μM Pb Research Articles

Effects of Cd 2+, Pb 2+ and CH 3Hg + on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency, reversibility, interactions with extracellular Ca 2+ and mechanisms of block

Effects of the neurotoxic heavy metals Cd 2+, Pb 2+ and CH 3Hg + on current carried by Ca 2+ ions ( I Ca) through high-voltage activated Ca 2+ channels in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells were examined to characterize possible differences in the mechanism of action of these metals on Ca 2+ channel function. Specifically, the potency and reversibility of effect on I Ca by each metal was examined, as well as the relationship between extracellular [Ca 2+] and potency of block of I Ca by Cd 2+ and Pb 2+. In addition, the effect of each of these metals on Ca 2+ channels when applied to the intracellular side of the membrane was also examined. When extracellular solution contained 20, 10 or 5 mM Ca 2+, the estimated IC 50 values (total metal concentration) for block of I Ca were 15, 10, and 6.5 μM for Cd 2+ and 7.5, 2.0 and 1.1 μM for Pb 2+, respectively. CH 3Hg + (1–10 μM) blocked I Ca (20 mM Ca 2+) in a time- and concentration-dependent manner. When cells were washed with metal-free solutions, block of I Ca by Cd 2+ was reversed rapidly, whereas block by Pb 2+ was reversed only partially, and block of I Ca by CH 3Hg + was not reversed. When Pb 2+ and CH 3Hg + treated cells were washed in metal-free solutions containing 50 μM d-penicillamine (DPEN), block of I Ca by 10 μM Pb 2+ was rapidly and completely reversed, whereas, block of I Ca by 5 μM CH 3Hg + was not reversed. Higher concentrations (500 μM) of 2,3-dimercapto-1-propane sulfonic acid (DMPS) did reverse partially the block of I Ca by 5 and 10 μM CH 3Hg +. When Cd 2+, Pb 2+ or CH 3Hg + was present in the intracellular solution, Ca 2+ channel currents were significantly reduced. These results characterize effects of Cd 2+ on Ca 2+ channels and demonstrate that Cd 2+, Pb 2+ and CH 3Hg + differ in their actions on Ca 2+ channels.

Effect of lead on cytoskeletal proteins expressed in E14 mesencephalic primary cultures

Several lines of evidence indicated that Pb exposure in vivo and in vitro altered neurite morphology in central and peripheral neurons. The present report shows that neurite length in mesencephalic primary cultures, consisting of neurons and glia, was decreased by Pb exposure when serum factors, presumably essential for glial functions, were absent in the culture medium. We studied whether a serum factor might control the mechanisms involved in the uptake and accumulation of Pb and its effect on cytoskeleton proteins. The total amount of Pb taken up in cell cultures was measured by atomic absorption spectroscopy and appeared to be down-regulated by a non-albumin-like serum component. In presence of serum, Pb exposure failed to alter cytoskeletal proteins. Instead, in serum-free neurobasal medium, Pb uptake failed to reach saturation within 6 h. Western blot analysis showed that the τ, 280 kDa MAP-2b, 70 kDa MAP-2c and GAP-43 protein bands were decreased 24 h after a 3 h exposure to 3 or 6 μM Pb in absence of serum. However, if cultures were maintained in serum-containing media after a 3 h Pb exposure without serum, the immunoblots did not differ from those of controls. It can be inferred that a serum factor prevents cytoskeletal protein alterations by Pb. In serum free medium, Pb that is primarily scavenged by the metallothionein I/II isoforms present in glial cells, may bind to thiol residues of proteins involved in either oxidative stress response or transcriptional regulation of cytoskeletal proteins.

