Article date: August 2001
By: Bozena Kaminska, Izabela Figiel, Beata Pyrzynska, Rafal Czajkowski, Grazyna Mosieniak in Volume 133, Issue 7, pages 997-1004
Calcineurin is a ubiquitous calcium/calmodulin dependent protein phosphatase that has been shown to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In the present study we show that CsA, a specific inhibitor of calcineurin, affects the survival of cultures developed from hippocampal dentate gyrus. Mixed neuronal‐glial cultures exposed to 8–40 μM CsA undergo cell death characterized by apoptotic changes in cellular and nuclear morphology.
TUNEL‐positive staining was observed only in neurons that developed pyknotic morphology after treatment with 8 μM CsA for 24–72 h.
Immunocytochemical staining with an anti‐GFAP monoclonal antibody revealed that astrocytes from mixed neuronal/glial cultures were unaffected by exposure to CsA at doses toxic for neurons and all TUNEL‐positive cells were neurons.
MK‐801, a noncompetitive inhibitor of glutamate receptor, does not inhibit the appearance of TUNEL‐positive neurons and apoptotic changes in nuclear morphology.
Preincubation of cells with 8 μM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 μM MK‐801 had no effect on CsA‐induced changes in Ca2+ level.
Our findings suggest that the neuronal death after CsA treatment is not a result of glutamate excitotoxicity and the increase in intracellular calcium concentration in neurons is not dependent on calcium influx via NMDA channel.
Calcineurin is a ubiquitous calcium/calmodulin dependent protein phosphatase that has been shown to regulate the activity of ion channels, glutamate release, and synaptic plasticity. In the present study we show that CsA, a specific inhibitor of calcineurin, affects the survival of cultures developed from hippocampal dentate gyrus. Mixed neuronal‐glial cultures exposed to 8–40 μM CsA undergo cell death characterized by apoptotic changes in cellular and nuclear morphology.
TUNEL‐positive staining was observed only in neurons that developed pyknotic morphology after treatment with 8 μM CsA for 24–72 h.
Immunocytochemical staining with an anti‐GFAP monoclonal antibody revealed that astrocytes from mixed neuronal/glial cultures were unaffected by exposure to CsA at doses toxic for neurons and all TUNEL‐positive cells were neurons.
MK‐801, a noncompetitive inhibitor of glutamate receptor, does not inhibit the appearance of TUNEL‐positive neurons and apoptotic changes in nuclear morphology.
Preincubation of cells with 8 μM CsA increased basal intracellular calcium level in time dependent manner and decreased relative calcium response to glutamate. Application of 1 μM MK‐801 had no effect on CsA‐induced changes in Ca2+ level.
Our findings suggest that the neuronal death after CsA treatment is not a result of glutamate excitotoxicity and the increase in intracellular calcium concentration in neurons is not dependent on calcium influx via NMDA channel.
British Journal of Pharmacology (2001) 133, 997–1004; doi:10.1038/sj.bjp.0704177
DOI: 10.1038/sj.bjp.0704177
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