Differential Effect of Adrenalectomy on Rat Liver Metallothionein mRNA Levels in Basal and Stress Conditions J. Hidalgo*, S. J. Rhee**, P. C. Huang and J. S. Garvey Department of Biology, Syracuse University, Syracuse, New York, and Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland, U. S. A.
Liver metallothionein (MT) mRNA and serum M T levels of adrenalectomized (ADX) and shamADX rats in basal and stress (1, 3 or 6 h of restraint) conditions have been measured. Serum MT levels were overall lower in ADX than in sham-ADX rats. Basal liver MT mRNA levels were increased in ADX rats, suggesting that glucocorticoids have an inhibitory role on the regulation of liver MT synthesis. In contrast, liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats, suggesting a stimulatory role for glucocorticoids. These results suggest that glucocorticoids have a different role in liver MT regulation depending on the physiological situation.
MT appears to be under complex endocrine control {Cousins 1985; Dunn, Blalock and Cousins 1987). Among the different hormones considered to have an important role on MT regulation, glucocorticoids have been the most studied. Thus, the synthetic glucocorticoid dexamethasone appears to be an effective inducer of MT both in vivo and in vitro {Cousins 1985; Dunn, Blalock and Cousins 1987). In addition, it has been demonstrated that dexamethasone is a direct inducer, like metals, of the MT gene {Karin, Andersen, Slater, Smith and Herschman 1980; Mayo and Palmiter 1981). However, the role of glucocorticoids on MT synthesis in physiological conditions remains conflicting in rodents. Whereas natural glucocorticoids appear to be MT inducers in HeLa Key words cells {Karin and Herschman 1980), the administration of Cortisol to hamsters {Klaassen 1981) and that of corticosterone to Serum and Liver Metallothionein — Liver either normal {Brady 1981; Bracken and Klaassen 1987a; LehMetallothionein mRNA — Adrenalectomy — Glucocorticoids — Dexamethasone — Corticosterone — Aldosterone — man-McKeeman, Andrews and Klaassen 1988) or adrenalectomized {Brady and Burger 1979) rats failed to alter substanStress tially liver MT levels at doses well above physiological levels. Furthermore, the administration of ACTH {McCormick 1987) and that of a long-lastingACTH-(1-24)preparation {Hidalgo et al. 1988b) did not alter liver MT levels either. Moreover, stuIntroduction dies with rat primary hepatocyte cultures indicated that corticosterone was able to (modestly) increase MT levels only at Metallothionein (MT) is a low-molecular concentrations at least 100 times higher than those achievable weight, cysteine- and heavy metal-rich protein. Initially MT in the plasma of severely stressed rats {Bracken and Klaassen was thought to be involved in heavy metal detoxification 1987b). The results with adrenalectomized (ADX) rats also {Webb 1986), but it now appears more likely that MT is inargue against a positive role of glucocorticoids on liver MT volved in Zn and Cu metabolism {Cousins 1985; Dunn, regulation, as ADX rats show consistently increased liver MT Blalock and Cousins 1987) and in the adaptation of the orlevels {Brady and Burger 1979; Hidalgo et al. 1988b; DiSilvesganism to stress {Brady 1981; Oh, Deagen, Whanger and tro and Cousins 1984; Failla, Caperna and Dougherty 1986). Weswig 1978; Hidalgo, Campmany, Borras, Garvey and Armaria 1988a; Hidalgo, Giralt, Garvey and Armario 1988b). MT has been demonstrated to be an effective free radical Therefore, it appears that the role of glucocorscavenger in vitro {Thornalley and Vasak 1985; Thomas, Baticoids on liver MT regulation in physiological conditions is at present unclear. In addressing this problem, we have previously examined in detail the role of glucocorticoids on liver *Present address: and serum MT levels in the rat under a spectrum of experimenDepartamento de Biologia Celular y Fisiologia tal conditions {Hidalgo et al. 1988b). The results indicated that Facultad de Ciencias, Universidad Autonoma de Barcelona corticosterone, the most important glucocorticoid in the rat, Bellaterra, Barcelona, Spain. had in fact an inhibitory role on the maintenance of MT levels **Present address: in the liver; glucocorticoids, in contrast, appeared to mediate Hyosung Women's University the release of M T into the serum at least in some circumKyungbuk, Taegu, Korea. Horm.metab.Res.24(1992)233-336 © Georg Thieme Verlag Stuttgart • New York
Received: 7 Dec. 1990
Accepted: 30 Sept. 1991
Downloaded by: NYU. Copyrighted material.
Summary
chowski and Girotti 1986) and an antioxidant role for liver MT during stress has recently been suggested {Hidalgo et al. 1988a).
Horm. metab. Res. 24 (1992)
J. Hidalgo, S. J. Rhee, P. C. Huang and J. S. Garvey
Fig. 2 Serum MT levels of adrenalectomized (ADX) and shamadrenalectomized (sham-ADX) rats in basal and stress conditions. Stressed rats were subjected to 1, 3 or 6 h of restraint stress. Data are mean ± standard error. For statistical significances see Results.
(Garvey 1984). Results were analyzed with the analysis of the variance (ANOVA) with one (time of stress) or two factors (time of stress and experimental condition, i. e., ADX vs sham-ADX). Results
Fig. 1 Liver MT mRNA accumulation of adrenalectomized (ADX) and sham-adrenalectomized (sham-ADX) rats in basal and stress conditions. Stressed rats were subjected to 1, 3 or 6 h of restraint stress. MT mRNA levels were measured in pools of 4 rats per group (A) and also in a single liver from a 5th rat per group (B) as described in Materials and Methods.
stances. The aim of the present study was to obtain further insight concerning the physiological role of glucocorticoids on liver M T synthesis by measuring the effect of adrenalectomy (ADX) on rat liver MT mRNA accumulation in basal and stress situations.
Figure 1 shows MT mRNA accumulation in the liver of ADX and sham-ADX rats in basal and stress situations. The results shown are those measured in pooled samples from 4 rats per group (Figure 1 A) as well as those obtained in a separate assay performed with a single liver from a 5th rat (Figure 1B), which are shown as representative of the variation between individuals. Essentially the same results were obtained in both assays, namely (a) liver MT mRNA levels were higher in ADX than in sham-ADX rats, and (b) liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats.
Figure 2 shows serum MT levels of all the experimental groups. Two-way ANOVA with time of stress and experimental condition (ADX vs sham-ADX) as main factors indicated that the time of stress did not affect significantly Materials and Methods serum MT levels (p < 0.282) and that these were significantly (p < 0.001) lower overall in ADX rats. The interaction beADX and sham-ADX male Sprague-Dawley rats tween both factors was significant (p < 0.03), suggesting that were purchased from Charles River Breeding Laboratories and were the adrenalectomy was modifying the response of serum MT 47 days old when killed. They were maintained in groups of four per to stress, as could be deduced easily seeing Figure 2. This was cage under standard conditions. ADX rats received saline solution confirmed by analyzing the effect of the time period of stress (0.9 % NaCl) as their drinking fluid. on serum MT separately for ADX and sham-ADX rats. OneOne week after their arrival, the rats were subjected way ANOVA followed by the Duncan multiple range test indito 0, 1, 3 or 6 hours of restraint stress caused by having each rat encated that serum MT levels were significantly (p < 0.05) lower closed in a wrapping of metallic net. Six to eight rats comprised each in 6 h-stressed, ADX rats, compared to basal and 1 and 3 fagroup. The rats were killed by decapitation; the trunk blood was collected at 4 °C and the serum stored at — 20 °C. The livers were immedi- stressed rats. No effect was observed in sham-ADX rats (p < 0.68). In a separate experiment, we have observed that ately removed and a piece of =250 mg, selected uniformly from a ADX rats maintained with corticosterone, but not with allobe, was wrapped in aluminum foil and submerged into liquid nitrogen. The packages were then stored at — 70 °C in a Revco ultra-freezer. dosterone, therapy have higher serum MT levels than ADX Liver MT mRNA levels were measured in those samples essentially as rats (see below). previously described (Thomas, Morris and Huang 1988). A preliminary assay was performed with a single liver from each of the 8 groups Discussion (i. e. 0,1,3,6 h for ADX and sham-ADX). Then pieces of liver of 4 rats per group were pooled for a second assay. Autoradiography of cytodot blot filters was followed by excision and counting of the dots to quanIn basal conditions, liver MT mRNA levels tify hybridizable mRNA. Serum MT levels were analyzed by a highly were clearly higher in ADX rats than in sham-ADX rats. This is sensitive and specific radioimmunoassay as previously described consistent with the protein levels found in the liver of ADX
Downloaded by: NYU. Copyrighted material.
