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Effect of two potent calmodulin antagonists on calcium transport of brush border and basolateral vesicles from human duodenum
Author(s) -
STOLL R.,
STERN H.,
RUPPIN H.,
DOMSCHKE W.
Publication year - 1987
Publication title -
alimentary pharmacology and therapeutics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.308
H-Index - 177
eISSN - 1365-2036
pISSN - 0269-2813
DOI - 10.1111/j.1365-2036.1987.tb00642.x
Subject(s) - vesicle , calmodulin , brush border , epithelial polarity , chemistry , biochemistry , membrane , biophysics , biology , enzyme
SUMMARY In the present in‐vitro study we investigated the possible role of the calmodulin‐antagonistic drugs loperamide and calmidazolium in the regulation of transepithelial Ca 2+ transport of human duodenum. Brush border membrane vesicles and basolateral membrane vesicles were simultaneously prepared from surgically resected pieces of morphologically intact human duodenum with a modified Percoll‐gradient centrifugation method. Brush border and basolateral membrane vesicles were characterized using enzyme marker analysis and electron microscopy: alkaline phosphatase was enriched 20‐fold in brush border membrane vesicles, whereas [Na + + K + ]‐stimulated adenosine triphosphatase was enriched 15‐fold in basolateral membrane vesicles. Calmodulin activity was determined by a specific radioimmunoassay after solubilizing brush border and basolateral membrane vesicles in 1% Triton X‐100. In basolateral membrane vesicles, we found no calmodulin activity. In brush border membrane vesicles calmodulin activity was impaired by 50% after pre‐incubation with loperamide or calmidazolium. We measured calcium, sodium, d ‐glucose and d ‐mannitol uptake with a rapid filtration technique. Before the transport experiments, brush border and basolateral membrane vesicles were pre‐incubated with 5 μM loperamide or 5 μM calmidazolium for 60 min at 5 °C. In drug‐pretreated, brush border membrane vesicles calcium uptake was signficantly reduced after 1 min incubation (– 25%±5%, P < 0.05); this effect was completely reversed in the presence of 5 μM calmodulin. In basolateral membrane vesicles, we found two Ca 2+ transport systems: (1) Na + /Ca 2+ exchange and (2) ATP‐dependent Ca 2+ transport. In basolateral membrane vesicles loperamide had no effect. Calmidazolium had no effect on Na + /Ca 2+ exchange, but significantly inhibited ATP‐dependent Ca 2+ transport. This effect could not be reversed by calmodulin.