Calcitonin increases fatty acid synthetase activity dependently of calcium-calmodulin in the hepatic cytosol of fed rats

The effect of calcitonin (CT) on fatty acid synthetase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu1,7] eel CT; 80 MRC mU/100 g body weight) produced significant increases in fatty acid synthetase activity and calcium concentration in the hepatic cytosol of intact and thyroparathyroidectomized rats. The hormonal effect on the enzyme activity was not observed in rats fasted for 24 h. The increase in fatty acid synthetase activity by CT administration was completely inhibited by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The increased enzyme activity of CT-treated rats was markedly reduced by addition of W-7 (15 microM), a calmodulin inhibitor, in the enzyme assay system. Moreover, the cytosolic enzyme activity of normal rat liver was markedly raised by in vitro addition of both calcium ion (5 microM) and calmodulin (2.5 micrograms). These results suggest that CT increases fatty acid synthetase activity in the hepatic cytosol of fed rats, and that this hormonal regulation may depend on calmodulin, and be mediated through raised calcium in the cytosol.     

Calcitonin action on ATP citrate lyase activity in the hepatic cytosol of fed rats and its calcium-calmodulin dependency

The effect of calcitonin (CT) on ATP citrate lyase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu107] eel CT; 80 MRC mU/100 g body weight) produced significant increases in ATP citrate lyase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The increased cytosolic ATP citrate lyase activity resulting from CT administration was prevented by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The rise in enzyme activity of CT-treated rats was markedly reduced by the presence of W-7 (10 and 100 microM), a calmodulin inhibitor, in the enzyme assay system, while that of control rats was not significantly altered by the drug. These results suggest that CT increases ATP citrate lyase activity in the hepatic cytosol of fed rats, and that this hormonal regulation may depend on calmodulin, and be mediated through raised calcium in the cytosol.     

Calcitonin and hepatic fatty acid synthesis: effect of thyroparathyroidectomy on the elevation of ATP citrate lyase activity by refeeding of starved rats

The effect of thyroparathyroidectomy (TPTX) on ATP citrate lyase regulation, a rate-limiting enzyme of fatty acid synthesis in hepatic cytosol, was investigated in rats refed after a 24 h fast. ATP citrate lyase activity in the hepatic cytosol was increased 2-fold by refeeding. This increase was suppressed about 50% by TPTX. The suppression of the enzyme activity by TPTX was completely restored by administration of calcitonin (CT; 80 MRC mU/100 g body weight). This hormonal effect was also observed at 20 MRC mU/100 g dose of CT. CT administration to refeeding-TPTX rats produced a significant increase in the calcium content of the liver tissue and the cytosol. The cytosolic ATP citrate lyase activity increase with CT administration was completely blocked by treatment of cytosol with EGTA (10 microM). This inhibition was clearly reversed by addition of calcium ion (1.25-5.0 microM). In addition, CT-induced rise in enzyme activity was markedly reduced by the presence of W-7 (5 and 50 microM), a calmodulin inhibitor, in the enzyme assay system. The present results suggest that CT plays a role in the elevation of hepatic ATP citrate lyase activity brought about by refeeding of fasted rats, and that this hormonal regulation might depend on Ca2+-calmodulin.     

Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in liver cytosol of normal and regucalcin transgenic rats

The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the liver cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1-20 microM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 microM). NO synthase activity was significantly increased by the addition of calcium (5-20 microM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10-50 microM), an antagonist of Ca(2+)/calmodulin. The addition of regucalcin (0.1-0.5 microM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 microM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA, 1 mM) or TFP (20 microM), indicating that regucalcin acts independent on Ca(2+)/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 microM) in elevating NO synthase activity in the liver cytosol of normal rats was not seen in the liver cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the liver cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the liver cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the liver cytosol of rats.     

