Veteran Affairs Healthcare System, Long Beach; and College of Medicine, University of California, Irvine, Irvine, California
Submitted 11 January 2005 ; accepted in final form 15 February 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
intestinal transport; transport mechanism; transport regulation
The mechanism involved in the intestinal absorption of dietary niacin, and its regulation, is not well understood. Previous studies in laboratory animals have reported the mechanism to function either via simple diffusion of the undissociated form of nicotinic acid (according to the pH partition hypothesis and assisted by acid microclimate at the luminal surface of the intestine) (3) or via a carrier-mediated mechanism (4, 14, 18, 20). The latter studies, however, reported an apparent Michaelis-Menten constant (Km) for the carrier-mediated process from 3.52 to 17.0 mM. The high apparent Km values reported in the previous studies raise a concern regarding the physiological relevance of the described system, because intestinal luminal concentration of niacin under physiological conditions is estimated to be in the micromolar but not the millimolar range (6, 19).
Chemically, nicotinic acid is a monocarboxylic acid with a pKa of 4.9. Transport of monocarboxylic acids (i.e., lactate, pyruvate, and the ketone bodies acetoacetate and -hydroxybutyrate) is mediated in mammalian cells by a family of monocarboxylate transporters (MCTs) (7). MCTs are low-affinity carriers that display apparent Km values in the millimolar range (7). Previous studies have reported inhibition of niacin uptake by monocarboxylic acids (18, 20), raising the possibility of involvement of the MCT systems in niacin uptake. Our aims in the present study were to investigate the mechanism of niacin uptake in the human intestine using as models cultured, human-derived intestinal epithelial Caco-2 cells and purified isolated brush-border membrane vesicles (BBMVs) isolated from the jejunum of organ donors. Our results demonstrate for the first time the existence of a highly specialized, acidic pH-dependent, high-affinity, carrier-mediated system for niacin uptake by human intestinal epithelial cells. Also, evidence was obtained suggesting that the system may be under the regulation of an intracellular protein tyrosine kinase (PTK)-mediated pathway.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell Culture and Uptake Studies
Human-derived intestinal epithelial Caco-2 cells (passage 20; American Type Culture Collection, Manassas, VA; these cells were derived from colorectal adenocarcinoma obtained from a 72-year-old Caucasian male) were grown in Dulbeccos modified Eagles medium supplemented with 10% (vol/vol) fetal bovine serum in 75-cm2 plastic flasks at 37°C in a 5% CO2-95% air atmosphere, with medium changed every 23 days. The cells were subcultured and plated onto 24-well plates. Uptake studies were performed on confluent cell monolayers (between passages 23 and 36) 35 days after confluence. Cells were fed fresh incubation medium the day before uptake experiments were performed.
[3H]Nicotinic acid uptake was examined in Caco-2 cells incubated in Krebs-Ringer buffer containing (in mM) 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES, pH 5.0 (unless otherwise stated). [3H]Nicotinic acid was added to the incubation medium at the onset of the uptake experiment, and the reaction was terminated after 3 min (unless otherwise stated) by the addition of 1 ml of ice-cold buffer, followed by immediate aspiration. Cells were then rinsed twice with ice-cold buffer and lysed with 1 ml of 1 N NaOH. Lysates were neutralized with HCl, and then radioactivity was measured using a scintillation counter. The protein content of cell digests was measured in parallel wells using the Bio-Rad Dc protein assay kit (Bio-Rad, Hercules, CA).
The metabolic form of the transported substrate after 3-, 7-, and 10-min incubation with 60 nM [3H]nicotinic acid was determined using thin-layer chromatography with silica-precoated thin-layer plates and a solvent system comprising a 4:4:2 ratio (vol/vol) of n-butanol to acetic acid to water.
Human Intestinal BBMVs and Uptake Studies
Human intestinal BBMVs were kindly provided by Dr. P. K. Dudeja (University of Illinois at Chicago) and were isolated from the jejunum of organ donors with the use of a well-established procedure (2) and in accordance with the institutional protocols approved by the Institutional Review Board of the University of Illinois at Chicago. Uptake of [3H]niacin into intestinal BBMVs was measured using a rapid-filtration technique described previously (12). Briefly, BBMVs were preloaded with a buffer containing (in mM) 280 mannitol and 20 HEPES-Tris, pH 7.4, and then incubated in a buffer containing (in mM) 140 mannitol, 100 NaCl, 10 HEPES, and 10 MES (pH 5.0 or 7.4) in the presence of [3H]niacin. Uptake studies were performed at 10 s (initial rate; Ref. 18) at 37°C.