234
Effect of Adrenalectomy on MT Regulation
To study the effect of stress on liver MT mRNA accumulation, some rats were subjected to 1, 3 or 6 h of restraint stress. As expected, liver MT mRNA levels were clearly increased by restraint stress in sham-ADX rats. They were approximately twice those of basal rats in 1 h-stressed rats, remained elevated in 3 h-stressed rats, and returned to basal levels in 6 h-stressed rats. In contrast, liver MT mRNA levels were not increased by stress in ADX rats. In fact, there was a tendency for liver MT mRNA levels to be decreased by stress throughout the stress period. This suggests that glucocorticoids are needed for liver M T induction by stress. Therefore, these results suggest that glucocorticoids may have a different role on the regulation of the M T gene depending on the physiological situation, i. e. inhibiting it in basal conditions, and activating it in stress conditions. The latter is particularly noteworthy, since a role for glucocorticoids mediating liver MT induction by stress has long been speculated (Cousins 1985; Mayo and Palmiter 1985) but never demonstrated. The above results for MT mRNA would indicate that MT concentration in the liver should be higher in ADX rats than in sham-ADX rats in basal conditions, and lower in stress conditions. However, liver MT levels are higher in ADX than in sham-ADX rats not only in basal but also during acute and chronic stress situations (Hidalgo et al. 1988b). This discrepancy between MT mRNA and protein accumulation levels could be explained by the fact that glucocorticoids appear to mediate the MT flux from the liver into the circulation during stress (Hidalgo et al. 1988b). The present results also indicate that, in general, ADX rats had lower serum MT levels than sham-ADX rats. ADX rats failed especially to maintain normal serum MT levels after 6 hours of stress. This failure extends to longer time periods of stress, although serum MT levels are then increased compared to basal levels (Hidalgo et al. 1988b), likely because of increased liver MT levels. In a separate experiment, we have observed that ADX rats maintained with corticosterone, but not aldosterone, therapy have higher serum MT levels than ADX rats (2.3 + 0.21, 3.2 ±0.22, and 2.3 + 0.23 ng MT/ml serum, mean + SE, n = 7 - 9 , for ADX, ADX + corticosterone, and ADX + al-
235
dosterone, respectively). Therefore, taken together these results suggest a role for glucocorticoids on the regulation of serum MT levels. On the light of the above results, liver MT regulation might be regarded differently in basal and stress situations. In the former, the inhibitory role of glucocorticoids would likely counteract and/or balance the positive role of Zn (which is a primary inducer of the MT gene) and/or other hormones on MT gene regulation; in the latter, glucocorticoids in addition to Zn and/or stress hormones that are known to induce liver MT levels, such as catecholamines (Brady and Helvig 1984), would increase liver MT synthesis. This synthesis would lead not to an increase in liver MT concentration but rather to an increased export of the protein, which might participate in the redistribution/metabolism of Zn during stress. Acknowledgements The authors acknowledge Betty C. Smith for her able assistance and a PDF fellowship from the Korean Engineering and Science Foundation (to SJR). J. Hidalgo and J. S. Garvey acknowledge the support of NIH grant ES 01629. J. Hidalgo also acknowledges the support of the Fondo de Investigaciones Sanitarias de la Seguridad Social, Grant 90/0065-2-D. References Bracken, W. M., C. D. Klaassen: Induction of hepatic metallothionein by alcohols: evidence for an indirect mechanism. Toxicol. Appl. Pharmacol. 87: 257-263 (1987a) Bracken, W. M., C. D. Klaassen: Induction of metallothionein by steroids in rat primary hepatocyte cultures. Toxicol. Appl. Pharmacol. 87: 381-388 (1987b) Brady, F. O.: Synthesis of rat hepatic zinc thionein in response to the stress of sham operation. Life Sci. 28:1647—1655 (1981) Brady, F. O., P. C. Burger: The effect of adrenalectomy on zinc thionein levels in rat liver. Biochem. Biophys. Res. Commun. 91: 911-918(1979) Brady, F. O., B. Helvig: Effect of epinephrine and norepinephrine on zinc thionein levels and induction in rat liver. Am. J. Physiol. 