodium concentrations were found to be slightly increased in the Li-treated rats compared with those in controls. Plasma concentration of AVP and transcripts of AVP gene in the PVN and SON were significantly increased in the Li-treated rats compared with controls. These results suggest that dehydration andror the activation of visceral afferent inputs may contribute to the elevation of plasma AVP and the upregulation of AVP gene expression in the PVN and the SON of the Li-induced diabetes insipidus rat. q 1997 Elsevier Science B.V. Keywords: Lithium; Arginine vasopressin; Supraoptic nucleus; Paraventricular nucleus; In situ hybridization; Rat
1. Introduction Lithium ŽLi. is commonly used in the clinical management of manic-depressive psychosis w13x. However, Litreated patients often have polydipsia and hypotonic polyuria that resists the exogenous administration of arginine vasopressin ŽAVP. w6,15x. In the rat as well as the human, the chronic oral administration of Li induces diabetes insipidus ŽDI. syndrome w6,17,18x. The DI syndrome is caused by an AVP-resistant urinary concentrating defect in the kidney. It has been demonstrated that plasma concentration of AVP is elevated in Li-treated rats w17x and human w1,2x, and the plasma osmolality, plasma protein and hematocrit in Li-induced DI rats were not significantly altered from control levels w4,10,11,17x. However, the finding of elevated plasma AVP level in the Li-induced DI rat
is controversial as it has been recently shown that AVP is not elevated in this experimental model w10x. The present study was done to evaluate the effects of Li on the secretion and production of AVP in vivo, using a combination of measuring plasma concentration of AVP by radioimmunoassay ŽRIA. and the semi-quantitative analysis of the expression of AVP gene in the hypothalamic paraventricular ŽPVN. and supraoptic ŽSON. nuclei by in situ hybridization histochemistry, since it is well known that AVP is produced in the PVN and SON, transported to the posterior pituitary and secrete into the systemic circulation w3x.
2. Materials and methods 2.1. Animals
)
Corresponding author. Fax: q81 Ž93. 691-6913.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 8 7 - 1
Five week old male Wistar rats, weighing approximately 100 g, were used in all experiments. They were
162
H. Anai et al.r Brain Research 772 (1997) 161–166
housed in an air-conditioned room Ž23–258C. under a 12-h light and 12-h dark cycle. One group of animals was fed a standard diet ŽCE-2, Clea Japan Inc., Tokyo, Japan. and allowed to drink tap water ad libitum. The other group was fed a standard diet containing LiCl Ž60 mmolrkg. and allowed to drink both tap water and a 0.9% NaCl solution ad libitum. In order to compensate for the lack of plasma sodium and body fluid caused by polyuria in Li-treated rats, they became polydipsia and preferred 0.9% NaCl solution to tap water. Li-treated animals and the corresponding controls were handled in the same way. Both groups were sacrificed by decapitation at 10:00–11:00 h on the same day, after 4 weeks, and immediately the blood and tissues were collected and analyzed simultaneously for both groups. The urine was collected for a 24 h period from each animal that was housed in a standard metabolic cage. All experimental procedures in the present study were done in accordance with the guidelines on the use and care of laboratory animals as set out by the Physiological Society of Japan and approved by the animal care committee as this institution.
Table 1 Plasma electrolytes, osmolalities in plasma and urine at the end of 4 weeks treatment
Plasma Li ŽmEqrl. Na ŽmEqrl. K ŽmEqrl. Plasma Osm ŽmOsmrkg H 2 O. Urine Osm ŽmOsmrkg H 2 O.
Control
Li-treated
n.d. 141.2"0.6 5.9"0.2 303"2.2 939"132
0.63"0.08 143.3"0.8 a 5.8"0.1 306"1.6 320"45 b
Values are the means"S.E.M.; ns13–14 rats per group. a P - 0.05 and b P - 0.01, versus control Žunpaired Student’s t-test.. n.d.s not detectable.
2.2. Measurement of electrolytes and Li in plasma and urine Plasma concentrations of Naq and Kq were measured using standard methods. The osmolalities of plasma and urine were measured using an Osmotic Pressure AUTO & STAT ŽOM-6030, Kyoto Daiichi Pure Chemicals Co, Ky-
Fig. 1. Bright ŽA and C. and dark ŽB and D. field photomicrographs of emulsion-dipped slides hybridized to a 35 S-labelled oligodeoxynucleotide probe complementary to AVP mRNA in the PVN. Panels A and B are sections from control rats; panels C and D from Li-treated rats. dp, dorsal parvocellular component of PVN; mp, medial parvocelular component of PVN; pm, posterior magnocellular component of PVN; 3 V, third ventricle. Scale bar is 100 mm in length.
H. Anai et al.r Brain Research 772 (1997) 161–166
oto, Japan.. In addition, plasma and urine concentrations of Li in both the Li-treated and control rats were measured using an automatic electrolyte analyzer ŽHitachi 710, Hitachi Inc., Ibaraki, Japan.. 2.3. Radioimmunoassay (RIA) for AVP Plasma AVP levels were determined using an RIA with a specific AVP-antiserum and w 125 Ix-AVP tracer Žspecific activity, 14.8 kBqrkit. available as a commercial kit ŽAVP RIA ‘Mitsubishi’, Yuka Medias Co., Ltd., Ibaraki, Japan.. All plasma samples from an individual animal were assayed simultaneously and in duplicate. The volume of each plasma sample was 1 ml for the assay. The sensitivity of the assay was 0.2 pgrml. The within- and between-assay coefficients of variation for AVP determination were 10% and 10%, respectively. 2.4. In situ hybridization histochemistry for AVP The brains were carefully removed, frozen on powdered dry ice, and stored at y808C until in situ hybridization analysis. In situ hybridization histochemistry was per-
163
formed on frozen coronal brain sections cut at 12 mm on a cryostat at y208C, thawed and mounted onto gelatinrchrome alum-coated slides and stored at y808C until used. The PVN and SON were localized by referring to a standard atlas of the rat brain by Paxinos and Watson w12x. The brain sections, including magnocellular parts of the PVN and the SON were chosen from 4 sections, 8 sites per rat to measure the density of autoradiography. The slides were warmed to room temperature and allowed to dry for 10 min, then fixed in 4% formaldehyde in phosphate buffered saline ŽPBS. for 5 min, washed twice in PBS, and incubated in 0.9% NaCl containing 0.25% acetic anhydride Žvrv. and 0.1 M triethanolamine at room temperature for 10 min. The sections were then dehydrated through 70% Ž1 min., 80% Ž1 min., 95% Ž2 min. and 100% Ž1 min. ethanol and delipidated in 100% chloroform for 5 min. The slides were then partially rehydrated in 100% Ž1 min. followed by 95% Ž1 min. ethanol and allowed to dry briefly in air. Hybridization was carried out at 378C overnight in 45 ml of buffer consisting of 50% formamide and 4 = SSC Ž1 = SSC s 150 mM NaCl, 15 mM sodium citrate. containing 500 mgrml sheared salmon sperm DNA ŽSigma, St. Louis, MO, USA., 250 mgrml
Fig. 2. Bright ŽA and C. and dark ŽB and D. field photomicrographs of emulsion-dipped slides hybridized to a 35 S-labelled oligodeoxynucleotide probe complementary to AVP mRNA in the SON. Panels A and B are sections from control rats; panels C and D from Li-treated rats. OC, optic chiasma. Scale bar is 100 mm in length.
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Fig. 3. Bright ŽA and C. and dark ŽB and D. field photomicrographs of emulsion-dipped slides hybridized to a 35 S-labelled oligodeoxynucleotide probe complementary to AVP mRNA in the SCN. Panels A and B are sections from control rats; panels C and D from Li-treated rats. The allow indicate the neurosecretory cells expressing AVP gene. The expression of the AVP gene was strong. The expression of AVP gene in the SCN was no difference between control and Li-treated rat. 3 V, third ventricle. Scale bar is 100 mm in length.
baker’s yeast total RNA ŽBoehringer Mannheim GmbH, Mannheim, Germany., 1 = Denhardt’s solution and 10% dextran sulfate Ž500 000 MWt, Sigma, St. Louis, MO, USA., under a Nescofilm ŽBando Chemical IMD, Ltd., Osaka, Japan. coverslip. The probe used was 35 S 3X endlabelled deoxyoligonucleotides complementary to transcripts coding for AVP Žprobe sequences 5X CAG CTC CCG GGC TGG CCC GTC CAG CT-3X , complementary to bases 1843–1868 of rat AVP.. The specificity of the probe was confirmed by competition with a 100-fold excess of the unlabelled probe in the present study. A total of 5 = 10 5 cpmrslide was used. After hybridization, the sections were washed for 1 h in 4 changes of 1 = SSC at 558C and for a further 1 h in 2 changes of 1 = SSC at room temperature. All experimental sections were treated simultaneously throughout to minimize the effects of variations in hybridization and wash stringency. Hybridized sections of the PVN and SON were apposed to autoradiography film ŽHyperfilm, Amersham, Bucks, UK. for 8 h. The resulting images were analyzed by computerized densitometry using an MCID imaging analyzer ŽImaging Research Inc., Ontario, Canada.. The mean optical density of auto-
radiographs was measured by comparison with simultaneously exposed w 14 Cx micro-scale ŽAmersham, Bucks, UK.. Slides hybridized to the AVP probe were dipped in nuclear emulsion ŽK-5, Ilford, Cheshire, UK. and further exposed for 32 h. And they were developed in D-19 developer
Fig. 4. Effects of Li treatment on AVP transcript prevalence in the PVN, the SON and the SCN. Values represent the means"S.E.M., ns6; ) P - 0.01 compared to control Žunpaired Student’s t-test..
H. Anai et al.r Brain Research 772 (1997) 161–166
ŽKodak, New York, USA., fixed with Fujifix ŽFuji Photo Film Co., Ltd., Tokyo, Japan., dehydrated and covered with a coverslip. 2.5. Statistics All data shown are means" S.E.M. The changes in plasma and urine electrolytes, and plasma AVP after Li treatment were statistically analyzed by using Student’s t-tests Žunpaired.. The data obtained from in situ hybridization histochemistry were also analyzed by using Student’s t-test Žunpaired.. P - 0.05 was considered to indicate statistical significance. 3. Results The body weights changed from 103 " 3.0 to 176 " 6.1 g in Li-treated rats Ž n s 21. and from 103 " 4.5 to 266 " 9.6 g in control Ž n s 14. after 4 weeks. At the end of 4 weeks daily volume of urine was 42.7 " 5.3 ml in Litreated rats Ž n s 19. and 18.7 " 1.3 ml in controls Ž n s 14.. The plasma concentrations of Li, Na and K, the plasma osmolalities and the urine osmolalities in control and Li-treated rats were summarized in Table 1. Plasma sodium concentrations were slightly increased in the Li-treated rats compared with those in controls Ž P - 0.05.. Although the osmolalities of urine in Li-treated rats were significantly lower than those in controls Ž P - 0.01., plasma osmolalities in Li-treated rats were not different from those of controls. The plasma concentrations of AVP were 16.5 " 3.6 pgrml in Li-treated rats Ž n s 14. and 5.0 " 1.9 in controls Ž n s 14.. The plasma levels of AVP in Li-treated rats were significantly higher than those in controls Ž P 0.05.. Treatment with Li for 4 weeks induced a significant increase in AVP transcripts in the PVN Ž138 " 9.0%. and the SON Ž178 " 23%. compared to control values Ž n s 6, P - 0.01. ŽFig. 4.. Microscopic examination revealed that the number of cells bound to AVP probe and intensity of the signals in both magnocellular and the parvocellular parts of the PVN and the SON were remarkably increased ŽFigs. 1 and 2.. As it is well known that AVP-producing cells exist in the suprachismatic nucleus ŽSCN., the transcripts of the AVP gene were also observed in the SCN. The expression of the AVP gene in the SCN of control rats were similar with that of the Li-treated rats ŽFig. 3.. The signals were completely abolished by competition with a 100-fold excess of the unlabelled probe. 4. Discussion The results of the present study show that chronic oral administration of Li results in a significant increase in the expression of AVP gene in the PVN and SON, as well as an increase in plasma concentrations of AVP in rats.
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In Li-treated rats, it was found that the plasma concentration of sodium was slightly but significantly increased compared with that of controls. Therefore, it is possible to explain that stimulations of sodium receptors or osmoreceptors in the brain or in the hepatorportal circulation may contribute to the secretion of AVP and the upregulation of AVP gene expression in the PVN and SON in Li-treated rats. However, the observed ranges of the plasma osmolality and sodium concentration in the present study are unlikely to cause a significant increase of plasma AVP level according to a previous study w16x. The other possible explanation is that afferent inputs to the AVP-producing cells in the PVN and SON originating from visceral receptors may be activated in the Li-induced DI rats. It has been demonstrated that renal afferents excited AVP-producing cells in the SON w5,7x and stimulate secretion of AVP and oxytocin w14x. Afferents, thought to originate from renal chemoreceptors andror mechanoreceptors may be activated by the hypotonic polyuria and hemodynamic changes in the kidney in the Li-treated DI rats. The possibility also exists that in Li-treated rats plasma volume was decreased as a result of the polyuria. This change in the circulation would activate either low pressure receptors or remove the inhibition on magnocellular neurons in the PVN and SON from high pressure baroreceptors. Both of these mechanisms would contribute to the elevated plasma AVP and the upregulation of AVP gene expression in the PVN and SON in Li-treated rats. Clinical and experimental studies have demonstrated that plasma AVP levels are increased in Li-treated patients w9x and rats w17x. However, Baylis et al. reported that plasma concentration of AVP was very low in some patients treated by Li w2x and Hensen et al. also reported that plasma AVP and osmolality did not change in Litreated rats w10x. In the present study we showed that plasma concentrations of AVP were significantly increased in Li-treated rats compared with control. This result was in agreement with that of previous studies w9,17x. It has been demonstrated that content of AVP in the hypothalamus and the posterior pituitary were decreased in Li-induced polyuria w17x, indicating that the elevated plasma AVP level is due to an increase of the release of AVP from the posterior pituitary. As the swelling of the cells in the SON were also reported in Li-induced DI rats w8x, secretion and production of AVP in the neurosecretory cells in the hypothalamus may be increased in Li-induced DI. Although we did not attempt to examine the size of the AVP-producing cells in the PVN and the SON, our microscopic observation showed that the area of those nuclei was bigger than that of control rats ŽFigs. 3 and 4.. In situ hybridization histochemistry revealed that the expression of AVP gene in the magnocellular parts of the PVN and the SON was significantly increased in Li-treated rats compared with control. It also revealed that the expression of AVP gene in the medial parvocellular parts of the PVN were remarkably increased in Li-treated rats. This observa-
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tion may represent increased activity in neurons in this subnucleus that project to cardioregulatory areas in the brainstem that may be activated by the changes in arterial pressure in Li-treated rats. It would be expected that low arterial pressure would activate PVN-medullary pathways that control sympathoexcitatory drive in Li-treated rats. In the SCN there was no difference between control and Li-treated rats. These results indicate that the increase of production of AVP in the PVN and the SON and the effects of Li on the expression of the AVP gene were specific in those nuclei. In conclusion, we have demonstrated that chronic administration of Li increases plasma concentration of AVP with upregulation of the expression of AVP gene in the PVN and SON of rats. Acknowledgements We express our appreciation to Ms Rieko Nishi, Akiko Sugimoto and Rika Suematsu for their technical assistance. This work was supported in part by Grant-in Aids for Scientific Research, Nos. 08457022 and 07507004 for H.Y. from the Ministry of Education, Science, Sports and Culture, Japan, Special Grant by the Ministry of Labor for ‘Occupational Health Studies’ and another from the Salt Science Research Foundation. References w1x M. Abramow, E. Cogan, Role of lithium-ADH interaction in lithium-induced polyuria, Year Book, 1984, pp. 29–34. w2x P.H. Baylis, D.A. Heath, Water disturbances in patients treated with oral lithium carbonate, Ann. Int. Med. 88 Ž1978. 607–609. w3x M.J. Brownstein, J.T. Russell, H. Gainer, Synthesis, transport, and release of posterior pituitary hormones, Science 207 Ž1980. 373–378.
w4x S.L. Carney, C. Ray, A.H.B. Gillies, Mechanism of lithium-induced polyuria in the rat, Kidney Int. 50 Ž1996. 377–383. w5x M.M. Caverson, J. Ciriello, Contribution of paraventricular nucleus to afferent renal nerve pressor response, Am. J. Physiol. 254 Ž1988. R531–543. w6x S. Christensen, E. Kusano, A.N.K. Yusufi, N. Murayama, T.P. Dousa, Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats, J. Clin. Invest. 75 Ž1985. 1869– 1879. w7x T.A. Day, J. Ciriello, Effects of renal receptor activation on neurosecretory vasopressin cells, Am. J. Physiol. 253 Ž1987. R234–241. w8x G.L. Ellman, G.L. Gan, Lithium ion and water balance in rats, Toxicol. Appl. Pharmacol. 25 Ž1973. 617–620. w9x P.W. Gold, G.L. Robertson, R.M. Post, W. Kaye, J. Ballenger, D. Rubinow, F.K. Goodwin, The effect of lithium on the osmoregulation of arginine vasopressin secretion, J. Clin. Endocrinol. Metab. 56 Ž1983. 295–299. w10x J. Hensen, M. Haenelt, P. Gross, Lithium induced polyuria and renal vasopressin receptor density, Nephrol. Dial. Transplant. 11 Ž1996. 622–627. w11x D. Marples, S. Chritensen, E. Cristensen, P.D. Ottosen, S. Nielsen, Lithium-induced down regulation of aquaporin-2 water channel expression in rat kidney medulla, J. Clin. Invest. 95 Ž1995. 1838–1845. w12x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. w13x M. Peet, J.P. Pratt, Lithium. Current status in psychiatric disorders, Drugs 46 Ž1993. 7–17. w14x J.K. Simon, N.W. Kasting, J. Ciriello, Afferent renal nerve effects on plasma vasopressin and oxytocin in conscious rats, Am. J. Physiol. 256 Ž1989. R1240–1244. w15x I. Singer, Lithium and the kidney, Kidney Int. 19 Ž1981. 374–387. w16x E.M. Stricker, J.G. Verbalis, Interaction of osmotic and volume stimuli in regulation of neurohypophyseal secretion in rats, Am. J. Physiol. 250 Ž1986. R267–R275. w17x M. Sugawara, K. Hashimoto, Z. Ota, Involvement of prostaglandin E2, cAMP, and vasopressin in lithium-induced polyuria, Am. J. Physiol. 254 Ž1988. R863–R869, ŽRegulatory Integrative Comp. Physiol. 23.. w18x M. Yamaki, E. Kusano, T. Tetsuka, S. Takeda, S. Homma, N. Murayama, Y. Asano, Cellular mechanism of lithium-induced nephrogenic diabetes insipidus in rats, Am. J. Physiol. 261 Ž1991. F505–F511, ŽRenal Fluid Electrolyte Physiol. 30..
