Title

Nitric Oxide Increases Calcium Oscillations in Response to Muscarinic Receptor Stimulation

Presenter Information

Casey Anna Growcock

Department

Biological Sciences

Major

Biology

Research Advisor

Aronstam, Robert

Advisor's Department

Biological Sciences

Funding Source

Missouri S&T cDNA Resource Center

Abstract

The effect of NO on calcium oscillations elicited by low concentrations of a muscarinic agonist in CHO cells stably transfected with the gene for the human M3 receptor. Nitric oxide’s targets include several synaptic receptor-mediated signaling processes. Agonist stimulation of M3 muscarinic acetylcholine receptors leads to release Ca2+ into the cytosol; this depletion of calcium stores stimulates calcium influx. At near threshold agonist concentrations, a cyclical release and re-uptake of calcium from intracellular stores (calcium oscillations) is the predominant response. Calcium influx was eliminated by the use of calcium-free medium; fura-2 fluorescence was used to quantify cytosolic calcium concentration; carbamylcholine was used to activate muscarinic receptors; nitric oxide was provided by varying concentrations of S-nitrosylglutathione (SNG) before measuring muscarinic responses. Calcium release from intracellular stores was characterized by carbamylcholine sensitivity, maximum initial response, and the presence, amplitude and decay of calcium oscillations. Pretreatment with nitric oxide donor decreased the amplitude of the initial Ca2+ peak by ≈15%, but increased the proportion of cells displaying oscillatory behavior from < 10% to > 90%. The threshold for the elicitation of Ca2+ responses by carbamylcholine increased, consistent with a NOmediated subsensitivity. The average delay in the manifestation of the Ca2+ response increased markedly from 5.3 sec to 8.0 and 12.2 sec in the presence of 4.3 and 8.5 mM SNG, respectively. Nitric oxide modulates the ability of M3 muscarinic receptors to activate calcium signaling pathways as indicated by an enhanced development of calcium oscillations. These changes may reflect changes in the potency of the muscarinic ligand.

Biography

Anna Growcock plans on attending graduate school and pursue her doctorate in neurobiology. She participates in Scrubs, Joe’s P.E.E.R.S. and coaches the local USA Swim team.

Research Category

Sciences

Presentation Type

Poster Presentation

Document Type

Poster

Award

Sciences poster session, Third place

Location

Upper Atrium/Hallway

Presentation Date

08 Apr 2009, 9:00 am - 11:45 am

Comments

Joint project with Barbara Wheelden

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Apr 8th, 9:00 AM Apr 8th, 11:45 AM

Nitric Oxide Increases Calcium Oscillations in Response to Muscarinic Receptor Stimulation

Upper Atrium/Hallway

The effect of NO on calcium oscillations elicited by low concentrations of a muscarinic agonist in CHO cells stably transfected with the gene for the human M3 receptor. Nitric oxide’s targets include several synaptic receptor-mediated signaling processes. Agonist stimulation of M3 muscarinic acetylcholine receptors leads to release Ca2+ into the cytosol; this depletion of calcium stores stimulates calcium influx. At near threshold agonist concentrations, a cyclical release and re-uptake of calcium from intracellular stores (calcium oscillations) is the predominant response. Calcium influx was eliminated by the use of calcium-free medium; fura-2 fluorescence was used to quantify cytosolic calcium concentration; carbamylcholine was used to activate muscarinic receptors; nitric oxide was provided by varying concentrations of S-nitrosylglutathione (SNG) before measuring muscarinic responses. Calcium release from intracellular stores was characterized by carbamylcholine sensitivity, maximum initial response, and the presence, amplitude and decay of calcium oscillations. Pretreatment with nitric oxide donor decreased the amplitude of the initial Ca2+ peak by ≈15%, but increased the proportion of cells displaying oscillatory behavior from < 10% to > 90%. The threshold for the elicitation of Ca2+ responses by carbamylcholine increased, consistent with a NOmediated subsensitivity. The average delay in the manifestation of the Ca2+ response increased markedly from 5.3 sec to 8.0 and 12.2 sec in the presence of 4.3 and 8.5 mM SNG, respectively. Nitric oxide modulates the ability of M3 muscarinic receptors to activate calcium signaling pathways as indicated by an enhanced development of calcium oscillations. These changes may reflect changes in the potency of the muscarinic ligand.