Calcium transport by rat duodenal villus and crypt basolateral membranes

1987 ◽  
Vol 252 (2) ◽  
pp. G170-G177 ◽  
Author(s):  
J. R. Walters ◽  
M. M. Weiser
Keyword(s):  
Vitamin D ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Cell Fraction ◽  
Basolateral Membranes ◽  
Pump Activity ◽  
Crypt Cells

Rat duodenal cells were isolated sequentially to give fractions enriched for villus and crypt cells. From each of these fractions, basolateral-enriched membrane vesicles were prepared and ATP-dependent calcium uptake was studied. Calcium uptake was sensitive to temperature, was inhibited by vanadate and by A23187, and was lower in vitamin D-deficient animals. In normal animals, calcium transport was approximately twofold greater in villus-tip than in crypt cell-fraction basolateral membranes though the affinity of the uptake for calcium was similar (Km = 0.3 microM). In vitamin D-deficient animals, the crypt-to-villus gradient was reduced, and in all fractions, calcium transport was similar to or lower than that in the crypts of normal animals. Six hours after vitamin D-deficient animals were repleted with 1,25-dihydroxycholecalciferol, a significant increase in calcium transport by everted gut sacs was present; however, basolateral calcium transport was significantly increased in only the mid-villus fractions, and no change was seen in the villus-tip fractions. Thus vitamin D appears necessary for the development of increased basolateral membrane calcium pump activity in duodenal villus cells, but not all cells in vitamin D-deficient rats are able to respond to 1,25-dihydroxycholecalciferol.

scholarly journals Identification and isolation of the phosphorylated intermediate of the calcium pump in rat intestinal basolateral membranes

1988 ◽  
Vol 256 (2) ◽  
pp. 593-598 ◽  
Author(s):  
R Wajsman ◽  
J R F Walters ◽  
M M Weiser
Keyword(s):  
Vitamin D ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Basolateral Membrane Vesicles ◽  
Basolateral Membranes ◽  
Calmodulin Binding ◽  
Pump Activity ◽  
Acyl Phosphate ◽  
Crypt Cells

Transport of Ca2+ by the ATP-dependent Ca2+ pump has been demonstrated previously in rat intestinal basolateral-membrane vesicles. To identify the Ca2+-pump protein, duodenal basolateral membranes were phosphorylated with [gamma-32P]ATP in the presence of Ca2+ and La3+, under conditions conducive for maximal formation of the phosphorylated intermediate of the Ca2+ pump. Four major phosphoprotein bands were seen on autoradiograms of acidic SDS/polyacrylamide gels; the properties of a phosphoprotein (pp) at 130 kDa (pp130) were consistent with those expected for the plasma-membrane Ca2+ pump. This phosphoprotein was markedly enhanced by La3+, exhibited the characteristics of an acyl-phosphate bond, was preferentially phosphorylated from ATP and inhibited by micromolar concentrations of vanadate. Another phosphoprotein of 115 kDa possibly represented the endoplasmic reticulum Ca2+ pump or a fragment of pp130. Other phosphoproteins of 75 and 95 kDa were predominantly expressions of alkaline phosphatase. Formation of pp130 was highest in duodenal basolateral-membrane preparations when compared with those of jejunum and ileum or other subcellular fractions. A similar correlation between Ca2+-pump activity and pp130 formation was not found in membranes from villus-tip and crypt cells or in vitamin D-deficient animals. pp130 was isolated as a single phosphoprotein by calmodulin-affinity chromatography. We conclude that pp130 represents the phosphorylated intermediate of the rat intestinal basolateral-membrane Ca2+ pump, which can be separated from other phosphoproteins using its properties as a calmodulin-binding protein.

Depression of calcium pump activity in renal cortex of vitamin D-deficient rats with secondary hyperparathyroidism

1990 ◽  
Vol 123 (4) ◽  
pp. 438-444 ◽  
Author(s):  
Yusuke Tsukamoto ◽  
Teiichi Tamura ◽  
Michiyo Saitoh ◽  
Yumiko Takita ◽  
Toshiaki Nakano
Keyword(s):  
Vitamin D ◽  
Secondary Hyperparathyroidism ◽  
Hormonal Regulation ◽  
Serum Calcium Level ◽  
Renal Cortex ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Basolateral Membrane Vesicles ◽  
Pump Activity

Abstract. To examine the hormonal regulation of the ATP-dependent Ca2+ pump in the kidneys, the ATP-dependent Ca2+ uptake by the basolateral membrane vesicles in the renal cortex was measured using radioactive calcium (45Ca2+) in rats with vitamin D deficiency or rats undergoing thyroparathyroidectomy. The Vmax of the Ca2+ pump activity was increased not only by administering calcitriol, but also by normalizing the serum calcium level in vitamin D-deficient rats. PTH suppressed the Ca2+ pump activity in normocalcemic vitamin D-deficient rats. Thyroparathyroidectomy did not affect the Ca2+ pump activity in the kidneys of normal rats. It was concluded that the ATP-dependent Ca2+ pump activity was depressed by secondary hyperparathyroidism in vitamin D-deficient rats.

