Quantification of Ca(2+)-ATPases in porcine duodenum. Effects of 1,25(OH)2D3 deficiency.
Previous studies have identified a calmodulin-stimulated ATP-dependent Ca2+ pump as the major Ca2+ efflux pathway in enterocytes. Here, we developed methods to quantify the number of Ca2+ pumps in basolateral and intracellular membranes from porcine duodenum. By the use of a pig strain with a genetic defect in renal 1 alpha-hydroxylase, we were able to investigate the influence of 1,25(OH)2D3-deficiency on the number of Ca(2+)-ATPases in porcine duodenum. The amount of Ca(2+)-ATPase in isolated basolateral membranes was 5.5 +/- 0.7 micrograms/mg protein, while the Vmax of ATP-dependent Ca2+ transport into inside-out resealed basolateral membrane vesicles was 2.6 +/- 0.4 nmol/mg protein per min. From these data we estimated roughly about 95 x 10(3) plasma membrane Ca2+ pump sites per enterocyte. In addition, the amount of intracellular Ca(2+)-ATPase in microsomal fractions was 0.41 +/- 0.02 microgram/mg protein. Comparison of these parameters between control and rachitic animals showed that Ca2+ pump capacities in both basolateral membranes and microsomal fractions of porcine duodenum are not influenced by 1,25(OH)2D3-deficiency. In conclusion, stimulatory effects of 1,25(OH)2D3 on intestinal Ca2+ transport most likely result from specific effects on apical influx and facilitation of cytosolic Ca2+ diffusion by Ca(2+)-binding proteins and not from an increase in Ca2+ pumping capacity in basolateral membranes.
Timmermans
J A
JA
Department of Physiology, University of Nijmegen, The Netherlands.
Kaune
R
R
Bindels
R J
RJ
van Os
C H
CH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D015153:Blotting, Western; D002117:Calcitriol / Q000172:deficiency*; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000032:analysis / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004386:Duodenum / Q000201:enzymology*; D004797:Enzyme-Linked Immunosorbent Assay; D007700:Kinetics; D012279:Rickets / Q000201:enzymology; D013552:Swine
D015220:Calcium Channels; D002147:Calmodulin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002117:Calcitriol; D002118:Calcium