Effects of different dietary carbohydrates on hepatic enzymes of copper-deficient rats

The present study was undertaken to measure the activities of several hepatic enzymes of regulatory importance in the pathways of lipogenesis and gluconeogenesis in rats fed diets marginally deficient in copper (1.2 micrograms Cu/g of diet) and containing either fructose, glucose, or starch as the carbohydrate sources. Although all copper-deficient rats exhibited the characteristic signs of copper deficiency, they were more pronounced in rats fed the diet containing fructose. Except for the activity of phosphoenolpyruvate carboxykinase which was unaffected either by copper deficiency or by the type of dietary carbohydrate, the hepatic activities of glucose-6-phosphate dehydrogenase, malic enzyme, L-alpha-glycerophosphate dehydrogenase and fructose 1,6-diphosphatase were unaffected by copper deficiency but were affected by the type of carbohydrate in the diet. Fructose produced the greatest increase in enzymatic activities, whereas starch produced the least activity and glucose induced an intermediate effect. These results indicate that the deleterious effects of a fructose diet deficient in copper on biochemical and physiological indices could not be due to an immediate metabolite of fructose. However, the involvement of a subsequent metabolite of fructose in the mechanism of copper utilization and/or requirement cannot be excluded.     

Some properties of fructose 1,6-diphosphatase of rat liver and their relation to the control gluconeogenesis

1. Fructose 1,6-diphosphatase has been purified tenfold from rat liver. The final preparation was not contaminated by either glucose 6-phosphatase or phosphofructokinase. The properties of the enzyme have been investigated in an attempt to define factors that could be of revelance to metabolic control of fructose 1,6-diphosphatase activity. 2. The metal ions Fe²⁺, Fe³⁺ and Zn²⁺ inhibited the activity of fructose 1,6-diphosphatase even in the presence of an excess of mercaptoethanol; other metal ions tested had no effect. The inhibition produced by Zn²⁺ was reversed by EDTA, but that produced by either Fe²⁺ or Fe³⁺ was not reversible. 4. The enzyme has a very low K_m for fructose 1,6-diphosphate (2·0μm). Concentrations of fructose 1,6-diphosphate above 75μm inhibited the activity; however, even at very high fructose 1,6-diphosphate concentrations only 70% inhibition was obtained. 5. The activity was also inhibited by low concentrations of AMP, which lowered V_max. and increased K_m for fructose 1,6-diphosphate. Evidence is presented that suggests that AMP can be defined as an allosteric inhibitor of fructose 1,6-diphosphatase. 6. The inhibitions by both fructose 1,6-diphosphate and AMP were extremely specific. Also, the degree of inhibition was not affected by the presence of intermediates of glycolysis, of the tricarboxylic acid cycle, of amino acid metabolism or of fatty acid metabolism. 7. It is suggested that the intracellular concentrations of AMP and fructose 1,6-diphosphate could be of significance in controlling the activity of fructose 1,6-diphosphatase in the liver cell. The possible relationship between these intermediates and the control of gluconeogenesis is discussed.     

Calcitonin increases pyruvate carboxylase activity in hepatic mitochondria of rats

The effect of calcitonin (CT) on calcium content and enzyme activity in the hepatic mitochondria of intact rats was investigated. A single subcutaneous administration of CT (80 MRC mU/100 g BW) produced a significant increase in the content of calcium, the activity of pyruvate carboxylase, succinate dehydrogenase and ATPase 15 min after the hormone treatment. The significant increases in calcium content and pyruvate carboxylase activity were also observed 30 min after CT administration, while succinate dehydrogenase and ATPase activity began to decrease. A physiological dose of CT (20 MRC mU/100 g BW) caused a marked increase in calcium content and pyruvate carboxylase activity but not succinate dehydrogenase of ATPase-activity. The removal of calcium by 10 mM EGTA washing of the mitochondria produced a remarkable reduction in pyruvate carboxylase activity increased by CT administration. The addition of calcium ion of 2.5 x 10(-2) - 2.5 x 10(1) nmoles Ca2+ per mg mitochondrial protein produced a marked increase in pyruvate carboxylase activity. The present results suggest that calcium taken up by the hepatic mitochondria after CT administration activates pyruvate carboxylase.     

A comparison of the effects of diabetes induced with either alloxan or streptozotocin and of starvation on the activities in rat liver of the key enzymes of gluconeogenesis

1. Measurements of the activities in rat liver of the four key enzymes involved in gluconeogenesis, i.e. pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate carboxykinase (EC 4.1.1.32), fructose 1,6-diphosphatase (EC 3.1.3.11) and glucose 6-phosphatase (EC 3.1.3.9), have been carried out, all four enzymes being measured in the same liver sample. Changes in activities resulting from starvation and diabetes have been studied. Changes in concentration (activity/unit wet weight of tissue) were compared with changes in the hepatic cellular content (activity/unit of DNA). 2. Each enzyme was found to increase in concentration during starvation for up to 3 days, but only glucose 6-phosphatase and phosphoenolpyruvate carboxykinase showed a significant rise in content. Fructose 1,6-diphosphatase appeared to decrease in content somewhat during the early stages of starvation. 3. There was a marked increase in the concentration of all four enzymes in non-starved rats made diabetic with alloxan or streptozotocin, for the most part similar responses being found for the two diabetogenic agents. On starvation, however, the enzyme contents in the diabetic animals tended to fall, often with streptozotocin-treated animals to values no greater than for the normal overnight-starved rat. Deprivation of food during the period after induction of diabetes with streptozotocin lessened the rise in enzyme activity. 4. The results are compared with other published values and factors such as substrate and activator concentrations likely to influence activity in vivo are considered. 5. Lack of correlation of change in fructose 1,6-diphosphatase with the other enzymes questions whether it should be included in any postulation of control of gluconeogenic enzymes by a single gene unit.     

Expression of calcium-binding protein regucalcin mRNA in rat liver is stimulated by calcitonin: The hormonal effect is mediated through calcium

The involvement of a hypocalcemic hormone calcitonin (CT) in the expression of hepatic Ca²⁺-binding protein regucalcin mRNA was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). A single oral administration of calcium chloride (100 mg Ca/100 g body weight) to rats induced a remarkable increase in the serum calcium concentration and a corresponding elevation of the liver calcium content during 120 min after the administration. Thyroparathyroidectomy (TPTX) did not cause a significant increase in the liver calcium content after calcium administration. Hepatic regucalcin mRNA level was markedly elevated by calcium administration; the level was about 180% of controls at 60 min after the administration. This increase was completely abolished by TPTX. A single subcutaneous administration of CT (synthetic eel CT; 25–100 MRC mU/100 g) to TPTX rats received oral administration of calcium (100 mg/100 g) produced a remarkable increase in hepatic regucalcin mRNA levels; the level was about 280% of controls with the dose of 25 MRC mU CT/100 g. The present finding suggests that the expression of hepatic mRNA is stimulated by CT, and that the hormonal effect is mediated through Ca²⁺ in rat liver.     

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.     

Effect of a high fructose diet on the activities of enzymes implicated in the conversion of fructose to glucose by the liver and the intestinal mucosa of the rat

The activity of enzymes implicated in the metabolic pathway of fructose to glucose conversion was shown in rat liver and intestine. In rats on normal diet, the specific activity of glucose-6-phosphatase, fructokinase, fructose-1,6-diphosphatase and triokinase was low in the intestine confirming that sugar conversion is not operative in this organ. In rats on a fructose diet, all the specific enzymatic activities tested were increased except for the hepatic triokinase and triose phosphate isomerase and for the intestinal triose phosphate isomerase. The intestine acquires the possibility to transform fructose to glucose by modifying the activities of enzymes implicated in the same metabolic pathway as that intervening in the liver.     

Effect of calcitonin on Ca-ATPase activity of plasma membrane in liver of rats.

The effect of calcitonin (CT) on Ca-ATPase activity in the plasma membrane fraction of rat liver was investigated. CT (80 MRC mU/100 g BW) administered subcutaneously to rats, caused a significant decrease in serum calcium, while increasing liver calcium. The administration of CT produced a rapid decrease of Ca-ATPase activity in the plasma membrane fraction of liver, whereas CT did not cause a significant alteration of p-nitrophenyl phosphatase activity. The maximal response of CT was obtained with 80 MRC mU/100 g BW. Meanwhile, the administration of imidazole (30 mg/100 g BW) which has a hypocalcemic effect, like CT, produced a significant increase in liver calcium and a corresponding fall in Ca-ATPase activity of the plasma membrane fraction. The reduction of Ca-ATPase activity produced by imidazole was significantly potentiated by the simultaneous administration of CT, and the rise in liver calcium was enhanced slightly. The present results suggest that the action of CT on liver calcium involves the decrease of Ca-ATPase activity in the plasma membrane of rat liver.     

Involvement of cytosol proteins in oleate activation of rabbit liver fructose-1,6-diphosphatase.

