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Effects of Thyroid and Glucocorticoid Hormones on Kv1.5 Potassium Channel Gene Expression in the Rat Left Ventricle
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
237, 521–526 (1997)
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Effects of Thyroid and Glucocorticoid Hormones on Kv1.5 Potassium Channel Gene Expression in the Rat Left Ventricle Atsushi Nishiyama,* Fukushi Kambe,†,1 Kaichiro Kamiya,* Shunsuke Yamaguchi,† Yoshiharu Murata,† Hisao Seo,† and Junji Toyama* *Department of Circulation and †Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan 464-01
Received July 24, 1997
The effects of thyroid and glucocorticoid hormones on the expression of the Kv1.5 potassium channel gene were studied in the rat left ventricle. Rats were rendered hypothyroid by oral administration of methimazole (MMI). Hyperthyroidism was induced in the MMItreated rats by administration of L-thyroxine (T4). Kv1.5 mRNA levels decreased markedly in the hypothyroid rats, whereas they increased in the hyperthyroid rats. Propranolol, a b-adrenergic blocker, did not inhibit the T4-dependent increase in Kv1.5 mRNA, indicating that the increase is not due to the increased badrenergic stimuli under hyperthyroidism. Accordingly, treatment of the MMI-treated hypothyroid rats with isoproterenol, a b-adrenergic receptor agonist, did not increase the mRNA. The Kv1.5 mRNA levels positively correlated with the thyroid hormone levels in sera. When rats were adrenalectomized and rendered hypothyroid, Kv1.5 mRNA became undetectable. Administration of 3,3 *,5-triiodothyronine (T3) at a dose to induce hyperthyroidism did not restore the mRNA level. However, T3 significantly increased the mRNA level when dexamethasone was co-administered at a physiological dose. These results for the first time demonstrate that thyroid hormone up-regulates Kv1.5 mRNA levels in the rat left ventricle and they demonstrate that glucocorticoid is required for this induction. q 1997 Academic Press
Kv1.5 is one of the voltage-gated potassium channels expressed in the cardiac myocytes (1). Recently, it has been shown that Kv1.5 mRNA is decreased in hypertro1 Address all correspondence and requests for reprints to: Fukushi Kambe, M.D. Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan 464-01. Fax: 81-52-789-3887. E-mail: [email protected].
phic cardiac myocytes induced by renovascular hypertension and in spontaneously hypertensive rats (2). It is also known that a synthetic glucocorticoid, dexamethasone, increases the expression of Kv1.5 gene expression in the rat heart ventricle (3). These studies prompted us to study the effects of thyroid and glucocorticoid hormones on the expression of Kv1.5 gene, since it is well known that cardiac hypertrophy is induced by hyperthyroidism (4-7) and glucocorticoid potentiates thyroid hormone action (8-11). MATERIALS AND METHODS Animal treatments. The first experiment aimed to study the effects of thyroid status on cardiac Kv1.5 expression. Eight-week-old male Wistar rats were assigned to 6 groups (control, hypothyroid and 4 hyperthyroid groups). Each group contained 4 rats. Rats were rendered hypothyroid by addition of 0.025% methimazole (2-mercapto-1-methyl-imidazole, MMI) to the drinking water for 5 weeks (12). Hyperthyroidism was induced in MMI-treated hypothyroid rats by daily intraperitoneal injection of various doses (1, 2, 5 and 20 mg/ 100g body weight/day) of L-thyroxine (T4 ; Sigma Chemical Co., St. Louis, MO) for the last 7 days (13). Control and hypothyroid rats were given daily intraperitoneal injections of vehicle (0.03 % bovine serum albumin, 0.9 % NaCl) for the last 7 days. The second experiment aimed to study the effects of propranolol, a b-adrenergic blocker, and isoproterenol, a b-adrenergic receptor agonist, on the expression of the Kv1.5 mRNA. Rats were assigned to 5 groups: control, hypothyroid, hyperthyroid, hyperthyroid plus propranolol, and hypothyroid plus isoproterenol groups. Each group contained 4 rats. Rats were rendered hypothyroid by the administration of MMI for 5 weeks. Hyperthyroidism was induced in MMItreated hypothyroid rats by daily intraperitoneal injection of 20 mg/ 100g body weight/day of T4 for the last 7 days. Propranolol (Sigma) was added to the drinking water at 750 mg/liter and administered to the MMI-treated rats together with T4 for the last 7 days (5). Isoproterenol (Sigma) was injected intraperitoneally to the MMItreated hypothyroid rats at a dose of 500 mg/100g body weight/day for the last 7 days (14). The third experiment examined the effects of dexamethasone (Dex) as well as thyroid hormone on the Kv1.5 expression. Rats were adrenalectomized under ether anesthesia. After a dorsal midline incision, the skin was pulled laterally and the adrenal on each side was ex-
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Changes in Body and Weights, Ventricular Weights, and Concentrations of Serum T4 and T3 Body Weight (g)
Groups Control MMI MMI / T4 MMI / T4 MMI / T4 MMI / T4
1 mg 2 mg 5 mg 20mg
Initial 310 300 316 301 320 318
{ { { { { {
8 5 3 5 4 8
4-week 476 355 379 370 384 362
{ { { { { {
10 8* 14* 10* 10* 8*
5-week (Sacrifice) 475 350 385 377 384 361
{ { { { { {
Ventricular weight (mg/g body weight)
12 8* 10* 11* 11* 9*
3.85 3.50 3.61 3.68 4.45 5.00
{ { { { { {
0.19 0.08 0.29 0.12 0.18† 0.17*†
T4 (mg/dl) 2.8 { 0.3 õ1.00* 5.3 { 0.3*† 7.2 { 0.6*† 7.4 { 0.7*† 10.9 { 2.3*†
T3 (ng/dl) 55 39 69 93 155 259
{ { { { { {
2 1* 3*† 8*† 5*† 24*†
Note. Rats were rendered hypothyroid by giving MMI for 5 weeks. Hyperthyroidism was induced by daily injection of various doses of T4 (1, 2, 5 and 20 mg/100g body weight/day) for the last 7 days to the MMI-treated rats. The initial body weights (Initial) and body weights before and after the T4 administration (4-week and 5-week, respectively) are shown. The left ventricles and blood samples were obtained from 4 rats in each group 12 hours after the last T4 or vehicle injection. The data are expressed as mean { S.E. Statistical significance was determined by Student’s t-test and p values less than 0.05 were considered as significantly different. *: vs control. †: vs MMI group.
