Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms
Epithelial sodium channels (ENaC) located in the apical membranes of transporting epithelia, such as cells of the renal distal nephron, constitute the rate limiting step in the transcellular transport of sodium. The regulation of sodium ion reabsorption in the kidney, which is essential for salt and...
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | English |
| Published: |
Department of Human Biology
2023
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613226139648001 |
|---|---|
| access_status_str | Open Access |
| author | Butterworth, Michael Bruce |
| author2 | Els, Callie |
| author_browse | Butterworth, Michael Bruce Els, Callie |
| author_facet | Els, Callie Butterworth, Michael Bruce |
| author_sort | Butterworth, Michael Bruce |
| collection | Thesis |
| description | Epithelial sodium channels (ENaC) located in the apical membranes of transporting epithelia, such as cells of the renal distal nephron, constitute the rate limiting step in the transcellular transport of sodium. The regulation of sodium ion reabsorption in the kidney, which is essential for salt and water homeostasis, is therefore governed by the cellular control of ENaC activity. It is well established that the neurohypophysial hormone vasopressin (antidiuretic hormone) is responsible for both water and sodium regulation and, in renal cells, increases sodium permeability by regulating ENaCs at their apical membranes. The mechanisms involved in acute ENaC regulation have however not been demonstrated unequivocally. Evidence exists to suggest that sodium ion transport rates could be increased either by direct activation of membrane-resident ENaCs, or recruitment of channels from cytoplasmic storage pools. In order to address the issue, this study aimed to investigate the hypothesis that vasopressin regulation of ENaC occurred by targeted exocytic delivery of channels to the apical membrane from sub-apical pools. All investigations were carried out on the sodium transporting A6 cultured cell line derived from Xenopus laevis kidney. The secondary messenger pathway of vasopressin stimulation had not been extensively characterised in these cells and this was the first study undertaken. Once it was established that an adenosine-3',5'-cyclic monophosphate (cAMP) secondary messenger pathway was in operation, attention was shifted to ENaC regulation in response to a raised intracellular cAMP level. The electrophysiological technique of current-fluctuation analysis was employed to characterise the single channel response to cAMP production. These studies demonstrated that the main factor responsible for increased sodium transport was a dramatic increase in the number of channels at the apical surface despite declines in both single channel conductance and channel open probability. Investigating intracellular membrane traffic by marker-uptake and confocal microscopic techniques demonstrated that cAMP pre-stimulation significantly altered the rates of membrane endocytosis from the apical surface implicating regulated recruitment and retrieval mechanisms for ENaC regulation. These methods provided indirect evidence for the shuttling hypothesis and established that the vesicle trafficking machinery was responsive to cAMP, but did not associate ENaC directly with the process. In order to demonstrate the movement of ENaC in conjunction with membrane trafficking mechanisms, a green fluorescent protein (GFP) tagged-ENaC was employed. By using live cell confocal microscopic techniques, the dynamic movement of GFP-ENaC in response to cAMP was demonstrated. Shuttling of ENaC to the apical surface was observed on cAMP stimulation with endocytic retrieval on removal of stimulus. For the first time in live cells these data directly demonstrate the dynamic regulation of ENaC by shuttling mechanisms. |
| format | Thesis |
| id | oai:open.uct.ac.za:11427/38392 |
| institution | University of Cape Town (South Africa) |
| language | eng |
| last_indexed | 2026-06-10T12:32:46.693Z |
| license_str | Not specified — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UCTD — University of Cape Town Open Access Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Department of Human Biology |
| publisherStr | Department of Human Biology |
| record_format | dspace |
| source_str | UCTD — University of Cape Town Open Access Repository |
| spelling | oai:open.uct.ac.za:11427/38392 Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms Butterworth, Michael Bruce Els, Callie Anatomy and Cell Biology Epithelial sodium channels (ENaC) located in the apical membranes of transporting epithelia, such as cells of the renal distal nephron, constitute the rate limiting step in the transcellular transport of sodium. The regulation of sodium ion reabsorption in the kidney, which is essential for salt and water homeostasis, is therefore governed by the cellular control of ENaC activity. It is well established that the neurohypophysial hormone vasopressin (antidiuretic hormone) is responsible for both water and sodium regulation and, in renal cells, increases sodium permeability by regulating ENaCs at their apical membranes. The mechanisms involved in acute ENaC regulation have however not been demonstrated unequivocally. Evidence exists to suggest that sodium ion transport rates could be increased either by direct activation of membrane-resident ENaCs, or recruitment of channels from cytoplasmic storage pools. In order to address the issue, this study aimed to investigate the hypothesis that vasopressin regulation of ENaC occurred by targeted exocytic delivery of channels to the apical membrane from sub-apical pools. All investigations were carried out on the sodium transporting A6 cultured cell line derived from Xenopus laevis kidney. The secondary messenger pathway of vasopressin stimulation had not been extensively characterised in these cells and this was the first study undertaken. Once it was established that an adenosine-3',5'-cyclic monophosphate (cAMP) secondary messenger pathway was in operation, attention was shifted to ENaC regulation in response to a raised intracellular cAMP level. The electrophysiological technique of current-fluctuation analysis was employed to characterise the single channel response to cAMP production. These studies demonstrated that the main factor responsible for increased sodium transport was a dramatic increase in the number of channels at the apical surface despite declines in both single channel conductance and channel open probability. Investigating intracellular membrane traffic by marker-uptake and confocal microscopic techniques demonstrated that cAMP pre-stimulation significantly altered the rates of membrane endocytosis from the apical surface implicating regulated recruitment and retrieval mechanisms for ENaC regulation. These methods provided indirect evidence for the shuttling hypothesis and established that the vesicle trafficking machinery was responsive to cAMP, but did not associate ENaC directly with the process. In order to demonstrate the movement of ENaC in conjunction with membrane trafficking mechanisms, a green fluorescent protein (GFP) tagged-ENaC was employed. By using live cell confocal microscopic techniques, the dynamic movement of GFP-ENaC in response to cAMP was demonstrated. Shuttling of ENaC to the apical surface was observed on cAMP stimulation with endocytic retrieval on removal of stimulus. For the first time in live cells these data directly demonstrate the dynamic regulation of ENaC by shuttling mechanisms. 2023-09-05T14:09:04Z 2023-09-05T14:09:04Z 2001 2023-09-05T14:08:41Z Doctoral Thesis Doctoral PhD http://hdl.handle.net/11427/38392 eng application/pdf Department of Human Biology Faculty of Health Sciences |
| spellingShingle | Anatomy and Cell Biology Butterworth, Michael Bruce Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms |
| thesis_degree_str | Doctoral |
| title | Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms |
| title_full | Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms |
| title_fullStr | Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms |
| title_full_unstemmed | Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms |
| title_short | Regulation of sodium channel densities in A6 renal epithelial cells by hormonally-dependent mechanisms |
| title_sort | regulation of sodium channel densities in a6 renal epithelial cells by hormonally dependent mechanisms |
| topic | Anatomy and Cell Biology |
| url | http://hdl.handle.net/11427/38392 |
| work_keys_str_mv | AT butterworthmichaelbruce regulationofsodiumchanneldensitiesina6renalepithelialcellsbyhormonallydependentmechanisms |