Calcium Signalling in a Fluid Transporting Epithelium

MacPherson, Matthew (2001) Calcium Signalling in a Fluid Transporting Epithelium. PhD thesis, University of Glasgow.

Abstract

The Drosophila Malpighian tubule is an ideal model epithelium for the study of fluid transport and cell signalling. This thesis has aimed to develop further the tubule for the study of calcium signals in response to a variety of stimuli, and their relation to fluid transport. To do this an 'integrative' approach combining molecular genetic and pharmacological studies with physiological assays of tubule function was used. The expression of several calcium channel genes (DmcalA, DmcalD, trp, trpl and trp?), and several genes encoding molecules involved in the regulation of calcium signalling (inaC, pkc53e, inaD, norpA and plc-21e) was surveyed by Reverse Transcriptase-PCR. All these genes, with the exception of inaD, are expressed in tubules. CAP2b is a neuropeptide that stimulates an increase in fluid secretion rate and also a sharp increase in [Ca2+]i, exclusively in the principal cells of the tubule. Through the examination of CAP2b-stimulated fluid secretion an d CAP2b-stimulated rises in [Ca2+]i, as well as studies using fluorescently labelled calcium channel antagonists, verapamil- and nifedipine-sensitive calcium channels were shown to be involved in stimulated fluid secretion and calcium signalling. By immunocytochemistry, alpha1 subunits were localised to main segment principal cells. Additionally, cGMP was shown to stimulate a prolonged rise in [Ca2+]i through the influx of extracellular calcium via nifedipine and verapamil sensitive channels. The role of transient receptor potential channels (TRP, TRPL) in CAP2b-and thapsigargin-stimulated [Ca2+]i rises was examined using Drosophila trp and trpl mutants expressing aequorin. Analysis of the CAP2b-stimulated calcium signal, found an attenuation of the signal elicited in trpl mutants, hut both attenuation and potentiation of signal in different trp mutants. Alongside results obtained using TRP and TRPL antagonists, this suggested a major role for TRPL in the production of the CAP2b calcium response. Furthermore, TRP and TRPL were localised to main segment principal cells by immunocytochemistry. Finally, the effect on calcium signalling of molecules further downstream in the signal transduction pathway was examined using mutants containing the nitric oxide synthase gene under heat shock control. Although no effect on calcium responses to either CAP2b or cGMP was observed following heat shock-induced ectopic expression of NOS, there was a significant if inconsistent rise in unstimulated fluid secretion. Further experiments suggested this to be due to a rise in intracellular cGMP. Together the results presented here suggest an involvement of phenylalkylamine (PAA)- and dihydropyridine (DHP)-sensitive calcium channels and TRP and TRPL in the production of the CAP2b calcium response. Overall, this thesis demonstrates the amenability of the Malpighian tubule for the study of calcium signalling and goes some way to revealing the calcium regulatory mechanisms lying within.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Shireen Davies
• Keywords: Physiology
• Date of Award: 2001
• Depositing User: Enlighten Team
• Unique ID: glathesis:2001-76003
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 17:08
• Last Modified: 19 Nov 2019 17:08
• URI: https://theses.gla.ac.uk/id/eprint/76003