Long-term activation of capacitative Ca2+ entry in mouse microglial cells.
The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.
Department of Physiology, Birmingham University, Edgbaston, UK.
E C
EC
Toescu
T
T
Möller
H
H
Kettenmann
A
A
Verkhratsky
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology*; D002462:Cell Membrane / Q000502:physiology; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology*; D007700:Kinetics; D007811:Lanthanum / Q000494:pharmacology; D051379:Mice; D008815:Mice, Inbred Strains; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000502:physiology*; D012044:Regression Analysis; D013997:Time Factors; D014544:Uridine Triphosphate / Q000494:pharmacology
D015220:Calcium Channels; D018048:Receptors, Purinergic P2; D007811:Lanthanum; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate