Inhibitors of Ca2+ channels, calmodulin and protein kinases prevent A23187 and other inductions of metallothionein mRNA in EC3 rat hepatoma cells.

The role of calcium in the induction of MT mRNA has been studied in EC3 rat hepatoma cells, using various inducers (A23187, TPA, norepinephrine, and 2-chloroadenosine) and inhibitors (H7:PK-A and PK-C; W7:calmodulin; verapamil:calcium channel blocker; and TMB-8; cytosolic calcium chelator). The inhibitions of inductions observed in this study were consistent with calcium playing an important role in MT mRNA induction by itself and via crosstalk among the PK-A, PK-C, and calmodulin-dependent protein kinase pathways. Calcium has an important role in the complicated second messenger pathways which result in the positive interaction of transcription factors with the promoters of MT genes.

Purinergic agonist induction of metallothionein.

Metallothionein (MT) protein is readily induced in vivo in rat liver by adenosine and adenosine agonists (2-chloroadenosine, 5-(N-ethyl) carboxamido adenosine, and 5-chloro-5-deoxyadenosine). These presumably operate via AMP/adenosine receptors of the P1 (A2) type, which use the cAMP pathway. ATP was ineffective as an inducer for MT. 2-Chloroadenosine was the most effective inducer (7.27-fold at 11 hr). This induction was blockable by the adenosine antagonists, caffeine and theophylline. MT protein induction by 2-chloroadenosine in primary cultured rat hepatocytes was modest (1.55-fold), but this was also blocked by theophylline. MT mRNA induction was assessed using dot blot and Northern gel assays. Large inductions by 2-chloroadenosine (5.1- to 41-fold) were seen, and these were detectable as early as 2 hr in vivo. Two rat hepatoma cell lines (EC3 and 2M) were studied in vitro. Modest inductions of MT mRNA were seen: 2.10-fold for EC3 and 4.12-fold for 2M. Our studies implicate the potential role of the purinergic system in the modulation of transcription of MT genes in rat liver. The sources of adenosine in vivo that might cause induction of MT mRNA and protein are not well defined, but adenosine may be important as a signal in stress response situations involving tissue damage, such as ischemia, hypoxia, and hemorrhagic shock.

Phorbol ester induction of rat hepatic metallothionein in vivo and in vitro.

1. The induction of metallothionein (MT) protein by TPA (O-tetradecanoyl phorbol acetate), a protein kinase C activator, was demonstrated in vivo in rat liver and in vitro in rat hepatocytes in primary culture. In vivo half maximal induction at 25 hr was seen at 26 nmol TPA/kg body wt. Five- to seven-fold inductions were seen in vivo. De novo protein synthesis was required for this induction as demonstrated by cycloheximide inhibition of [35S]cysteine incorporation into MT protein. 2. TPA induction of MT protein in primary cultures of rat hepatocytes reached levels of 2.6-4.1-fold, as assessed by [35S]cysteine incorporation, 1.34-2.20-fold, as assessed by 109Cd binding in a metal displacement/HPLC assay, and 2.5-5-fold, as assessed by 109Cd binding in a metal displacement/Sephadex G-75 Superfine assay. 3. The induction of MT mRNA by TPA was demonstrated in vivo in rat liver and in vitro in 2 rat hepatoma cell lines, EC3 and 2M. MT mRNA was quantitated using dot blot and Northern gel assays. In vivo TPA induced hepatic MT mRNA 2.36-5.88-fold (dot blot) and 7.4-22-fold (Northern gels). In vitro TPA induced MT mRNA 1.71-15.26-fold in EC3 cells and 2.23-8.43-fold in 2M cells. MT mRNA was 0.54 kb, and alpha-tubulin mRNA was 1.62 kb in size on Northern gels. 4. TPA induction of MT protein and mRNA in vivo and in vitro is rapid and persistent and occurs at low concentrations. The 2 rat hepatoma cell lines provide a useful system in which to study MT induction in vitro without confounding secondary effects which can occur in vivo.

Induction of zinc metallothionein by calcium ionophore in vivo and in vitro.

The calcium ionophore, A23187, can induce rat hepatic metallothionein (MT) when administered in vivo (5.8-fold, 5.0 microM, 11 h) and rat hepatocyte MT when administered in vitro (10.70-fold, 1.0 microM, 24 h). Several rat hepatoma cell lines (2M, 4.55-fold; JM2, 12.29-fold; EC3, 14.12-fold; HTC, 7.99-fold) and a normal rat liver cell line (Clone 9, 39.67-fold) were tested for their inducibility of MT mRNA by Cd2+ (10 microM, 8 h). Quantitatively, JM2 and 2M made the most MT mRNA, while HTC made the least. A23187 (0.1-7.0 microM) was studied as an inducer of MT mRNA in these cell lines (except for HTC) and in HeLa. A variety of responses and tolerances were seen with inductions ranging up to 32.11-fold. Quantitatively, the best responding cell lines were EC3 and 2M. A combination induction experiment, using TPA, a protein kinase C activator, and A23187 in EC3 cells revealed an additive effect of the two inducers on MT mRNA levels: TPA (10 nM), 11.71-fold; A23187 (3.0 microM), 6.71-fold; and TPA + A23187, 20.00-fold. These studies have implicated perturbations in cytosolic calcium ion concentrations, caused by the ionophore A23187, as being involved in the complicated signaling systems which can lead to induction of MT mRNA and protein.

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies.

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.

The involvement of catecholamines and polypeptide hormones in the multihormonal modulation of rat hepatic zinc thionein levels.

Catecholamines can induce rat hepatic zinc thionein to high levels via alpha 1- and beta 2-adrenoceptors. Polypeptide hormones (glucagon and angiotensin II) are also inducers, but only to the moderate levels attained by glucocorticoids (dexamethasone). Turpentine induced inflammation stimulates the synthesis of ZnMT, but this process is not mediated by catecholamines. Phorbol esters, which are tumor promoters, can stimulate protein kinase C. Angiotensin II and alpha 1-agonists activate protein kinase C via diacylglycerol release from phosphatidylinositol-4,5-diphosphate. Phorbol esters can also stimulate the synthesis of rat hepatic zinc thionein, implicating protein kinase C activation in this induction. The multihormonal modulation of metallothionein gene activation has become increasingly more complex.

Effects of glucagon, Arg-vasopressin, and angiotensin II on rat hepatic zinc thionein levels.

Rat hepatic zinc thionein levels can be modulated by a variety of external and internal stimuli. Metals, such as zinc or copper, induce levels 20 to 50 fold over controls. Catecholamines can increase levels 10 to 20 fold, while glucocorticoids, such as dexamethasone, can increase levels modestly by 2-6 fold. We have investigated the ability of additional hormones, which have receptors on hepatocytes, to modulate the levels of hepatic zinc thionein. Glucagon, angiotensin II, and Arg-vasopressin were administered intravenously and intraperitoneally, one time and three times, over an 11 hour period. Zinc thionein levels in rat liver were increased 1.7 to 5.6 fold by glucagon and 1.7 to 3.6 fold by angiotensin II, but not at all by Arg-vasopressin, as compared to appropriate controls. Glucagon and angiotensin II, when administered in vivo, can modulate zinc thionein levels in rat liver to an extent similar to glucocorticoids. Hepatic zinc thionein levels must now be recognized to be affected in vivo by metals, glucocorticoids, catecholamines, and polypeptide hormones.

Effect of epinephrine and norepinephrine on zinc thionein levels and induction in rat liver.

Hepatic zinc metallothionein (MT) levels are increased in response to a variety of stresses. Glucocorticoid induction of zinc thionein is insufficient in accounting for the levels attained. The potential involvement of catecholamines in the modulation of rat hepatic zinc metabolism and zinc thionein levels has been systematically studied. Eleven hours after multiple injections (6) of epinephrine, norepinephrine, or isoproterenol, zinc thionein levels of 4.01 +/- 0.74, 6.83 +/- 0.67, and 11.75 +/- 0.96 micrograms Zn in MT/g liver, respectively, were attained (untreated, 1.04 +/- 0.14). The levels of hepatic zinc thionein thus reached the range of stress response-induced levels (4-10 micrograms Zn in MT/g liver), attained 11 h after the onset of the stress. Multiple injections of isoproterenol and norepinephrine induced the formation of isoforms MT-I and MT-II in roughly equal amounts. The alpha-adrenoceptor blocker phentolamine blocked the 11-h increase in norepinephrine-stimulated (6) zinc thionein levels by 88%. The beta-adrenoceptor blocker propranolol blocked the 11-h increase in isoproterenol-stimulated (6) zinc thionein levels by 55%. This inhibition could be increased to 72% by previous administration of both phentolamine and propranolol. Catecholamines stimulated increases in both the zinc and the protein of MT, the latter as assessed by [35S]cysteine incorporation. Both of these increases were blocked by cycloheximide, confirming the requirement for de novo protein synthesis in this induction response.

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury.

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.

Metabolism of zinc and copper in the neonate: zinc thionein in developing rat brain, heart, lung, spleen, and thymus.

In a continuing study of the importance of metallothionein (MT) in the growth and development of neonates, zinc and copper metabolism in rat brain, heart, lung, spleen, and thymus has been analyzed in 5, 10, 15, 20, and 25 day old rats. Total, cytosol, and MT zinc and copper concentrations and organ contents were determined. Zinc, but very little, if any copper was associated with MT in these organs. Concentrations ranged from 0.03 to 3.3 micrograms Zn in MT/g; organ contents ranged from 0.003 to 2.2 micrograms Zn in MT/organ. Brain exhibited the highest concentrations and contents of zinc in MT, approaching the levels found in kidneys. Rank order of organ contents of zinc in MT was brain greater than lung greater than heart, spleen, thymus, during this neonatal growth period. When organ growth was rapid, a large percentage (20-95%) of the cytosolic zinc present in these organs was associated with MT, as has been previously observed with liver, kidneys, and testes. None of these organs undergoes the dramatic changes in zinc and copper metabolism previously observed in neonatal rat liver and gastrointestinal tract, and in maturing testes. They are more comparable to kidneys in their concentrations of zinc in MT. Like testes, little copper is found in these organs.

Metabolism of zinc and copper in the neonate: accumulation of Cu in the gastrointestinal tract of the newborn rat.

1. The concentration of copper in the rat intestine was found to increase rapidly after birth to a maximum greater than 140 microgram/g wet weight at 2 d of age and then to decline, at first slowly to 90 microgram/g wet weight on day thirteen and then rapidly to 40 microgram/g and 3 . 4 microgram/g wet weight on the 15th and 19th day respectively. The intestinal concentration of Zn, which doubled between 1 d prepartum and 2 d post partum, also fell slowly until 10 d of age, but thereafter remained constant. 2. From the 2nd to the 15th day post partum approximately 60% of the total Cu and 50% of the total zinc in the intestine was located in the soluble fraction of the tissue. Most of the Zn in this fraction was bound by proteins of molecular weights greater than 13700 daltons, whereas most of the Cu was present as an extremely polydisperse complex of lower molecular weight. This complex in the intestine of the 5-d-old rat, in contrast with the soluble proteins of higher molecular weight, did not incorporate either 3H or 35S within 4 h of the administration of L-[4,5-3H]leucine and L-[35S]cystine. 3. The loss of Cu from the intestine between the 13th and 15th day of post-natal age occurred mainly from this complex and was accompanied by the transient appearance of Cu in a fraction of low molecular weight. 4. At 21 d of age the soluble fraction of the intestine contained only a small amount of Cu. This was distributed between two protein fractions, one of which contained Zn and appeared to be a metallothionein. 5. The results are discussed in relation to the control of Zn and Cu absorption.

Reactivation in vitro of zinc-requiring apo-enzymes by rat liver zinc-thionein.

The ability of rat liver zinc-thionein to donate its metal to the apo-enzymes of the zinc enzymes horse liver alcohol dehydrogenase, yeast aldolase, thermolysin, Escherichia coli alkaline phosphatase and bovine erythrocyte carbonic anhydrase was investigated. Zinc-thionein was as good as, or better than, ZnSO(4), Zn(CH(3)CO(2))(2) or Zn(NO(3))(2) in donating its zinc to these apo-enzymes. Apo-(alcohol dehydrogenase) could not be reactivated by zinc salts or by zinc-thionein. Incubation of the other apo-enzymes with near-saturating amounts of zinc as ZnSO(4), Zn(CH(3)CO(2))(2), Zn(NO(3))(2), or zinc-thionein resulted in reactivation of the apo-enzymes. With apo-aldolase zinc-thionein gave 100% reactivation within 30min. Reactivation by ZnSO(4) and Zn(CH(3)CO(2))(2) was complete and instantaneous. Zinc-thionein was somewhat better than Zn(NO(3))(2) in completely reactivating apo-thermolysin. With apo-(alkaline phosphatase) 43% reactivation was obtained with Zn(CH(3)CO(2))(2) and 18% with zinc-thionein. With apo-(carbonic anhydrase) zinc-thionein was better than ZnSO(4), Zn(CH(3)CO(2))(2) or Zn(NO(3))(2), with a maximal reactivation of 54%. That zinc was really being transferred from zinc-thionein to apo-(carbonic anhydrase) was shown by the fact that 2,6-pyridine dicarboxylic acid and 1,10-phenanthroline had minimal effects on the reactivation of apo-(carbonic anhydrase) when added after the incubation {[apo-(carbonic anhydrase)+zinc thionein]+chelator}, but inhibited reactivation when added before the incubation {apo-(carbonic anhydrase)+[zinc-thionein+chelator]}. These observations support the idea that zinc-thionein can function in zinc homeostasis as a reservoir of zinc, releasing the metal to zinc-requiring metalloenzymes according to need.

The influence of zinc status on the levels of metallothionein in isolated perfused rat liver.

The levels of cadmium and zinc metallothionein in isolated perfused livers of zinc depleted and repleted rats were investigated. Rats (160-200 g or 90-120 g) were fed either a zinc-deficient or zinc-supplemented diet for 62 or 42 days. The 90 to 120 g rats were repleted with 30 ppm Zn2+ in the drinking water for 1, 4 and 10 days after 35 days of depletion, and another group after depletion for 21 days was repleted for 15 days on a 21% casein diet. At appropriate times livers were cannulated, removed and perfused +/- 25 mug Cd2+ as CdCl2, for 2 hours through the portal vein in a perfusion apparatus. Zinc depletion resulted in significant decreases in growth rate (1.4 versus 5.7 g/day) and liver weights. Repletion with Zn for 10 days increased the weight gain to 7.1 g/day. Depletion decreased the incorporation of Zn and Cd into metallothionein by 57 to 60%, while repletion for 1, 4 and 10 days increased the incorporation to nearly 500% of control levels. But, as repletion progressed, zinc in metallothionein fell to normal levels after 15 days. Cadmium in metallothionein did not decrease between 1 and 10 days of repletion. Zinc deficiency also reduced the zinc content of the liver by 44%, which increased to normal levels after 10 days of repletion. These data suport the conclusion that zinc status can influence the incorporation of cadmium and zinc into metallothionein.