Mechanism of regulation of calcium-pumping activity in chick intestine

1992 ◽  
Vol 262 (5) ◽  
pp. G797-G805
Author(s):  
J. Takito ◽  
T. Shinki ◽  
H. Tanaka ◽  
T. Suda
Keyword(s):  
Vitamin D ◽  
Calcium Transport ◽  
Calcium Uptake ◽  
Early Stage ◽  
Basolateral Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Intestinal Calcium Transport ◽  
Pumping Activity

The role of the calcium pump in the stimulation of intestinal calcium transport activity by 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] was examined in chicks. The in situ intestinal absorption of calcium increased approximately threefold in the duodenum, jejunum, and ileum 6 h after a single injection of 625 ng of 1 alpha,25(OH)2D3 into vitamin D-deficient chicks. The same treatment also increased approximately twofold the rate of ATP-dependent calcium uptake by the basolateral membrane vesicles (BL) isolated from those three sites. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that a Mg(2+)-dependent calcium-stimulated phosphorylated intermediate with an apparent molecular mass of 105 kDa appeared in the BL. The 1 alpha,25(OH)2D3 treatment gave no change in the levels of the intermediate. Pretreatment of the BL with alkaline phosphatase decreased the calcium uptake by the BL isolated from 1 alpha,25(OH)2D3-treated chicks, but it had little effect on the uptake by the BL from vitamin D-deficient chicks. These results suggest that at an early stage of the 1 alpha,25(OH)2D3-induced intestinal calcium transport process, the vitamin regulates the calcium-pumping activity of chick intestinal BL by phosphorylation and dephosphorylation but not by a stoichiometric change in the pump.

Calbindin-D9k stimulates the calcium pump in rat enterocyte basolateral membranes

1989 ◽  
Vol 256 (1) ◽  
pp. G124-G128 ◽  
Author(s):  
J. R. Walters
Keyword(s):  
Vitamin D ◽  
Specific Activity ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Basolateral Membrane Vesicles ◽  
Calbindin D9k ◽  
Uptake Rates ◽  
Micromolar Range ◽  
Stimulation Of

Calbindin-D9k, a vitamin D-dependent Ca2+-binding protein, is closely associated with the transcellular absorption of calcium by mammalian enterocytes. Studies were performed to determine whether physiological concentrations of calbindin-D9k altered Ca2+ transport by the ATP-dependent Ca2+ pump in rat duodenal basolateral membrane vesicles. In solutions where free Ca2+ was buffered by EGTA, only a small stimulation of Ca2+ uptake rates could be demonstrated, and it was likely that this was secondary to changes in free Ca2+ concentration. However, a threefold stimulation of uptake by 30 microM calbindin-D9k was found when EGTA-free solutions were used, and changes in free Ca2+ activity or 45Ca2+ specific activity were avoided. The affinity for Ca2+ was reduced in this system but appeared to be stimulated by either calbindin-D9k or EGTA. Other Ca2+-binding proteins that bind Ca2+ in the micromolar range were found to increase Ca2+ uptake in the absence of EGTA. These experiments suggest that one of the actions of calbindin-D9k is to stimulate the rate of extrusion of Ca2+ from the enterocyte by increasing Ca2+ transport by the Ca2+ pump.

Effect of vitamin D deficiency on lipid composition and calcium transport in basolateral membrane vesicles from chick intestine

IUBMB Life ◽  
1997 ◽  
Vol 42 (2) ◽  
pp. 339-347 ◽  
Author(s):  
Arturo Alisio ◽  
Fernando Cañas ◽  
Delia de Bronia ◽  
Rodolfo Pereira ◽  
Nori Tolosa de Talamoni
Keyword(s):  
Vitamin D ◽  
Vitamin D Deficiency ◽  
Lipid Composition ◽  
Calcium Transport ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Basolateral Membrane Vesicles

Effects of 1,25(OH)2D3 on enterocyte basolateral membrane Ca transport in rats

1989 ◽  
Vol 256 (3) ◽  
pp. G613-G617 ◽  
Author(s):  
M. J. Favus ◽  
V. Tembe ◽  
K. A. Ambrosic ◽  
H. N. Nellans
Keyword(s):  
Vitamin D ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Cellular Transport ◽  
Dihydroxyvitamin D3 ◽  
Ca Transport ◽  
Intracellular Ca ◽  
Net Flux ◽  
Energy Dependent

