James Mwanjewe

Proliferating brain cells are a target of neurotoxic CSF in systemic autoimmune disease.

Brain atrophy, neurologic and psychiatric (NP) manifestations are common complications in the systemic autoimmune disease, lupus erythematosus (SLE). Here we show that the cerebrospinal fluid (CSF) from autoimmune MRL-lpr mice and a deceased NP-SLE patient reduce the viability of brain cells which proliferate in vitro. This detrimental effect was accompanied by periventricular neurodegeneration in the brains of autoimmune mice and profound in vivo neurotoxicity when their CSF was administered to the CNS of a rat. Multiple ionic responses with microfluorometry and protein peaks on electropherograms suggest more than one mechanism of cellular demise. Similar to the CSF from diseased MRL-lpr mice, the CSF from a deceased SLE patient with a history of psychosis, memory impairment, and seizures, reduced viability of the C17.2 neural stem cell line. Proposed mechanisms of cytotoxicity involve binding of intrathecally synthesized IgG autoantibodies to target(s) common to different mammalian species and neuronal populations. More importantly, these results indicate that the viability of proliferative neural cells can be compromised in systemic autoimmune disease. Antibody-mediated lesions of germinal layers may impair the regenerative capacity of the brain in NP-SLE and possibly, brain development and function in some forms of CNS disorders in which autoimmune phenomena have been documented.

Role of transient receptor potential canonical 6 (TRPC6) in non-transferrin-bound iron uptake in neuronal phenotype PC12 cells.

Cells take up transferrin-bound iron or NTBI (non-transferrin-bound iron). After treatment with NGF (nerve growth factor), PC12 cells exhibited a neuronal phenotype and an increase in the NTBI uptake (55Fe2+ or 55Fe3+). We loaded the cells with the dye calcein, whose fluorescence increases in the presence of Ca2+ but is quenched with Fe2+ or Fe3+. When examined using calcein fluorescence or radioactive iron, DAG (diacylglycerol)-stimulated NTBI entry was more in NGF-treated PC12 cells compared with untreated cells. All experiments were performed at 1.5 mM extracellular Ca2+. Nramp2 (natural-resistance-associated macrophage protein 2) mRNA expression did not change after the NGF treatment. Expression of the bivalent cation entry protein TRPC6 (transient receptor potential canonical 6) was detected only in the NGF-treated cells. To verify that increased NTBI uptake depended on TRPC6, we examined whether transfecting HEK-293 (human embryonic kidney 293) cells with TRPC6 also increased the NTBI (55Fe) uptake. We also cotransfected HEK-293 cells with two plasmids, one expressing TRPC6 and the other expressing the fluorescent protein DsRED2 to identify the transfected cells. Challenging the calcein-loaded HEK-293 cells (which intrinsically express the a1-adrenergic receptors) with phenylephrine or a cell-permeant DAG increased the fluorescence signal more rapidly in transfected cells compared with untransfected cells. However, when iron (Fe2+ and Fe3+) was added before adding phenylephrine or DAG, the fluorescence intensity decreased more rapidly in transfected cells compared with untransfected cells, thereby indicating a greater stimulation of the NTBI uptake in cells expressing TRPC6. We postulate that the increase in the NTBI entry into neuronal PC12 cells is through TRPC6, a pathway that is unique since it is receptor-stimulated. Since neuronal cells express TRPC6, this pathway may have a role in neurotoxicity.

Ascorbate uptake in pig coronary artery endothelial cells

Although smooth muscle and endothelial cells in pig coronary artery are morphologically and functionally distinct, ascorbate uptake has been characterized only in smooth muscle cells. Ascorbate transporters in kidney and intestinal epithelial cells differ from those in smooth muscle. We examined ascorbate transport and mRNA expression of sodium-dependent vitamin C transporters (SVCT) by RT-PCR in the pig coronary artery endothelial cell cultures. When 14C-ascorbate uptake in endothelial cells was examined as 14C or by HPLC, the two values did not differ from each other. 14C-ascorbate uptake was Na+-dependent, stereoselective for l-ascorbate and inhibited by sulfinpyrazone. The kinetic characteristics of the uptake were: Km = 27± 3 μM (Hill coefficient = 1) for ascorbate and Km = 73± 14 mM (Hill coefficient = 2) for Na+. Surprisingly, endothelial cells had similar kinetic parameters as smooth muscle cells, except for a slightly lower uptake velocity in endothelial cells. Comparison with the smooth muscle showed that both tissue types expressed mRNA for SVCT2. Endothelial cells differ from epithelial cells which express mainly SVCT1 but resemble smooth muscle cells in this respect. (Mol Cell Biochem 271: 43–49, 2005)

Dehydroascorbic acid uptake by coronary artery smooth muscle: effect of intracellular acidification

Dehydroascorbic acid (DHAA) enters cells via Na(+)-independent glucose transporters (GLUT) and is converted to ascorbate. However, we found that Na(+) removal inhibited [(14)C]DHAA uptake by smooth-muscle cells cultured from pig coronary artery. The uptake was examined for 2-12 min at 10-200 microM DHAA in either the presence of 134 mM Na(+) or in its absence (N-methyl D-glucamine, choline or sucrose replaced Na(+)). This inhibition of DHAA uptake by Na(+) removal was paradoxical because it was inhibited by 2-deoxyglucose and cytochalasin B, as expected of transport via the GLUT pathway. We tested the hypothesis that this paradox resulted from an inefficient intracellular reduction of [(14)C]DHAA into [(14)C]ascorbate upon intracellular acidosis caused by the Na(+) removal. Consistent with this hypothesis: (i) the Na(+)/H(+)-exchange inhibitors ethylisopropyl amiloride and cariporide also decreased the uptake, (ii) Na(+) removal and Na(+)/H(+)-exchange inhibitors lowered cytosolic pH, with the decrease being larger in 12 min than in 2 min, and (iii) less of the cellular (14)C was present as ascorbate (determined by HPLC) in cells in Na(+)-free buffer than in those in Na(+)-containing buffer. This inability to obtain ascorbate from extracellular DHAA may be detrimental to the coronary artery under hypoxia-induced acidosis during ischaemia/reperfusion.

Transient receptor potential protein mRNA expression in rat substantia nigra.

Receptor gated Ca2+ entry has been associated with transient receptor potential (TRP) proteins encoded by several different genes. Here, we compare expression of mRNA for TRP isoforms encoded by genes TRP1-6 in the rat substantia nigra and whole brain. The substantia nigra and the whole brain expressed mRNA predominantly for TRP3 and TRP6. The levels of TRP1, 2, 4 and 5 were very low in both. The TRP6 mRNA levels in substantia nigra and the whole brain were comparable while those for TRP3 were significantly lower in substantia nigra than in the whole brain. Thus substantia nigra differs from the whole brain in its TRP expression.

Treatment of PC12 cells with nerve growth factor increases iron uptake.

Phaeochromocytoma PC12 cells treated with nerve growth factor (NGF) differentiate into a neuronal phenotype. Here we compare the uptake of transferrin-bound and non-transferrin-bound iron in NGF-treated (neuronal phenotype) and control (proliferating) PC12 cells. The non-transferrin-bound iron uptake was greater in the NGF-treated cells than in the control, independently of the uptake time, the iron-chelating agents used, the oxidation state of iron (Fe(2+) or Fe(3+)) and the iron concentration tested. The NGF-treated cells expressed L-type and N-type voltage-operated Ca(2+) channels. Nitrendipine (an L-type inhibitor) and possibly omega-conotoxin (an N-type inhibitor) inhibited the iron uptake by 20%. Thapsigargin inhibits the endoplasmic reticulum Ca(2+) pump and allowed Mn(2+) entry into cells. Preincubating PC12 cells with thapsigargin increased the iron uptake. The rate of transferrin-bound iron uptake was less than 1% of the non-transferrin-bound iron uptake and the maximum transferrin-bound iron uptake was also very low. We conclude that an increase in the iron uptake by multiple pathways accompanies the transition of PC12 cells from the proliferating to the neuronal phenotype.

On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells.

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca2+-dependent and Ca2+-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909–10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139–3145). Studies reporting the two pathways have ignored the weak interactions of Ca2+ with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe2+ to NTA with and without Ca2+. We observed Ca2+ activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe2+ to NTA with or without Ca2+. However, Ca2+ did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca2+ and Fe2+ by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe2+, and total Ca2+concentrations). In solutions based on these constants and taking Ca2+ chelation into account, Ca2+ did not affect NTBI uptake over a range of free Fe2+concentrations. Thus, the Ca2+ activation of NTBI uptake observed using the constant total Fe2+ to NTA ratio was because of Ca2+-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca2+ dependence of NTBI uptake be re-evaluated.

Iron promotes cadmium binding to citrate

Iron‐cadmium interactions are important in cadmium toxicity. Dietary iron supplements may decrease cadmium retention after oral cadmium exposure but the underlying mechanism is not known. Using a CdS/AgS ion selective electrode to measure [Cd2+] in physiological saline solution at pH 7.4, we show that Fe2+ promotes Cd2+ binding to citrate thereby decreasing the availability of free Cd2+. This suggests the formation of high molecular weight Cd2+‐Fe2+‐citrate complexes. We confirm this suggestion by showing that 109Cd2+ is retained by 1 kDa cut off filters when present with total 50 μM Fe2+ plus 1 mM citrate but not when present with citrate alone. The formation of high molecular weight complexes may prevent Cd2+ absorption. As citrate is part of the diet, we suggest that these iron‐cadmium interactions may contribute to the protective effect of iron against cadmium toxicity.