Signal transduction via ion fluxes : a cell imaging study with emphasis on calcium oscillations

Signal transduction via ion fluxes is of vital importance for all mammalian cells. Cells can exploit ion regulatory mechanisms to generate and transmit intracellular signals. Improvements in digital microscopic imaging technology have made it possible to explore micro-environments and complex intracellular machineries. By using various cell imaging strategies, this thesis investigates cellular regulation and responses to intracellular signalling.

Spatial and temporal changes in intracellular calcium concentration represent one of the most versatile signalling mechanisms since frequency and amplitude of calcium oscillations can be encoded to control infinite cellular processes. The aim of this thesis was to identify inducers and to reveal the cellular mechanisms that generate calcium signalling.

Two physiological inducers of calcium oscillations have been identified. Spectral analysis of alpha-haemolysin and ouabain-induced calcium oscillations revealed a remarkably constant but significantly different frequency of 1.4±0.1 mHz and 3.6±0.2 mHz respectively.

Alpha-haemolysin, a toxin secreted from Escherichia coli, induced calcium oscillations that were dependent on L-type voltage-gated calcium channels and release of calcium from InsP3 receptor-sensitive internal stores. This signalling event caused increased production of cytokines IL-6 and IL-8.

Ouabain, a steroid hormone and the endogenous ligand of Na,K-ATPase, induced calcium oscillations that also depended on L-type voltage-gated calcium channels and InsP3 receptors. Capacitative calcium entry via CRAC-channels was additionally involved. Downstream cellular sensitivity to ouabain was evidenced by activation of the transcription factor NF-kappa B. This indicates that Na,K-ATPase may act as a receptor for ouabain. In addition, ion fluxes mediated by hormonal regulation of neuronal Na,K-ATPase were examined.

Regulation of a novel calcium signalling mechanism by physical interaction between Na,K-ATPase and InsP3 receptor was revealed. Fluorescent resonance energy transfer was used to determine the proximity between the two molecules. The results demonstrate a new role for Na,K-ATPase and a new InsP3-independent mechanism for activation of the InsP3 receptor.

In conclusion, this thesis presents two physiological inducers of calcium oscillations: alpha-haemolysin, a bacterial toxin that induces cytokine production; and ouabain, a steroid hormone that leads to NF-kappa B activation. This work also demonstrates that Na,K-ATPase acts as receptor for its endogenous ligand ouabain. Furthermore, a novel calcium signalling mechanism via physical interaction between Na,K-ATPase and InsP3 receptor was established.

List of scientific papers

I. Aizman O, Brismar H, Uhlen P, Zettergren E, Levey AI, Forssberg H, Greengard P, Aperia A (2000). Anatomical and physiological evidence for D1 and D2 dopamine receptor colocalization in neostriatal neurons. Nat Neurosci. 3(3): 226-30.
https://pubmed.ncbi.nlm.nih.gov/10700253

II. Uhlen P, Laestadius A, Jahnukainen T, Soderblom T, Backhed F, Celsi G, Brismar H, Normark S, Aperia A, Richter-Dahlfors A (2000). Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells. Nature. 405(6787): 694-7.
https://pubmed.ncbi.nlm.nih.gov/10864327

III. Aizman O, Uhlen P, Lal M, Brismar H, Aperia A (2001). Ouabain, a steroid hormone that signals with slow calcium oscillations. Proc Natl Acad Sci U S A. 98(23): 13420-4.
https://pubmed.ncbi.nlm.nih.gov/11687608

IV. Uhlen P, Miyakawa-Naito A, Aizman O, Zelenin S, Mikoshiba K, Brismar H, Aperia A (2002). Calcium oscillations triggered by physical interaction between Na,K-ATPase and InsP3 receptor. [Manuscript]