247: E318-E322(1984) Cousins, R. J.: Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65:238-309(1985) DiSilvestro, R. A., R. J. Cousins: Glucocorticoid independent mediation of interleukin-1 induced changes in serum zinc and liver metallothionein levels. Life Sci. 35:2113-2118 (1984) Dunn, M. A., T. L. Blalock, R. J. Cousins: Metallothionein. Proc. Soc. Exp. Biol. Med. 185: 107-199 (1987) Failla, M. L., T. J. Caperna, J. M. Dougherty: Influence of pancreatic and adrenal hormones on altered trace metal metabolism in the STZ-diabetic rat. In: Trace elements in man and animals; Mills C. F., I. Bremner, J. K. Chesters (eds). Commonwealth Agricultural Bureaux, Aberdeen (1986), pp. 330—333 Garvey, J. S.: Metallothionein: structure/antigenicity and detection/quantitation in normal physiological fluids. Environ. Health Perspect. 54: 117-127(1984) Hidalgo, J., L. Campmany, M. Borras, J. S. Garvey, A. Armario: Metallothionein response to stress in rats: role in free radical scavenging. Am. J. Physiol. 255: E518-E524(1988a) Hidalgo, J., M. Giralt, J. S. Garvey, A. Armario: Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions. Am. J. Physiol. 254: E71-E78 (1988b) Karin, M., R. D. Andersen, E. Slater, K. Smith, H. R. Herschman: Metallothionein mRNA induction in HeLa cells in response to
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and sham-ADX rats, i. e., we (Hidalgo et al. 1988b) and other laboratories (Brady and Burger 1979; DiSilvestro and Cousins 1984; Failla, Caperna and Dougherty 1986) have found that liver MT levels are increased by ADX. This suggests that the MT protein levels in the liver directly reflect MT synthesis, and that a factor present in the adrenal gland is regulating (inhibiting) liver MT synthesis in basal conditions. Previous studies (Hidalgo et al. 1988b) indicated that corticosterone but not aldosterone accounted for the effect of ADX on liver MT levels. Therefore, taken together, these data suggest that corticosterone is the adrenal factor that inhibits liver MT synthesis in basal conditions. This is in contrast to the results obtained with the synthetic glucocorticoid, dexamethasone, which exerts a direct positive effect on MT genes (Karin et al. 1980; Mayo and Palmiter 1981). However, the role of glucocorticoids on liver MT regulation might be different in basal situations compared to their role in other physiological situations (i. e. Quaife, Hammer, Mottet and Palmiter (1986) have suggested that corticosterone might be important in fetal liver MT regulation). This could indeed be the case in the MT response to acute stress, which is undoubtedly one of the most important physiological roles of MT.
Horm. metab. Res. 24 (1992)
zinc or dexamethasone is a primary induction response. Nature London 286: 295-297 (1980) Karin, M., H. R. Herschman: Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells. J. Cell. Physiol. 103:35-40(1980) Klaassen, C. D.: Induction of metallothionein by adrenocortical steroids. Toxicology 20: 275-279 (1981) Lehman-McKeeman, L. D., G. K. Andrews, C. D. Klaassen: Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats. Biochem. J. 249: 4 2 9 - 4 3 3 (1988) Mayo, K. E., R. D. Palmiter: Glucocorticoid regulation of metallothionein-I mRNA synthesis in cultured mouse cells. J. Biol. Chem. 256: 2621-2624 (1981) Mayo, K. E., R. D. Palmiter: Glucocorticoid regulation of the mouse metallothionein gene expression. In: Biochemical Actions of Hormones; Litwack G. (ed). Academic, New York (1985), pp. 6 9 - 88 McCormick, C. C: Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement. Proc. Soc. Exp. Biol. Med. 185: 413-419 (1987) Oh, S. H., J. T. Deagen, P. D. Whanger, P. H. Weswig: Biological function of metallothionein. V. Its induction by various stresses. Am. J. Physiol. 234: E282-E285 (1978) Quaife, C, R. E. Hammer, N. K. Mottet, R. D. Palmiter: Glucocorticoid regulation of metallothionein during murine development. Develop. Biol. 118: 549-555 (1986)
J. Hidalgo, S. J. Rhee, P. C. Huang and J. S. Garvey Thomas, J. P., G. J. Bachowski, A. W. Girotti: Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins. Biochim.Biophys. Acta 884:448-461 (1986) Thomas, D. J., S. Morris, P. C. Huang: Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium. Develop. Genet. 9: 1 3 - 2 2 (1988) Thornalley, P. J., M. Vasak: Possible role for metallothionein in protection against radiation induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim.Biophys. Acta 827:36-44(1985) Webb, M.: Role of metallothionein in cadmium metabolism. In: Handbook of experimental pharmacology; Foulkes E. C. (ed). Ed. Springer Verlag,Berlin(1986),pp.281-337
Requests for reprints should be addressed to: Dr. Juan Hidalgo Departamento de Biologia Celular y Fisiologia Facultad de Ciencias Universidad Autonoma de Barcelona Bellaterra 08193 Barcelona (Spain)
Downloaded by: NYU. Copyrighted material.
