Regulation of the rabbit ileal brush-border Na+/H+ exchanger by an ATP-requiring Ca++/calmodulin-mediated process.

Rood, Richard P.; Emmer, E.; Wesolek, John H.; McCullen, J.; Husain, Zakir; Cohen, Michael E.; Braithwaite, R. Scott; Murer, Heini; Sharp, Geoffrey W. G.; Donowitz, Mark

doi:10.1172/jci113665

Cited by 42 publications

(32 citation statements)

References 21 publications

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“…Using these cells we then tested the possible involvement of specific protein kinase-and Ca 2ϩ /calmodulin-mediated pathways in the regulation of folic acid uptake by colonocytes. We focused on pathways that involve protein kinases (PKC and PKA) for which consensus sequences have been shown to exist in the recently cloned folate carriers (13)(14)(15)32) as well as those pathways that have been shown to play an important role in the regulation of uptake of other nutrients by epithelial cells (PTK-and Ca 2ϩ /calmodulin-mediated pathways) (33)(34)(35)(36)(37)(38)(39)(40)(41). When specific modulators of these pathways were used, we found that PKC-mediated pathways had no role in regulating folic acid uptake by NCM460 cells.…”

Section: Discussionmentioning

confidence: 99%

“…We focused on pathways that involve protein kinases for which consensus sequences have been shown to exist in recently cloned folate transporters (namely protein kinase C (PKC) and A (PKA)) (13-15, 31) and on pathways that have been shown to play a role in the regulation of uptake of other nutrients by intestinal and other epithelia (namely, PTK and Ca 2ϩ /calmodulin) (32)(33)(34)(35)(36)(37)(38)(39)(40).…”

Section: Regulation Of Folic Acid Uptake In Ncm460 Cells and The Rolementioning

confidence: 99%

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A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Kumar

Moyer

Dudeja

et al. 1997

Journal of Biological Chemistry

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A significant proportion of the bacterially synthesized folate in the large intestine exists in the form of folate monoglutamate. Recent studies in our laboratory using human colonic apical membrane vesicles have shown the existence of an efficient carrier-mediated system for folate uptake. Nothing, however, is known about the cellular regulation of the colonic uptake process. In this study, we used a recently established human normal colonic epithelial cell line NCM460 to address this issue. Uptake of folic acid by NCM460 cells was: 1) linear with time for 4 min of incubation and occurred with minimal metabolic alterations, 2) temperature-and pH-(but not Na ؉ ) dependent, 3) saturable as a function of concentration (apparent K m of 1.4 M), 4) inhibited by structural analogs and anion transport inhibitors, and 5) energydependent. These characteristics of folic acid uptake by NCM460 cells are similar to those seen with apical membrane vesicles derived from human native colonic tissue. Using these cells, we found that protein kinase Cand Ca 2؉ /calmodulin-mediated pathways have no role in regulating folic acid uptake. On the other hand, cAMP (through a mechanism independent of protein kinase A) and protein-tyrosine kinase-mediated pathways were found to play a role in the regulation of folic acid uptake by these cells. These results establish the suitability of NCM460 cells as an in vitro model system for investigating the details of the mechanism of colonic folate uptake and its regulation. Folic acid uptake by these cells appears to involve a carrier-mediated system, which is temperature-, pH-, and energy-dependent and appears to be under the regulation of cAMP and protein tyrosine kinase.Folate is an essential micronutrient, which acts as a coenzyme in the synthesis of DNA and RNA and the interconversion and degradation of several amino acids (1-4). An adequate supply of folate is therefore necessary for normal cellular function, growth, and development. Folate deficiency has been suggested as one of the most common vitamin deficiencies in the Western Hemisphere (5, 6). Humans and other mammals cannot synthesize folate and rely on exogenous sources to meet their metabolic requirements. Folates are presented to the host from the diet and are also synthesized in the large intestine by normal microflora. The mechanism of absorption of dietary folate has been intensively examined over the past two decades at the tissue, cellular, subcellular, and more recently, molecular levels (6 -15). Absorption of dietary folate has been shown to occur mainly in the proximal small intestine and involves a specialized, carrier-mediated system (6 -12).As to the bacterially synthesized folate in the large intestine, a significant amount of that folate exists in the monoglutamate, i.e. the absorbable form. Using [ 3 H]p-aminobenzoic acid to label the newly synthesized folate by the intestinal flora, Rong et al. (16) have shown that a portion of this folate is indeed absorbed by the rat and is incorporated into its various tissues. ...