Dialyzed rabbit liver cytosol was specifically freed of endogenous fructose-1,6-diphosphatase by immunoadsorption on a column of Sepharose-immobilized anti-fructose-1,6-diphosphatase. This material increased the specific activity of homogeneous enzyme to the maximal rate observed with EDTA and shifted the pH optimum from 8.4 to 7.4. With oleate or other fatty acids as activators, the hydrolysis of fructose-1,6-diphosphatase by enzyme, at neutral pH, showed nonlinear initial rates dropping to lower linear rates. Cytosol activator acted synergistically with oleate both to increase neutral enzyme activity and to maintain the high initial catalytic rates. After sucrose density centrifugation or gel filtration, the cytosol had no effect by itself, but still potentiated oleate activation. The factor was destroyed by treatment with subtilisin or trypsin, but all attempts to identify a unique protein component in cytosol were unsuccessful. The presence of Na dodecyl-SOJ, deoxycholate, or urea did not improve the resolution of the factor, but these compounds did lower the K50 for activation by cytosol. Since fatty acids are the only unique compounds which have been isolated from cytosol which activated fructose-1,6-diphosphatase, it appears that soluble proteins can act as natural carriers for the fatty acids. This was supported by the fact that both dialyzed rabbit alpha-globulins and muscle phosphofructokinase also acted synergistically with oleate in a manner similar to cytosol. Phosphatidic acid and phosphatidylserine activated fructose-1,6-diphosphatase, and their action was synergistic with oleate. Glutathione (1 mM) activated the enzyme 5-fold at pH 7.3 and its effects were additive with oleate and cytosol or alpha-globulins.     

Suppressive role of endogenous regucalcin in the regulation of protein phosphatase activity in rat renal cortex cytosol

The role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the cytosol of rat renal cortex was investigated. Protein phosphatase activity toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol of kidney cortex. The addition of regucalcin (50-250 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity toward three phosphoamino acids. The effect of calcium (25 microM) and calmodulin (2.5 microg/ml) in increasing protein phosphatase activity toward three phosphoamino acids was significantly decreased by the addition of regucalcin (100 nM). Protein phosphatase activity toward three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The effect of antibody (25 ng/ml) in increasing the enzyme activity was significantly inhibited by cyclosporin A (10(-5) M) or vanadate (10(-5) M). Regucalcin in the kidney cortex cytosol was clearly decreased by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity toward three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin antibody (25 ng/ml) in increasing protein phosphatase activity toward three phosphoamino acids was not seen in the renal cortex cytocol of saline-administered rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the cytoplasm of rat kidney cortex.     

Comparison of calcitonin, epinephrine and glucagon effects on plasma membrane Ca-ATPase activity and calcium content in liver of rats

The effects of calcitonin (CT), epinephrine and glucagon on the plasma membrane Ca-ATPase activity and the calcium content in the liver were investigated 30 min after a single subcutaneous administration of hormones to rats. Ca-ATPase activity in the plasma membrane fraction was significantly decreased by CT (80 MRC mU/100 g BW), while it was not significantly lowered by insulin (100 mU/100 g BW), epinephrine (100 micrograms/100 g BW), glucagon (50 micrograms/100 g BW), or parathyroid hormone (25 U/100 g BW). The calcium content in the liver was markedly increased by CT, while it was not significantly elevated by epinephrine or glucagon. Meanwhile, the decrease of Ca-ATPase activity in the plasma membrane fraction produced by CT was significantly prevented by simultaneous administration of epinephrine or glucagon, and also the increase in liver calcium was noticeably interfered with. The present results suggests that the action of CT on liver calcium may differ from that of epinephrine or glucagon which causes an increase in cyclic AMP in the liver cells.     

Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.     

