Omega-3 fatty acids modulate ATPases involved in duodenal Ca absorption

doi:10.1016/S0952-3278(03)00067-X

Prostaglandins, Leukotrienes and Essential Fatty Acids

Volume 68, Issue 6, June 2003, Pages 423-429

https://doi.org/10.1016/S0952-3278(03)00067-X Get rights and content

Abstract

Dietary supplementation with fish oil that contains omega-3 polyunsaturated fatty acids has been shown to enhance bone density as well as duodenal calcium uptake in rats. The latter process is supported by membrane ATPases. The present in vitro study was undertaken to test the effect of omega-3 fatty acids on ATPase activity in isolated basolateral membranes from rat duodenal enterocytes. Ca-ATPase in calmodulin-stripped membranes was activated in a biphasic manner by docosahexanoic acid (DHA) (10–30 μg/ml) but not by eicosapentanoic acid (EPA). This effect was blocked partially by 0.5 μM calphostin (a protein kinase C blocker). DHA inhibited Na,K-ATPase (−49% of basal activity, [DHA]=30 μg/ml, P<0.01). This effect could be reversed partially by 50 μM genistein, a tyrosine kinase blocker. EPA also inhibited Na,K-ATPase: (−47% of basal activity, [EPA]=30 μg/ml, P<0.01), this effect was partially reversed by 100 μM indomethacin, a cyclo-oxygenase blocker. Omega-3 fatty acids are thus involved in multiple signalling effects that effect ATPases in BLM.

Introduction

Duodenal mineral absorption has been linked with dietary fat content for several decades [1]. As infant feeding formulas became available in the 1960s, it soon became apparent that the inclusion of a high ratio of unsaturated to saturated fatty acids in the formula had beneficial effects on Ca absorption [2], [3]. The relevance of this principle on the adult diet was documented by Van Bokkum et al. [4] who reported decreased faecal Ca levels (indicating a positive Ca balance) after increasing dietary linoleic acid (LA), the parent omega-6 fatty acid. Song and co-workers [5] reported that oral intake of arachidonic acid (AA) (likewise an omega-6 fatty acid) could increase Ca absorption in vivo while Buck and co-workers [6] have shown that supplementation with primrose oil, sunflower oil (containing mainly omega-6) as well as fish oil (containing mainly omega-3's), all caused an increase in Ca absorption, as measured with a radioactive Ca tracer technique.

The mechanism of essential fatty acid (EFA) action, however, remained unclear. Duodenal Ca absorption is a multifaceted process consisting of at least three steps [7]: uptake through Ca channels in the brush border membrane, transport through the cell by a carrier protein (calcium binding protein, CaBP, stimulated by Vitamin D) as well as extrusion of calcium through the basolateral membrane by Ca-ATPase and a Na–Ca exchanger. The latter is driven by Na⁺,K⁺-ATPase (NKA) also present in the basolateral membrane. Early experiments in the seventies on microvillar membranes suggested that EFAs potentiated Vitamin D action on duodenal Ca absorption. Increases in Ca-ATPase activity was ascribed to changes in the lipid environment of the enzyme [8]. Subsequent studies by the groups of Bikle, Kreutter and Brasitus [9], [10], [11] confirmed these observations.

Our own laboratory investigated the effects of EFAs on Ca uptake and Ca-ATPase activity in basolateral membranes (BLM) isolated from duodenal enterocytes. Dietary supplementation for 12 weeks with evening primrose oil (EPO) and fish oil (FO) both increased Ca uptake into BLM vesicles, EPO reaching slightly higher levels than FO [12]. Furthermore, supplementation with a EPO plus FO mix yielding a ratio of EPA+DHA:GLA of 1:3 caused an increase Ca-ATPase activity over controls that had been fed the parent omega-6 and omega-3 fatty acids in corn oil (containing LA) and linseed oil (containing α-LA) in a ratio of 3:1 [13]. Further in vitro studies in which BLM were exposed to AA, however, could not show an influence of the fatty acid on Ca-ATPase activity [14].

The present in vitro study was undertaken in an attempt to elucidate the mechanism by which supplementation with omega-3 fatty acids, had achieved an increase in Ca-ATPase activity. Parallel studies were done on the influence of omega-3 fatty acids on Na,K-ATPase, since this enzyme drives the Ca–Na exchanger in BLM [7].

Section snippets

Materials

Docosahexanoic acid (DHA), eicosapentanoic acid (EPA), calphostin (CP), indomethacin (IDM), genistein (GEN), ouabain, ethyleneglycolbis-(β-aminoethyl ether) N,N′-tetraacetic acid (EGTA), phenyl-methyl-sulphonyl fluoride (PMSF), dithiothreitol (DTT), dimethylsulfoxide (DMSO) and Tris-ATP were purchased from Sigma Chemical Co, St Louis, Mo. USA. All other reagents were of reagent grade and obtained from Saarchem Chemicals, Johannesburg.