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Section: Discussionmentioning

confidence: 99%

Section: Regulation Of Folic Acid Uptake In Ncm460 Cells and The Rolementioning

confidence: 99%

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Kumar

Moyer

Dudeja

et al. 1997

Journal of Biological Chemistry

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“…The final two pellets, control and carbachol treated, were needle homogenized with a 1-ml syringe and 25-gauge needle in the same buffer and the suspensions brought to a protein concentration of 5 (20). Uptake of Na' was measured over 8 s after mixing the membranes with transport buffer, which is during the period when 22Na' uptake, with and without a pH gradient, is linear with time in these vesicles (2 changes occurred in the homogenates. In Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Drugs or hormones which raise cytosol free by affecting the activity ofthe Na+/H' exchanger (2). The regulation by Ca2l is somewhat complex.…”

Section: Introductionmentioning

confidence: 99%

Carbachol- and elevated Ca(2+)-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells.

et al. 1991

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Protein kinase C is involved in mediating the effects of elevated Ca2+ in ileal villus Na+ absorbing cells to inhibit NaCI absorption. The present studies were undertaken to understand the mechanism by which this occurs. The effects of carbachol and the calcium ionophore A23187, agents which elevate intracellular Ca2" and inhibit NaCl absorption in ileal villus cells, were studied. Carbachol treatment of villus cells caused a rapid decrease in protein kinase C activity in cytosol, with an accompanying increase in microvillus membrane C kinase. Exposure of the villus cells to calcium ionophore also caused a quantitatively similar decrease in cytosol C kinase and increase in C kinase activity in the microvillus membrane. This increase caused by carbachol and Ca2+ ionophore was specific for the microvillus membrane. In fact, 30 s and 10 min after exposure of the cells to carbachol, basolateral membrane protein kinase C decreased, in a time-dependent manner, whereas 10 min of Ca2" ionophore exposure did not alter basolateral C kinase. Exposure of villus cells to Ca2" ionophore or carbachol caused similar increases in microvillus membrane diacylglycerol content. As judged by the ability to inhibit Na+/H+ exchange measured in ileal villus cell brush border membrane vesicles, the protein kinase C which translocated to the microvillus membrane was functionally significant. Inhibition of Na+/H+ exchange required ATP and was reversed by the protein kinase C antagonist H-7. In conclusion, the effect of carbachol and Ca2" ionophore in regulation of ileal NaCl absorption is associated with an increase in microvillus membrane diacylglycerol content and functionally active protein kinase C. The effects of both carbachol and Ca2" ionophore are different on brush border and basolateral membrane distribution of protein kinase C. (J. Clin. Invest. 1991. 88:855-863.)

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“…Collected pellets (basolateral membrane fraction) were phosphorylated in vitro, using the technique of Ruiz & Arruda (1992), by transient exposure to a hypotonic solution containing 10 mM MgCl 2 , 10 mM KCl and 5 mM Mes/Tris buffer pH 6.8, as well as either 0.8 mM ATP, 8 U l _1 creatine kinase, 8 mM creatine phosphate, 1.2 mM CaCl 2 and 1.2 mM PMA (phosphorylating solution), or 0.8 mM AMP-PNP (nonphosphorylating solution). Creatine kinase and creatine phosphate constitute an ATP-regenerating system, while AMP-PNP is a non-hydrolysable analogue of ATP (Rood et al 1988). The treatment lasted 5 min at 30°C, and this procedure resulted in opening up the vesicles.…”

Section: Protein Kinase C Regulation Of Rat Jejunal Transport Systemsmentioning

confidence: 99%

Protein Kinase C Regulation of Rat Jejunal Transport Systems: Mechanisms Involved in Lactate Movement

Tosco¹,

Orsenigo²,

Gastaldi³

et al. 2002

Experimental Physiology

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We examined whether protein kinase C (PKC) modulates the transport systems involved in lactate movements across the plasma membranes of rat jejunum. In vitro phosphorylated membrane vesicles were used to perform uptake studies, the results of which suggested that PKC activation exerts an inhibitory effect on basolateral H+‐lactate symport, as well as on apical Na+‐glucose cotransport. The specificity of the response to PKC was confirmed by using staurosporine, chelerythrine or 4‐α‐PMA. Experiments performed using the whole tissue incubated in vitro confirmed the reduction of lactate transport elicited by PKC and gave evidence for an associated inhibition of fluid transport. Na+,K+‐ATPase activity seems to be unaffected by the kinase and inhibited by Ca2+. Taken together, our results suggest that the overall action of PKC results from the simultananeous modulation of multiple pathways, targeted to a reduction of both lactate and bicarbonate transports without altering cell pH homeostasis.

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Regulation of the rabbit ileal brush-border Na+/H+ exchanger by an ATP-requiring Ca++/calmodulin-mediated process.

Cited by 42 publications

References 21 publications

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

A Protein-tyrosine Kinase-regulated, pH-dependent, Carrier-mediated Uptake System for Folate in Human Normal Colonic Epithelial Cell Line NCM460

Carbachol- and elevated Ca(2+)-induced translocation of functionally active protein kinase C to the brush border of rabbit ileal Na+ absorbing cells.

Protein Kinase C Regulation of Rat Jejunal Transport Systems: Mechanisms Involved in Lactate Movement

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