Effects of the novel thyroid hormone analogues, SKF L-94901, DIBIT, and 3'-AC-T2 on mitochondrial function.

The effects of L-T3 (3,3',5-triiodo-L-thyronine) and three novel analogues, SKF L-94901 (3,5-Dibromo-3'-pyridazinone-L-thyronine), Dibit (3,5-Dibromo-3'-isopropyl-L-thyronine), and 3'-Ac-T2(3'-Acetyl-3,5,-Diiodo-L-thyronine), on mitochondrial parameters were determined in hypothyroid rats. The parameters include the 24 hour hormone-induced changes in the bc1 complex and in the proton permeability of the mitochondrial inner membrane. The cardiac sparing analogue, SKF L-94901, had no effect on mitochondrial respiration or proton permeability; but the analogue did increase a-glycerophosphate dehydrogenase activity, mitochondrial ubiquinone content, and altered the bypass respiration in the bc1 complex. Dibit also did not increase respiration significantly but did change the other parameters. 3'-Ac-T2 increased respiration, mitochondrial ubiquinone content, proton permeability, enzyme activity and altered the bypass of the Antimycin A blockage in the bc1 complex.

Effects of 3,3',5-triiodo-L-thyronine (L-T3) and T3 analogues on mitochondrial function.

The effects of L-T3 and several analogues on mitochondrial parameters were determined in hypothyroid rats. These parameters include the 24 hour hormone-induced changes in the bc1 complex and in the inner membrane's proton permeability. L-T3, and all analogues except rT3, increased mitochondrial ubiquinone to euthyroid levels. L-T3, D-T3 and 3'IpT2 but not rT3, Triac, Triprop, or Dimit, altered the bypass respiration in the bc1 complex. L-T3, D-T3, Triac, 3'IpT2, and Triprop, but not rT3 or Dimit, increased the membrane's proton permeability. Actinomycin D did not prevent the increase in mitochondrial ubiquinone or the permeability change. The results show the selective thyromimetic properties of the analogues and that some of the mitochondrial changes do not require protein synthesis.

The early triiodothyronine-induced changes in state IV respiration is not regulated by the proton permeability of the mitochondrial inner membrane.

The time course of changes in mitochondrial respiration, alpha-glycerophosphate dehydrogenase activity, and inner membrane proton permeability and the effects of protein synthesis inhibition was determined in hypothyroid rats treated with triiodothyronine. Respiratory rates, alpha-glycerophosphate dehydrogenase activity and proton permeability were decreased in hypothyroid rats and rose to euthyroid levels 9-12 hours after triiodothyronine treatment. Some rats received actinomycin D with the hormone to inhibit protein synthesis. Sixteen hours after actinomycin D and hormone treatment, the increases in respiratory rates and in alpha-glycerophosphate dehydrogenase activity, were completely inhibited. However, actinomycin D did not inhibit the increase in the membrane's permeability.

Effects of thyroid hormones on the bypasses of the antimycin A block in the bc1 complex of rat liver mitochondria.

The effect of thyroid hormones on the electron flow through the bc1 complex of rat liver mitochondria was studied using two dye bypasses of the Antimycin A block of the bc1 complex by the method of Alexandre and Lehninger (Biochim. Biophys. Acta 767:120; 1984). Bypass respiration rates with both DCIP (2,6-dichlorophenolindophenol) and TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) were elevated in the hyperthyroid rats and depressed in the hypothyroid groups compared to the euthyroid controls. T3 treatment of hypothyroid rats returned the bypass rates to control levels in 24 hours with the TMPD dye but not for the DCIP. This further demonstrates that different portions of the bc1 complex respond individually to the thyroid state.

Thyroid hormone effects on the State IV proton motive force in rat liver mitochondria.

In order to further investigate the mechanisms regulating the control of mitochondrial respiration by thyroid hormone, the proton motive force was measured during State IV respiration in liver mitochondria isolated from euthyroid, hyperthyroid, hypothyroid and T3-treated hypothyroid rats. The proton motive force was significantly higher in the hyperthyroid group due to an increased delta pH. The proton motive force of hypothyroid mitochondria was lower than controls due to a decreased membrane potential. The proton motive force for the T3-treated hypothyroid group did not differ from the euthyroid group due to negating changes in the pH gradient and the membrane potential. The intramitochondrial volume was decreased in the hyperthyroid group and unchanged in the other groups. The results indicate that the thyroid status alters the proton motive force in State IV through individual changes in the pH and membrane potential components of the force. The component that changes in hyperthyroid mitochondria is different from that changing in hypothyroid mitochondria.

Interaction of heparin cofactor II with neutrophil elastase and cathepsin G.

We investigated the interaction of the human plasma proteinase inhibitor heparin cofactor II (HC) with human neutrophil elastase and cathepsin G in order to examine 1) proteinase inhibition by HC, 2) inactivation of HC, and 3) the effect of glycosaminoglycans on inhibition and inactivation. We found that HC inhibited cathepsin G, but not elastase, with a rate constant of 6.0 x 10(6) M-1 min-1. Inhibition was stable, with a dissociation rate constant of 1.0 x 10(-3) min-1. Heparin and dermatan sulfate diminished inhibition slightly. Both neutrophil elastase and cathepsin G at catalytic concentrations destroyed the thrombin inhibition activity of HC. Inactivation was accompanied by a dramatic increase in heat stability, as occurs with other serine proteinase inhibitors. Proteolysis of HC (Mr 66,000) produced a species (Mr 58,000) that retained thrombin inhibition activity, and an inactive species of Mr 48,000. Amino acid sequence analysis led to the conclusion that both neutrophil elastase and cathepsin G cleave HC at Ile66, which does not affect HC activity, and at Val439, near the reactive site Leu444, which inactivates HC. Since cathepsin G is inhibited by HC and also inactivates HC, we conclude that cathepsin G participates in both reactions simultaneously so that small amounts of cathepsin G can inactivate a molar excess of HC. High concentrations of heparin and dermatan sulfate accelerated inactivation of HC by neutrophil proteinases, with heparin having a greater effect. Heparin and dermatan sulfate appeared to alter the pattern, and not just the rate, of proteolysis of HC. We conclude that while HC is an effective inhibitor of cathepsin G, it can be proteolyzed by neutrophil proteinases to generate first an active inhibitor and then an inactive molecule. This two-step mechanism might be important in the generation of chemotactic activity from the amino-terminal region of HC.

Thyroid hormone effects on the proton permeability of rat liver mitochondria.

The effect of thyroid status on passive permeability of liver mitochondrial inner membrane was studied in euthyroid (EU), hyperthyroid (HYPER), hypothyroid (HYPO) rats and hypothyroid rats treated with a single dose of 3,3',5-triiodothyronine (HYPO + T3). Respiration was titrated with uncoupler and plotted against uncoupler concentration to calculate proton permeability. The passive permeability was the same in EU, HYPER, and HYPO + T3 rats, but was significantly lower in HYPO mitochondria. The State IV respiration rates were the same as the oligomycin-inhibited rats for EU, HYPO and HYPO + T3 rats, but in HYPER rats the oligomycin rate was less than the State IV rate. The data show that hypothyroid mitochondria have decreased permeability of the inner membrane which can be normalized by a single dose of T3. Hyperthyroid mitochondria show increased oligomycin-sensitive ATPase activity in State IV, a possible mechanism of the enhanced respiratory rate.