In cortical cultures of trisomy 16 mouse brain the upregulated metallothionein-I/II fails to respond to H 2O 2 exposure or glutamate receptor stimulation

To assess whether a defective oxidative defense may contribute to Down's syndrome, we studied the regulation of the metallothionein(MT)-I/II isoforms in primary cultures of cerebral cortex from fetal trisomy 16 mice and their euploid littermates. Western blot analysis showed that MT-I/II was upregulated and the protein carbonyl content was higher in trisomy 16 compared with euploid cultures. Addition of N-acetyl- l-cysteine to the culture medium reduced the increment of MT-I/II in trisomy 16 cortical cells. In euploid, but not trisomic cortical cultures, kainic acid, trans-(±)-ACPD, or H 2O 2 exposure elicited a dose-dependent increase of the MT-I/II immunoblots. In trisomic cells, the MT-I/II immunoblot densities were not increased beyond their elevated basal levels. In contrast, 25 μM Pb induced MT-I/II, to a similar extent, in cortical cultures from euploid and trisomy 16 mice. This suggests that the antioxidant—but not the metal—response element of the MT-I/II promoter was altered by increased oxidative stress. Our data suggest that, in the trisomy 16 mouse, the effects of increased production of reactive oxygen species, due to the increased SOD-1, GluR5, or amyloid precursor protein gene dosage, is exacerbated by an insufficient or missing antioxidant response.

Adsorption of metal cations precisely quantified by surface resistance of thin epitaxial silver film electrodes

dc-resistance measurements of metal thin films can be used to investigate processes at the solid-vacuum and solid-electrolyte boundary. In electrochemistry this is a new method to determine the quantity of adsorbed species on solid electrodes. We have grown an epitaxial Ag(111) thin film electrode on a Si(111) substrate with stable characteristics under electrochemical conditions [1]. The adsorption and desorption of small quantities of metal cations from aqueous solutions ( eg 1 μM Pb 2+) are detected by changes of the dc-resistance, resulting from a diffuse scattering of conduction electrons by the adsorbates. In contrast to polycrystalline silver films epitaxial Ag(111) thin film electrodes allow us to perform many deposition and stripping cycles of lead—without changing the properties of the thin film.

Dual, non-competitive interaction of lead with neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells

In cultured mouse neuroblastoma N1E-115 cells, inorganic lead (Pb 2+) affects inward currents induced by activation of neuronal type nicotinic acetylcholine receptors (nAChR) in a biphasic manner. At nanomolar concentrations a blocking action is observed, while at submillimolar concentrations this blocking effect is reversed, resulting in a U-shaped concentration–effect curve. Maximal block by 90% is observed at 1–3 μM Pb 2+. The interactions of Pb 2+ with nAChR were examined at the blocking concentration of 10 nM Pb 2+ and at 10 μM Pb 2+, presenting the reversal of block. The fitted E max for nAChR receptor activation by ACh was attenuated at both high and low Pb 2+ concentrations by 24% and 54%, respectively. The EC 50 values of the activation curves were not significantly altered; amounting to 53 μM, 64 μM and 86 μM ACh in the control situation and in the presence of 10 nM and 10 μM Pb 2+, respectively. Further, receptor desensitization and ion channel block by ACh were also not affected by Pb 2+. The results indicate that Pb 2+ affects nAChR in a dual manner that involves inhibition and potentiation, both by non-competitive interactions. Neuronal nAChR expressed in N1E-115 cells resemble a combination of α4 and β2 subunits, that constitute the predominant subunits in the central nervous system. The differential inhibition and potentiation of nAChR, together with the high sensitivity, are of interest with respect to Pb 2+ neurotoxicity.

Dynamics of lead accumulation in mycorrhizal and non-mycorrhizal Norway spruce (Picea abies (L.) Karst.)

Twelve-week-old seedlings of Norway spruce (Picea abies (L.) Karst), non-mycorrhizal or mycorrhizal with Laccaria laccata, Paxillus involutus or Pisolithus tinctorius were exposed to 5 μM Pb for either 32 or 42 days in a quartz sand-nutrient solution system. Ultrathin sections of mycorrhizal and non-mycorrhizal short roots were examined by X-ray microanalysis. After 42 days Pb treatment, the Pb content of the cortex cell walls was lower in the non-mycorrhizal short roots and in the P. involutus mycorrhizae than in the mycorrhizae of L. laccata or P. tinctorius. The Pb content of the cell walls of the hyphal mantle was higher in P. involutus than in L. laccata or P. tinctorius. The short term experiment over 32 days showed that the Pb content of the cortex cell walls strongly increased during the first 16 days in the non-mycorrhizal roots and the L. laccata mycorrhizae, whereas it increased more slowly in the P. involutus mycorrhizae. After 32 days Pb treatment, the Pb content in the cortex cell walls in the P. involutus mycorrhizae was similar to that in the non-mycorrhizal roots. P. involutus also decreased Pb translocation from the roots to the stems. Mycorrhizal infection was not affected by Pb but with P. involutus, the amount of extramatrical mycelium was reduced by 50% on day 32 compared to day 16. The extramatrical mycelium of L. laccata was not reduced by Pb. It is concluded that ectomycorrhizal fungi differ in their effect on Pb accumulation in the roots of Norway spruce. The binding capacity of the extramatrical mycelium seems to be an important factor.

