Thyroid hormone affects the hydrolysis of inositol phospholipids in the rat hypothalamus

Neuroscience Letters, 134 (1992)275-278

275

© 1992ElsevierScientificPublishers Ireland Ltd. All rights reserved0304-3940/92/$03.50 NSL 08320

Thyroid hormone affects the hydrolysis of inositol phospholipids in the rat hypothalamus T o k u j i Iriuchijima, H a r u o M i z u m a , T o s h i o M i c h i m a t a , T a k a y u k i Ogiwara, M a s a n o b u Y a m a d a , Masami Murakami and Masatomo Mori First Department of Internal Medicine, Gunma University School of Medicine, Maebashi (Japan)

(Received28 August 1991;Revisedversionreceived4 October 199I; Accepted7 October 1991) Key words: Thyroid hormone;Thyroxine;Hypothyroidism;Inositolphospholipid hydrolysis;Inositol phosphate; Ouabain; Hypothalamus

We have attempted to elucidatethe effectof thyroid hormone on phospholipaseC-linkedinositolphospholipidhydrolysisin the rat hypothalamus. Hypothalamic slices of each animal, euthyroid control, hypothyroid,and thyroxine(T4)-supplementedhypothyroid rats were labeled with [3H]myoinositol in the presence of 5 mM LiCI, and then incubated for 60 rain in KHG buffer containing either vehicleor 1 mM ouabain, a Na-K ATPase inhibitor. Hypothyroidismcaused a significant increase in both basal and ouabain-stimulated accumulation of [3H]inositolphosphate ([3H]IP)in hypothalamic slices, whereas supplement with T4 to hypothyroid rats resulted in a complete restoration of hypothalamic [3H]IPformation to the value of euthyroid control. The present results indicate that thyroid hormone affects phospholipase C-linked inositol phospholipid hydrolysisin the hypothalamus, suggestingthat negativefeedbackaction of thyroid hormone may occur at a post-receptorsite in the hypothalamus.

Although several brain neurotransmitters are well known to stimulate the hydrolysis of inositol phospholipids to generate two possible second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglyclerol [3, 8, 15], depolarizing stimuli can also induce phosphatidylinositol breakdown in neuronal tissues [1, 2, 9, 13]. Audigier et al. [1] reported that not only M1 muscarinic receptor activation, but Ca 2+ influx induced by membrane depolarization stimulates phospholipase C activity in synaptosomes. In addition, Gusovsky et al. [9] indicate that Na + influx may play an important role in regulating a phosphatidylinositol turnover in nerve terminals. On the other hand, Kendall and Nahorski [13] observed that the release of endogenous acetylcholine stimulated by depolarization induced receptor-linked inositol phospholipid hydrolysis. However, the precise mechanism underlying depolarization-induced inositol phospholipid hydrolysis remains to be defined. In recent years, we reported that hypothalamic thyrotropin-releasing hormone (TRH) could be stimulated by depolarizing agents in vitro [11], and that thyroid hormones directly changed in vitro depolarization-induced release of T R H from the rat hypothalamus [10]. Furthermore, Bruhn et al. [6] also observed that the release of Correspondence: T. Iriuchijima, First Department of Internal Medicine, Gunma UniversitySchoolof Medicine,Maebashi 371, Japan.

T R H and p r e p r o T R H from median eminence in response to K + were enhanced in the hydrothyroid state. Since depolarization stimulates both release of neurotransmitters and/or neurohormones and inositol phospholipid hydrolysis in neuronal tissues, these observations have prompted the hypothesis that thyroid hormone affects the hydrolysis of inositol phospholipids induced by depolarization in the hypothalamus. However, little information is available concerning this hypothesis. In the present study, therefore, we examined the influences of thyroid hormone on the hydrolysis of inositol phospholipids in response to ouabain, a depolarizing agent, in the rat hypothalamus. Adult male Wistar rats, weighing between 180 and 200 g, were decapitated and each hypothalamus was sliced (350 x 350 gm) using a Mcllwain tissue chopper as described previously [12]. The slices were preincubated in 1 ml modified Krebs-Henseleit glucose ( K H G ) buffer (NaC1 118, KCI 4.7, CaCI2 1.3, KH2PO4 1.2, MgSO4 1.2, NaHCO3 25, glucose 11.7 mM, saturated with 95% 02/ 5% CO2, pH, 7.4) at 37°C for 60 min with two exchanges of fresh medium. Equilibrated hypothalamic slices (ca. 50 mg) were then incubated in 0.24 ml fresh K H G buffer containing 0.3/~M [34H]myoinositol (specific activity = 12.8-17.1 Ci/mmol, New England Nuclear, Boston, MA, U.S.A.) and 5 mM LiCI for 30 min. At the end of incubation, 10 /tl ouabain (Sigma Chemical Co. St.