posed by gently splitting the external and internal oblique muscles. Each adrenal was then excised with the surrounding fat. The adrenalectomized rats were given water containing 150 mM NaCl. Administration of MMI started one week after the operation. After 3week treatment with MMI, Dex (0, 1, 10 and 100 mg/100g body weight/day; Banyu Pharmaceutical Co. Ltd., Tokyo, Japan) alone or in combination with 3,3*,5-triiodothyronine (T3 ; 50 mg/100g body weight/day; Sigma) were intraperitoneally administered to the rats for 7 days (13, 15). Each group contained 3 rats. Twelve hours after the last injections of hormones and agents except for isoproterenol, the left ventricles were dissected out, immediately frozen in liquid nitrogen and stored at 080 7C until extraction of total RNA. An additional injection of isoproterenol was performed 2 hours before the sacrifice. Sera were separated and frozen at 030 7C until the analysis of T4 and T3 . The hormones were measured by radioimmunoassays [T4 : Magnetic T4 radioimmunoassay kit (CibaCorning, Tokyo, Japan), T3 : Gamma Coat T3 radioimmunoassay kit (Baxter Healthcare Corp., Cambridge, MA)]. All rats were treated in accord with the principles and procedures outlined by the committee for animal experiment of Nagoya University. Northern blot analysis. Total RNA was extracted from the frozen left ventricles after pulverization in liquid nitrogen using the protocol described by Chomczynski and Sacchi (16). The protocol of Northern blot analysis was described in our previous report (17). The rat Kv1.5 cDNA was cloned by the reverse transcription-polymerase chain reaction (RT-PCR) method described previously (18) using rat heart mRNA as a template. The sense (5*-GCCTGGAGACTCTGCCTGAGTTCAGGGATG-3*) and antisense (5*-GGTGTAAAGCAGATGCCCAGGCTCAAGGGG-3*) oligonucleotides were used for PCR. The PCR product was ligated into pCR II plasmid (Invitrogen Corporation, San Diego, CA). The nucleotide sequence of the cloned cDNA was verified by sequencing analysis. The EcoRI-EcoRI fragment corresponding to the 3 *-end of Kv1.5 cDNA (nucleotide 1024-1886 when translation start site was assigned as 1 (1)) was isolated and labeled with 32P-dCTP (Du Pont-New England Nuclear, Boston, MA) using a random-primed DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). The mRNA levels were determined by Fujix Bioimage Analyzer (BAS 2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) and were corrected by the mRNA level for GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The cloning of GAPDH cDNA was described in our previous report (19). Ribonuclease protection assay (RPA). For RPA, another rat Kv1.5 cDNA fragment was amplified by RT-PCR using the sense (5*-CCGAGTATTTAAGCCCACCTG-3*) and antisense (5*-CTAAGCTTT-
TTAAAGTCAAATTTG-3*) oligonucleotides. The amplified cDNA (nucleotides 1888-2117 (1)) was cloned into pGEM-T vector using TA cloning system (Promega Corporation, Madison, WI). The plasmid containing Kv1.5 cDNA was linearized by digestion with a restriction enzyme NcoI, and then antisense cRNA probes were prepared using MAXIscript kit (Ambion Inc., Austin, TX) and [a-32P]UTP (New England Nuclear). The cyclophilin cRNA probe was also prepared from the cDNA purchased from Ambion (pTRI-cycloph-
FIG. 1. Effect of thyroid status on the expression of cardiac Kv1.5 mRNA. Rats were rendered hypothyroid by the addition of MMI for 5 weeks. Hyperthyroidism was induced in the MMI-treated hypothyroid rats by the addition of various doses (1, 2, 5 and 20 mg/100g body weight/day) of T4 for the last 7 days. Vehicle was daily given to the control and hypothyroid rats. Aliquots of 20 mg total RNA extracted from the left ventricles were subjected to Northern blot analysis using 32P-labeled Kv1.5 cDNA as a probe. Rehybridization was performed using 32P-labeled GAPDH cDNA. A representative autoradiogram of control, MMI-treated hypothyroid and 20 mg T4treated hyperthyroid rats is shown. The positions of 3.5-kb Kv1.5 mRNA and 1.3-kb GAPDH mRNA are indicated by the arrowheads. The positions of 28 S and 18 S ribosomal RNA are also indicated.