Pb 2+ reduces the current from NMDA receptors expressed in Xenopus oocytes

We compared the effects of Pb 2+ on four types of NMDA receptors expressed in Xenopus ooeytes. Pb 2+ reduced the currents evoked by glutamate and glycine. The K i values of the receptors, ε1/ζ1, ε2/ζ1, ε3/ζ1 and ε4/ζ1, were 39, 34, 54 and 42 μM, respectively, and their Hill coefficients were 0.53, 4.6, 0.52 and 0.37, respectively. The ε2/ζ1 receptor that was inhibited in the presence of over 30 μM Pb 2+ was not recovered to the control level after a Pb 2+ washout for over 30 min, suggesting that ε2/ζ1 is responsible for the chronic Pb 2+ intoxication in the nervous system.

Potentiation and inhibition of subtypes of neuronal nicotinic acetylcholine receptors by Pb 2+

Effects of inorganic lead (Pb 2+) on defined subtypes of neuronal nicotinic acetylcholine receptors have been investigated. Voltage clamp experiments have been performed on Xenopus oocytes expressing α3β2, α3β4 and α4β2 neuronal nicotinic acetylcholine receptor subunit combinations. In oocytes expressing the α3β2 subunit combination Pb 2+ enhances the peak amplitude of nicotinic acetylcholine receptor-mediated inward currents evoked by superfusion with 100 μM acetylcholine. At concentrations of 1–250 μM, Pb 2+ potentiates α3β2 receptor-mediated inward current concentration dependently by a factor of 1.1–11.0. Inward currents evoked by low (3 μM) and high (1 mM) concentrations of acetylcholine are potentiated to a similar extent. Conversely, in oocytes expressing the α3β4 subunit combination Pb 2+ inhibits the nicotinic receptor-mediated inward currents evoked with μM acetylcholine. Inhibitory effects are observed in the concentration range of 1 nM–100 μM Pb 2+, but the degree of inhibition varies between oocytes. A similar inhibition of the α4β2 nicotinic receptor-mediated inward current by Pb 2+ indicates that α as well as β subunits are involved in the potentiating and inhibitory effects. Possible reasons for the variation in the inhibitory effects of Pb 2+ on α3β4 and α4β4 nicotinic receptor-mediated inward currents have been investigated and are discussed. The divalent cations Ca 2+ and Mg 2+ potentiate both α3β2 and α3β4 nicotinic receptor-mediated inward currents. The distinct modulation of receptor function by Pb 2+ and by Ca 2+ and Mg 2+ and the dependence of the modulatory effect of Pb 2+ on subunit composition suggest that Pb 2+ interacts with multiple sites on the α and β subunits of neuronal nicotinic acetylcholine receptors.

Porphyrin production and excretion by long-term cultures of adult rat hepatocytes and effect of lead exposure

