Effect of two potent calmodulin antagonists on calcium transport of brush border and basolateral vesicles from human duodenum.
In the present in-vitro study we investigated the possible role of the calmodulin-antagonistic drugs loperamide and calmidazolium in the regulation of transepithelial Ca2+ 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 microM loperamide or 5 microM calmidazolium for 60 min at 5 degrees C. In drug-pretreated, brush border membrane vesicles calcium uptake was significantly reduced after 1 min incubation (-25% +/- 5%, P less than 0.05); this effect was completely reversed in the presence of 5 microM calmodulin. In basolateral membrane vesicles, we found two Ca2+ transport systems: (1) Na+/Ca2+ exchange and (2) ATP-dependent Ca2+ transport. In basolateral membrane vesicles loperamide had no effect. Calmidazolium had no effect on Na+/Ca2+ exchange, but significantly inhibited ATP-dependent Ca2+ transport. This effect could not be reversed by calmodulin.
Stoll
R
R
Department of Medicine, University of Erlangen-Nuremberg, FRG.
Stern
H
H
Ruppin
H
H
Domschke
W
W
D001693:Biological Transport, Active / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors*; D004386:Duodenum / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D066298:In Vitro Techniques; D008139:Loperamide / Q000494:pharmacology*; D008871:Microvilli / Q000187:drug effects / Q000378:metabolism
D002147:Calmodulin; D007093:Imidazoles; C031938:calmidazolium; D008139:Loperamide; D002118:Calcium