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

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.

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

Acta Endocrinologica ◽

10.1530/acta.0.1230438 ◽

1990 ◽

Vol 123 (4) ◽

pp. 438-444 ◽

Cited By ~ 1

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.

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

Biochemical Journal ◽

10.1042/bj2560593 ◽

1988 ◽

Vol 256 (2) ◽

pp. 593-598 ◽

Cited By ~ 5

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.

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Calcium transport by rat duodenal villus and crypt basolateral membranes

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1987.252.2.g170 ◽

1987 ◽

Vol 252 (2) ◽

pp. G170-G177 ◽

Cited By ~ 4

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.

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Proton-lactate co transport in basolateral membrane vesicles from rat jejunum

Bioscience Reports ◽

10.1007/bf01198466 ◽

1996 ◽

Vol 16 (6) ◽

pp. 521-527

Author(s):

Maria Novella Orsenigo ◽

Marisa Tosco ◽

Umberto Laforenza ◽

Alide Faelli

Keyword(s):

Basolateral Membrane ◽

Membrane Vesicles ◽

Rat Jejunum ◽

Basolateral Membrane Vesicles ◽

Strong Inhibition ◽

Lactate Transport ◽

Basolateral Membranes ◽

Lactate Uptake ◽

Stimulation Of

Proton-coupled lactate transport across the basolateral membrane of rat jejunal enterocyte was studied using well purified membrane vesicles. L-lactate uptake is stimulated by an inwardly directed H+ gradient; the effect of the pH difference is drastically reduced by FCCP and by pCMBS; unlabelled L-lactate causes a strong inhibition, whilst furosemide is uneffective. The H+ gradient-dependent stimulation of L-lactate uptake is significantly inhibited also by SCN−: this finding could explain results recently reported in the literature in which H+-lactate symport was not evidenced in basolateral membranes from rat jejunum.

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Stimulation of intestinal basolateral membrane calcium-pump activity by recombinant synthetic calbindin-D9k and specific mutants

Biochemical and Biophysical Research Communications ◽

10.1016/0006-291x(90)92134-l ◽

1990 ◽

Vol 170 (2) ◽

pp. 603-608 ◽

Cited By ~ 15

Author(s):

Julian R.F. Walters ◽

Alison Howard ◽

Maud V. Charpin ◽

Kathy C. Gniecko ◽

Peter Brodin ◽

...

Keyword(s):

Basolateral Membrane ◽

Calcium Pump ◽

Calbindin D9k ◽

Pump Activity ◽

Stimulation Of

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Indirect Na+ dependency of urate and p-aminohippurate transport in pig basolateral membrane vesicles

AJP Renal Physiology ◽

10.1152/ajprenal.1991.261.2.f265 ◽

1991 ◽

Vol 261 (2) ◽

pp. F265-F272 ◽

Cited By ~ 7

Author(s):

D. Werner ◽

F. Roch-Ramel

Keyword(s):

Proximal Tubule ◽

Basolateral Membrane ◽

Membrane Vesicles ◽

Organic Anions ◽

Transport Processes ◽

Basolateral Membrane Vesicles ◽

Pig Kidney ◽

Different Characteristics ◽

Stimulation Of

Membrane vesicles were used to study the basolateral transport of urate and p-aminohippurate (PAH) in the proximal tubule of the pig kidney. Consistent with a cooperation between a Na(+)-2-oxoglutarate cotransporter and a 2-oxoglutarate-urate or a 2-oxoglutarate-PAH exchanger, urate and PAH uptakes were stimulated in presence of extravesicular 2-oxoglutarate when an inwardly directed Na+ gradient was applied. Both transports exhibited, however, different characteristics. The optimal 2-oxoglutarate concentration for stimulating uptakes was 10 microM for PAH and 150 microM for urate. Extravesicular chloride was required to observe a stimulation of PAH uptake but not of urate uptake. Transports of both PAH and urate exhibited different affinity sequences for various organic anions. Stimulated PAH uptake was inhibited by probenecid greater than cold PAH greater than urate = pyrazinoate greater than lactate, whereas stimulated urate uptake was inhibited by probenecid greater than cold urate greater than PAH and not by pyrazinoate or lactate. These results are consistent with independent transport processes for urate and PAH in pig basolateral membrane vesicles, both being indirectly driven by an inwardly directed Na+ gradient.