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Fig. 1. Time- and ouabain dose-dependent hydrolysis ofinositol phospholipids in rat hypothalamic slices. The left panel shows the time course of inositol phospholipid hydrolysis. Hypothalamic slices were labeled with pH]myoinositol and incubated with 1 m M ouabain at 37°C for 5--90 min. The right panel shows effects of increasing doses of ouabain on [3H]IP accumulation. Hypothalamic slices labeled with [3H]myoinositol were incubated with ouabain in doses of 0 I m M at 37°C for 60 rain. Each bar represents the mean + S.E.M. (n=5).

Louis, MO, U.S.A.) in a concentration of l mM was added and then incubation continued for 5-90 rain. Hypothalamic slices labeled with [3H]myoinositol were incubated with varying concentrations of ouabain (final concentrations, 0-1 mM) for 60 min. Three groups of rats, euthyroid control, hypothyroid (two weeks after total thyroidectomy), and thyroxine (T4)-supplemented hypothyroid (thyroidectomy followed by supplement with T4 [1.5/tg/100 g body weight x 14 days]), were used as described elsewhere [14]. Blood samples were collected for determination of plasma TSH by RIA using a NIDDK RIA kit (NIDDK, Bethesda, MD, U.S.A.) with rTSH-RPI used as standards. Hypothalamic slices of each animal were preincubated in KHG buffer for 60 min, and then labeled with [3H]myoinositol for 30 min. After addition of either vehicle or 1 mM ouabain, the incubation was further continued for 60 min. Because the accumulation of [3H]inositol phosphate ([3H]IP) reflects the hydrolysis of inositol phospholipids, [3H]IP were determined according to the method of Brown et al. [5] as described previously [12]. In brief, at the end of incubation, reaction was terminated by addition of methanol/chloroform. After the mixture was centrifuged at 1000 g for 5 rain, 750/21 of upper aqueous phase was diluted with 2.25 ml distilled water, and 0.5 ml of a 50% slurry of anion exchange resin (AGI-X8 formate form, 100-200 mesh, Bio-Rad, Tokyo, Japan) was added to each tube followed by centrifugation at 1000 g for 5 rain. The precipitate was washed 4 times with 3 ml of 5 mM unlabelled myoinositol in distilled water. Five hundred/d of 1 M ammonium formate in 0.1 M formic acid was added to each precipitate followed by centrifugation as described above. Four hundred/~1 of the supernatant was trans-

ferred to a scintillation vial containing 5 ml Aquasol-2 to determine [3H]IP. Statistical significance was assessed by Duncan's multiple range test. Fig. 1 shows a time- and dose-related increase of [3H]IP accumulation in response to ouabain in hypothalamic slices. One millimolar ouabain stimulated the formation of [3H]IP in hypothalamic tissues during the 90 rain incubation (5 rain, 23.78_+0.48, 15 min, 25.86+0.88, 30 min, 27.10_+0.44, 60 min, 31.86+0.98, 90 min, 30.60-+0.80 dpm/mg of wet weight in response to 1 mM ouabain in the left panel). Furthermore, hypothalamic [3H]IP accumulated in an ouabain dosedependent manner (ouabain, 0 mM, 12.98+1.06; 0.1 raM, 20.88_+0.20; 0.2 mM, 24.40-+ 1.60; 0.5 raM,

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Fig. 2. Effects of thyroidectomy (Tx) and T 4 supplement (Tx + T4) on blood TSH and pH]IP accumulation in hypothatamic slices. Hypothalamic slices of each animal were labeled with [3H]myoinositol for 30 min, followed by addition of either vehicle (0) or 1 m M ouabain (OB). Each bar represents the mean + S.E.M. (n=5). The statistical significance of differences from the control was analyzed by Duncan's multiple range test: * P < 0.05, ** P < 0.01.

277 26.86__ 1.12; 1 m M 28.86+0.28 d p m / m g of wet weight in the right panel). Fig. 2 depicts the effects of thyroidectomy and supplement with T4 tO thyroidectomized rats on plasma T S H concentrations and the formation of [3H]IP in the rat hypothalamus. Thyroidectomy caused a striking elevation in plasma TSH concentrations (the control group, 0.49+0.04 vs the thyroidectomized group, 2.93+0.22 /~g/ml; P < 0 . 0 1 ) , which represents severe thyroid deficiency. Hypothyroidism resulted in a marked increase in both basal and ouabain-induced [3H]IP formation in the hypothalamus (basal accumulation of [3H]IP: the control group, 16.24+0.78 vs the hypothyroid group, 20.84___ 0.98 d p m / m g of wet weight, P < 0.05; ouabainstimulated accumulation of [3H]IP: the control group, 24.72+1.22 vs the hypothyroid group, 32.32+1.62 d p m / m g of wet weight, P < 0.01). In contrast, in vivo administration of T4 to thyroidectomized rats produced significant decreases in both plasma TSH level and [3H]IP formation to the values of euthyroid control rats (TSH, 0.92 + 0.12/~g/ml; basal and ouabain-stimulated accumulation of [3H]IP, 15.50+1.76 and 25.26+2.60 d p m / m g of wet weight, respectively). According to the hypothesis of stimulus-secretion coupling by Douglas and Rubin [7], the arrival of an action potential at a nerve terminal induces membrane depolarization followed by opening of the voltage sensitive Ca 2+ channel. The resulting increase in cytoplasmic Ca 2+ induces the release of neurotransmitters [4]. Besides the stimulation of neurotransmitter release, membrane depolarization has recently become apparent to induce the hydrolysis of inositol phospholipids in neuronal tissues [1, 2, 9, 13]. Although the precise biochemical mechanisms underlying depolarization-induced inositol phospholipid hydrolysis are poorly understood, it is suggested that Ca 2+ influx [1] or the elevation of cytoplasmic Na + [9] participates in the regulation of inositol phospholipid breakdown in neuronal tissues. In the present study, we demonstrated that ouabain, which can elevate intracellular N a + by inhibiting a N a - K ATPase, caused a time- and dose-dependent increase in the formation of [3H]IP in hypothalamic slices. These findings are compatible with the report by Gusovsky et al. [9]. In recent years, it has been reported that thyroid hormone influences the release of T R H [6, 10] and several brain neurotransmitters such as norepinephrine and dopamine [16, 17] from the hypothalamus. Since not only the release of these neurotransmitters and/or neurohormones, but also the hydrolysis of inositol phospholipids could be stimulated by membrane depolarization, this evidence has raised the possibility that thyroid hormone may influence the hydrolysis ofinositol phospholipids in hypothalamic neurons. In the present study, we

clearly demonstrated that experimental hypothyroidism resulted in a marked enhancement of ouabain-induced [3H]IP formation in the hypothalamic tissues, whereas supplement with T4 to hypothyroid rats caused a complete restoration in the formation of [3H]IP. In addition, it should be noted that basal accumulation of [3H]IP in the hypothalamus significantly increased in the hypothyroid state. Therefore, the present data indicate that the activity of phospholipase C to hydrolyze inositol phospholipids could be enhanced by thyroid deficiency in both resting and depolarizing condition of hypothalamic neurons. Although the reason why phosphatidylinositol turnover in the hypothalamus could be accelerated in the hypothyroid state is unknown, the present results imply that negative feedback action of thyroid hormone may occur at post-receptor sites in the hypothalamus. However, the mechanism responsible for thyroid hormone regulation of the hydrolysis of inositol phospholipids in the hypothalamus remains to be elucidated.

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