Subcellular Localization of Sulfated Glucuronic Acid-Containing Glycolipids Reacting with Anti-Myelin-Associated Glycoprotein Antibody

Peripheral nerve glycolipids, with which anti-myelin-associated glycoprotein (MAG) antibodies from patients with demyelinating neuropathy and plasma cell dyscrasia cross-react, proved to be novel glycosphingolipids containing a sulfated glucuronyl residue. Consequently, there has been much interest in the immunological role that these sulfated glucuronyl-glycosphingolipids (SGGLs) may play in the pathogenesis of this disorder. For the determination of the distribution of these glycolipids in various nervous tissues and, thereby, the elucidation of their pathogenicity, a quantitative immunostaining-TLC method for their detection has been devised. Using this method, we demonstrated that these glycolipids were distributed in greatly different amounts in the peripheral nerves from human, bovine, chicken, rat, and rabbit. Subcellular localization studies of bovine peripheral nerve also demonstrated that they were enriched in the axolemma-enriched fraction and present in glial-related membranes in lower concentrations. In addition, these glycolipids were present in bovine dura mater and transformed rat Schwann cells. These biochemical results suggest that not only myelin but also axons could be involved as targets of the anti-MAG antibody in macroglobulinemia neuropathy, and it may also be necessary to examine anti-SGGL activity in patients with axonal neuropathy associated with plasma cell dyscrasia.     

Effect of Lithium on Schwann Cell Proliferation Stimulated by Axolemma- and Myelin-Enriched Fractions

Cultured Schwann cells stimulated with an axolemma- or myelin-enriched fraction incorporated 2.5 to three times as much [3H]thymidine when 10 mM lithium was added to the extracellular medium. The ability of lithium to enhance the mitogenic activity of either fraction was dose dependent. This result was not due to an increase in osmolarity, because addition of 10 mM NaCl had no effect on the amount of labeled thymidine accumulated by Schwann cells treated with either membrane fraction. In an earlier study, the effect of either membrane fraction could be potentiated with active phorbol esters. Lithium significantly enhanced the incorporation of [3H]thymidine into Schwann cells treated with a myelin-enriched fraction and phorbol esters. In contrast, lithium slightly increased the amount of labeled thymidine incorporated into Schwann cells stimulated with an axolemma-enriched fraction and phorbol esters. The mitogenic activity of either membrane fraction was impaired when the calcium channel blockers Mn2+ and nifedipine were added. Addition of lithium stimulated an increase in the amount of [3H]thymidine accumulated by Schwann cells treated with either the axolemma- or myelin-enriched fraction in the presence of either Mn2+ or nifedipine.     

Role of Intracellular pH in the Axolemma- and Myelin-Induced Proliferation of Schwann Cells

In order to provide additional information on the biochemical events that interact to cause Schwann cells to proliferate, we have monitored the intracellular pH of Schwann cells that have been stimulated to divide with myelin-enriched fractions (MEF) or axolemma-enriched fractions (AEF). The intracellular pH of Schwann cells was monitored using 2',7'-bis(carboxymethyl)-5(6)-carboxyfluorescein (BCECF), which displays an increase in fluorescence upon alkalinization. Both AEF and MEF caused dose-dependent increases in the intracellular fluorescence of the Schwann cell cultures. At their maximum doses, AEF and MEF stimulation resulted in a 260 and 300% increase in intracellular fluorescence, respectively. The increase in intracellular fluorescence was abolished when cells were stimulated in Na+-free media, suggesting a role for the Na+/H+ exchanger. Mitotic stimulation required integrity of the Na+/H+ exchanger, as inhibition of the Na+/H+ exchanger for periods up to 1 h after addition of mitogen caused a significant inhibition of subsequent mitosis. Phorbol esters, which can potentiate AEF- and MEF-induced Schwann cell proliferation, increased intracellular fluorescence fivefold, an effect which was also dependent upon the presence of Na+ in the culture media. The specificity of the increase in intracellular pH for AEF and MEF was tested by incubating Schwann cells with liver microsomes and a biologically inactive phorbol alcohol, neither of which is significantly mitogenic for Schwann cells. Neither liver microsomes nor phorbol alcohol had a significant effect on intracellular pH. The implications of the increase in intracellular pH in Schwann cells with respect to inositol phospholipid metabolism, protein kinase C activation, and cellular proliferation are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)