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A modification of a cell isolation technique used in animal studies was developed to remove enterocytes from duodenal biopsy specimens. Citrate-ethylenediaminetetra-acetic acid treatment removed enterocytes from any underlying lamina propria and produced single cells and strips of cells. A mean (SEM) of 4-39 (2.06) x 106 cells was obtained from nine duodenal biopsy specimens. Enterocyte recovery was estimated enzymatically using alkaline phosphatase activity and was found to be 61%. Cytological assessment of the cells with CAM 5*2 showed that 98% of the cells isolated were enterocytes with an intact brush border. The cells responded well to vasoactive intestinal peptide stimulation in the absence of an exogenously added adenosine triphosphate regenerating system. The addition of vasoactive intestinal peptide to duodenal enterocytes produced a biphasic dose dependent increase in cyclic adenosine monophosphate production. Stimulation of these cells with 10-13M vasoactive intestinal peptide resulted in a 50% stimulation over basal value while 10-6M vasoactive intestinal peptide led to a fivefold increase in cyclic adenosine monophosphate production. We conclude that duodenal biopsy specimens are a good source of human intestinal cells for the study of enterocyte physiology. The cells were viable and highly responsive to vasoactive intestinal peptide.

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This study demonstrates that the Inhibition of adenylate cyclase activity by Ca" is enhanced m the presence of mcreasmg [EGTA] (0, 0.3. 1. 2.5 mM) by 2 orders of magnitude.It has been established that this effect is not because of poor Ca" buffering by low [EGTA] or high Ca*' bmdmg by the membrane preparation.It is present irrespective of stimulus. We suggest the enhanced sensitivity of adenylate cyclase to Ca" induced by EGTA is caused by the Ca-EGTA complex being a more Inhibitory spectes than Ca". Thus consideration of the effects of the CapEGTA complex should be made when interpreting the results from expertments mvolving Ca" and EGTA.