Structural and functional properties of human alpha-thrombin, phosphopyridoxylated alpha-thrombin, and gamma T-thrombin. Identification of lysyl residues in alpha-thrombin that are critical for heparin and fibrin(ogen) interactions.

alpha-Thrombin derivatives obtained either by site-specific modification at lysyl residues (phosphopyridoxylated) or by limited trypsinolysis (gamma T-thrombin) were compared to correlate structural modifications with the functional reactivity toward fibrin(ogen) and heparin. alpha-Thrombin phosphopyridoxylated in the absence of heparin (unprotected) showed approximately 2 mol of label incorporated/mol of thrombin, but only 1 mol of label incorporated/mol of proteinase when modified in the presence of added heparin (protected). In contrast to native alpha-thrombin, both phosphopyridoxylated alpha-thrombin derivatives failed to interact with a fibrin monomer-agarose column and had reduced fibrinogen clotting activity, which is very similar to gamma T-thrombin. Heparin accelerated the rate of antithrombin III inhibition of alpha-thrombin, heparin-protected modified-alpha-thrombin, and gamma T-thrombin in a manner consistent with a template mechanism but was without effect on unprotected modified alpha-thrombin. In a heparin-catalyzed antithrombin III inhibition assay of alpha-thrombin, we found that D-Phe-Pro-Arg chloromethyl ketone-active site-inactivated gamma T-thrombin competed for heparin binding. It has been shown that limited proteolysis/autolysis of the B-chain of alpha-thrombin in the area around Arg-B73 (in beta T/beta- and gamma T/gamma-thrombin), but not that around Lys-B154 (in gamma T/gamma-thrombin), diminishes specific interactions with fibrinogen (Hofsteenge, J., Braun, P. J., and Stone , S. R. (1988) Biochemistry 27, 2144-2151). In unprotected modified alpha-thrombin, lysyl residues B21, B65, B174, and B252 were phosphopyridoxylated. In heparin-protected modified alpha-thrombin, only lysyl residues B21 and B65 were phosphopyridoxylated. These observations suggest that lysyl residues 21/65 of the B-chain of alpha-thrombin are involved in fibrin(ogen) interactions, and lysyl residues 174/252 of the B-chain are important in heparin interactions.

Thyroxine-induced changes in rat liver mitochondrial ubiquinone.

Ubiquinone was extracted from liver mitochondria isolated from euthyroid and hyperthyroid rats. The redox state of ubiquinone was determined during States III and IV respiration with succinate or glutamate-malate substrates. Ubiquinone was more reduced during State III or IV in the hyperthyroid mitochondria with either substrate. Furthermore, the concentration of ubiquinone increased in the hyperthyroid rats.

Altered tissue oxygen tension responsiveness in diabetes.

Subcutaneous oxygen tension (tissue PO2) was measured by a polarographic method in the legs of insulin-dependent diabetics (IDDM) and controls. Current flow was measured continuously using a five-stage protocol: baseline; 4 min of complete arterial occlusion; during recovery from ischemia; baseline approximately re-established; induction of hyperemia by local application of heat. Eleven patients with IDDM of 4-32 years of duration, without peripheral arterial disease, were studied and compared with 10 controls. The mean baseline subcutaneous PO2 in diabetics was less than controls; however, the difference was not statistically significant. At the end of arterial occlusion the mean decrease in tissue PO2 was less (P less than 0.025) in diabetics (4.7 +/- 0.9 mm Hg, SEM) compared to controls (10.2 +/- 1.6 mm Hg). With induction of hyperemia the increase in tissue PO2 was lower (P less than 0.001) in diabetics (7.4 +/- 0.4 mm Hg) than in controls (18.6 +/- 1.7 mm Hg). The observed differences provide for the first time direct evidence of altered tissue PO2 responses in diabetes.

Inhibition of respiration in mitochondria and in digitonin-treated rat hepatocytes by podophyllotoxin.

The effects of the microtubular inhibitor, podophyllotoxin, on mitochondrial respiration were determined using isolated, digitonin-permeabilized hepatocytes and isolated mitochondria. In hepatocytes, podophyllotoxin (1.5 mM) inhibited coupled and uncoupled respiration of both FAD and NAD-linked substrates. In mitochondria, podophyllotoxin inhibited State III respiration, prevented the return to State IV respiration, and inhibited uncoupled respiration. There was no inhibition of ascorbate/TMPD oxidation in either the hepatocytes or the mitochondria. Podophyllotoxin had no effect upon oligomycin inhibition of coupled respiration. Oligomycin had no effect on the podophyllotoxin-inhibition of uncoupled respiration in either hepatocytes or mitochondria. The results indicate that podophyllotoxin alters electron flow at a site early in the electron transport chain.

Inhibition of the effects of thyroid hormone on rat liver by 5,5'-diphenylthiohydantoin.

Metabolites were measured in freeze-clamped livers from rats that had been maintained for 3 weeks on a stock diet supplemented with 0.1% 5,5'-diphenylthiohydantoin (DPTH). Compared with control animals, DPTH-treated animals had lower levels of phosphoenolypyruvate and 3-phosphoglycerate and elevated ratios of [ATP]/[ADP][Pi] and [NADP+]/[NADPH], suggesting mild hypothyroidism. Conversely, the administration of thyroxine (T4) for 5 days to animals fed the control diet resulted in elevated levels of phosphoenolpyruvate, 3-phosphoglycerate, and ketone bodies and lowered ratios of [ATP]/[ADP][Pi] and [NADP+]/[NADPH], consistent with the known effects of thyroid hormones on liver tissues. In animals simultaneously treated with DPTH and T4, the effects of thyroxine on the [NADP+]/[NADPH] ratio and the levels of phosphoenolypyruvate, 2-phosphoglycerate and ketone bodies were reversed. However, the calculated free cytoplasmic [ATP]/[ADP][Pi] ratio and the calculated cytochrome c3+/cytochrome c2+ ratio did not return to control values. This suggests that those actions of thyroid hormone which are mediated by potentiation of adrenergic effects are reversed by DPTH. These actions include a decrease in peripheral lipolysis, a reduction of the free cytoplasmic [NADP+]/[NADPH] ratio, and an apparent inhibition of the pyruvate kinase reaction, but DPTH apparently does not reverse the effects of thyroid hormone on mitochondrial O2 consumption and ATP generation.

Thyroxine-induced changes in rat liver mitochondrial cytochromes.

The effects of 10 days of thyroxine injection (15 micrograms/100 g body weight) on rat liver mitochondrial cytochrome concentration and on the percent reduction of the individual cytochromes during succinate-driven state III and IV respiration was spectrophotometrically determined at cytochrome-specific wave-length pairs. The concentrations of cytochromes b, c, total c (c + c1) and a a3 increased in hyperthyroid rats. The concentration of cytochrome c1 remained constant in euthyroid and hyperthyroid rats. Changes in the concentration of the membrane-bound cytochromes were also determined by difference spectra in cytochrome c-depleted mitochondrial membranes. Cytochromes b and a a3 showed increased concentrations in hyperthyroid rats while the concentration of cytochrome c1 remained unchanged. Hyperthyroid mitochondria showed increased reduction of cytochromes b, c1, c and total c during state III respiration and cytochromes c1, c, and total c during state IV respiration. The percent reduction of cytochrome b decreased during state IV respiration in the hyperthyroid mitochondria. These results suggest that the increase in respiration observed in the hyperthyroid state may be related to changes both in the mitochondrial cytochrome concentration and in the cytochrome reduction level.

Effects of thyroidectomy, propylthiouracil, and thyroxine on pituitary content and immunocytochemical staining of thyrotropin (TSH) and thyrotropin releasing hormone (TRH).