One, twenty-five dihydroxyvitamin D3 [1,25(OH)2D3], commonly known as calcitriol, stimulates intestinal Ca absorption through increased activity of a cellular transport process. To determine whether transcellular Ca transport involves energy-dependent Ca efflux across enterocyte plasma membrane in vitamin D-sufficient rats, in vitro bidirectional Ca fluxes were measured under short-circuited conditions across proximal duodenum from rats fed diets adequate in vitamin D and containing a normal Ca diet (NCD), a low Ca diet (LCD), or fed NCD and injected with 50 ng of 1,25(OH)2D3 daily for 4 days before study. LCD or 1,25(OH)2D3 increased Ca net flux [Jnet, mucosal-to-serosal flux minus the serosal-to-mucosal flux] by increasing Ca mucosal-to-serosal flux (Jm----s) (mean +/- SE, NCD vs. LCD vs. 1,25(OH)2D3, 16 +/- 4 vs. 179 +/- 18 vs. 82 +/- 21 nmol.cm-2. h-1, P less than 0.0001). Initial ATP-dependent Ca uptake rates by duodenal basolateral membrane vesicles (BLMV) was greater in vesicles from rats fed NCD compared with LCD and not different from NCD injected with 1,25(OH)2D3. These studies suggest that in vitamin D-replete animals, 1,25(OH)2D3 increases epithelial Ca Jm----s by mechanisms that do not involve ATP-dependent BLM Ca efflux. ATP-dependent Ca exit from the cell under these conditions may play a role in intracellular Ca homeostasis rather than Ca absorption.

Characterization of a distinct Na(+)-H+ exchanger in basolateral membranes of human jejunum

1993 ◽  
Vol 264 (1) ◽  
pp. G45-G50
Author(s):  
S. Acra ◽  
W. Dykes ◽  
W. Nylander ◽  
F. K. Ghishan
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Kinetic Studies ◽  
Maximal Velocity ◽  
Nacl Transport ◽  
Human Organ ◽  
Basolateral Membranes ◽  
Plot Analysis ◽  
Gradient Technique ◽  
Na Uptake

Kinetically distinct Na(+)-H+ exchangers exist on the apical and basolateral membranes of rabbit ileal enterocytes. The apical Na(+)-H+ exchanger appears to function in electroneutral NaCl transport, whereas the basolateral Na(+)-H+ exchanger may function in homeostatic intravesicular pH (pHi) regulation and volume regulation. This study is designed to determine the presence and characteristics of the Na(+)-H+ exchanger in basolateral membrane vesicles (BLMV) prepared from jejunal tissues of human organ donors. A well-validated Percoll-gradient technique was used to prepare BLMV. An outwardly directed H+ gradient [pHi/extravesicular pH (pHo) = 5.2/7.5] resulted in a Na+ uptake overshoot (1.45 +/- 0.21 nmol/mg protein) 2.5-fold above equilibrium values (0.59 +/- 0.13 nmol/mg protein). Na+ uptake at equilibrium represented transport into an osmotically sensitive intravesicular space as validated by an osmolality study. Na+ uptake represented an electroneutral process, as shown by studies in which negative membrane potentials were induced by K+ and the ionophore valinomycin. Na+ uptake was linear for the first 15 s of transport as depicted by y = 0.042x + 0.002, r2 = 0.98. Dixon plot analysis of amiloride sensitivity revealed an ID50 value for amiloride of 29 microM (fourfold lower than ID50 for brush-border Na(+)-H+ exchanger). Kinetic studies of amiloride-sensitive Na+ uptake revealed a maximal velocity = 1.53 +/- 0.19 nmol.mg protein-1.5 s-1 and Michaelis constant = 9.83 +/- 3.5 mM. By varying pHi a sigmoidal effect of internal H+ on Na+ uptake was noted consistent with an internal modifier site for protons. To confirm this finding, the effect of pHi on Na+ efflux and Na(+)-Na+ exchange was studied.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium

1988 ◽  
Vol 252 (1) ◽  
pp. 215-220 ◽  
Author(s):  
A Enyedi ◽  
J Minami ◽  
A J Caride ◽  
J T Penniston
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Calcium Uptake ◽  
Limited Proteolysis ◽  
Membrane Vesicles ◽  
Calcium Pump ◽  
Ionophore A23187 ◽  
Plasma Membrane Vesicles ◽  
Active Calcium ◽  
Stimulation Of

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.

scholarly journals Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport

1993 ◽  
Vol 294 (2) ◽  
pp. 529-534 ◽  
Author(s):  
S A Pinches ◽  
S M Gribble ◽  
R B Beechey ◽  
A Ellis ◽  
J M Shaw ◽  
...  
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Independent Manner ◽  
Basolateral Membranes ◽  
Luminal Membrane ◽  
C Terminus ◽  
Normal Human

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

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