236 Horm. metab. Res. 24 (1992)
Liver metallothionein (MT) mRNA and serum M T levels of adrenalectomized (ADX) and shamADX rats in basal and stress (1, 3 or 6 h of restraint) conditions have been measured. Serum MT levels were overall lower in ADX than in sham-ADX rats. Basal liver MT mRNA levels were increased in ADX rats, suggesting that glucocorticoids have an inhibitory role on the regulation of liver MT synthesis. In contrast, liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats, suggesting a stimulatory role for glucocorticoids. These results suggest that glucocorticoids have a different role in liver MT regulation depending on the physiological situation.
MT appears to be under complex endocrine control {Cousins 1985; Dunn, Blalock and Cousins 1987). Among the different hormones considered to have an important role on MT regulation, glucocorticoids have been the most studied. Thus, the synthetic glucocorticoid dexamethasone appears to be an effective inducer of MT both in vivo and in vitro {Cousins 1985; Dunn, Blalock and Cousins 1987). In addition, it has been demonstrated that dexamethasone is a direct inducer, like metals, of the MT gene {Karin, Andersen, Slater, Smith and Herschman 1980; Mayo and Palmiter 1981). However, the role of glucocorticoids on MT synthesis in physiological conditions remains conflicting in rodents. Whereas natural glucocorticoids appear to be MT inducers in HeLa Key words cells {Karin and Herschman 1980), the administration of Cortisol to hamsters {Klaassen 1981) and that of corticosterone to Serum and Liver Metallothionein — Liver either normal {Brady 1981; Bracken and Klaassen 1987a; LehMetallothionein mRNA — Adrenalectomy — Glucocorticoids — Dexamethasone — Corticosterone — Aldosterone — man-McKeeman, Andrews and Klaassen 1988) or adrenalectomized {Brady and Burger 1979) rats failed to alter substanStress tially liver MT levels at doses well above physiological levels. Furthermore, the administration of ACTH {McCormick 1987) and that of a long-lastingACTH-(1-24)preparation {Hidalgo et al. 1988b) did not alter liver MT levels either. Moreover, stuIntroduction dies with rat primary hepatocyte cultures indicated that corticosterone was able to (modestly) increase MT levels only at Metallothionein (MT) is a low-molecular concentrations at least 100 times higher than those achievable weight, cysteine- and heavy metal-rich protein. Initially MT in the plasma of severely stressed rats {Bracken and Klaassen was thought to be involved in heavy metal detoxification 1987b). The results with adrenalectomized (ADX) rats also {Webb 1986), but it now appears more likely that MT is inargue against a positive role of glucocorticoids on liver MT volved in Zn and Cu metabolism {Cousins 1985; Dunn, regulation, as ADX rats show consistently increased liver MT Blalock and Cousins 1987) and in the adaptation of the orlevels {Brady and Burger 1979; Hidalgo et al. 1988b; DiSilvesganism to stress {Brady 1981; Oh, Deagen, Whanger and tro and Cousins 1984; Failla, Caperna and Dougherty 1986). Weswig 1978; Hidalgo, Campmany, Borras, Garvey and Armaria 1988a; Hidalgo, Giralt, Garvey and Armario 1988b). MT has been demonstrated to be an effective free radical Therefore, it appears that the role of glucocorscavenger in vitro {Thornalley and Vasak 1985; Thomas, Baticoids on liver MT regulation in physiological conditions is at present unclear. In addressing this problem, we have previously examined in detail the role of glucocorticoids on liver *Present address: and serum MT levels in the rat under a spectrum of experimenDepartamento de Biologia Celular y Fisiologia tal conditions {Hidalgo et al. 1988b). The results indicated that Facultad de Ciencias, Universidad Autonoma de Barcelona corticosterone, the most important glucocorticoid in the rat, Bellaterra, Barcelona, Spain. had in fact an inhibitory role on the maintenance of MT levels **Present address: in the liver; glucocorticoids, in contrast, appeared to mediate Hyosung Women's University the release of M T into the serum at least in some circumKyungbuk, Taegu, Korea. Horm.metab.Res.24(1992)233-336 © Georg Thieme Verlag Stuttgart • New York
Received: 7 Dec. 1990
Accepted: 30 Sept. 1991
Downloaded by: NYU. Copyrighted material.
Summary
chowski and Girotti 1986) and an antioxidant role for liver MT during stress has recently been suggested {Hidalgo et al. 1988a).
Horm. metab. Res. 24 (1992)
J. Hidalgo, S. J. Rhee, P. C. Huang and J. S. Garvey
Fig. 2 Serum MT levels of adrenalectomized (ADX) and shamadrenalectomized (sham-ADX) rats in basal and stress conditions. Stressed rats were subjected to 1, 3 or 6 h of restraint stress. Data are mean ± standard error. For statistical significances see Results.
(Garvey 1984). Results were analyzed with the analysis of the variance (ANOVA) with one (time of stress) or two factors (time of stress and experimental condition, i. e., ADX vs sham-ADX). Results
Fig. 1 Liver MT mRNA accumulation of adrenalectomized (ADX) and sham-adrenalectomized (sham-ADX) rats in basal and stress conditions. Stressed rats were subjected to 1, 3 or 6 h of restraint stress. MT mRNA levels were measured in pools of 4 rats per group (A) and also in a single liver from a 5th rat per group (B) as described in Materials and Methods.
stances. The aim of the present study was to obtain further insight concerning the physiological role of glucocorticoids on liver M T synthesis by measuring the effect of adrenalectomy (ADX) on rat liver MT mRNA accumulation in basal and stress situations.
Figure 1 shows MT mRNA accumulation in the liver of ADX and sham-ADX rats in basal and stress situations. The results shown are those measured in pooled samples from 4 rats per group (Figure 1 A) as well as those obtained in a separate assay performed with a single liver from a 5th rat (Figure 1B), which are shown as representative of the variation between individuals. Essentially the same results were obtained in both assays, namely (a) liver MT mRNA levels were higher in ADX than in sham-ADX rats, and (b) liver MT mRNA levels were increased by stress in sham-ADX but not in ADX rats.
Figure 2 shows serum MT levels of all the experimental groups. Two-way ANOVA with time of stress and experimental condition (ADX vs sham-ADX) as main factors indicated that the time of stress did not affect significantly Materials and Methods serum MT levels (p < 0.282) and that these were significantly (p < 0.001) lower overall in ADX rats. The interaction beADX and sham-ADX male Sprague-Dawley rats tween both factors was significant (p < 0.03), suggesting that were purchased from Charles River Breeding Laboratories and were the adrenalectomy was modifying the response of serum MT 47 days old when killed. They were maintained in groups of four per to stress, as could be deduced easily seeing Figure 2. This was cage under standard conditions. ADX rats received saline solution confirmed by analyzing the effect of the time period of stress (0.9 % NaCl) as their drinking fluid. on serum MT separately for ADX and sham-ADX rats. OneOne week after their arrival, the rats were subjected way ANOVA followed by the Duncan multiple range test indito 0, 1, 3 or 6 hours of restraint stress caused by having each rat encated that serum MT levels were significantly (p < 0.05) lower closed in a wrapping of metallic net. Six to eight rats comprised each in 6 h-stressed, ADX rats, compared to basal and 1 and 3 fagroup. The rats were killed by decapitation; the trunk blood was collected at 4 °C and the serum stored at — 20 °C. The livers were immedi- stressed rats. No effect was observed in sham-ADX rats (p < 0.68). In a separate experiment, we have observed that ately removed and a piece of =250 mg, selected uniformly from a ADX rats maintained with corticosterone, but not with allobe, was wrapped in aluminum foil and submerged into liquid nitrogen. The packages were then stored at — 70 °C in a Revco ultra-freezer. dosterone, therapy have higher serum MT levels than ADX Liver MT mRNA levels were measured in those samples essentially as rats (see below). previously described (Thomas, Morris and Huang 1988). A preliminary assay was performed with a single liver from each of the 8 groups Discussion (i. e. 0,1,3,6 h for ADX and sham-ADX). Then pieces of liver of 4 rats per group were pooled for a second assay. Autoradiography of cytodot blot filters was followed by excision and counting of the dots to quanIn basal conditions, liver MT mRNA levels tify hybridizable mRNA. Serum MT levels were analyzed by a highly were clearly higher in ADX rats than in sham-ADX rats. This is sensitive and specific radioimmunoassay as previously described consistent with the protein levels found in the liver of ADX
Downloaded by: NYU. Copyrighted material.
