Adenylate cyclase in the human duodenum

Smith, J.A., 1991. Adenylate cyclase in the human duodenum. PhD, Nottingham Trent University.

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The studies undertaken In this work take advantage of a source of human duodenal tissue, In the form of biopsies, from patients attending the endoscopy clinic at the City Hospital, Nottingham. The work utilises two different biopsy preparations, a particulate membrane preparation and isolated mucosal epithelial cells. The regulation of adenylate cyclase was examined with respect to the effects of calcium, calmodulin and EGTA in membrane preparations and the regulation of VIP stimulated cAMP synthesis was studied in cells.

The concentration dependent effects of Ca2+ on adenylate cyclase activity in particulate membranes were measured. Adenylate cyclase activity was inhibited by Ca2+ concentrations greater than 90nM. There was no stimulatory phase indicative of calmodulin stimulated adenylate cyclase activity. Addition of calmodulin to EGTA washed membranes did not stimulate enzyme activity. Inhibition of adenylate cyclase activity by the calmodulin antagonist TFP and IODO 8 did not inhibit enzyme activity at concentrations specific for calmodulin antagonism. The Ca2+ chelator EGTA increased the enzyme's sensitivity to Ca2+. In the presence of 1μM free Ca2+, EGTA inhibited adenylate cyclase activity Irrespective of stimulus. In the absence of added Ca2+, EGTA inhibited NaF stimulated enzyme activity but stimulated basal, forskolin and GMP-PNP stimulated enzyme activity. These data suggested EGTA removes membrane bound Ca2+ associated with adenylate cyclase activity which was not associated with calmodulin.

Greater than 80% of isolated epithelial cells were viable based on trypan blue exclusion. VIP stimulated cAMP production in isolated epithelial cells (EC50=l .78pmol). Maximum stimulation was elicited by 100nM VIP. VIP stimulated cAMP production was not inhibited by TFP or IODO 8 at concentrations as high as 100μM.

The results from both approaches suggest that human duodenal adenylate cyclase is not calmodulin dependent and the physiological significance of Ca2+ inhibition of adenylate cyclase is unclear in this tissue.

Item Type: Thesis
Creators: Smith, J.A.
Date: 1991
ISBN: 9781369324228
Divisions: Schools > School of Science and Technology
Record created by: Linda Sullivan
Date Added: 11 Nov 2020 12:25
Last Modified: 11 Oct 2023 09:27

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