Research report
Upregulation of the expression of vasopressin gene in the paraventricular and supraoptic nuclei of the lithium-induced diabetes insipidus rat Hirofumi Anai a, ) , Yoichi Ueta b , Ryota Serino b , Masayoshi Nomura b , Narutoshi Kabashima b , Izumi Shibuya b , Masayuki Takasugi a , Yasuhide Nakashima a , Hiroshi Yamashita b a
b
Second Department of Internal Medicine, School of Medicine, UniÕersity of Occupational and EnÕironmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan Department of Physiology, School of Medicine, UniÕersity of Occupational and EnÕironmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807, Japan Accepted 8 July 1997
Abstract The expression of arginine vasopressin ŽAVP. gene in the paraventricular ŽPVN. and supraoptic nuclei ŽSON. was investigated in rats with lithium ŽLi.-induced polyuria, using in situ hybridization histochemistry and radioimmunoassay. The male Wistar rats consuming a diet that contained LiCl Ž60 mmolrkg. for 4 weeks developed marked polyuria. The Li-treated rats produced a large volume of hypotonic urine with low ionic concentrations. Plasma sodium concentrations were found to be slightly increased in the Li-treated rats compared with those in controls. Plasma concentration of AVP and transcripts of AVP gene in the PVN and SON were significantly increased in the Li-treated rats compared with controls. These results suggest that dehydration andror the activation of visceral afferent inputs may contribute to the elevation of plasma AVP and the upregulation of AVP gene expression in the PVN and the SON of the Li-induced diabetes insipidus rat. q 1997 Elsevier Science B.V. Keywords: Lithium; Arginine vasopressin; Supraoptic nucleus; Paraventricular nucleus; In situ hybridization; Rat
1. Introduction Lithium ŽLi. is commonly used in the clinical management of manic-depressive psychosis w13x. However, Litreated patients often have polydipsia and hypotonic polyuria that resists the exogenous administration of arginine vasopressin ŽAVP. w6,15x. In the rat as well as the human, the chronic oral administration of Li induces diabetes insipidus ŽDI. syndrome w6,17,18x. The DI syndrome is caused by an AVP-resistant urinary concentrating defect in the kidney. It has been demonstrated that plasma concentration of AVP is elevated in Li-treated rats w17x and human w1,2x, and the plasma osmolality, plasma protein and hematocrit in Li-induced DI rats were not significantly altered from control levels w4,10,11,17x. However, the finding of elevated plasma AVP level in the Li-induced DI rat
is controversial as it has been recently shown that AVP is not elevated in this experimental model w10x. The present study was done to evaluate the effects of Li on the secretion and production of AVP in vivo, using a combination of measuring plasma concentration of AVP by radioimmunoassay ŽRIA. and the semi-quantitative analysis of the expression of AVP gene in the hypothalamic paraventricular ŽPVN. and supraoptic ŽSON. nuclei by in situ hybridization histochemistry, since it is well known that AVP is produced in the PVN and SON, transported to the posterior pituitary and secrete into the systemic circulation w3x.
2. Materials and methods 2.1. Animals
)
Corresponding author. Fax: q81 Ž93. 691-6913.
0006-8993r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 0 8 8 7 - 1
Five week old male Wistar rats, weighing approximately 100 g, were used in all experiments. They were
162
H. Anai et al.r Brain Research 772 (1997) 161–166
housed in an air-conditioned room Ž23–258C. under a 12-h light and 12-h dark cycle. One group of animals was fed a standard diet ŽCE-2, Clea Japan Inc., Tokyo, Japan. and allowed to drink tap water ad libitum. The other group was fed a standard diet containing LiCl Ž60 mmolrkg. and allowed to drink both tap water and a 0.9% NaCl solution ad libitum. In order to compensate for the lack of plasma sodium and body fluid caused by polyuria in Li-treated rats, they became polydipsia and preferred 0.9% NaCl solution to tap water. Li-treated animals and the corresponding controls were handled in the same way. Both groups were sacrificed by decapitation at 10:00–11:00 h on the same day, after 4 weeks, and immediately the blood and tissues were collected and analyzed simultaneously for both groups. The urine was collected for a 24 h period from each animal that was housed in a standard metabolic cage. All experimental procedures in the present study were done in accordance with the guidelines on the use and care of laboratory animals as set out by the Physiological Society of Japan and approved by the animal care committee as this institution.
Table 1 Plasma electrolytes, osmolalities in plasma and urine at the end of 4 weeks treatment
Plasma Li ŽmEqrl. Na ŽmEqrl. K ŽmEqrl. Plasma Osm ŽmOsmrkg H 2 O. Urine Osm ŽmOsmrkg H 2 O.
Control
Li-treated
n.d. 141.2"0.6 5.9"0.2 303"2.2 939"132
0.63"0.08 143.3"0.8 a 5.8"0.1 306"1.6 320"45 b
Values are the means"S.E.M.; ns13–14 rats per group. a P - 0.05 and b P - 0.01, versus control Žunpaired Student’s t-test.. n.d.s not detectable.
2.2. Measurement of electrolytes and Li in plasma and urine Plasma concentrations of Naq and Kq were measured using standard methods. The osmolalities of plasma and urine were measured using an Osmotic Pressure AUTO & STAT ŽOM-6030, Kyoto Daiichi Pure Chemicals Co, Ky-
Fig. 1. Bright ŽA and C. and dark ŽB and D. field photomicrographs of emulsion-dipped slides hybridized to a 35 S-labelled oligodeoxynucleotide probe complementary to AVP mRNA in the PVN. Panels A and B are sections from control rats; panels C and D from Li-treated rats. dp, dorsal parvocellular component of PVN; mp, medial parvocelular component of PVN; pm, posterior magnocellular component of PVN; 3 V, third ventricle. Scale bar is 100 mm in length.