234
Effect of Adrenalectomy on MT Regulation
To study the effect of stress on liver MT mRNA accumulation, some rats were subjected to 1, 3 or 6 h of restraint stress. As expected, liver MT mRNA levels were clearly increased by restraint stress in sham-ADX rats. They were approximately twice those of basal rats in 1 h-stressed rats, remained elevated in 3 h-stressed rats, and returned to basal levels in 6 h-stressed rats. In contrast, liver MT mRNA levels were not increased by stress in ADX rats. In fact, there was a tendency for liver MT mRNA levels to be decreased by stress throughout the stress period. This suggests that glucocorticoids are needed for liver M T induction by stress. Therefore, these results suggest that glucocorticoids may have a different role on the regulation of the M T gene depending on the physiological situation, i. e. inhibiting it in basal conditions, and activating it in stress conditions. The latter is particularly noteworthy, since a role for glucocorticoids mediating liver MT induction by stress has long been speculated (Cousins 1985; Mayo and Palmiter 1985) but never demonstrated. The above results for MT mRNA would indicate that MT concentration in the liver should be higher in ADX rats than in sham-ADX rats in basal conditions, and lower in stress conditions. However, liver MT levels are higher in ADX than in sham-ADX rats not only in basal but also during acute and chronic stress situations (Hidalgo et al. 1988b). This discrepancy between MT mRNA and protein accumulation levels could be explained by the fact that glucocorticoids appear to mediate the MT flux from the liver into the circulation during stress (Hidalgo et al. 1988b). The present results also indicate that, in general, ADX rats had lower serum MT levels than sham-ADX rats. ADX rats failed especially to maintain normal serum MT levels after 6 hours of stress. This failure extends to longer time periods of stress, although serum MT levels are then increased compared to basal levels (Hidalgo et al. 1988b), likely because of increased liver MT levels. In a separate experiment, we have observed that ADX rats maintained with corticosterone, but not aldosterone, therapy have higher serum MT levels than ADX rats (2.3 + 0.21, 3.2 ±0.22, and 2.3 + 0.23 ng MT/ml serum, mean + SE, n = 7 - 9 , for ADX, ADX + corticosterone, and ADX + al-
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dosterone, respectively). Therefore, taken together these results suggest a role for glucocorticoids on the regulation of serum MT levels. On the light of the above results, liver MT regulation might be regarded differently in basal and stress situations. In the former, the inhibitory role of glucocorticoids would likely counteract and/or balance the positive role of Zn (which is a primary inducer of the MT gene) and/or other hormones on MT gene regulation; in the latter, glucocorticoids in addition to Zn and/or stress hormones that are known to induce liver MT levels, such as catecholamines (Brady and Helvig 1984), would increase liver MT synthesis. This synthesis would lead not to an increase in liver MT concentration but rather to an increased export of the protein, which might participate in the redistribution/metabolism of Zn during stress. Acknowledgements The authors acknowledge Betty C. Smith for her able assistance and a PDF fellowship from the Korean Engineering and Science Foundation (to SJR). J. Hidalgo and J. S. Garvey acknowledge the support of NIH grant ES 01629. J. Hidalgo also acknowledges the support of the Fondo de Investigaciones Sanitarias de la Seguridad Social, Grant 90/0065-2-D. References Bracken, W. M., C. D. Klaassen: Induction of hepatic metallothionein by alcohols: evidence for an indirect mechanism. Toxicol. Appl. Pharmacol. 87: 257-263 (1987a) Bracken, W. M., C. D. Klaassen: Induction of metallothionein by steroids in rat primary hepatocyte cultures. Toxicol. Appl. Pharmacol. 87: 381-388 (1987b) Brady, F. O.: Synthesis of rat hepatic zinc thionein in response to the stress of sham operation. Life Sci. 28:1647—1655 (1981) Brady, F. O., P. C. Burger: The effect of adrenalectomy on zinc thionein levels in rat liver. Biochem. Biophys. Res. Commun. 91: 911-918(1979) Brady, F. O., B. Helvig: Effect of epinephrine and norepinephrine on zinc thionein levels and induction in rat liver. Am. J. Physiol. 