Data Presentation and Statistical Analysis
Values are means ± SE of multiple individual uptake determinations and are expressed in picomoles or femtomoles per milligram of protein per unit of time. Students t-test and ANOVA were used in statistical analysis. P < 0.05 was considered statistically significant. Kinetic parameters of the saturable uptake process of nicotinic acid [i.e., maximal velocity (Vmax) and apparent Km] were calculated as described by Wilkinson (21). Uptake by the saturable component was calculated at each concentration subtracting uptake by simple diffusion (determined from the slope of the line between the point of origin and uptake at high pharmacological concentration of niacin; 1 mM) from total uptake.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The time-dependent uptake of nicotinic acid (0.106 µM) by Caco-2 cells at pH 5.0 was examined. Uptake was found to be linear up to 10 min at the rate of 159 fmol·mg of protein1·min1 (Fig. 1). Thus 3-min incubation was chosen as the standard incubation time for which to study the effect of different factors and/or conditions on the initial rate of niacin uptake. Uptake of a high niacin concentration, 10 µM, also was found to be linear for the selected incubation time of 3 min (data not shown).
|
|
The effect of incubation temperature on the initial rate of niacin (6 nM) uptake was also investigated. The results showed niacin uptake to be significantly (P < 0.01) higher at 37°C compared with 27°C (224.8 ± 8.0 and 82.3 ± 0.5 fmol·mg of protein1·3 min1, respectively), with a calculated Q10 value (ratio of transport rate at 37°C to transport rate at 27°C) of 2.7.
The role of Na+ dependence in intestinal niacin uptake was tested by examining the effect of isosmotic replacement of Na+ in the incubation medium with K+ or Li+ on the initial rate of niacin (6 nM) uptake. Similar niacin uptake was observed in the presence and absence of Na+ (233.2 ± 6.5, 225.3 ± 4.8, and 226.9 ± 4.5 fmol·mg of protein1·3 min1 for control, K+, and Li+, respectively). In a related study, we tested the effect of pretreatment (30 min) of cells with the Na+-K+-ATPase inhibitor ouabain (1 mM) on the initial rate of niacin (6 nM) uptake. Ouabain-pretreated cells showed no significant decrease in niacin uptake (234.74 ± 13.4 and 228.2 ± 12.8 fmol·mg of protein1·3 min1 for control and after pretreatment with ouabain, respectively). Therefore, uptake of niacin by Caco-2 cells was regarded as a Na+-independent process.
We further examined the effect of pretreating (30 min) the cells with the metabolic inhibitors iodoacetate (at 1 mM) and 2,4-dinitrophenol (DNP; 0.5 mM) on the initial rate of niacin (6 nM) uptake. Both compounds were found to cause significant (P < 0.01) inhibition of niacin uptake (230.54 ± 7.6, 48.14 ± 2.6, and 102.66 ± 8.62 fmol·mg of protein1·3 min1 for control, iodoacetate, and DNP, respectively).
