The adenosine triphosphate-dependent calcium pump in rat small intestine: effects of vitamin D deficiency and cell isolation methods.
Duodenal villus cells from vitamin D-deficient (-D) and replete rats (+D) were isolated either in citrate buffers according to the method of Stern or by vibration in EDTA-containing solutions according to the method of Harrison and Webster. Basolateral plasma membrane vesicles (BLMV) were purified, and active Ca2+ transport was studied. The rates of ATP-dependent Ca2+-transport in BLMV from -D and +D animals were 2.6 +/- 1.0 and 10.6 +/- 0.6 nmol Ca2+/min X mg protein when cells had been isolated in citrate buffers. These transport rates were 9.2 +/- 0.7 and 9.6 +/- 0.5, respectively, when cells were isolated by vibration. The specific activities of other enzyme markers and vesicle parameters, such as the degree of resealing or purification factors, were not influenced by the cell isolation procedure. Addition of lipase and protease inhibitors to the citrate buffer or fasting the animals before death increase the active Ca2+ transport rates in BLM from -D rats to control levels. These results indicate that the ATP-driven Ca2+ pump in duodenal plasma membranes from vitamin D-deficient rats is more prone to inactivation during enterocyte isolation procedures. With the vibration technique, six populations of cells could be obtained that are sequentially released from villus tip to crypt base. A similar villus-crypt gradient of active Ca2+ transport was present in duodenal BLMV from -D and +D animals. In ileal BLMV from vitamin D-deficient rats a significantly lower Ca2+ transport rate was found compared to that in +D animals, even when villus cells were isolated by vibration. It is thought that the mechanisms by which 1,25-dihydroxyvitamin D3 regulate transcellular Ca2+ fluxes in duodenum and ileum must be different.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D004386:Duodenum / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism* / Q000473:pathology; D007700:Kinetics; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D014808:Vitamin D Deficiency / Q000378:metabolism*
D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium