Related Experiment Videos
N-methyl-D-aspartate receptors expressed in a nonneuronal cell line mediate subunit-specific increases in free
E R Grant1, B J Bacskai, D E Pleasure
1Department of Pharmacology, University of Pennsylvania School of Medicine, Children's Seashore House, Philadelphia 19104, USA.
Abstract:
N-methyl-D-aspartate (NMDA) receptors can mediate cell death in neurons and in non-neuronal cells that express recombinant NMDA receptors. In neurons, increases in intracellular calcium correlate with NMDA receptor-mediated death, supporting a key role for loss of cellular calcium homeostasis in excitotoxic cell death. In the present study, free intracellular calcium concentrations were examined in response to activation of recombinant NMDA receptors expressed in human embryonic kidney 293 cells. Intracellular calcium was measured in transfected cell populations by cotransfection with the calcium-sensitive, bioluminescent protein aequorin and by single cell imaging with the fluorescent calcium indicator fluo-3. Agonist application to NR1/2A or NR1/2B-transfected cells elicited robust rises in intracellular calcium. NR1/2A responses were inhibited by the noncompetitive antagonists MK-801 and dextromethorphan and were dependent on extracellular calcium but not on intracellular calcium stores. In contrast, no detectable intracellular calcium responses were observed in NR1/2C-transfected cells. These findings indicate that NMDA receptors in the absence of other neuron-specific factors can mediate increases in intracellular calcium with subunit specificity and extracellular calcium dependence.
Insights
N-methyl-D-aspartate (NMDA) receptors, when expressed in non-neuronal cells, can increase intracellular calcium levels. This calcium increase is dependent on specific NMDA receptor subunits and external calcium, but not internal stores.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- N-methyl-D-aspartate (NMDA) receptors are implicated in neuronal cell death.
- Loss of cellular calcium homeostasis is a key factor in excitotoxic cell death.
- Recombinant NMDA receptors can be expressed in non-neuronal cells.
Purpose of the Study:
- To investigate intracellular calcium changes mediated by recombinant NMDA receptors in human embryonic kidney 293 cells.
- To determine the subunit specificity and calcium source dependence of NMDA receptor-mediated calcium influx.
Main Methods:
- Transfection of human embryonic kidney 293 cells with NMDA receptor subunits (NR1/2A, NR1/2B, NR1/2C).
- Measurement of intracellular calcium using the bioluminescent protein aequorin and the fluorescent indicator fluo-3.
- Application of NMDA receptor agonists and antagonists (MK-801, dextromethorphan).
Main Results:
- Activation of NR1/2A and NR1/2B subunits caused significant increases in intracellular calcium.
- These responses were blocked by NMDA receptor antagonists and required extracellular calcium.
- No detectable calcium response was observed in cells expressing NR1/2C subunits.
- Intracellular calcium stores were not essential for the observed calcium increases.
Conclusions:
- NMDA receptors, even without neuron-specific factors, can regulate intracellular calcium levels.
- The calcium-mediating capacity of NMDA receptors is dependent on specific subunits.
- Extracellular calcium is crucial for NMDA receptor-mediated calcium influx in these non-neuronal cells.