The effect on some enzymes of rat tissue of diets low in fat content

1. Rats of two strains were kept on three different diets; one was a commercial diet of rat pellets, one contained about 80% of sucrose and 20% of casein and was supplemented with corn oil, and the third was a similar diet without the corn oil. 2. On the commercial diet, the specific activities of pyruvate kinase, glucose 6-phosphate dehydrogenase and fructose 1,6-diphosphatase in the livers of one strain of rats (strain A) were 1.5-3 times those in the other strain (strain B). When the diet high in sucrose and supplemented with corn oil was given, there were large increases in the specific activity of pyruvate kinase, glucose 6-phosphate dehydrogenase and fructose 1,6-diphosphatase in the livers of strain A rats. With strain B rats the increases were much smaller. Omission of corn oil from the diet caused a threefold increase in the specific activity of glucose 6-phosphate dehydrogenase in strain B rats, but had little effect on other enzymes. 3. The enzymes of the kidneys and hearts of strain A rats were also more active than those of strain B rats. In strain A rats, the specific activities of pyruvate kinase and fructose 1,6-diphosphatase in the kidney increased when the sucrose content of the diet was high, but in the kidneys of strain B rats there was little change. 4. In strain A rats, the specific activity of pyruvate kinase in the heart more than doubled with the high-sucrose-corn oil diet and increased threefold when corn oil was omitted. No changes were seen in strain B rats. 5. In strain A rats, omission of corn oil from the diet increased the ability of the kidneys to synthesize glucose from lactate. 6. In strain B rats, addition of corn oil to the diet resulted in a decrease in the liver in the specific activity of ATP citrate lyase and in the ability to incorporate acetate into lipid.     

Inhibitory effect of regucalcin on protein phosphatase activity in the nuclei of rat kidney cortex

The role of regucalcin, which is a regulatory protein of calcium signaling, in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex was investigated. Protein phosphatase activity towards phosphotyrosine, phosphoserine, and phosphothreonine was found in the nuclei. The enzyme activity towards three phosphoamino acids was significantly increased by the addition of calcium chloride (10-50 microM) in the enzyme reaction mixture. This increase was significantly inhibited by trifluoperazine (25 or 50 microM), an antagonist of calmodulin. The presence of regucalcin (50 or 100 nM) in the enzyme reaction mixture caused a significant decrease in protein phosphatase activity towards three phosphoamino acids. This effect was also seen in the presence of calcium (25 microM) and/or calmodulin (5 microg/ml). Protein phosphatase activity towards three phosphoamino acids was significantly increased in the presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture. This effect was completely blocked by the addition of regucalcin (100 nM). The effect of antibody (25 ng/ml) in increasing protein phosphatase activity towards phosphotyrosine was significantly inhibited by vanadate (10(-4) M). Also, the antibody's effect towards phosphoserine and phosphothreonine was significantly inhibited by cyclosporin A (10(-5) M). Endogenous regucalcin was found in the nuclei of rat kidney cortex using Western blot analysis. Nuclear regucalcin level was significantly reduced by the administration of saline (0.9% NaCl) for seven days in rats. Protein phosphatase activity towards three phosphoamino acids was significantly decreased by saline administration. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing protein phosphatase activity towards three phosphoamino acids was weakened in the renal cortex nuclei of saline-administrated rats. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in the nuclei of rat kidney cortex cells.     

Changes in hepatic lipogenesis during development of the rat

1. Changes in the activities of ATP citrate lyase, ;malic' enzyme, glucose 6-phosphate dehydrogenase, pyruvate kinase and fructose 1,6-diphosphatase, and in the ability to incorporate [1-(14)C]acetate into lipid have been measured in the livers of developing rats between late foetal life and maturity. 2. In male rats the activities of those systems directly or indirectly concerned in lipogenesis (acetate incorporation into lipid, ATP citrate lyase and glucose 6-phosphate dehydrogenase) fall after birth and are maintained at a low value until weaning. After weaning these activities rise to a maximum between 30 and 40 days and then decline, reaching adult values at about 60 days. ;Malic' enzyme activity follows a similar course, except that none could be detected in the foetal liver. Pyruvate kinase activity is lower in foetal than in adult livers and rises to slightly higher than the adult value in the post-weaning period. Fructose 1,6-diphosphatase activity rises from a very low foetal value to reach a maximum at about 10 days but falls rapidly after weaning to reach adult values at about 30 days. 3. Weaning rats on to a high-fat diet caused the low activities of acetate incorporation, ATP citrate lyase, glucose 6-phosphate dehydrogenase and pyruvate kinase, characteristic of the suckling period, to persist. ;Malic' enzyme and fructose 1,6-diphosphatase activities were not altered appreciably. 4. No differences could be detected in hepatic enzyme activities between males and females up to 35 days, but after this time female rats gave higher values for acetate incorporation, glucose 6-phosphate dehydrogenase activity and ;malic' enzyme activity. 5. The results are discussed in relation to changes in alimentation and hormonal influences.     

Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.