Evidence for Existence of a Carrier-Mediated Mechanism for Niacin Uptake by Caco-2 Cells and Purified Native Human Intestinal BBMVs
In these investigations, we examined the initial rate of niacin uptake (i.e., 3 min) as a function of concentration over a wide range of concentrations spanning nanomolar and micromolar ranges (we also included the nanomolar range because recent studies with other water-soluble vitamins such as thiamin and biotin have suggested the existence of a high-affinity system in the nanomolar range in addition to the well-characterized systems that operate at the micromolar ranges of these vitamins; Refs. 5, 17). Our findings show that niacin uptake over the nanomolar range to be linear (R = 0.997) as a function of concentration (Fig. 3A). On the other hand, saturation was observed in niacin uptake over the micromolar concentration range (Fig. 3B). Kinetic parameters of the saturable component were calculated as described in MATERIALS AND METHODS and found to be 0.53 ± 0.08 µM and 13.32 ± 0.58 pmol·mg protein1·3 min1 for apparent Km and maximal velocity (Vmax), respectively. These findings suggest the existence of a carrier-mediated uptake system for niacin that operates in the micromolar range. To confirm this conclusion and to determine the specificity of the niacin uptake system, we examined the effect of unlabeled niacin and that of its structural analogs isonicotinic acid, nicotinamide, isonicotinic acid hydrazide, nicotinyl alcohol, and nicotinuric acid on the initial rate of [3H]niacin (6 nM) uptake by confluent monolayers of Caco-2 cells. We also examined the effect of the niacin related compound 5-methyl-1H-pyrazole-3-carboxylic acid (which has been shown to act as a high-affinity ligand for the recently described niacin receptor HM74A; Ref. 22) on the initial rate of [3H]niacin uptake. The results (Table 1) show that none of the compounds other than unlabeled niacin significantly affected niacin uptake.
|
|
To establish the relevance of these findings on the existence of a carrier-meditated uptake system for niacin to the native human intestine, we examined the effect of unlabeled niacin (1 mM) on the initial rate of uptake of [3H]niacin (12 nM) by purified BBMVs isolated from the jejunum of human organ donors. The study was performed at incubation buffer pH 5.0 and 7.4 (intravesicular pH was kept at 7.4 in both cases). The results showed unlabeled niacin to cause a significant (P < 0.01) inhibition in [3H]niacin uptake in BBMVs incubated at pH 5.0, while no inhibition was observed at pH 7.4 (Fig. 4).
|
Effect of sulfhydryl group reagents on [3H]niacin uptake by Caco-2 cells. The effect of pretreatment (30 min) of Caco-2 cells with different concentrations of the sulfhydryl group inhibitor p-chloromercuribenzene sulfonate (p-CMBS) on the initial rate of niacin (6 nM) uptake by Caco-2 cells was examined. Cells pretreated with the compound were found to have a significantly (P < 0.01) inhibited niacin uptake (238.61 ± 7.4, 162.11 ± 10.0, and 113.01 ± 3.2 fmol·mg of protein1·3 min1 for control, and pretreatment with 0.1 mM and 0.5 mM p-CMBS, respectively). To test the specificity and reversibility of the inhibition in niacin uptake by p-CMPS, we first pretreated the Caco-2 cells with the inhibitor (0.5 mM for 30 min), removed the inhibitor, and added the reducing agent dithiothreitol (10 mM for 30 min). We then examined initial rate of niacin (6 nM) uptake. The results show that such treatment led to a significant (P < 0.01) reversal of the inhibitory effect of p-CMBS (238.61 ± 7.4, 113.01 ± 3.2, and 153.29 ± 4.4 fmol·mg of protein1·3 min1 for control cells, those pretreated with p-CMBS, and those pretreated with p-CMBS and then dithiothreitol, respectively).
Effect of monocarboxylic acids on [3H]niacin uptake by Caco-2 cells.
As mentioned earlier, niacin is a weak monocarboxylic acid with a pKa value of 4.9. Previous studies have suggested that niacin may be transported by the MCT systems. To test this possibility, we examined the effect of different model substrates for the MCT systems on the initial rate of [3H]niacin (6 nM) uptake by Caco-2 cells. The results (Table 2) show that all of the monocarboxylic acids tested (25 mM) had no effect on niacin uptake. In a related study, we examined whether the inhibitors of MCTs, -cyano-4-hydroxycinnamate and phloretin, can affect the initial rate of niacin uptake by Caco-2 cells. The results show that neither
-cyano-4-hydroxycinnamate nor phloretin to affect niacin uptake significantly (228.18 ± 6.7, 221.49 ± 5.1, and 223.31 ± 10.27 fmol·mg of protein1·3 min1 for control,
-cyano-4-hydroxycinnamate, and phloretin, respectively).
|
We examined the possible cellular regulation of the niacin uptake process into Caco-2 cells by specific intracellular regulatory pathways. Pathways shown to be involved in the regulation of uptake of other water-soluble vitamins and other nutrients in intestinal and other cellular systems, i.e., protein kinase A (PKA)-, PTK-, protein kinase C (PKC)-, and Ca2+/calmodulin-mediated pathways, (13, 15, 16), were chosen. These studies were performed by pretreating (for 1 h) the cells with specific modulators of the individual pathway, followed by determination of the initial rate of niacin (6 nM) uptake. The results were compared with simultaneously performed control experiments.
