Proceedings of The Physiological Society

University of Edinburgh (2011) Proc Physiol Soc 25, PC39

Poster Communications

Nicotinic acid adenine dinucleotide phosphate evokes global calcium signals in mouse pulmonary arterial smooth muscle cells by activating Two Pore Segment Channel 2

E. O. Agbani1, O. A. Ogunbayo1, J. Parrington2, A. Galione2, J. Ma3, M. X. Zhu4, M. A. Evans1

1. Centre for Integrative Physiology, University Of Edinburgh, Edinburgh, United Kingdom. 2. University Department of Pharmacology, University of Oxford, Oxford, United Kingdom. 3. Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey, United States. 4. Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas, United States.


Increasing evidence provides strong support for our proposal that the Two Pore Segment Channels (TPCs, TPCN for gene name) (TPCs) mediate intracellular calcium (Ca2+) release from endolysosomal stores in response to nicotinic acid adenine dinucleotide phosphate (NAADP)(1). Studies have identified and characterized 3 sub-types of this channel family, with TPC2 targeted to lysosomes and TPC1 and TPC3 targeted to endosomes (2). In this study, we investigated the effects of TPC2 knockout on lysosomal distribution and Ca2+ signaling in pulmonary arterial smooth muscle cells (PASMCs) derived by explant culture. All procedures were in accordance with animal handling and procedure regulations of the United Kingdom. Consistent with our previous findings on acutely isolated rat pulmonary arterial smooth muscle cells (3), intracellular dialysis from a patch pipette (voltage-clamp mode, -40 mV holding potential) of 10 nM NAADP induced a global Ca2+ transient in PASMCs developed from pulmonary artery explants of wild type (WT) mice on day 4 of culture. The Fura-2 fluorescence ratio (F340/F380) increased from 0.36 ± 0.05 to 1.38 ± 0.18 (n=10). Evoked transients were markedly attenuated by pre-incubation of cells with thapsigargin (1µM) and abolished by pre-incubation with bafilomycin (1µM). In marked contrast, 10 nM NAADP failed to induce a Ca2+ transient in PASMCs developed by 4 day culture of artery explants from TPCN2 knockout mice; TPC2 knockout being confirmed by genotyping and by quantitative RT-PCR. Surprisingly, however, when the concentration of applied NAADP was raised to 1µM, small and spatially restricted Ca2+ transients were evoked in PASMCs (Day 4) from TPCN2 knockout mice; F340/F380 increasing from 0.32 ± 0.02 to a peak of 0.75 ± 0.04 (n=8). Semi-quantitative analysis of confocal images suggests that TPC2 knockout was not accompanied by an identifiable change in the size of Lysotracker labeled acidic organelles (WT day 4 = 1.3 ± 0.06μm3; KO Day 4 = 1.4 ± 0.1µm3). Nor did we see any change in the organization of Lysotracker labeled acidic organelles, which remained largely perinuclear in both WT and knockout PASMCs. We conclude that NAADP mediates global Ca2+ transients in PASMCs by activating the lysosome targeted TPC2.

Where applicable, experiments conform with Society ethical requirements