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FIG. 2. Relationship between serum levels of thyroid hormones and levels of cardiac Kv1.5 mRNA. The abundances of cardiac Kv1.5 mRNA in individual rats are normalized by the GAPDH mRNA levels and are plotted with the logarithm of the serum levels of thyroid hormones. Closed and open circles indicate MMI-treated and control rats, respectively. Open triangle, closed triangle, open square and closed square indicate rats treated with 1, 2, 5 and 20 mg T4 , respectively. The levels of Kv1.5 mRNA in the left ventricle of 24 rats correlate positively with the logarithm of the serum levels of thyroid hormones (T3 vs. Kv1.5 mRNA: r Å 0.729, p õ 0.01; T4 vs. Kv1.5 mRNA: r Å 0.742, p õ 0.01).
ilin-rat antisense control template, nucleotide 38 to 142) to detect cyclophilin mRNA as an internal control. RPA was performed using HybSpeed RPA kit (Ambion) according to manufacturer’s protocol. Hybridization of the two probes (2 1 104 cpm Kv1.5 cRNA and 2 1 104 cpm cyclophilin cRNA) with 10 mg total RNA was carried out at 68 7C for 10 min, followed by digestion with RNase A and RNase T1 at 37 7C for 30 min. The reaction was terminated by addition of sodium-dodecyl sulfate and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. The protected fragments were visualized by autoradiography after electrophoresis on a 5% polyacrylamide/8 M urea gel. Quantitative analysis was carried out using Fujix Bioimage Analyzer. The mRNA levels were normalized by the levels for cyclophilin.
RESULTS AND DISCUSSION Effects of Thyroid Status on Body and Ventricular Weights and on Serum T4 and T3 Concentrations As shown in Table 1, induction of hypothyroidism was ascertained by a marked decrease in T4 and T3 in the rats treated with MMI alone. Administration of various doses of T4 to the MMI-treated rats for the final 7 days increased the serum concentrations of T4 and T3 in a dose-dependent manner. Initial body weights before MMI administration were similar in all the groups. After the administration of MMI for 4 weeks, body weights in all the MMI-treated groups were significantly lower than that of the control group. Various doses of T4 administration for 7 days did not affect the body weights. Although statistically not significant, the left ventricular weight of rats treated with MMI alone was lower than that of the control group. Administration of T4 at doses of 5 mg and 20 mg resulted in a significant increase in ventricular weight above that in the group treated with MMI alone, suggesting that hyperthyroidism induced a modest degree of cardiac hypertrophy in the left ventricle as previously reported (4-6).
Thyroid Hormone Increases the Kv1.5 mRNA Level in Rat Left Ventricle Fig. 1 shows the representative autoradiograms of Kv1.5 and GAPDH mRNAs in rat left ventricle. Kv1.5 mRNA in the left ventricle of control rats was detected as a single band of approximately 3.5 kb. The size was compatible with the report by Takimoto et al (20). Administration of MMI resulted in a marked decrease in the Kv1.5 mRNA level; its expression was not detected in 3 rats out of 4 hypothyroid rats. Administration of T4 20 mg to the MMI-treated hypothyroid rats markedly increased the mRNA level more than that of the control. On the other hand, GAPDH mRNA changed little. The Kv1.5 mRNA levels normalized by GAPDH mRNA and the logarithm of the serum levels of thyroid hormones in individual rats are plotted in Fig. 2. A significant positive correlation was observed between the Kv1.5 mRNA levels and the thyroid hormone levels (T3 vs. Kv1.5 mRNA: r Å 0.729, p õ 0.01; T4 vs. Kv1.5 mRNA: r Å 0.742, p õ 0.01). These results demonstrate that the change in thyroid status alters the expression of cardiac Kv1.5 gene. Since several studies showed that the changes in mRNA level of Kv1.5 channel were associated with its protein level (3, 20, 21), it is likely that thyroid hormone consequently alters the number of Kv1.5 channel protein in rat left ventricle. Increase in Kv1.5 channel may cause increased outward current in the repolarization phase of the cardiac myocytes. This could, at least partially, account for shortening of the action potential duration under hyperthyroidism reported by S. Di Meo et al (22). The finding that the increase in Kv1.5 mRNA level was associated with cardiac hypertrophy in the hyperthyroid rats was unexpected, since a decrease in the
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FIG. 3. Effects of propranolol and isoproterenol on the thyroid hormone-dependent expression of cardiac Kv1.5 mRNA. Rats were rendered hypothyroid by the addition of MMI. Hyperthyroidism was induced in the MMI-treated hypothyroid rats by the addition of T4 (20 mg/100g body weight/day) for the last 7 days. Propranolol (Pro) was added to the drinking water at 750 mg/liter and administered to the MMI-treated rats together with T4 for the last 7 days. Isoproterenol (Iso, 500 mg/100g body weight/day) was injected to the MMItreated rats for the last 7 days. Aliquots of 10 mg total RNA extracted from the left ventricles were subjected to RPA using 32P-labeled Kv1.5 and cyclophilin cRNAs as probes. A representative autoradiogram is shown in the upper panel. Open arrowheads indicate the positions of undigested cRNA probes for Kv1.5 and cyclophilin (Cyclo). The positions of protected cRNAs are indicated by the closed arrowheads. The abundances of Kv1.5 mRNA are normalized by cyclophilin mRNA levels and are expressed as arbitrary units in the lower panel. Values are the mean { S.E. (nÅ4). *: põ0.01.
mRNA level had been shown in the cardiac hypertrophy induced by renovascular hypertension (2). This might imply that the Kv1.5 mRNA level is regulated independently from the intracellular signals leading to hypertrophy. The Increase in Kv1.5 mRNA by Thyroid Hormone Is Not Mediated through b-Adrenergic Receptor It has been shown that thyroid hormone increases the number of b-adrenergic receptor (23-26), by increasing the transcription of its gene (27). Increased sensitivity of the heart to b-adrenergic stimuli in the
hyperthyroid rats may enhance the intracellular cAMP production. Recently, cAMP has been shown to increase the steady state levels of Kv1.5 mRNA in primary cardiac cells (28). Therefore, there is a possibility that the increased b-adrenergic stimuli might cause the increase in expression of Kv1.5 mRNA by thyroid hormone. To address this possibility, we examined the effects of propranolol, a b-adrenergic blocker, and isoproterenol, a b-adrenergic receptor agonist, on the expression of the mRNA. Propranolol was administered together with T4 to the MMI-treated hypothyroid rats for the last 7 days. Isoproterenol was injected to the MMI-treated hypothyroid rats for the last 7 days. As shown in Fig. 3, administration of T4 to the hypothyroid rats markedly increased the Kv1.5 mRNA in the rat left ventricle. However, the treatment of the hyperthyroid rats with pharmacological dose of propranolol did not inhibit the increase by T4 , indicating that the increase is not due to the increased b-adrenergic stimuli under hyperthyroidism. Accordingly, the treatment of the MMI-treated hypothyroid rats with isoproterenol did not increase the Kv1.5 mRNA. These results indicate that thyroid hormone regulates the Kv1.5 gene expression by a mechanism independent of a b-adrenergic receptor-mediating mechanism. It is possible that thyroid hormone directly increase the transcription of Kv1.5 gene as it activates the transcription of the genes for b-adrenergic receptor (27), myosin heavy chain a and b (29, 30), and sarcoplasmic reticulum calcium ATPase (31) in the cardiac myocytes. Glucocorticoid Is Required for the Up-Regulation of Kv1.5 mRNA by Thyroid Hormone It was reported that glucocorticoid agonist Dex increases Kv1.5 mRNA level in the rat ventricle (3). We thus examined the effects of Dex on the thyroid hormone-mediated induction of Kv1.5 mRNA. Rats were adrenalectomized and rendered hypothyroid by administration of MMI (ADX-MMI rats). Reduction of endogenous glucocorticoids and thyroid hormones was ascertained by measurement of serum hormone levels (data not shown). In this experiment, T3 was administered to the rats, since Dex modulates the expression of iodothyronine type I 5*-deiodinase in the liver as described in our previous report (10). As shown in Fig. 4, Kv1.5 mRNA was not detected in ADX-MMI rats. Interestingly, administration of T3 50 mg alone (a supraphysiological dose) to the ADX-MMI rats did not increase the mRNA level. Treatment with physiological dose of Dex 1 mg (15) neither restored the mRNA. However, administration of T3 50 mg together with Dex 1 mg markedly increased the Kv1.5 mRNA level. These results indicate that glucocorticoids are required for the T3-mediated induction of
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FIG. 4. Effect of thyroid and glucocorticoid hormones on the expression of cardiac Kv1.5 mRNA. Rats were adrenalectomized and given MMI. After 3-week treatment with MMI, Dex (0, 1, 10 and 100 mg/100g body weight/day) alone or in combination with T3 (50 mg/100g body weight/day) were administered to the rats for the last 7 days. A representative autoradiogram of cardiac Kv1.5 and GAPDH mRNA is shown in the upper panel. The abundances of Kv1.5 mRNA are normalized by GAPDH mRNA levels and are expressed as arbitrary units in the lower panel. Values are the mean { S.E. (nÅ3). *: põ0.05, **: põ0.01.
cardiac Kv1.5 gene expression. The supraphysiological doses of Dex 10 mg and 100 mg increased the mRNA even in the absence of T3 administration. Treatment with T3 together with Dex further increased the mRNA level compared to the level of Dex alone. Synergistic or additive effects of thyroid and glucocorticoid hormones have been reported in the regulation of growth hormone and iodothyronine type I 5*deiodinase genes (8-10). Our results showed that the expression of cardiac Kv1.5 gene is also cooperatively up-regulated by thyroid and glucocorticoid hormones. In addition, it is of interest that glucocorticoid is necessary for the thyroid hormone-mediated induction of cardiac Kv1.5 gene expression. A similar finding was reported in somatotroph development in the fetal rat pituitary gland. Nogami et al showed that Dex treatment of dams induces growth hormone mRNA in the fetal rat pituitary and that additional T4 treatment enhanced the effect of Dex (11). However, this T4 effect was not observed in the absence of Dex. Molecular mechanism underlying the cooperative effects of thyroid and glucocorticoid hormones has not been fully understood. In conclusion, the present study for the first time demonstrated that thyroid hormone increases the Kv1.5 mRNA in the rat left ventricle and that this effect is not mediated through b-adrenergic stimuli. Furthermore, we showed that glucocorticoid is indispensable for the thyroid-hormone-mediated induction. Our study showed that the expression of one of the voltage-gated potassium channel genes is under control of thyroid hormone. We are currently studying whether the expression of other potassium channel genes is altered by the hormone, in order to clarify overall effects
of thyroid hormone on the voltage-gated potassium channels. ACKNOWLEDGMENTS We thank Dr. Devanand Sarkar for critically reading the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (07670774) from the Ministry of Education, Science, Sports and Culture, Japan.
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14. Meszaros, J., Ryder, K. O., and Hart, G. (1996) Am. J. Physiol. 271, H2360–H2367. 15. Walker, B. R., Williams, B. C., and Edwards, C. R. (1994) J. Endocrinol. 141, 467–472. 16. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 17. Kambe, F., Seo, H., Murata, Y., and Matsui, N. (1988) Mol. Endocrinol. 2, 181–185. 18. Kambe, F., Tsukahara, S., Kato, T., and Seo, H. (1993) Biochim. Biophys. Acta 1171, 307–310. 19. Miyazaki, T., Sato, M., Murata, Y., Maeda, K., and Seo, H. (1995) Am. J. Nephrol. 15, 48–56. 20. Takimoto, K., Fomina, A. F., Gealy, R., Trimmer, J. S., and Levitan, E. S. (1993) Neuron 11, 359–369. 21. Gidh-Jain, M., Huang, B., Jain, P., and El-Sherif, N. (1996) Cir. Res. 79, 669–675. 22. Di Meo, S., de Martino Rosaroll, P., Piro, M. C., and De Leo, T. (1994) Arch. Int. Physiol. Biochim. Biophys. 102, 153–159.
