Bettaiya Rajanna

Modulation of protein kinase C by heavy metals

Protein kinase C (PKC) regulates a variety of intracellular and extracellular signals across the neuronal membrane. PKC requires calcium and phospholipid, particularly phosphatidylserine (PS) for its activation. The data indicates that mercury (Hg), lead (Pb) and methyl mercury (CH3Hg) in vitro inhibited the PKC activity at micromolar concentrations in a concentration-dependent manner with IC50 values of 1.5, 2.12 and 0.22 microM, respectively. The IC50 values indicate that CH3Hg was more potent in inhibiting the enzyme activity than Hg or Pb. The basal PKC activity was also inhibited by Pb or Hg. However, the PS-stimulated PKC activity was more sensitive to Pb or Hg than the basal enzyme. The phorbol ester binding to PKC was also found to be inhibited by micromolar concentrations of these metals in vitro. Hg and CH3Hg were more potent inhibitors of phorbol ester binding than Pb. Dithiothreitol (DTT), a dithiol, but not glutathione (GSH) a monothiol, protected the activities of both PS-stimulated and basal PKC from metal-inhibition in a concentration-dependent manner. The present study suggests that the dithiols but not monothiols effectively protect metal-inhibited activity of PKC in rat brain.

Effect of cadmium chloride on rat brain synaptosomal ATPases

Cadmium has been shown to alter membrane-bound ATPases both in vitro and in rats treated with the metal. However, cadmium effects on the substrate and ionic activation kinetics of Na+-K+ ATPase activity were determined by a coupled enzymatic method. A concentration-response curve was determined using 5-30 microM cadmium chloride in the reaction medium. The data showed a 50% inhibition at 15 microM cadmium chloride. Cadmium effects on ATP, Na+ and K+ activation of Na+-K+ ATPase were determined by varying the concentration of these substrates at 15 microM cadmium chloride. The double-reciprocal plots showed that cadmium inhibition of Na+-K+ was competitive with ATP and Na+ in that the Km values were increased but not the Vmax values. In contrast, cadmium inhibition was noncompetitive with K+ activation where both Km and Vmax values were decreased. The present data suggest that cadmium may be competing with ATP and Na+ sites on Na+-K+ ATPase in rat brain synaptosomes.

Effects of cadmium and mercury on Na+-K+ ATPase and uptake of 3H-dopamine in rat brain synaptosomes

Effects in vivo of cadmium (Cd), mercury (Hg) and methylmercury (CH3Hg) on Na(+)-K+ ATPase and uptake of 3H-dopamine (DA) in rat brain synaptosomes were studied. These heavy metals significantly inhibited the Na(+)-K+ ATPase activity in a dose-dependent manner. Similarly, inhibition of DA uptake by synaptosomes was also observed in rats treated with these metals. Intraperitoneal route of metal administration was found to be more effective than per os treatment. Mercuric compounds compared to Cd elicited a higher inhibition of Na(+)-K+ ATPase and DA uptake in rat brain synaptosomes.

In vitro metal inhibition of N-methyl-D-aspartate specific glutamate receptor binding in neonatal and adult rat brain

The in vitro effect of methyl mercury (MM) and lead (Pb) on N-methyl-D-aspartate (NMDA)-specific glutamate receptor binding in neonatal (10 days old) and adult rat brain was investigated. The cerebral cortex was isolated from the neonatal and adult male Sprague-Dawley rats and the synaptic plasma membranes were prepared to study the NMDA-specific glutamate receptor binding by using (3H)-glutamic acid. The metal salts such as methyl mercury chloride and lead acetate were used to study the effect of MM and Pb. Both MM and Pb significantly inhibited the receptor binding in neonatal and adult rat brain in a concentration-dependent manner. MM (IC50:0.95 +/- 0.08 microM) was more potent in inhibiting the receptor binding than Pb (IC50:60 +/- 7 microM) in neonatal rat brain. A similar high potency was observed for MM than Pb in adult rat brain but the IC50 values are very high (70 +/- 6 microM and 300 +/- 24 microM respectively) indicating less effect compared to neonatal brain. The data suggest that NMDA-receptor binding was more sensitive to MM and Pb in neonatal brain than in adult. MM was more effective than Pb because of its more lipophilicity.

Effect of Intraperitoneally Injected Cadmium on Renal and Hepatic Gluconeogenic Enzymes in Rats

Male Sprague-Dawley rats were injected intraperitoneally with 0, 0.25, 0.75 and 1.25 mg/kg/day for 14 days. At the end of 7 and 14 days treatment period, body weight gain, serum protein, serum glucose, serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were measured. Glucose-6-phosphatase (G6-Pase), fructose-1, 6-di-phosphatase (FD-Pase), phosphoenol pyruvate carboxykinase (PEPCK) and pyruvate carboxylase (PC) in kidney and liver were determined. A significant decrease in body weight gain in rats treated with 1.25 mg cadmium for 7 and 14 days was observed. Serum glucose, serum protein, SGOT and SGPT were increased in cadmium treated rats. A significant increase in all four key gluconeogenic enzymes were observed in both kidney and liver tissues of rats treated with cadmium. The results of this study suggest that cadmium induces gluconeogenesis which is dose and time dependent.

Effects of chronic treatment with cadmium on ATPases, uptake of catecholamines, and lipid peroxidation in rat brain synaptosomes

The chronic effects of cadmium on specific activities of oligomycin-sensitive (OS) and -insensitive Mg2+ ATPase, Na(+)-K+ ATPase, lipid peroxidation, and uptake of catecholamines in brain synaptosomes of rats treated daily for 2 or 4 months were studied. Cadmium significantly decreased the specific activities of OS-Mg2+ ATPase, Na(+)-K+ ATPase, and uptake of [3H]-dopamine (3H-DA) and [3H]norepinephrine (3H-NE) and increased the lipid peroxidation.

Effect of mercuric chloride on the kinetics of cationic and substrate activation of the rat brain microsomal ATPase system

Mercuric chloride (HgCl2), a neurotoxic compound, inhibited the adenosine triphosphatase (ATPase) system in a concentration-dependent manner. Hydrolysis of ATP was linear with time with or without HgCl2 in the reaction mixtures. Higher inhibition of (Na(+)-K+)ATPase activity by HgCl2 was observed in alkaline (8.0 to 9.0) pH and at lower temperatures (17 to 32 degrees). Activation energy values were increased slightly in the presence of HgCl2. Activation of (Na(+)-K+)ATPase by ATP in the presence of HgCl2 showed a decrease in Vmax from 15.29 to 5.0 mumol of inorganic phosphate (Pi)/mg protein/hr with no change in Km. Similarly, activation of K(+)-stimulated p-nitrophenyl phosphatase (K(+)-PNPPase) in the presence of HgCl2 showed a decrease in Vmax from 3.26 to 1.35 mumols of p-nitrophenol (PNP)/mg protein/hr with no change in Km. K(+)-activation kinetic studies indicated that HgCl2 decreased Vmax from 14.01 to 4.30 mumols Pi/mg protein/hr in the case of (Na(+)-K+)ATPase and from 3.45 to 2.40 mumols PNP/mg protein/hr in the case of K(+)-PNPPase with no changes in Km. Na(+)-activation of (Na(+)-K+)ATPase in the presence of HgCl2 showed a decrease in Vmax from 11.06 to 3.23 mumols Pi/mg protein/hr and an increase in Km from 1.06 to 2.08 mM. Preincubation of microsomes with sulfhydryl (SH) agents dithiothreitol, cysteine and glutathione protected HgCl2-inhibition of (Na(+)-K+)ATPase. The data suggest that HgCl2 inhibited (Na(+)-K+)ATPase by interfering with the dephosphorylation of the enzyme-phosphoryl complex.

Effect of cadmium on hepatic and renal gluconeogenic enzymes in female rats

Earlier, we have reported that cadmium (Cd) induced gluconeogenesis in male rats. Since females are as much exposed to cadmium as are males, this study was conducted to determine Cd effects on gluconeogenesis in female rats. Adult female rats were injected intraperitoneally (i.p.) with Cd at dose levels of 0.25, 0.75 and 1.25 mg/kg body weight per day for 4 weeks. The controls received saline for the same length of time. Daily food consumption and body weight gain were recorded. At the end of 2 and 4 weeks, 4 rats from each group were killed. Extension of treatment with 1.25 mg Cd for 4 weeks resulted in extreme Cd toxicity killing all animals before the completion of full treatment period. There were no significant changes in total body weight gain and weights of liver and kidney due to Cd. Serum protein increased significantly in animals receiving 0.75 and 1.25 mg Cd for 4 and 2 weeks, whereas serum glucose increased only in animals injected with 1.25 mg Cd for 2 weeks. SGOT and SGPT were elevated (P less than 0.01) in dose- and time-dependent fashion. Activities of three key gluconeogenic enzymes glucose-6-phosphatase (G-6-Pase), fructose-1,6-diphosphatase (FD-Pase), and phosphoenolpyruvate carboxykinase (PEPCK) in liver and kidney were induced significantly (P less than 0.01) in animals injected with 0.75 mg for 2 and 4 weeks and 1.25 mg for 2 weeks, and these increases were dose- and time-related. These results suggest that Cd alters hepatic and renal gluconeogenesis in female rats also.

The effects of cadmium in vitro on adenosine triphosphatase system and protection by thiol reagents in rat brain microsomes

Cadmium (Cd) inhibited the activities of Na+-K+ ATPase (IC50=5.0×10−5 M), K+-p-nitrophenyl phosphatase (PNPPase) (IC50=4.0×10−5 M) and 3H-ouabain binding (IC50=7.5×10−5 M) in rat brain microsomes. Monothiols (cysteine but not glutathione and D-pencillamine) and dithiols (dimercaprol, dimercaptosuccinic acid and dithiothreitol) offered varied levels of protection against Cd-inhibition of Na+-K+ ATPase. Protection of Na+-K+ ATPase by these sulfhydryl (SH) agents was higher at 7.5 as compared to 8.5 pH. The present data suggest that Cd-inhibited Na+-K+ ATPase, by interfering with phosphorylation of enzyme molecule and dephosphorylation of the enzyme-phosphoryl complex and exerts a similar effect to that of SH-blocking agents.

Inhibition of rat brain microsomal Na+/K(+)-ATPase and ouabain binding by mercuric chloride.

This study concerned the effects of mercuric chloride on Na+/K(+)-ATPase and [3H]ouabain binding in rat brain microsomes in vitro. The data showed that HgCl2 inhibited Na+/K(+)-ATPase effectively at micromolar concentrations. The degree of inhibition was decreased with increases in enzyme concentration and incubation time. Variations in the ionic strength of Na+ and K+ did not alter the percent inhibition of Na+/K(+)-ATPase activity by HgCl2. Repeated washings partially restored enzyme activity. The binding of [3H]ouabain to microsomal membranes was inhibited by HgCl2 in a concentration-dependent manner. Cumulative inhibition studies with HgCl2 and ouabain indicated that these inhibitors did not act concurrently and independently on Na+/K(+)-ATPase.