H. Anai et al.r Brain Research 772 (1997) 161–166
oto, Japan.. In addition, plasma and urine concentrations of Li in both the Li-treated and control rats were measured using an automatic electrolyte analyzer ŽHitachi 710, Hitachi Inc., Ibaraki, Japan.. 2.3. Radioimmunoassay (RIA) for AVP Plasma AVP levels were determined using an RIA with a specific AVP-antiserum and w 125 Ix-AVP tracer Žspecific activity, 14.8 kBqrkit. available as a commercial kit ŽAVP RIA ‘Mitsubishi’, Yuka Medias Co., Ltd., Ibaraki, Japan.. All plasma samples from an individual animal were assayed simultaneously and in duplicate. The volume of each plasma sample was 1 ml for the assay. The sensitivity of the assay was 0.2 pgrml. The within- and between-assay coefficients of variation for AVP determination were 10% and 10%, respectively. 2.4. In situ hybridization histochemistry for AVP The brains were carefully removed, frozen on powdered dry ice, and stored at y808C until in situ hybridization analysis. In situ hybridization histochemistry was per-
163
formed on frozen coronal brain sections cut at 12 mm on a cryostat at y208C, thawed and mounted onto gelatinrchrome alum-coated slides and stored at y808C until used. The PVN and SON were localized by referring to a standard atlas of the rat brain by Paxinos and Watson w12x. The brain sections, including magnocellular parts of the PVN and the SON were chosen from 4 sections, 8 sites per rat to measure the density of autoradiography. The slides were warmed to room temperature and allowed to dry for 10 min, then fixed in 4% formaldehyde in phosphate buffered saline ŽPBS. for 5 min, washed twice in PBS, and incubated in 0.9% NaCl containing 0.25% acetic anhydride Žvrv. and 0.1 M triethanolamine at room temperature for 10 min. The sections were then dehydrated through 70% Ž1 min., 80% Ž1 min., 95% Ž2 min. and 100% Ž1 min. ethanol and delipidated in 100% chloroform for 5 min. The slides were then partially rehydrated in 100% Ž1 min. followed by 95% Ž1 min. ethanol and allowed to dry briefly in air. Hybridization was carried out at 378C overnight in 45 ml of buffer consisting of 50% formamide and 4 = SSC Ž1 = SSC s 150 mM NaCl, 15 mM sodium citrate. containing 500 mgrml sheared salmon sperm DNA ŽSigma, St. Louis, MO, USA., 250 mgrml
Fig. 2. Bright ŽA and C. and dark ŽB and D. field photomicrographs of emulsion-dipped slides hybridized to a 35 S-labelled oligodeoxynucleotide probe complementary to AVP mRNA in the SON. Panels A and B are sections from control rats; panels C and D from Li-treated rats. OC, optic chiasma. Scale bar is 100 mm in length.
164
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Fig. 3. Bright ŽA and C. and dark ŽB and D. field photomicrographs of emulsion-dipped slides hybridized to a 35 S-labelled oligodeoxynucleotide probe complementary to AVP mRNA in the SCN. Panels A and B are sections from control rats; panels C and D from Li-treated rats. The allow indicate the neurosecretory cells expressing AVP gene. The expression of the AVP gene was strong. The expression of AVP gene in the SCN was no difference between control and Li-treated rat. 3 V, third ventricle. Scale bar is 100 mm in length.
baker’s yeast total RNA ŽBoehringer Mannheim GmbH, Mannheim, Germany., 1 = Denhardt’s solution and 10% dextran sulfate Ž500 000 MWt, Sigma, St. Louis, MO, USA., under a Nescofilm ŽBando Chemical IMD, Ltd., Osaka, Japan. coverslip. The probe used was 35 S 3X endlabelled deoxyoligonucleotides complementary to transcripts coding for AVP Žprobe sequences 5X CAG CTC CCG GGC TGG CCC GTC CAG CT-3X , complementary to bases 1843–1868 of rat AVP.. The specificity of the probe was confirmed by competition with a 100-fold excess of the unlabelled probe in the present study. A total of 5 = 10 5 cpmrslide was used. After hybridization, the sections were washed for 1 h in 4 changes of 1 = SSC at 558C and for a further 1 h in 2 changes of 1 = SSC at room temperature. All experimental sections were treated simultaneously throughout to minimize the effects of variations in hybridization and wash stringency. Hybridized sections of the PVN and SON were apposed to autoradiography film ŽHyperfilm, Amersham, Bucks, UK. for 8 h. The resulting images were analyzed by computerized densitometry using an MCID imaging analyzer ŽImaging Research Inc., Ontario, Canada.. The mean optical density of auto-
radiographs was measured by comparison with simultaneously exposed w 14 Cx micro-scale ŽAmersham, Bucks, UK.. Slides hybridized to the AVP probe were dipped in nuclear emulsion ŽK-5, Ilford, Cheshire, UK. and further exposed for 32 h. And they were developed in D-19 developer
Fig. 4. Effects of Li treatment on AVP transcript prevalence in the PVN, the SON and the SCN. Values represent the means"S.E.M., ns6; ) P - 0.01 compared to control Žunpaired Student’s t-test..