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Basolateral tetraethylammonium transport in intact tubules: specificity and trans-stimulation

AJP Renal Physiology ◽

10.1152/ajprenal.1991.261.3.f386 ◽

1991 ◽

Vol 261 (3) ◽

pp. F386-F392 ◽

Cited By ~ 4

Author(s):

W. H. Dantzler ◽

S. H. Wright ◽

V. Chatsudthipong ◽

O. H. Brokl

Keyword(s):

Organic Cation ◽

Basolateral Membrane ◽

Physiological Role ◽

Membrane Vesicles ◽

Cation Transport ◽

Organic Cations ◽

Basolateral Membrane Vesicles ◽

Bathing Medium ◽

First Time ◽

Stimulation Of

To examine the specificity of proximal renal basolateral organic cation transport, the effects of unlabeled organic cation substrates in the bathing medium on the rate of uptake [14C]tetraethylammonium ([14C]TEA) by intact nonperfused proximal tubules and isolated basolateral membrane vesicles (BLMV) from rabbit kidneys were explored. The pattern of inhibition of transport by a battery of unlabeled organic cations was similar in intact tubules and BLMV. To determine if trans-stimulation could be demonstrated across the basolateral membrane of intact tubules, the effects of preloading tubules with unlabeled substrates on the rate of uptake of [14C]TEA and the effects of unlabeled substrates in the bathing medium on the rate of efflux of [14C]TEA from tubules preloaded with this labeled substrate were examined. Trans-stimulation was clearly demonstrated for the first time in intact tubules. However, of the compounds that significantly inhibited [14C]TEA uptake (TEA, amiloride, tetrapropylammonium, mepiperphenidol, isopropyl pyridinium, and choline), only TEA itself and choline produced a trans-stimulation of [14C]TEA uptake. Moreover, choline appeared to be at least as effective as TEA itself as a counter ion for TEA transport. Such trans-stimulation could play a physiological role in the net reabsorption of choline and the net secretion of most other organic cations.

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Stimulation of basolateral Na(+)-HCO3- cotransporter by angiotensin II in rabbit renal cortex

AJP Renal Physiology ◽

10.1152/ajprenal.1993.265.2.f195 ◽

1993 ◽

Vol 265 (2) ◽

pp. F195-F203 ◽

Cited By ~ 6

Author(s):

S. Eiam-Ong ◽

S. A. Hilden ◽

C. A. Johns ◽

N. E. Madias

Keyword(s):

Renal Cortex ◽

Basolateral Membrane ◽

Enzyme Reaction ◽

Membrane Vesicles ◽

Independent Effect ◽

Ang Ii ◽

Basolateral Membrane Vesicles ◽

Michaelis Constant ◽

Direct Stimulation ◽

Stimulation Of

Angiotensin (ANG) II is now recognized as a powerful direct controller of Na+ reabsorption in the proximal convoluted tubule, a property that predominantly reflects stimulation of the transepithelial NaHCO3 flux. Numerous studies have established that this effect of ANG II represents stimulation of the apical Na+/H+ exchanger, but a single microperfusion study has also suggested direct stimulation of the basolateral Na(+)-HCO3- cotransporter. We have carried out studies in basolateral membrane vesicles from rabbit renal cortex to examine directly whether ANG II exerts an independent effect on the Na(+)-HCO3- cotransporter. Preincubation of vesicles with ANG II (10(-11) to 10(-9) M) for 15 min enhanced the activity of the cotransporter, the greatest effect occurring at 10(-11) M (41 +/- 1.1%, P < 0.005). This stimulation reflected an increase in the maximal enzyme reaction velocity of the cotransporter but no change in the Michaelis constant for Na+. ANG II had no effect on Na(+)-dependent succinate transport. ANG I (10(-9) M) and ANG III (10(-10) M) also stimulated the Na(+)-HCO3- cotransporter, and captopril (10(-4) M) attenuated the ANG I stimulation by 68 +/- 3.5% (P < 0.01) but not that of ANG II and III. Saralasin (10(-11) to 10(-8) M) by itself behaved as an agonist, and its stimulation was additive to that by ANG II. The nonpeptide ANG II receptor antagonist, losartan potassium (10(-6) M), and the disulfide-reducing agent, dithiothreitol (10 mM), each by itself had no effect on the cotransporter but each markedly attenuated the ANG II effect (by 77 +/- 1.4%, P < 0.01 and 74 +/- 1.6%, P < 0.005, respectively) in accord with the view that the basolateral receptor belongs to subtype 1. These results identify physiological concentrations of ANG II as a potent, direct, and specific stimulator of the basolateral Na(+)-HCO3- cotransporter.

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