The possible involvement of a PTK-mediated pathway was assessed by examining the effect of pretreatment with genistein and tyrphostin A25 on the initial rate of niacin uptake. The results (Table 3) show that both of the inhibitors caused significant inhibition of niacin uptake; the negative control experiments with genistin and tyrphostin A1, however, were without effect. Next, we determined the effect of genistein on the kinetic characteristics of the niacin transport in Caco-2 cells. For this purpose, we measured the initial rate of niacin uptake as a function of concentration in genistein (50 µM)-pretreated cells and compared the results with that of untreated control cells. Our results show (Fig. 5) a significant (P < 0.01) decrease in the Vmax of niacin uptake in genistein-pretreated cells (12.88 ± 0.14 and 7.27 ± 0.25 pmol·mg protein1·3 min1 for control and genistein-treated cells, respectively), whereas the apparent Km was found to increase slightly with genistein pretreatment (0.52 ± 0.02 µM and 0.67 ± 0.06 µM for control and genistein-treated cells, respectively).
|
|
The potential role of PKC-mediated pathway in regulating niacin uptake by Caco-2 cells was also examined by testing the effect of pretreating the cells with the PKC activator phorbol 12-myristate 13-acetate (PMA; 10 µM) or the PKC inhibitors staurosporine and chelerythrine (both 1 µM) on the initial rate of niacin uptake. None of the modulators led to a significant effect on niacin uptake (228.0 ± 5.5, 222.8 ± 7.3, 227.7 ± 9.7, and 221.6 ± 5.3 fmol·mg of protein1·3 min1 for control, PMA, staurosporine, and chelerythrine, respectively).
We also examined the potential involvement of a cAMP- or PKA-mediated pathway in the regulation of niacin uptake by Caco-2 cells. Pretreatment of cells with a compound known to increase intracellular cAMP level, i.e., dibutyryl cAMP (1 mM) or forskolin (100 µM), as well as an inhibitor of PKA, i.e., H-89 (50 µM), caused no significant effect on niacin uptake (230.8 ± 10.4, 238.2 ± 10.8, 221.0 ± 2.8, and 231.0 ± 9.6 fmol·mg of protein1·3 min1 for control, dibutyryl cAMP, H-89, and forskolin, respectively).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The studies with confluent monolayers of Caco-2 cells showed niacin uptake to be both temperature and energy dependent and to occur with minor metabolic alterations in the transported substrate. Also, the niacin uptake process was Na+ independent as indicated by the lack of effect of Na+ replacement with other monovalent cations on the initial rate of niacin uptake, as well as by the inability the Na+-K+-ATPase inhibitor ouabain to affect the substrate uptake. Niacin uptake by Caco-2 cells, however, was found to be highly dependent on acidic incubation buffer pH. Decreasing the incubation buffer pH from 8.0 to 5.0 led to a marked (5 fold) increase in niacin uptake. This finding suggests the possible involvement of the niacin-H+ cotransport system, which is supported by the finding of significant inhibition in niacin uptake after pretreatment of Caco-2 cells with the protonophore FCCP (see also below).
Uptake of niacin as a function of concentration showed saturation when tested in the micromolar range but not in the nanomolar range. This finding suggests the involvement of a carrier-mediated system for substrate uptake that operates in the former concentration range. The apparent Km of the saturable process was calculated to be 0.53 µM, suggesting that this system is most likely responsible for the absorption of dietary niacin (estimated to be in the micromolar range; Refs. 6, 19). The existence of a carrier-mediated system for [3H]niacin uptake in Caco-2 cells was further confirmed by the findings of significant cis inhibition and trans stimulation by unlabeled niacin. The relevance of these findings to the native human intestine was also established by demonstrating significant inhibition in [3H]niacin uptake by unlabeled niacin in purified BBMVs isolated from the jejunum of human organ donors. Our studies with intestinal BBMVs have also shown the niacin uptake process to be similarly dependent on very acidic pH. The inability of previous studies to identify the existence of a high-affinity niacin uptake system is most probably due to the use of high working niacin concentrations (18, 20). Previous studies have reported an apparent Km at 3.52 and 17.0 mM (4, 14, 18, 20), which is rather high, considering that the luminal concentration of niacin was in the micromolar range. It is possible that the latter system (which is not specific for niacin but appears to be shared by other monocarboxylates; Refs. 18 and 20) is responsible for the uptake of pharmacological concentrations of niacin, which are used clinically for the prevention of atherosclerotic cardiovascular disease (10, 11).