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ŽKodak, New York, USA., fixed with Fujifix ŽFuji Photo Film Co., Ltd., Tokyo, Japan., dehydrated and covered with a coverslip. 2.5. Statistics All data shown are means" S.E.M. The changes in plasma and urine electrolytes, and plasma AVP after Li treatment were statistically analyzed by using Student’s t-tests Žunpaired.. The data obtained from in situ hybridization histochemistry were also analyzed by using Student’s t-test Žunpaired.. P - 0.05 was considered to indicate statistical significance. 3. Results The body weights changed from 103 " 3.0 to 176 " 6.1 g in Li-treated rats Ž n s 21. and from 103 " 4.5 to 266 " 9.6 g in control Ž n s 14. after 4 weeks. At the end of 4 weeks daily volume of urine was 42.7 " 5.3 ml in Litreated rats Ž n s 19. and 18.7 " 1.3 ml in controls Ž n s 14.. The plasma concentrations of Li, Na and K, the plasma osmolalities and the urine osmolalities in control and Li-treated rats were summarized in Table 1. Plasma sodium concentrations were slightly increased in the Li-treated rats compared with those in controls Ž P - 0.05.. Although the osmolalities of urine in Li-treated rats were significantly lower than those in controls Ž P - 0.01., plasma osmolalities in Li-treated rats were not different from those of controls. The plasma concentrations of AVP were 16.5 " 3.6 pgrml in Li-treated rats Ž n s 14. and 5.0 " 1.9 in controls Ž n s 14.. The plasma levels of AVP in Li-treated rats were significantly higher than those in controls Ž P 0.05.. Treatment with Li for 4 weeks induced a significant increase in AVP transcripts in the PVN Ž138 " 9.0%. and the SON Ž178 " 23%. compared to control values Ž n s 6, P - 0.01. ŽFig. 4.. Microscopic examination revealed that the number of cells bound to AVP probe and intensity of the signals in both magnocellular and the parvocellular parts of the PVN and the SON were remarkably increased ŽFigs. 1 and 2.. As it is well known that AVP-producing cells exist in the suprachismatic nucleus ŽSCN., the transcripts of the AVP gene were also observed in the SCN. The expression of the AVP gene in the SCN of control rats were similar with that of the Li-treated rats ŽFig. 3.. The signals were completely abolished by competition with a 100-fold excess of the unlabelled probe. 4. Discussion The results of the present study show that chronic oral administration of Li results in a significant increase in the expression of AVP gene in the PVN and SON, as well as an increase in plasma concentrations of AVP in rats.
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In Li-treated rats, it was found that the plasma concentration of sodium was slightly but significantly increased compared with that of controls. Therefore, it is possible to explain that stimulations of sodium receptors or osmoreceptors in the brain or in the hepatorportal circulation may contribute to the secretion of AVP and the upregulation of AVP gene expression in the PVN and SON in Li-treated rats. However, the observed ranges of the plasma osmolality and sodium concentration in the present study are unlikely to cause a significant increase of plasma AVP level according to a previous study w16x. The other possible explanation is that afferent inputs to the AVP-producing cells in the PVN and SON originating from visceral receptors may be activated in the Li-induced DI rats. It has been demonstrated that renal afferents excited AVP-producing cells in the SON w5,7x and stimulate secretion of AVP and oxytocin w14x. Afferents, thought to originate from renal chemoreceptors andror mechanoreceptors may be activated by the hypotonic polyuria and hemodynamic changes in the kidney in the Li-treated DI rats. The possibility also exists that in Li-treated rats plasma volume was decreased as a result of the polyuria. This change in the circulation would activate either low pressure receptors or remove the inhibition on magnocellular neurons in the PVN and SON from high pressure baroreceptors. Both of these mechanisms would contribute to the elevated plasma AVP and the upregulation of AVP gene expression in the PVN and SON in Li-treated rats. Clinical and experimental studies have demonstrated that plasma AVP levels are increased in Li-treated patients w9x and rats w17x. However, Baylis et al. reported that plasma concentration of AVP was very low in some patients treated by Li w2x and Hensen et al. also reported that plasma AVP and osmolality did not change in Litreated rats w10x. In the present study we showed that plasma concentrations of AVP were significantly increased in Li-treated rats compared with control. This result was in agreement with that of previous studies w9,17x. It has been demonstrated that content of AVP in the hypothalamus and the posterior pituitary were decreased in Li-induced polyuria w17x, indicating that the elevated plasma AVP level is due to an increase of the release of AVP from the posterior pituitary. As the swelling of the cells in the SON were also reported in Li-induced DI rats w8x, secretion and production of AVP in the neurosecretory cells in the hypothalamus may be increased in Li-induced DI. Although we did not attempt to examine the size of the AVP-producing cells in the PVN and the SON, our microscopic observation showed that the area of those nuclei was bigger than that of control rats ŽFigs. 