23. Williams, L. T., Lefkowitz, R. J., Watanabe, A. M., Hathaway, D. R., and Besch, H. J. (1977) J. Biol. Chem. 252, 2787–2789. 24. Ciaraldi, T. P., and Marinetti, G. V. (1978) Biochim. Biophys. Acta 541, 334–346. 25. Scarpace, P. J., and Abrass, I. B. (1981) Endocrinology 108, 1276–1278. 26. Revelli, J. P., Pescini, R., Muzzin, P., Seydoux, J., Fitzgerald, M. G., Fraser, C. M., and Giacobino, J. P. (1991) Biochem. J. 277, 625–629. 27. Bahouth, S. W. (1991) J. Biol. Chem. 266, 15863–15869. 28. Mori, Y., Matsubara, H., Folco, E., Siegel, A., and Koren, G. (1993) J. Biol. Chem. 268, 26482–26493. 29. Izumo, S., Nadal, G. B., and Mahdavi, V. (1986) Science 231, 597–600. 30. Gustafson, T. A., Bahl, J. J., Markham, B. E., Roeske, W. R., and Morkin, E. (1987) J. Biol. Chem. 262, 13316–13322. 31. Rohrer, D., and Dillmann, W. H. (1988) J. Biol. Chem. 263, 6941–6944.
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Effects of Thyroid and Glucocorticoid Hormones on Kv1.5 Potassium Channel Gene Expression in the Rat Left Ventricle Atsushi Nishiyama,* Fukushi Kambe,†,1 Kaichiro Kamiya,* Shunsuke Yamaguchi,† Yoshiharu Murata,† Hisao Seo,† and Junji Toyama* *Department of Circulation and †Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan 464-01
Received July 24, 1997
The effects of thyroid and glucocorticoid hormones on the expression of the Kv1.5 potassium channel gene were studied in the rat left ventricle. Rats were rendered hypothyroid by oral administration of methimazole (MMI). Hyperthyroidism was induced in the MMItreated rats by administration of L-thyroxine (T4). Kv1.5 mRNA levels decreased markedly in the hypothyroid rats, whereas they increased in the hyperthyroid rats. Propranolol, a b-adrenergic blocker, did not inhibit the T4-dependent increase in Kv1.5 mRNA, indicating that the increase is not due to the increased badrenergic stimuli under hyperthyroidism. Accordingly, treatment of the MMI-treated hypothyroid rats with isoproterenol, a b-adrenergic receptor agonist, did not increase the mRNA. The Kv1.5 mRNA levels positively correlated with the thyroid hormone levels in sera. When rats were adrenalectomized and rendered hypothyroid, Kv1.5 mRNA became undetectable. Administration of 3,3 *,5-triiodothyronine (T3) at a dose to induce hyperthyroidism did not restore the mRNA level. However, T3 significantly increased the mRNA level when dexamethasone was co-administered at a physiological dose. These results for the first time demonstrate that thyroid hormone up-regulates Kv1.5 mRNA levels in the rat left ventricle and they demonstrate that glucocorticoid is required for this induction. q 1997 Academic Press
Kv1.5 is one of the voltage-gated potassium channels expressed in the cardiac myocytes (1). Recently, it has been shown that Kv1.5 mRNA is decreased in hypertro1 Address all correspondence and requests for reprints to: Fukushi Kambe, M.D. Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan 464-01. Fax: 81-52-789-3887. E-mail: [email protected].
phic cardiac myocytes induced by renovascular hypertension and in spontaneously hypertensive rats (2). It is also known that a synthetic glucocorticoid, dexamethasone, increases the expression of Kv1.5 gene expression in the rat heart ventricle (3). These studies prompted us to study the effects of thyroid and glucocorticoid hormones on the expression of Kv1.5 gene, since it is well known that cardiac hypertrophy is induced by hyperthyroidism (4-7) and glucocorticoid potentiates thyroid hormone action (8-11). MATERIALS AND METHODS Animal treatments. The first experiment aimed to study the effects of thyroid status on cardiac Kv1.5 expression. Eight-week-old male Wistar rats were assigned to 6 groups (control, hypothyroid and 4 hyperthyroid groups). Each group contained 4 rats. Rats were rendered hypothyroid by addition of 0.025% methimazole (2-mercapto-1-methyl-imidazole, MMI) to the drinking water for 5 weeks (12). Hyperthyroidism was induced in MMI-treated hypothyroid rats by daily intraperitoneal injection of various doses (1, 2, 5 and 20 mg/ 100g body weight/day) of L-thyroxine (T4 ; Sigma Chemical Co., St. Louis, MO) for the last 7 days (13). Control and hypothyroid rats were given daily intraperitoneal injections of vehicle (0.03 % bovine serum albumin, 0.9 % NaCl) for the last 7 days. The second experiment aimed to study the effects of propranolol, a b-adrenergic blocker, and isoproterenol, a b-adrenergic receptor agonist, on the expression of the Kv1.5 mRNA. Rats were assigned to 5 groups: control, hypothyroid, hyperthyroid, hyperthyroid plus propranolol, and hypothyroid plus isoproterenol groups. Each group contained 4 rats. Rats were rendered hypothyroid by the administration of MMI for 5 weeks. Hyperthyroidism was induced in MMItreated hypothyroid rats by daily intraperitoneal injection of 20 mg/ 100g body weight/day of T4 for the last 7 days. Propranolol (Sigma) was added to the drinking water at 750 mg/liter and administered to the MMI-treated rats together with T4 for the last 7 days (5). Isoproterenol (Sigma) was injected intraperitoneally to the MMItreated hypothyroid rats at a dose of 500 mg/100g body weight/day for the last 7 days (14). The third experiment examined the effects of dexamethasone (Dex) as well as thyroid hormone on the Kv1.5 expression. Rats were adrenalectomized under ether anesthesia. After a dorsal midline incision, the skin was pulled laterally and the adrenal on each side was ex-
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Changes in Body and Weights, Ventricular Weights, and Concentrations of Serum T4 and T3 Body Weight (g)
Groups Control MMI MMI / T4 MMI / T4 MMI / T4 MMI / T4
1 mg 2 mg 5 mg 20mg
Initial 310 300 316 301 320 318
{ { { { { {
8 5 3 5 4 8
4-week 476 355 379 370 384 362
{ { { { { {
10 8* 14* 10* 10* 8*
5-week (Sacrifice) 475 350 385 377 384 361
{ { { { { {
Ventricular weight (mg/g body weight)
12 8* 10* 11* 11* 9*
3.85 3.50 3.61 3.68 4.45 5.00
{ { { { { {
0.19 0.08 0.29 0.12 0.18† 0.17*†
T4 (mg/dl) 2.8 { 0.3 õ1.00* 5.3 { 0.3*† 7.2 { 0.6*† 7.4 { 0.7*† 10.9 { 2.3*†
T3 (ng/dl) 55 39 69 93 155 259
{ { { { { {
2 1* 3*† 8*† 5*† 24*†
Note. Rats were rendered hypothyroid by giving MMI for 5 weeks. Hyperthyroidism was induced by daily injection of various doses of T4 (1, 2, 5 and 20 mg/100g body weight/day) for the last 7 days to the MMI-treated rats. The initial body weights (Initial) and body weights before and after the T4 administration (4-week and 5-week, respectively) are shown. The left ventricles and blood samples were obtained from 4 rats in each group 12 hours after the last T4 or vehicle injection. The data are expressed as mean { S.E. Statistical significance was determined by Student’s t-test and p values less than 0.05 were considered as significantly different. *: vs control. †: vs MMI group.
posed by gently splitting the external and internal oblique muscles. Each adrenal was then excised with the surrounding fat. The adrenalectomized rats were given water containing 150 mM NaCl. Administration of MMI started one week after the operation. After 3week treatment with MMI, Dex (0, 1, 10 and 100 mg/100g body weight/day; Banyu Pharmaceutical Co. Ltd., Tokyo, Japan) alone or in combination with 3,3*,5-triiodothyronine (T3 ; 50 mg/100g body weight/day; Sigma) were intraperitoneally administered to the rats for 7 days (13, 15). Each group contained 3 rats. Twelve hours after the last injections of hormones and agents except for isoproterenol, the left ventricles were dissected out, immediately frozen in liquid nitrogen and stored at 080 7C until extraction of total RNA. An additional injection of isoproterenol was performed 2 hours before the sacrifice. Sera were separated and frozen at 030 7C until the analysis of T4 and T3 . The hormones were measured by radioimmunoassays [T4 : Magnetic T4 radioimmunoassay kit (CibaCorning, Tokyo, Japan), T3 : Gamma Coat T3 radioimmunoassay kit (Baxter Healthcare Corp., Cambridge, MA)]. All rats were treated in accord with the principles and procedures outlined by the committee for animal experiment of Nagoya University. Northern blot analysis. Total RNA was extracted from the frozen left ventricles after pulverization in liquid nitrogen using the protocol described by Chomczynski and Sacchi (16). The protocol of Northern blot analysis was described in our previous report (17). The rat Kv1.5 cDNA was cloned by the reverse transcription-polymerase chain reaction (RT-PCR) method described previously (18) using rat heart mRNA as a template. The sense (5*-GCCTGGAGACTCTGCCTGAGTTCAGGGATG-3*) and antisense (5*-GGTGTAAAGCAGATGCCCAGGCTCAAGGGG-3*) oligonucleotides were used for PCR. The PCR product was ligated into pCR II plasmid (Invitrogen Corporation, San Diego, CA). The nucleotide sequence of the cloned cDNA was verified by sequencing analysis. The EcoRI-EcoRI fragment corresponding to the 3 *-end of Kv1.5 cDNA (nucleotide 1024-1886 when translation start site was assigned as 1 (1)) was isolated and labeled with 32P-dCTP (Du Pont-New England Nuclear, Boston, MA) using a random-primed DNA labeling kit (Boehringer Mannheim, Mannheim, Germany). The mRNA levels were determined by Fujix Bioimage Analyzer (BAS 2000; Fuji Photo Film Co., Ltd., Tokyo, Japan) and were corrected by the mRNA level for GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The cloning of GAPDH cDNA was described in our previous report (19). Ribonuclease protection assay (RPA). For RPA, another rat Kv1.5 cDNA fragment was amplified by RT-PCR using the sense (5*-CCGAGTATTTAAGCCCACCTG-3*) and antisense (5*-CTAAGCTTT-
TTAAAGTCAAATTTG-3*) oligonucleotides. The amplified cDNA (nucleotides 1888-2117 (1)) was cloned into pGEM-T vector using TA cloning system (Promega Corporation, Madison, WI). The plasmid containing Kv1.5 cDNA was linearized by digestion with a restriction enzyme NcoI, and then antisense cRNA probes were prepared using MAXIscript kit (Ambion Inc., Austin, TX) and [a-32P]UTP (New England Nuclear). The cyclophilin cRNA probe was also prepared from the cDNA purchased from Ambion (pTRI-cycloph-
FIG. 1. Effect of thyroid status on the expression of cardiac Kv1.5 mRNA. Rats were rendered hypothyroid by the addition of MMI for 5 weeks. Hyperthyroidism was induced in the MMI-treated hypothyroid rats by the addition of various doses (1, 2, 5 and 20 mg/100g body weight/day) of T4 for the last 7 days. Vehicle was daily given to the control and hypothyroid rats. Aliquots of 20 mg total RNA extracted from the left ventricles were subjected to Northern blot analysis using 32P-labeled Kv1.5 cDNA as a probe. Rehybridization was performed using 32P-labeled GAPDH cDNA. A representative autoradiogram of control, MMI-treated hypothyroid and 20 mg T4treated hyperthyroid rats is shown. The positions of 3.5-kb Kv1.5 mRNA and 1.3-kb GAPDH mRNA are indicated by the arrowheads. The positions of 28 S and 18 S ribosomal RNA are also indicated.
