Endocrinology, Vol 120, 868-873, Copyright © 1987 by Endocrine Society
The adenosine triphosphate-dependent calcium pump in rat small intestine: effects of vitamin D deficiency and cell isolation methods
EJ van Corven, MD de Jong and CH van Os
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.