Porphyrin production and excretion and the effects of lead exposure were studied in long-term cultures of adult rat hepatocytes cultured on a feeder layer of 3T3 cells after addition of 5-aminolevulinic acid. Porphyrin excretion into the culture medium showed an irregular profile during the first 10 days, with a maximum increase of 50% at day 4 and at day 10 a value similar to that of day 1. Thereafter, porphyrin excretion decreased progressively to 18% of the initial value after 4 weeks. The cellular porphyrin content, after 7 and 28 days in culture, reached values 3.8 and 2.4-fold higher than the corresponding day 1 value. The exposure to 0.5 and 2.4 μM Pb 2+ for up to 28 days produced a biphasic effect on porphyrin excretion. Firstly, there was a progressive decrease up to 81% during the first 6 days of lead exposure and, secondly, this effect was followed by an increase reaching control values at day 15 and of up to 6.7-fold after 22 days of exposure to 2.4 μM Pb 2+. Similar changes were observed in cellular porphyrin content. The exposure to 0.5 and 2.4 μM Pb 2+ for 2 and 4 weeks also produced morphological alterations and release of cytoplasmic enzymes. Our results show that hepatocytes cultured on 3T3 cells produce and excrete porphyrins for 28 days and that exposure for 4 weeks to micromolar lead concentrations alters these functions and cell morphology and produces cytotoxic effects which are better evaluated by monitoring alterations in porphyrin excretion than by enzyme leakage. They also suggest that this culture system is a useful model for assessing the toxic effects of xenobiotics on the biosynthesis of heme by liver cells.

Mycorrhizal infection and ageing affect element localization in short roots of Norway spruce [Picea abies (L) Karst.

Norway spruce [Picea abies (L.) Karst.] seedlings, nonmycorrhizal of mycorrhizal with Laccaria laccata or Paxillus involutus were grown in a quartz sand-nutrient solution system for 6 months and then treated with 5 μM Pb for 4 days. Element contents of cortex cell wall of young, medium and old short roots were determined by X-ray microanalysis of longitudinal thin sections. The Pb content was influenced neither by age nor by the distance from the root tip (up to 1.7 mm) but was significantly lower in the P. involutus mycorrhizae than in the L. laccata mycorrhizae or in nonmycorrhizal short roots. In the P. involutus mycorrhizae, the P content of the cortex cell walls was twice as high in young mycorrhizae than in old mycorrhizae. In the nonmycorrhizal short roots and the L. laccata mycorrhizae, P content was influenced neither by age nor by distance from the root tip. The Ca and Fe contents of the cortex cell walls increased with age in the nonmycorrhizal short roots and the mycorrhizae. It is concluded that the element content of the cortex cell walls of short roots is strongly influenced by age, while the distance from the root tip seems to be of minor importance.

Lead perturbs 1,25 dihydroxyvitamin D 3 modulation of intracellular calcium metabolism in clonal rat osteoblastic (ROS [formula omitted]) cells

1,25-Dihydroxyvitamin D 3 (1,25(OH) 2D 3) is known to modulate Ca 2+ metabolism in several cell types. 1,25(OH) 2D 3 causes an increase in Ca 2+ influx and probably exerts many of its effects via the Ca 2+ messenger system. Lead (Pb 2+) interacts with and perturbs normal Ca 2+ signalling pathways; hence, the purpose of this work was to determine if Pb 2+ perturbs 1,25(OH) 2D 3 modulation of Ca 2+ metabolism in ROS 17 2.8 cells, which express receptors for and respond to 1,25(OH) 2D 3, and to determine the effect of 1,25(OH) 2D 3 on Pb 2+ metabolism in these cells. In both cases three kinetic compartments described the intracellular metabolism of the isotope. These data show that 1 μM Pb 2+ inhibits 1,25(OH) 2D 3 modulated increases in Ca 2+ flux, whereas 5 μM Pb 2+ increases membrane fluxes, all intracellular Ca 2+ pools, and total cell Ca 2+. In the Pb 2+ metabolism studies it was found that 10 nM 1,25(OH) 2D 3 increases intracellular Pb 2+. Pb 2+ appears to disrupt the modulation of intracellular steady-state Ca 2+ homeostasis by 1,25(OH) 2D 3 in a complex, biphasic manner and may therefore perturb functions that are modulated by 1,25(OH) 2D 3 via the Ca 2+ messenger system.