Preparation of basolateral membranes (BLM) from rat enterocytes

Male Sprague-Dawley rats obtained from the Pretoria

Omega-3 fatty acid effects on Ca-ATPase in BLM

Neither DHA nor EPA (both at 30 μg/ml) had any influence on Ca-ATPase activity in native BLM, as shown in Fig. 1A. This may be due to the fact that the enzyme was already fully activated by calmodulin that interacts with the carboxyl terminal of the molecule [16]. Upon removal of calmodulin by incubation with EGTA-imidazole, DHA, but not EPA (again at 30 μg/ml) could activate Ca-ATPase by 247% (Fig. 1B). The fatty acid concentrations used in this experiment (101–108 μM) are in the physiological

Discussion and conclusion

The rate-limiting step of Ca transport through the duodenal enterocyte is extrusion of Ca at the basolateral membrane by Ca-ATPase [1], [25]. Ca-ATPase activity is primarily regulated by the Ca-calmodulin complex that interacts with the enzyme near the carboxyl terminal of the molecule. The calmodulin-binding domain causes tonic auto-inhibition of the Ca pump due to its interaction with the catalytic domain of the enzyme, when this part of the peptide binds to the Ca-calmodulin complex, this

Acknowledgments

This research was funded by NAVKOM and RDP grants from the University of Pretoria to M. Haag as well as an MRC, South Africa grant to M.C. Kruger. We would also like to thank Prof E. Carafoli, Padua, Italy, for instructive communication.

References (43)

M.C. Kruger et al.
Calcium metabolism, osteoporosis and essential fatty acids: a review
Prog. Lipid Res.
(1997)
E.M. Widdowson
Absorption and excretion of fat, nitrogen and minerals from ‘filled’ milks by babies one week old
Lancet
(1965)
M.K. Song et al.
A new low-molecular weight calcium-binding ligand in rat small intestine
Life Sci.
(1983)
D.B.P. Goodman et al.
Vitamin D₃ induced alteration of microvillar membrane lipid composition
Biochem. Biophys. Res. Commun.
(1972)
D. Kreutter et al.
The role of fatty acids in the regulation of brush-border Ca²⁺ transport
Biochem. Biophys. Acta
(1984)
T.A. Brasitus et al.
Variations in dietary diacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes
Biochim. Biophys. Acta
(1985)
H. Coetzer et al.
Calcium transport by isolated brush border and basolateral membrane vesiclesrole of essential fatty acid supplementation
Prostaglandins Leukot. Essent. Fatty acids
(1994)
F. Leonard et al.
Modulation of intestinal vitamin D receptor availability and calcium ATPase activity by essential fatty acids
Prostaglandins Leukot. Essent. Fatty Acids
(2001)
M. Haag et al.
Effect of arachidonic acid on duodenal enterocyte ATPases
Prostaglandins Lipid Mediators
(2001)
B.N. Ames
Assay of inorganic phosphate total phosphate, and phosphatases
Methods Enzymol.
(1966)

V. Niggli et al.

Acidic phospholipids unsaturated fatty acids and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca²⁺-ATPase

J. Biol. Chem.

(1981)

E.A. Nunez

Free fatty acids as modulators of the steroid hormone message

Prostaglandins, Leukot. Essent. Fatty Acids

(1993)

M.G. Jack-Hays et al.

Activation of Na⁺/K⁺-ATPase by fatty acids acylglycerols and related amphiphilesstructure–activity relationship

Biochim. Biophys. Acta

(1996)

H.G.P. Swarts et al.

Binding of unsaturated fatty acids to Na⁺,K⁺-ATPase leading to inhibition and inactivation

Biochim. Biophys. Acta

(1990)

K. Oishi et al.

Inhibition of Na,K-ATPase and sodium pump by protein kinase C regulators sphingosine lysophosphatidylcholine and oleic acid

J. Biol. Chem.

(1990)

G.R. Monteith et al.

The plasma membrane calcium pump—a physiological perspective on its regulation

Cell Calcium

(1995)

M. Haag et al.

Upregulation of duodenal calcium absorption by polyunsaturated fatty acidsevents at the basolateral membrane

Med. Hypoth.

(2001)

P. Caroni et al.

Regulation of Ca²⁺-pumping ATPase of heart sarcolemma by a phosphorylation/dephosphorylation process

J. Biol. Chem.

(1981)

F. Hofmann et al.

Phosphorylation of the calmodulin-binding domain of the plasma membrne Ca²⁺ pump by protein kinase C reduces its interaction with calmodulin and its pump receptor site

J. Biol. Chem.

(1994)

L.C. Wright et al.

Regulation of the activity and phosphorylation of the plasma membrane Ca²⁺-ATPase by protein kinase C in intact human erythrocytes

Arch. Biochem. Biophys.

(1993)

B. Mirnikjoo et al.

Protein kinase inhibition by omega-3 fatty acids

J. Biol. Chem.

(2001)

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Omega-3 fatty acids modulate ATPases involved in duodenal Ca absorption

Abstract

Introduction

Section snippets

Materials

Preparation of basolateral membranes (BLM) from rat enterocytes

Omega-3 fatty acid effects on Ca-ATPase in BLM

Discussion and conclusion

Acknowledgments

Prog. Lipid Res.

Lancet

Life Sci.

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Acta

Biochim. Biophys. Acta

Prostaglandins Leukot. Essent. Fatty acids

Prostaglandins Leukot. Essent. Fatty Acids

Prostaglandins Lipid Mediators

Methods Enzymol.

J. Biol. Chem.

Prostaglandins, Leukot. Essent. Fatty Acids

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem.

Cell Calcium

Med. Hypoth.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.