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FIG. 2. Relationship between serum levels of thyroid hormones and levels of cardiac Kv1.5 mRNA. The abundances of cardiac Kv1.5 mRNA in individual rats are normalized by the GAPDH mRNA levels and are plotted with the logarithm of the serum levels of thyroid hormones. Closed and open circles indicate MMI-treated and control rats, respectively. Open triangle, closed triangle, open square and closed square indicate rats treated with 1, 2, 5 and 20 mg T4 , respectively. The levels of Kv1.5 mRNA in the left ventricle of 24 rats correlate positively with the logarithm of the serum levels of thyroid hormones (T3 vs. Kv1.5 mRNA: r Å 0.729, p õ 0.01; T4 vs. Kv1.5 mRNA: r Å 0.742, p õ 0.01).
ilin-rat antisense control template, nucleotide 38 to 142) to detect cyclophilin mRNA as an internal control. RPA was performed using HybSpeed RPA kit (Ambion) according to manufacturer’s protocol. Hybridization of the two probes (2 1 104 cpm Kv1.5 cRNA and 2 1 104 cpm cyclophilin cRNA) with 10 mg total RNA was carried out at 68 7C for 10 min, followed by digestion with RNase A and RNase T1 at 37 7C for 30 min. The reaction was terminated by addition of sodium-dodecyl sulfate and proteinase K, followed by phenol-chloroform extraction and ethanol precipitation. The protected fragments were visualized by autoradiography after electrophoresis on a 5% polyacrylamide/8 M urea gel. Quantitative analysis was carried out using Fujix Bioimage Analyzer. The mRNA levels were normalized by the levels for cyclophilin.
RESULTS AND DISCUSSION Effects of Thyroid Status on Body and Ventricular Weights and on Serum T4 and T3 Concentrations As shown in Table 1, induction of hypothyroidism was ascertained by a marked decrease in T4 and T3 in the rats treated with MMI alone. Administration of various doses of T4 to the MMI-treated rats for the final 7 days increased the serum concentrations of T4 and T3 in a dose-dependent manner. Initial body weights before MMI administration were similar in all the groups. After the administration of MMI for 4 weeks, body weights in all the MMI-treated groups were significantly lower than that of the control group. Various doses of T4 administration for 7 days did not affect the body weights. Although statistically not significant, the left ventricular weight of rats treated with MMI alone was lower than that of the control group. Administration of T4 at doses of 5 mg and 20 mg resulted in a significant increase in ventricular weight above that in the group treated with MMI alone, suggesting that hyperthyroidism induced a modest degree of cardiac hypertrophy in the left ventricle as previously reported (4-6).
Thyroid Hormone Increases the Kv1.5 mRNA Level in Rat Left Ventricle Fig. 1 shows the representative autoradiograms of Kv1.5 and GAPDH mRNAs in rat left ventricle. Kv1.5 mRNA in the left ventricle of control rats was detected as a single band of approximately 3.5 kb. The size was compatible with the report by Takimoto et al (20). Administration of MMI resulted in a marked decrease in the Kv1.5 mRNA level; its expression was not detected in 3 rats out of 4 hypothyroid rats. Administration of T4 20 mg to the MMI-treated hypothyroid rats markedly increased the mRNA level more than that of the control. On the other hand, GAPDH mRNA changed little. The Kv1.5 mRNA levels normalized by GAPDH mRNA and the logarithm of the serum levels of thyroid hormones in individual rats are plotted in Fig. 2. A significant positive correlation was observed between the Kv1.5 mRNA levels and the thyroid hormone levels (T3 vs. Kv1.5 mRNA: r Å 0.729, p õ 0.01; T4 vs. Kv1.5 mRNA: r Å 0.742, p õ 0.01). These results demonstrate that the change in thyroid status alters the expression of cardiac Kv1.5 gene. Since several studies showed that the changes in mRNA level of Kv1.5 channel were associated with its protein level (3, 20, 21), it is likely that thyroid hormone consequently alters the number of Kv1.5 channel protein in rat left ventricle. Increase in Kv1.5 channel may cause increased outward current in the repolarization phase of the cardiac myocytes. This could, at least partially, account for shortening of the action potential duration under hyperthyroidism reported by S. Di Meo et al (22). The finding that the increase in Kv1.5 mRNA level was associated with cardiac hypertrophy in the hyperthyroid rats was unexpected, since a decrease in the
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FIG. 3. Effects of propranolol and isoproterenol on the thyroid hormone-dependent expression of cardiac Kv1.5 mRNA. Rats were rendered hypothyroid by the addition of MMI. Hyperthyroidism was induced in the MMI-treated hypothyroid rats by the addition of T4 (20 mg/100g body weight/day) for the last 7 days. Propranolol (Pro) was added to the drinking water at 750 mg/liter and administered to the MMI-treated rats together with T4 for the last 7 days. Isoproterenol (Iso, 500 mg/100g body weight/day) was injected to the MMItreated rats for the last 7 days. Aliquots of 10 mg total RNA extracted from the left ventricles were subjected to RPA using 32P-labeled Kv1.5 and cyclophilin cRNAs as probes. A representative autoradiogram is shown in the upper panel. Open arrowheads indicate the positions of undigested cRNA probes for Kv1.5 and cyclophilin (Cyclo). The positions of protected cRNAs are indicated by the closed arrowheads. The abundances of Kv1.5 mRNA are normalized by cyclophilin mRNA levels and are expressed as arbitrary units in the lower panel. Values are the mean { S.E. (nÅ4). *: põ0.01.