Previous studies have demonstrated immunocytochemical staining for beta chains of thyroid stimulating hormone (TSH-beta) in rough endoplasmic reticulum of pituitary cells hypertrophied after thyroidectomy ("thyroidectomy cells") (Moriarty CG(1976): J Histochem Cytochem (24:846; Moriarty GC, Tobin RB (1976): J Histochem Cytochem 24:1140). Here we report the localization of thyrotropin releasing hormone (TRH) in serial sections of the same pituitaries to determine if it could be found at similar sites. No staining for TRH was found in hypertrophied TSH cells formed 42 days after the surgery, or after 14, 34, and 70 days of propylthiouracil (PTU) treatment. The loss in immunostaining in the PTU-treated rats was correlated with radioimmunoassay (RIA) measurements that showed a 65% reduction in anterior pituitary TRH content after 34, 70, and 98 days of PTU treatment (from 22.9--7.8 pg/mg wet wt) and a 50% reduction in TSH content after 34 days of treatment. When thyroxine was administered to hypothyroid rats for 3 days before death, our previous studies had demonstrated intense staining for TSH in granules inside the rough endoplasmic reticulum. In this study, the radioimmunoassay showed that TSH content rose dramatically in the hypothyroid animals treated with PTU for 77 days and thyroxine for 2 days before death (from 8.5--64.1 mU/mg wet wt); however, the rise in TRH content was minimal (5.8--9.8 pg/mg wet wt). The immunocytochemical stain for TRH correlated well with the RIA showing a weak reaction mainly on small granules in the cytoplasm. No reaction for TRH was found in rough endoplasmic reticulum. These results suggest that TRH and TSH storage sites are dissimilar in the hypothyroid rat. The presence of stain for TRH in granules in the cytoplasm suggests that it might play a role in the storage or packaging of TSH. Its absence in profiles of rough endoplasmic reticulum staining intensely for TSH suggests that it is not synthesized at this site. No definite conclusions about its origin can be drawn at this time.

Effect of thyroidectomy upon the activity of three mitochondrial shuttles in rats.

The activities of mitochondrial alpha-glycerophosphate, malate-asparte, and malate-citrate shuttles which oxidize NADH were determined in liver and kidney cortex mitochondria isolated from Tx and control rats. Weight gain, relative liver and kidney size, oxygen consumption, and serum thyroxine were also determined. Tx animals weighed less, had smaller livers and kidneys, and showed significantly lower oxygen consumption of the resting animal and a marked decrease in serum thyroxine levels. The activity of the three shuttles studies was significantly less in liver mitochondria from Tx animals, particularly when studied in the absence of added ADP. Thyroidectomy had less effect upon shuttle activity of mitochondria from the kidney cortex, with significant decrease in activity found only in the complete alpha-glycerophosphate shuttle and in the endogenous malate-aspartate and malate-citrate shuttles studied in the absence of ADP. Changes in shuttle activity caused by thyroid hormone are discussed in relation to other known metabolic effects of thyroxine, and the possible primary role of adenine nucleotide translocase activity in mediating other thyroxine-dependent metabolic changes is considered.

Effects of thyroxine treatment on the hepatic plasma membrane ATPase activity in rats.

The effects of T4 and T3 upon ATPase activity of plasma membrane preparations produced by different methods has been studied. Media without Na+ or K+ was used to identify Mg++ activated ATPase, and media containing 100-120 mM Na+ and 20-30 mM K+ in addition to Mg++ was used to identify the augmented activity of Na-K ATPase. We also utilized media containing Na+, K+, and Mg++ in the presence and absence of ouabain to identify the Na+-K+ activation. We found no effect of doses of thyroxine in the physiologic range upon the Na+-K+ ATPase activity. A dose-response study indicated that high doses of T4 may stimulate Mg++ activated ATPase. In the P2 preparation of the Touster method we found minimally enhanced Na+-K+ ATPase activity. The quantity of T4 needed to cause such enhancement is approximately 50 times the physiologic replacement dose needed to restore thyroidectomized animals to a euthyroid state. We conclude that thyroid hormones do not stimulate Na+-K+ ATPase of plasma membranes when administered in a physiologic quantity, but may enhance Mg++ atpase activity when administered in a pharmacologic or toxic quantity.

L-Thyroxine effects upon ATPase activities of several subcellular fractions of liver of the rat and the guinea pig.

The effect of thyroxine administration upon ATPase activity of several subcellular fractions of livers from rats and guinea pigs has been studied. To determine a patho-physiological dose of levo thyroxine [T4] for guinea pigs, a dose-response curve was examined of T4 effect upon oxidative phosphorylatin of guinea pig liver mitochondria. Maximum stimulation of mitochondrial respiration without uncoupling of oxidative phosphorylation was found with 15 microgram of T4 per 100 g body weight per day. This dose of T4 stimulated Mg++ activated ATPase of plasma membranes of guinea pigs and slightly stimulated Mg++ activated ATPase of guinea pig liver nuclear membranes. Rat liver nuclear membrane ATPase was not responsive to thyroxine at doses from 5 to 150 microgram per 100 g body weight. T4 significantly stimulated Ca++ or Mg++ ATPase of mitochondria and microsomes from both rat and guinea pig liver. Microsomes from both species were maximally activated by Mg++ and no significant additional stimulation with Ca++ was found. Mitochondrial ATPase from both species showed significantly greater Ca++ plus Mg++ ATPase activity than did Mg++ alone. Ca++ activated ATPase was approximately equal to dinitrophenol stimulated mitochondrial ATPase. Maximum activation of microsomal ATPase in both species was found with 1 mM calcium. We conclude that at physiological-intracellular concentrations of Ca++ and Mg++, thyroxine probably stimulates Mg++ activated microsomal ATPase and Ca++ activated mitochondrial ATPase. A potential role of Ca++ as a moderator of thyroxine stimulated activity in mitochondria and the relation of calcium to other metabolic reactions that are thyroxine sensitive is discussed.

Effect of L-thyroxine treatment on the levels of various hepatic metabolites in rats.

The effects of L-thyroxine (T4) treatment upon hepatic metabolism of control and T4 treated rats was studied. Livers were excised and freeze-clamped rapidly. The levels of adenine nucleotides and glycolytic and TCA cycle metabolites were determined. The redox ratios of free pyridine nucleotides of cytoplasm and mitochondria were calculated from selected substrate concentrations. T4 treatment resulted in lower phosphorylation states and caused reduction in ratios of cytoplasmic NAD and NADP and reduction in the ratio of mitochondrial NAD. T4 treatment changed the redox state of NADP to a greater degree than that of NAD, implying disequilibrium of the transhydrogenase reaction. Changes in glycolytic intermediates were minimal and did not indicate significant "crossover points" of enzymatic change. The most striking changes in metabolites caused by T4 were: increase in citrate, increase in ketones, and decrease in alpha-ketoglutarate. The metabolic consequences of changes in metabolites and of redox states of NAD and NADP were discussed. The equilibrium of reactions interrelating the phosphorylation potential and redox state of cytoplasmic NAD was not altered by T4 treatment. Reduction of cytoplasmic and mitochondrial NAD suggests a greater effect of T4 upon the transport of reducing equivalents into mitochondria than upon enhancement of oxidative capacity.

Studies on the control of lipogenesis: strain differences in hepatic metabolism.

Detailed studies of hepatic metabolism of lipemic BHE and nonlipemic Wistar rats were conducted. Hepatic lipogenic capacity was varied through the use of starvation or meal feeding. Livers were clamped in precooled copper plates and used for the assay of glycolytic, gluconeogenic, and lipogenic metabolites. Redox and phosphorylation states were calculated. Mitochondrial metabolism was evaluated through studies of the oxygen consumption of isolated mitochondria and through the study of the activities of the alpha-glycerophosphate and malate aspartate shuttles and ATPase. BHE rats have higher phosphorylation states, higher redox ratios, and lower shuttle activities and oxygen consumption by isolated mitochondria than their Wistar cohorts. The differences in oxidative phosphorylation, redox and phosphorylation states, and in the various shuttle activities suggest that BHE liver cells are geared towards lipogenesis at the expense of oxidative phosphorylation. It appears that the activity of the shuttles is controlled in part by phosphorylation state which in turn appears to affect respiration. We theorize from these data that genetically determined differences in the structure and function of the mitochondrial membrane (and perhaps the cell membrane as well) may affect the communication (via metabolites and adenine nucleotides) between the cytosol and mitochondria. Subtle differences in the exchange of metabolites and/or adenine nucleotides across the mitochondrial membrane could thus explain the lipogenic tendency of the liver of the BHE rat and the subsequent development of maturity onset hyperlipemia and hyperglycemia in this strain of rat.