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The Role of Intracellular Calcium Signaling in Regulating Transepithelial Ion Transport by the Malpighian Tubules of Rhodnius prolixus



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Intracellular ion and volume homeostasis is fundamental to epithelial cells during water and solute transport. When homeostasis is disrupted, it may result in pathological consequences, e.g. cystic fibrosis. We use Rhodnius prolixus Malpighian tubules as a model epithelium to study the mechanisms of intracellular homeostasis in epithelial cells. Previously published research indicates that intracellular calcium (Ca2+) signal may be involved in the intracellular homeostasis in some vertebrate epithelia by mediating the crosstalk between ion transporters. In this thesis, (I) we investigate if Ca2+ oscillations are part of crosstalk mechanism between apical and basolateral membrane transporters and, (II) use mass spectrometry (MS)-based proteomics to decipher proteins involved in crosstalk mechanism. Calcium imaging experiments revealed that the amplitude and the frequency of the intracellular Ca2+ oscillations displayed by Malpighian tubule cells correlated with the fluid transport rate. As the transport rate decreased with increasing concentration of the transport blocker, bumetanide, the Ca2+ oscillations displayed smaller amplitudes and frequency. Similarly, changes in transcellular potassium (K+) flux, while maintaining constant fluidsecretion rate, caused a concentration-dependent decrease in amplitude and frequency of Ca2+oscillations. These results are consistent with the hypothesis that Ca2+ oscillations may code information on ion transport flux that could be a part of the crosstalk mechanism. To further test the role of Ca2+ in regulating intracellular ion homeostasis, we measured intracellular potassium using imaging. Our results show that inhibiting intracellular Ca2+ oscillations blocked the ability of the tubule cells to maintain a constant intracellular K+ concentration after the cells were experimentally forced to instantaneously reduce transcellular K+ flux. This indicates that in the absence of Ca2+ oscillations, the cells are unable to maintain intracellular K+ homeostasis, suggesting that Ca2+ may be involved in regulating the ability of apical and basolateral transporters to crosstalk. We induced diuresis with serotonin to simulate crosstalk, which is fundamental to diuresis. MS-based proteomics were used to compare serotonin-stimulated samples with control. The results revealed several proteins of interest with a change in the proteome profile, suggesting a potential involvement in crosstalk mechanism. Of these proteins, we validated the roles of WNK and CAMKII, using conventional techniques. Inhibition of WNK significantly reduced fluid transport and reduced amplitude and frequency of Ca2+ oscillations in a dose dependent manner. Whereas, inhibition of CAMKII significantly reduced fluid transport but failed to affect the cell’s ability to keep an internal ion homeostasis with low K+ challenge. This suggests that the WNK pathway may be fundamental to the crosstalk mechanism, whereas CAMKII might not be involved. In conclusion, there exists a crosstalk mechanism between basolateral and apical membranes of R. prolixus Malpighian tubule that is mediated through Ca2+ oscillations. MS global proteomic profiling has revealed novel machinery that may be involved in ion transport and be potential markers for crosstalk. However, proteins identified must be further investigated for their physiological role in crosstalk.



Epithelial physiology, Epithelial cell crosstalk, Calcium oscillations, MS-based proteomics, Kinases, Calcium binding proteins



Master of Science (M.Sc.)







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