3 and 4.. In situ hybridization histochemistry revealed that the expression of AVP gene in the magnocellular parts of the PVN and the SON was significantly increased in Li-treated rats compared with control. It also revealed that the expression of AVP gene in the medial parvocellular parts of the PVN were remarkably increased in Li-treated rats. This observa-
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tion may represent increased activity in neurons in this subnucleus that project to cardioregulatory areas in the brainstem that may be activated by the changes in arterial pressure in Li-treated rats. It would be expected that low arterial pressure would activate PVN-medullary pathways that control sympathoexcitatory drive in Li-treated rats. In the SCN there was no difference between control and Li-treated rats. These results indicate that the increase of production of AVP in the PVN and the SON and the effects of Li on the expression of the AVP gene were specific in those nuclei. In conclusion, we have demonstrated that chronic administration of Li increases plasma concentration of AVP with upregulation of the expression of AVP gene in the PVN and SON of rats. Acknowledgements We express our appreciation to Ms Rieko Nishi, Akiko Sugimoto and Rika Suematsu for their technical assistance. This work was supported in part by Grant-in Aids for Scientific Research, Nos. 08457022 and 07507004 for H.Y. from the Ministry of Education, Science, Sports and Culture, Japan, Special Grant by the Ministry of Labor for ‘Occupational Health Studies’ and another from the Salt Science Research Foundation. References w1x M. Abramow, E. Cogan, Role of lithium-ADH interaction in lithium-induced polyuria, Year Book, 1984, pp. 29–34. w2x P.H. Baylis, D.A. Heath, Water disturbances in patients treated with oral lithium carbonate, Ann. Int. Med. 88 Ž1978. 607–609. w3x M.J. Brownstein, J.T. Russell, H. Gainer, Synthesis, transport, and release of posterior pituitary hormones, Science 207 Ž1980. 373–378.
w4x S.L. Carney, C. Ray, A.H.B. Gillies, Mechanism of lithium-induced polyuria in the rat, Kidney Int. 50 Ž1996. 377–383. w5x M.M. Caverson, J. Ciriello, Contribution of paraventricular nucleus to afferent renal nerve pressor response, Am. J. Physiol. 254 Ž1988. R531–543. w6x S. Christensen, E. Kusano, A.N.K. Yusufi, N. Murayama, T.P. Dousa, Pathogenesis of nephrogenic diabetes insipidus due to chronic administration of lithium in rats, J. Clin. Invest. 75 Ž1985. 1869– 1879. w7x T.A. Day, J. Ciriello, Effects of renal receptor activation on neurosecretory vasopressin cells, Am. J. Physiol. 253 Ž1987. R234–241. w8x G.L. Ellman, G.L. Gan, Lithium ion and water balance in rats, Toxicol. Appl. Pharmacol. 25 Ž1973. 617–620. w9x P.W. Gold, G.L. Robertson, R.M. Post, W. Kaye, J. Ballenger, D. Rubinow, F.K. Goodwin, The effect of lithium on the osmoregulation of arginine vasopressin secretion, J. Clin. Endocrinol. Metab. 56 Ž1983. 295–299. w10x J. Hensen, M. Haenelt, P. Gross, Lithium induced polyuria and renal vasopressin receptor density, Nephrol. Dial. Transplant. 11 Ž1996. 622–627. w11x D. Marples, S. Chritensen, E. Cristensen, P.D. Ottosen, S. Nielsen, Lithium-induced down regulation of aquaporin-2 water channel expression in rat kidney medulla, J. Clin. Invest. 95 Ž1995. 1838–1845. w12x G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, Sydney, 1986. w13x M. Peet, J.P. Pratt, Lithium. Current status in psychiatric disorders, Drugs 46 Ž1993. 7–17. w14x J.K. Simon, N.W. Kasting, J. Ciriello, Afferent renal nerve effects on plasma vasopressin and oxytocin in conscious rats, Am. J. Physiol. 256 Ž1989. R1240–1244. w15x I. Singer, Lithium and the kidney, Kidney Int. 19 Ž1981. 374–387. w16x E.M. Stricker, J.G. Verbalis, Interaction of osmotic and volume stimuli in regulation of neurohypophyseal secretion in rats, Am. J. Physiol. 250 Ž1986. R267–R275. w17x M. Sugawara, K. Hashimoto, Z. Ota, Involvement of prostaglandin E2, cAMP, and vasopressin in lithium-induced polyuria, Am. J. Physiol. 254 Ž1988. R863–R869, ŽRegulatory Integrative Comp. Physiol. 23.. w18x M. Yamaki, E. Kusano, T. Tetsuka, S. Takeda, S. Homma, N. Murayama, Y. Asano, Cellular mechanism of lithium-induced nephrogenic diabetes insipidus in rats, Am. J. Physiol. 261 Ž1991. F505–F511, ŽRenal Fluid Electrolyte Physiol. 30..