Biochemical and immunological properties of hepatic δ-aminolevulinic acid dehydratase in channel catfish (Ictalurus punctatus)

Selective inhibition of mammalian δ-aminolevulinic acid dehydratase (ALAD; porphobilinogen synthetase, EC 4.2.1.24) by Pb serves as a important biological marker of chemical exposure and injury. Water-borne exposure of Pb has been shown to inhibit ALAD in the blood and liver of several fish species. This study was undertaken to evaluate the relative susceptibility of fish hepatic ALAD to Pb inhibition in vitro and to characterize this enzyme as determined by a combination of biochemical and immunological techniques. The reaction rate and assay sensitivity were found to increase as a function of temperature and duration of incubation. The results of these studies demonstrated that the IC 50 (17.3 μM) for Pb inhibition of fish liver ALAD activity was 40-times higher than those values reported for rats (0.31–0.4 μM Pb). Kinetic analyses of fish hepatic ALAD activities indicated a K m of 0.043 ± 0.005 mM ALA and a V max of 2.57 ± 0.218 nmol porphobilinogen (PBG)/h/mg of protein. Further in vitro studies showed no activation of fish hepatic ALAD by Zn and only moderate inhibition by EDTA relative to rat ALAD. Western blot analyses using rabbit polyclonal antibodies directed to purified human erythrocyte ALAD suggested faint immunological cross-reactivity. These studies indicate that fish hepatic ALAD is biochemically distinct from the mammalian enzyme and that Zn is apparently not a cofactor for this enzyme and/or that the binding of any metal cofactor to the enzyme is extremely stable.

Cadmium Uptake by Primary Cultures of Rat Renal Cortical Epithelial Cells: Influence of Cell Density and Other Metal Ions

The uptake and accumulation of 1-5 μM cadmium (Cd) was studied in primary cultures of rat renal cortical epithelial cells under protein-free conditions at 4 or 37°C for up to 30 min. The cells were isolated from female rats by collagenase digestion and cultured for 3-7 days. Confluency of the culture, monitored morphologically as well as by total protein content, was achieved on Day 5. Cd accumulation at 1 μM concentration demonstrated an inverse relationship to the cell density; the Cd level in Day 5 culture was only 45% of that in Day 3 culture. In subconfluent cultures (Days 3 and 4) the Cd accumulation was temperature sensitive; on Day 3 the cells accumulated one-third less Cd at 4°C than at 37°C. In comparison, the confluent cells (Day 5) had the same Cd accumulation regardless of the incubation temperature. In these cells, preincubation with cyanide also had no significant effect on Cd accumulation, implying a lack of energy requirement for Cd uptake. As the transport of Cd may involve processes that exist for the essential metal ions, the effect of 30 μM zinc (Zn) and copper (Cu) on the accumulation of 1 μM Cd was studied in Day 5 cultures. Coincubation with Zn caused a 16% reduction in Cd levels at 37°C and even greater reduction (44% of control) at 4°C. Similarly, Cu inhibited Cd accumulation by 26 and 45% at 37 and 4°C, respectively, as compared to the temperature-matched controls. The V max for the initial Cd uptake (1 min) was 125 pmol/mg protein/mm and the K m was 7 μM. Both Zn and Cu exhibited competitive inhibition kinetics and doubled the K m for Cd uptake. The K i for Zn and Cu was 23 and 30 μM, respectively. Mercury (Hg) and lead (Pb) were also tested for their ability to affect Cd accumulation. As compared to the controls, 1 μM Hg caused an 11% reduction in Cd level at 37°C. In contrast, 1 μM Pb enhanced Cd accumulation by 20%. However, neither Hg nor Pb had any significant effect on Cd accumulation at 4°C. All four metals had no significant effect on the efflux of Cd from the cells. Thus, these metals affected Cd accumulation by changing its uptake rather than its efflux. The results of Cd accumulation at varying cell densities demonstrated that as the proximal renal epithelial cells became confluent, and supposedly formed tight junctions and developed brush border, the Cd uptake became more restricted than under subconfluent culture conditions where the uptake was apparently through the basolateral membrane. Lack of effect of cyanide and lower temperature on Cd accumulation in confluent cultures further suggests that Cd transport through the apical membrane might occur by diffusion. However, under subconfluent culture conditions, where Cd accumulation is temperature sensitive, the transport through the basolateral membrane could possibly occur via ion channels or carriers.

Lead Influences Root Growth and Mineral Nutrition of Picea abies Seedlings

Summary Seedlings of Picea abies (L.) Karst. were grown in nutrient solutions containing a range of Pb concentrations. A 4-week exposure to 0.5 μM Pb reduced the growth of primary, secondary and tertiary roots. The initiation of lateral roots appeared to be more sensitive to Pb than the growth of already formed roots. After a 9-week exposure to a range of Pb concentration (0.1-2 μM) the levels of Pb in roots increased with increasing Pb supply. The Pb levels in roots were higher than in needles by a factor of 100-300. The Pb levels in roots were strongly dependent upon the pH of the nutrient solution and decreased at lower pH. With the exception of decreased levels of Ca, Fe and Zn in root tips (5 mm) exposure to Pb only slightly affected the levels of other mineral elements in the roots. In needles, the Ca and Mn levels decreased after exposure to all Pb concentrations.

Pb 2+ blocks calcium currents of cultured dorsal root ganglion cells

The divalent cation lead (Pb 2+) blocks sustained and transient voltage sensitive calcium channel currents of cultured rat dorsal root ganglion cells. The IC 50 for inhibition of the total peak current evoked by a step depolarization from −80 to 0 mV was 0.6 μM, compared to an IC 50 of 2.2 μM for Cd 2+. The current activated by a depolarization from −40 to 0 mV was inhibited by 50% by 1.0 μM Pb 2+. Low threshold currents activated by a step from −100 to −30 mV were blocked by Pb 2+ at higher concentrations (IC 50=6 μM). The block progressed in the absence of channel activation and showed little voltage dependence. Peak sodium current was reduced by 6.6% at 1 μM Pb 2+ while at 20 μM the peak current was reduced by 40% with marked slowing of the time course of activation. The potassium rectifier current was reduced by 4.1% at 1 μM Pb 2+. Thus, Pb 2+ selectively blocks calcium currents at concentrations in the range of those causing toxicity in man.

19F-NMR study of the effect of lead on intracellular free calcium in human platelets

Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrationsof lead in the environment (≈1 μM) on the intracellular free Ca 2+ concentration in any human tissue. We have investigated the effect of lead on the intracellular free Ca 2+ concentration of human blood platelets using 19F-NMR and a fluorinated intracellular Ca 2+ indicator. We report a basal intracellular free Ca 2+ value of 172 ± 8 nM. Treatment with 1, 5, 10 and 25 μM Pb 2+ resulted in average increases in intracellular free Ca 2+ of 39%, 91%, 135% and 172%, respectively. The percent increase in intracellular free Ca 2+ was linearly and positively correlated with the log of Pb 2+ concentration. Using atomic absorption spectroscopy, a significant increase in total calcium of approx. 10 nmol/mg protein was found in 25 μM Pb 2+ treated platelets. This indicates that influx of external Ca 2+ contributes to the observed increase in free Ca 2+. The results provide an explanation for the previously reported effects of lead on platelet function, and suggest a mechanism for low level lead-induced hypertension.

Reduction of glutamine synthetase activity in astroglia exposed in culture to low levels of inorganic lead

Astroglia serve as a site of lead (Pb) deposition in Pb-exposed animals. Thus, the potential exists for astroglial function to be impaired by elevated intracellular Pb levels. We previously showed that the specific activity of glutamine synthetase (GS), an astroglial enzyme with a key role in glutamate and ammonia metabolism in the brain, is reduced in fetal guinea pigs exposed to low levels of lead. This observation indicates either a direct effect of Pb on astroglia or an indirect systemic effect. The purpose of the present study was to test the hypothesis that Pb directly reduces GS activity in astroglia. Cultured rat astroglia were fed three times per week with medium containing 0, 0.25, 0.5, or 1 μM Pb acetate for 7, 14, or 21 days. Trypan blue dye exclusion, cell number, and GS activity were measured. Lead treatment reduced the ability of cells to exclude trypan blue in a dose- and time-dependent manner, with the greatest reduction (28%) on day 21 in the group treated with 1 μM Pb. Cell numbers were reduced a maximum of 15% on day 15, but were unaffected on days 7 and 21. The effects of Pb exposure on GS activity were much more pronounced. For example, cultures exposed to 0.25 of 1 μM Pb for 7 days showed, respectively, 17 and 52% reduction in activity from the control value. Slightly greater reductions were measured on days 14 and 21. The much greater sensitivity in vitro of GS activity than dye exclusion or cell numbers to low level lead supports a specific enzymatic inhibition. In order to test the possibility that Pb might directly inhibit GS activity Pb-treated cells, we also measured the effect of Pb on GS activity in cytosolic extracts of the cells. Pb inhibited GS activity in a dose-dependent manner ranging from 27% inhibition at 0.1 nM to 67% at 0.1 μM and 100% at 10 μM Pb. These results indicate that astroglial function is vulnerable to Pb exposure, even at low lead levels.