247: E318-E322(1984) Cousins, R. J.: Absorption, transport and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol. Rev. 65:238-309(1985) DiSilvestro, R. A., R. J. Cousins: Glucocorticoid independent mediation of interleukin-1 induced changes in serum zinc and liver metallothionein levels. Life Sci. 35:2113-2118 (1984) Dunn, M. A., T. L. Blalock, R. J. Cousins: Metallothionein. Proc. Soc. Exp. Biol. Med. 185: 107-199 (1987) Failla, M. L., T. J. Caperna, J. M. Dougherty: Influence of pancreatic and adrenal hormones on altered trace metal metabolism in the STZ-diabetic rat. In: Trace elements in man and animals; Mills C. F., I. Bremner, J. K. Chesters (eds). Commonwealth Agricultural Bureaux, Aberdeen (1986), pp. 330—333 Garvey, J. S.: Metallothionein: structure/antigenicity and detection/quantitation in normal physiological fluids. Environ. Health Perspect. 54: 117-127(1984) Hidalgo, J., L. Campmany, M. Borras, J. S. Garvey, A. Armario: Metallothionein response to stress in rats: role in free radical scavenging. Am. J. Physiol. 255: E518-E524(1988a) Hidalgo, J., M. Giralt, J. S. Garvey, A. Armario: Physiological role of glucocorticoids on rat serum and liver metallothionein in basal and stress conditions. Am. J. Physiol. 254: E71-E78 (1988b) Karin, M., R. D. Andersen, E. Slater, K. Smith, H. R. Herschman: Metallothionein mRNA induction in HeLa cells in response to
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and sham-ADX rats, i. e., we (Hidalgo et al. 1988b) and other laboratories (Brady and Burger 1979; DiSilvestro and Cousins 1984; Failla, Caperna and Dougherty 1986) have found that liver MT levels are increased by ADX. This suggests that the MT protein levels in the liver directly reflect MT synthesis, and that a factor present in the adrenal gland is regulating (inhibiting) liver MT synthesis in basal conditions. Previous studies (Hidalgo et al. 1988b) indicated that corticosterone but not aldosterone accounted for the effect of ADX on liver MT levels. Therefore, taken together, these data suggest that corticosterone is the adrenal factor that inhibits liver MT synthesis in basal conditions. This is in contrast to the results obtained with the synthetic glucocorticoid, dexamethasone, which exerts a direct positive effect on MT genes (Karin et al. 1980; Mayo and Palmiter 1981). However, the role of glucocorticoids on liver MT regulation might be different in basal situations compared to their role in other physiological situations (i. e. Quaife, Hammer, Mottet and Palmiter (1986) have suggested that corticosterone might be important in fetal liver MT regulation). This could indeed be the case in the MT response to acute stress, which is undoubtedly one of the most important physiological roles of MT.
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zinc or dexamethasone is a primary induction response. Nature London 286: 295-297 (1980) Karin, M., H. R. Herschman: Glucocorticoid hormone receptor mediated induction of metallothionein synthesis in HeLa cells. J. Cell. Physiol. 103:35-40(1980) Klaassen, C. D.: Induction of metallothionein by adrenocortical steroids. Toxicology 20: 275-279 (1981) Lehman-McKeeman, L. D., G. K. Andrews, C. D. Klaassen: Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats. Biochem. J. 249: 4 2 9 - 4 3 3 (1988) Mayo, K. E., R. D. Palmiter: Glucocorticoid regulation of metallothionein-I mRNA synthesis in cultured mouse cells. J. Biol. Chem. 256: 2621-2624 (1981) Mayo, K. E., R. D. Palmiter: Glucocorticoid regulation of the mouse metallothionein gene expression. In: Biochemical Actions of Hormones; Litwack G. (ed). Academic, New York (1985), pp. 6 9 - 88 McCormick, C. C: Iron-induced accumulation of hepatic metallothionein: the lack of glucocorticoid involvement. Proc. Soc. Exp. Biol. Med. 185: 413-419 (1987) Oh, S. H., J. T. Deagen, P. D. Whanger, P. H. Weswig: Biological function of metallothionein. V. Its induction by various stresses. Am. J. Physiol. 234: E282-E285 (1978) Quaife, C, R. E. Hammer, N. K. Mottet, R. D. Palmiter: Glucocorticoid regulation of metallothionein during murine development. Develop. Biol. 118: 549-555 (1986)
J. Hidalgo, S. J. Rhee, P. C. Huang and J. S. Garvey Thomas, J. P., G. J. Bachowski, A. W. Girotti: Inhibition of cell membrane lipid peroxidation by cadmium- and zinc-metallothioneins. Biochim.Biophys. Acta 884:448-461 (1986) Thomas, D. J., S. Morris, P. C. Huang: Age-dependent variation for inducibility of metallothionein genes in mouse liver by cadmium. Develop. Genet. 9: 1 3 - 2 2 (1988) Thornalley, P. J., M. Vasak: Possible role for metallothionein in protection against radiation induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim.Biophys. Acta 827:36-44(1985) Webb, M.: Role of metallothionein in cadmium metabolism. In: Handbook of experimental pharmacology; Foulkes E. C. (ed). Ed. Springer Verlag,Berlin(1986),pp.281-337
Requests for reprints should be addressed to: Dr. Juan Hidalgo Departamento de Biologia Celular y Fisiologia Facultad de Ciencias Universidad Autonoma de Barcelona Bellaterra 08193 Barcelona (Spain)
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