Sharada 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.

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 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.

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.

In vitro interaction of heavy metals with ouabain receptors in rat brain microsomes

This study investigates the influence of heavy metals on ouabain-binding in presence of thiol (sulfhydryl) compounds. The data on in vitro effects of mercury (Hg), lead (Pb) and cadmium (Cd) showed significant inhibition of 3H-ouabain binding to microsomal membrane in a concentration-dependent manner. Maximum inhibition of 3H-ouabain binding was observed at 1 microM for Hg and 100 microM each for Pb and Cd. Preincubation with monothiol (L-cysteine or glutathione) or dithiol (dithiothreitol) protected inhibition of 3H-ouabain binding to the membranes by Hg or Pb. Dithiol but not monothiols partially protected Cd-inhibition. The present data confirm that the heavy metals interact with ouabain receptors in a manner similar to SH-blocking agents and protection of metal-inhibited 3H-ouabain binding by thiol compounds is metal specific.