The niacin uptake system of Caco-2 cells appears to be highly specific in nature. This conclusion is based on the observations that, with the exception of unlabeled niacin, none of the niacin structural analogs tested (i.e., isonicotinic acid, nicotinuric acid, niacinamide, nicotinyl alcohol, and isonicotinic acid hydrazide) had an effect on substrate uptake. Also, the related compound 5-methyl-1H-pyrazole-3-carboxylic acid, which represents a high-affinity ligand for the recently described niacin receptor HM74A (22), failed to affect niacin uptake by Caco-2 cells, suggesting that the intestinal Caco-2 uptake process does not involve this receptor.
An interesting observation in this study was the sensitivity of the niacin uptake system of Caco-2 cells to the inhibitory effect of the sulfhydryl group reagent p-CMBS. This inhibition was significantly reversed by treating the cells with the reducing agent dithiothreitol. Because p-CMBS can hardly penetrate the plasma membrane, the possibility exists that the affected sulfhydryl groups are located at the exofacial surface of the cell membrane. None of the other tested membrane transport inhibitors (SITS, probenecid, and amiloride) had an effect on niacin uptake.
Previous studies have suggested that niacin could be transported by the MCT system (7). Our findings in the present study argue against this concept. First, the affinity of most MCT systems is low (reportedly in the millimolar range), while the niacin uptake system described in this study functions in the micromolar range. Second, none of the model (prototype) ligands for these transporters, i.e., lactate, pyruvate, butyrate, and propionate, affected the initial rate of uptake of physiological concentrations of niacin by Caco-2 cells. Third, classic inhibitors of the MCT system, i.e., -cyano-4-hydroxycinnamate and phloretin (7), also failed to affect carrier-mediated niacin uptake by Caco-2 cells.
To further study the cellular uptake of niacin, we investigated the possible involvement of specific intracellular regulatory pathways in the regulation of niacin uptake by Caco-2 cells. We focused on pathways that have been shown to be involved in the regulation of uptake of other nutrients in intestinal and other epithelial systems (13, 15, 16). Our results show that while no role for PKA-, PKC- and Ca2+/calmodulin-mediated pathways is evident, a role for the PTK-mediated pathway is suggested. Inhibitors of this pathway (tyrphostin A25 and genistein, but not their negative controls tyrphostin A1 and genistin, respectively), were found to cause significant inhibition of niacin uptake. The inhibitory effect of one such inhibitor, genistein, was further tested and found to be mediated mainly via a decrease in the Vmax of the niacin uptake process, suggesting a decrease in the activity (and/or number) of the involved carriers. Further studies are required to determine the exact nature of the inhibitory mechanism involved.
In summary, the results of the present study demonstrate for the first time the existence of a highly specialized, acidic pH-dependent, high-affinity, carrier-mediated system for niacin uptake at the apical membrane of human intestinal epithelial cells. In addition, the results also suggest possible involvement of a PTK-mediated pathway in the regulation of niacin uptake by these cells.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
FOOTNOTES |
---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Dudeja PK, Tyagi S, Kavilaveettil RJ, Gill R, and Said HM. Mechanism of thiamine uptake by human jejunal brush-border membrane vesicles. Am J Physiol Cell Physiol 281: C786C792, 2001.
