ABSTRACT
We studied a novel function of the presenilins (PS1 and PS2) in governing capacitative calcium entry (CCE), a refilling mechanism for depleted intracellular calcium stores. Abrogation of functional PS1, by either knocking out PS1 or expressing inactive PS1, markedly potentiated CCE, suggesting a role for PS1 in the modulation of CCE. In contrast, familial Alzheimer's disease (FAD)-linked mutant PS1 or PS2 significantly attenuated CCE and store depletion-activated currents. While inhibition of CCE selectively increased the amyloidogenic amyloid beta peptide (Abeta42), increased accumulation of the peptide had no effect on CCE. Thus, reduced CCE is most likely an early cellular event leading to increased Abeta42 generation associated with FAD mutant presenilins. Our data indicate that the CCE pathway is a novel therapeutic target for Alzheimer's disease.
Subject(s)
Alzheimer Disease/physiopathology , Calcium Channels/metabolism , Calcium/metabolism , Membrane Proteins/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Calcium Channel Blockers/pharmacology , Calcium Channels/drug effects , Calcium Channels, L-Type/drug effects , Calcium Channels, L-Type/metabolism , Calcium Channels, N-Type/drug effects , Calcium Channels, N-Type/metabolism , Cells, Cultured , Cytochalasin D/pharmacology , Humans , Imidazoles/pharmacology , Ion Transport/drug effects , Ion Transport/genetics , Membrane Proteins/genetics , Membrane Proteins/pharmacology , Mice , Mice, Transgenic , Mutagenesis, Site-Directed , Neurons/cytology , Neurons/metabolism , Patch-Clamp Techniques , Peptide Fragments/metabolism , Presenilin-1 , Presenilin-2 , TransfectionABSTRACT
Previous investigations have shown that phorbol esters stimulate process extension in oligodendrocytes (OL), likely by the activation of protein kinase C (PKC). In this report, we demonstrate that treatment of OL with 4beta-phorbol-12, 13-dibutyrate (PDB; 0.1-1 microM) resulted in an increase in intracellular Ca2+ concentration ([Ca2+]i) from 94+/-2 nM (mean+/-S.E.M.) to 244+/-10 nM. This increase was produced by Ca2+ influx through a La3+-insensitive pathway. Changes in [Ca2+]i were also produced by modifying the extracellular Ca2+ concentration ([Ca2+]o) where [Ca2+]i was increased by elevations in [Ca2+]o. In parallel experiments we found that increased [Ca2+]o alone, without concurrent phorbol ester application, resulted in increased OL process extension as determined by the percent of OL with long processes (greater than 3 times the cell body diameter). These results demonstrate that increasing [Ca2+]o stimulates OL process outgrowth. Furthermore, both elevations in [Ca2+]o and PDB exposure increase [Ca2+]i, suggesting that some of the effects of phorbol esters on OL process extension are likely mediated by changes in [Ca2+]i.
Subject(s)
Calcium/metabolism , Oligodendroglia , Animals , Biological Transport/drug effects , Biological Transport/physiology , Calcium/analysis , Calcium Channels/metabolism , Carcinogens/pharmacology , Cell Division/drug effects , Cells, Cultured , Chelating Agents/pharmacology , Egtazic Acid/analogs & derivatives , Egtazic Acid/pharmacology , Enzyme Inhibitors/pharmacology , Fluorescent Dyes , Fura-2 , Indoles/pharmacology , Lanthanum/pharmacology , Mice , Mice, Inbred BALB C , Oligodendroglia/chemistry , Oligodendroglia/cytology , Oligodendroglia/enzymology , Phorbol 12,13-Dibutyrate/pharmacology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolismABSTRACT
The effects of protein kinase C (PKC) activation by phorbol ester on intracellular Ca2+ concentration ([Ca2+]i) and membrane currents in human microglia grown in culture were investigated. Treatment of microglia with phorbol myristate acetate (PMA) resulted in a large increase in [Ca2+]i in cells loaded with fura-2. The increased levels of [Ca2+]i were not altered following removal of the phorbol ester. In Ca(2+)-free medium, application of PMA did not increase [Ca2+]i. In addition, PMA application in standard Ca(2+)-solution containing lanthanum (1.8 mM) had no effect on the microglial response to PMA, suggesting that the phorbol ester actions were due to transmembrane influx of Ca2+ but not through voltage-gated Ca2+ channels. Whole-cell patch clamp measurements demonstrated that PMA potentiated an outward K+ current and inhibited an inward rectifier K+ current. This study is the first demonstration that PKC activation by phorbol ester leads to increased intracellular [Ca2+] and changes in membrane currents in human microglia.
