Manganese suppresses ATP-dependent intercellular calcium waves in astrocyte networks through alteration of mitochondrial and endoplasmic reticulum calcium dynamics.
The neurotoxicity of manganese [Mn] is due in part to glutamate excitotoxicity. Release of ATP by astrocytes is a critical modulator of glutamatergic neurotransmission, which is regulated by calcium (Ca(2+)) waves that propagate through astrocytic networks in response to synaptic activity. It was postulated that Mn alters ATP-dependent intracellular Ca(2+) dynamics in astrocytes, thereby suppressing Ca(2+) wave activity. Confluent primary cultures of cortical astrocytes were loaded with the Ca(2+)-sensitive dye fluo-4 and examined by fluorescence microscopy for Ca(2+) wave activity following micropipet mechanical stimulation of a single cell. Mitochondrial Ca(2+) was evaluated by fluorescence microscopy following addition of ATP using the mitochondrial-specific Ca(2+) dye rhod-2-AM. Imaging studies revealed that pretreatment of astrocytes with 1-10 microM Mn significantly reduced the rate, area, and amplitude of mechanically induced Ca(2+) waves. This attenuation was not a result of inhibited mitochondrial calcium uptake because robust calcium waves were still observed following pretreatment of astrocytes with Ru360, an inhibitor of mitochondrial Ca(2+) uptake, either in coupling or uncoupling conditions. However, determination of endoplasmic reticulum (ER) Ca(2+) levels in cells using the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin indicated that Mn reduced the available pool of releasable ER Ca(2+) at concentrations as low as 1 muM. Examination of ATP-stimulated changes in mitochondrial Ca(2+) indicated that, in cells pretreated with Mn, mitochondria retained high levels of Ca(2+). It is concluded that exposure of astrocytes to low concentrations of Mn(2+) results in sequestration of Ca(2+) within the mitochondria that reduces the available pool of releasable Ca(2+) within the ER, thereby inhibiting calcium wave activity.
Tjalkens
Ronald B
RB
Molecular, Cellular, and Integrative Neuroscience, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523-1680, USA. Ron.Tjalkens@colostate.edu
Zoran
Mark J
MJ
Mohl
Brianne
B
Barhoumi
Roula
R
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000648:ultrastructure*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000187:drug effects*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007211:Indoles; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D017975:Ruthenium Compounds / Q000494:pharmacology; D013997:Time Factors
D005904:Glial Fibrillary Acidic Protein; D007211:Indoles; C112020:Ru 360; D017975:Ruthenium Compounds; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D008345:Manganese; C007293:DAPI; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D002118:Calcium