mRNA level had been shown in the cardiac hypertrophy induced by renovascular hypertension (2). This might imply that the Kv1.5 mRNA level is regulated independently from the intracellular signals leading to hypertrophy. The Increase in Kv1.5 mRNA by Thyroid Hormone Is Not Mediated through b-Adrenergic Receptor It has been shown that thyroid hormone increases the number of b-adrenergic receptor (23-26), by increasing the transcription of its gene (27). Increased sensitivity of the heart to b-adrenergic stimuli in the
hyperthyroid rats may enhance the intracellular cAMP production. Recently, cAMP has been shown to increase the steady state levels of Kv1.5 mRNA in primary cardiac cells (28). Therefore, there is a possibility that the increased b-adrenergic stimuli might cause the increase in expression of Kv1.5 mRNA by thyroid hormone. To address this possibility, we examined the effects of propranolol, a b-adrenergic blocker, and isoproterenol, a b-adrenergic receptor agonist, on the expression of the mRNA. Propranolol was administered together with T4 to the MMI-treated hypothyroid rats for the last 7 days. Isoproterenol was injected to the MMI-treated hypothyroid rats for the last 7 days. As shown in Fig. 3, administration of T4 to the hypothyroid rats markedly increased the Kv1.5 mRNA in the rat left ventricle. However, the treatment of the hyperthyroid rats with pharmacological dose of propranolol did not inhibit the increase by T4 , indicating that the increase is not due to the increased b-adrenergic stimuli under hyperthyroidism. Accordingly, the treatment of the MMI-treated hypothyroid rats with isoproterenol did not increase the Kv1.5 mRNA. These results indicate that thyroid hormone regulates the Kv1.5 gene expression by a mechanism independent of a b-adrenergic receptor-mediating mechanism. It is possible that thyroid hormone directly increase the transcription of Kv1.5 gene as it activates the transcription of the genes for b-adrenergic receptor (27), myosin heavy chain a and b (29, 30), and sarcoplasmic reticulum calcium ATPase (31) in the cardiac myocytes. Glucocorticoid Is Required for the Up-Regulation of Kv1.5 mRNA by Thyroid Hormone It was reported that glucocorticoid agonist Dex increases Kv1.5 mRNA level in the rat ventricle (3). We thus examined the effects of Dex on the thyroid hormone-mediated induction of Kv1.5 mRNA. Rats were adrenalectomized and rendered hypothyroid by administration of MMI (ADX-MMI rats). Reduction of endogenous glucocorticoids and thyroid hormones was ascertained by measurement of serum hormone levels (data not shown). In this experiment, T3 was administered to the rats, since Dex modulates the expression of iodothyronine type I 5*-deiodinase in the liver as described in our previous report (10). As shown in Fig. 4, Kv1.5 mRNA was not detected in ADX-MMI rats. Interestingly, administration of T3 50 mg alone (a supraphysiological dose) to the ADX-MMI rats did not increase the mRNA level. Treatment with physiological dose of Dex 1 mg (15) neither restored the mRNA. However, administration of T3 50 mg together with Dex 1 mg markedly increased the Kv1.5 mRNA level. These results indicate that glucocorticoids are required for the T3-mediated induction of
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FIG. 4. Effect of thyroid and glucocorticoid hormones on the expression of cardiac Kv1.5 mRNA. Rats were adrenalectomized and given MMI. After 3-week treatment with MMI, Dex (0, 1, 10 and 100 mg/100g body weight/day) alone or in combination with T3 (50 mg/100g body weight/day) were administered to the rats for the last 7 days. A representative autoradiogram of cardiac Kv1.5 and GAPDH mRNA is shown in the upper panel. The abundances of Kv1.5 mRNA are normalized by GAPDH mRNA levels and are expressed as arbitrary units in the lower panel. Values are the mean { S.E. (nÅ3). *: põ0.05, **: põ0.01.
cardiac Kv1.5 gene expression. The supraphysiological doses of Dex 10 mg and 100 mg increased the mRNA even in the absence of T3 administration. Treatment with T3 together with Dex further increased the mRNA level compared to the level of Dex alone. Synergistic or additive effects of thyroid and glucocorticoid hormones have been reported in the regulation of growth hormone and iodothyronine type I 5*deiodinase genes (8-10). Our results showed that the expression of cardiac Kv1.5 gene is also cooperatively up-regulated by thyroid and glucocorticoid hormones. In addition, it is of interest that glucocorticoid is necessary for the thyroid hormone-mediated induction of cardiac Kv1.5 gene expression. A similar finding was reported in somatotroph development in the fetal rat pituitary gland. Nogami et al showed that Dex treatment of dams induces growth hormone mRNA in the fetal rat pituitary and that additional T4 treatment enhanced the effect of Dex (11). However, this T4 effect was not observed in the absence of Dex. Molecular mechanism underlying the cooperative effects of thyroid and glucocorticoid hormones has not been fully understood. In conclusion, the present study for the first time demonstrated that thyroid hormone increases the Kv1.5 mRNA in the rat left ventricle and that this effect is not mediated through b-adrenergic stimuli. Furthermore, we showed that glucocorticoid is indispensable for the thyroid-hormone-mediated induction. Our study showed that the expression of one of the voltage-gated potassium channel genes is under control of thyroid hormone. We are currently studying whether the expression of other potassium channel genes is altered by the hormone, in order to clarify overall effects
of thyroid hormone on the voltage-gated potassium channels. ACKNOWLEDGMENTS We thank Dr. Devanand Sarkar for critically reading the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (07670774) from the Ministry of Education, Science, Sports and Culture, Japan.
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