Protective role of chlorpromazine on lead-induced damage to heart mitochondria

1. The protective effect of chlorpromazine (CPZ) on the toxic effects of lead in mitochondrial functions was studied. 2. The findings indicate that CPZ at a concentration of 50μM protects heart mitochondria against lead-induced Ca 2+ uptake inhibition. 3. In addition, CPZ inhibits the drop of the transmembrane potential, as well as mitochondrial swelling as induced by 10 μM Pb 2+. 4. It is proposed that the protective effect of chlorpromazine can be due to its stabilizing action on biological membranes.

Effect of lead on the transport of transferrin-free and transferrin-bound iron into rabbit reticulocytes

The effects of Pb on iron transport into rabbit reticulocytes was investigated using two sources of iron, non-transferrin-bound ferrous iron, Fe(II), and transferrin-bound iron, and fractionating the cells into haem, cytosolic and stromal fractions. Uptake of Fe(II) into all three fractions was inhibited by low concentrations of Pb, 50% inhibition of uptake to the cytosol ( ic 50) occurring at 1 μM Pb. Fe(II) uptake could be divided into saturable and non-saturable components. The saturable component was inhibited at lower concentrations of Pb than the non-saturable component. Pb reduced the V max and increased the K m values for saturable Fe(II) transport. The effects of Pb on Fe(II) transport were reversible and were observed with PbCl 2 and Pb (NO 3) 2 as well as with lead acetate. Pb also inhibited the uptake of transferrin-bound iron but at higher concentrations ( ic 50, 4 μM) and the inhibition was less readily reversible. The effect was attributable to inhibition of transferrin endocytosis which resulted in a redistribution of transferrin receptors from intracellular to cell surface sites. These results show that Pb can inhibit transferrin endocytosis and iron transport across the cell membrane of reticulocytes and raise the possibility that these effects may contribute to the hypochromic anaemia associated with Pb poisoning, in addition to the previously established inhibition of enzymes of the haem synthesis pathway.

Control of [ 3H]ouabain binding to cerebromicrovascular (Na + + K +)-ATPase by metal ions and proteins

The (Na + + K +)-ATPase is localized to the cerebral endothelium, i.e. the blood-brain barrier, and is important for the maintenance of the brain electrolyte environment. Data from the present study indicate that Pb 2+ inhibits the binding of [ 3H]ouabain to the cerebral microvascular (Na + + K +)-ATPase in a time- and dose-dependent manner. Pb 2+-induced inhibition developed slowly with a maximum obtained after 40 min. Inhibition of [ 3H]ouabain binding to the enzyme was 48% at 10 μM Pb 2+ and appeared maximal (89%) at 100 μM Pb 2+ when compared to [ 3H]ouabain binding in untreated microvessels at 40 min. In contrast, 100 μM A1 3+ caused a 55% increase in [ 3H]ouabain binding to the (Na + + K +)-ATPase, relative to untreated microvessels at 40 min. Insulin or bovine serum albumin stimulated [ 3H]ouabain binding to the enzyme when added at similar concentrations. However, the addition of both insulin and bovine serum albumin did not result in an additive effect. These results show that insulin exerts a nonspecific effect on [ 3H]ouabain binding to the (Na + + K +)-ATPase similar to that evoked by bovine serum albumin. However, the metal ions Pb 2+ and Al 3+ provoke selective alterations in the cerebromicrovascular (Na + + K +)-ATPase with Pb 2+ inhibiting and Al 3+ stimulating [ 3H]ouabain binding.