3. Elbert J, Daniel H, and Rehner G. Intestinal uptake of nicotinic acid as a function of microclimate-pH. Int J Vitam Nutr Res 56: 8593, 1986.[ISI][Medline]
4. Fox KR, Adrian C, and Hogben M. Nicotinic acid active transport by in vitro bullfrog small intestine. Biochim Biophys Acta 332: 336340, 1974.[ISI]
5. Grafe F, Wohlrab W, Neubert RH, and Brandsch M. Transport of biotin in human keratinocytes. J Invest Dermatol 120: 428433, 2003.[CrossRef][ISI][Medline]
6. Guilarte TR and Pravlik K. Radiometric-microbiologic assay of niacin using Kloeckera brevis: analysis of human blood and food. J Nutr 113: 25872594, 1983.[ISI][Medline]
7. Halestrap AP and Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 343: 281299, 1999.[CrossRef][ISI][Medline]
8. Ijichi H, Ichiyama A, and Hayaishi O. Studies on the biosynthesis of nicotinamide adenine dinucleotide. III. comparative in vivo studies on nicotinic acid, nicotinamide, and quinolinic acid as precursors of nicotinamide adenine dinucleotide. J Biol Chem 241: 37013707, 1966.
9. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, and Hayaishi O. Studies on the biosynthesis of nicotinamide adenine dinucleotide: a role of picolinic carboxylase in the biosynthesis of nicotinamide adenine dinucleotide from tryptophan in mammals. J Biol Chem 240: 13951401, 1965.
10. Meyers CD and Kashyap ML. Management of the metabolic syndrome-nicotinic acid. Endocrinol Metab Clin North Am 33: 557575, 2004.[CrossRef][ISI][Medline]
11. Meyers CD, Kamanna VS, and Kashyap ML. Niacin therapy in atherosclerosis. Curr Opin Lipidol 15: 659665, 2004.[CrossRef][ISI][Medline]
12. Nabokina SM, Subramanian VS, and Said HM. Comparative analysis of ontogenic changes in renal and intestinal biotin transport in the rat. Am J Physiol Renal Physiol 284: F737F742, 2003.
13. Nabokina SM, Ma TY, and Said HM. Mechanism and regulation of folate uptake by human pancreatic epithelial MIA PaCa-2 cells. Am J Physiol Cell Physiol 287: C142C148, 2004.
14. Sadoogh-Abasian F and Evered DF. Absorption of nicotinic acid and nicotinamide from rat small intestine in vitro. Biochim Biophys Acta 598: 385391, 1980.[ISI][Medline]
15. Said HM, Ortiz A, Subramanian VS, Neufeld EJ, Moyer MP, and Dudeja PK. Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460. Am J Physiol Gastrointest Liver Physiol 281: G144G150, 2001.
16. Said HM, Ortiz A, and Ma TY. A carrier-mediated mechanism for pyridoxine uptake by human intestinal epithelial Caco-2 cells: regulation by a PKA-mediated pathway. Am J Physiol Cell Physiol 285: C1219C1225, 2003.
17. Said HM, Balamurugan K, Subramanian VS, and Marchant JS. Expression and functional contribution of hTHTR-2 in thiamin absorption in human intestine. Am J Physiol Gastrointest Liver Physiol 286: G491G498, 2004.
18. Simanjuntak MT, Tamai I, Terasaki T, and Tsuji A. Carrier-mediated uptake of nicotinic acid by rat intestinal brush-border membrane vesicles and relation to monocarboxylic acid transport. J Pharmacobiodyn 13: 301309, 1990.[Medline]
19. Spector R. Niacin and niacinamide transport in the central nervous system: in vivo studies. J Neurochem 33: 895904, 1979.[ISI][Medline]
20. Takanaga H, Maeda H, Yabuuchi H, Tamai I, Higashida H, and Tsuji A. Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane. J Pharm Pharmacol 48: 10731077, 1996.[ISI][Medline]
21. Wilkinson GN. Statistical estimation in enzyme kinetics. Biochem J 80: 324332, 1961.[ISI][Medline]
22. Wise A, Foord SM, Fraser NJ, Barnes AA, Elshourbagy N, Eilert M, Ignar DM, Murdock PR, Steplewski K, Green A, Brown AJ, Dowell SJ, Szekeres PG, Hassall DG, Marshall FH, Wilson S, and Pike NB. Molecular identification of high and low affinity receptors for nicotinic acid. J Biol Chem 278: 98699874, 2003.
|
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |