Am J Physiol Cell Physiol Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 293: C1773-C1778, 2007. First published October 10, 2007; doi:10.1152/ajpcell.00409.2007
0363-6143/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/C1773    most recent
00409.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Said, H. M.
Right arrow Articles by Kashyap, M. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Said, H. M.
Right arrow Articles by Kashyap, M. L.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Mechanism of nicotinic acid transport in human liver cells: experiments with HepG2 cells and primary hepatocytes

Hamid M. Said,1,2,,* Svetlana M. Nabokina,1,2 Krishnaswamy Balamurugan,1,2 Zainab M. Mohammed,2 Cecilia Urbina,1,2 and Moti L. Kashyap1,*

1Veterans Affairs Medical Center, Long Beach; and 2Department of Medicine and Department of Physiology and Biophysics, University of California College of Medicine, Irvine, California

Submitted 6 September 2007 ; accepted in final form 5 October 2007

ABSTRACT

This study reports on the functional expression of a specific, high-affinity carrier-mediated mechanism for the transport of niacin (nicotinic acid) in human liver cells. Both human-derived liver HepG2 cells and human primary hepatocytes were used as models in these investigations. The initial rate of transport of nicotinic acid into HepG2 cells was found to be acidic pH, temperature, and energy dependent; it was, however, Na+ independent in nature. Evidence for the existence of a carrier-mediated system that is specific for [3H]nicotinic acid transport was found and included the following: 1) saturability as a function of concentration with an apparent Km of 0.73 ± 0.16 µM and Vmax of 25.02 ± 1.45 pmol·mg protein–1·3 min–1, 2) cis-inhibition by unlabeled nicotinic acid and nicotinamide but not by unrelated organic anions (lactate, acetate, butyrate, succinate, citrate, and valproate), and 3) trans-stimulation of [3H]nicotinic acid efflux by unlabeled nicotinic acid. Transport of the vitamin into human primary hepatocytes occurs similarly via an acidic pH-dependent and specific carrier-mediated process. Inhibitors of the Ca2+-calmodulin-mediated pathway (but not modulators of the PKC-, PKA-, and protein tyrosine kinase-mediated pathways) inhibited nicotinic acid transport into both HepG2 cells and human primary hepatocytes. Maintenance of HepG2 cells (for 48 h) in growth medium oversupplemented with nicotinic acid (or nicotinamide) did not affect the subsequent transport of [3H]nicotinic acid into HepG2 cells. These results show, for the first time, the existence of a specific and regulated membrane carrier-mediated system for nicotinic acid transport in human liver cells.

niacin; transport mechanism

THE WATER-SOLUBLE VITAMIN NIACIN (nicotinic acid) serves as a precursor for the synthesis of the coenzymes NAD and NADP. NAD- and NADP-linked enzymes participate in a number of metabolic reactions that maintain the redox state of cells including the pentose phosphate shunt and glycolysis (13, 14). In pharmacological doses, nicotinic acid also has a lipid-regulating effect and is in use clinically for that purpose (8, 17, 18). The mechanism(s) of the lipid-regulating effect of nicotinic acid is not well defined, and several mechanisms have recently been described (8, 17). Frank deficiency of nicotinic acid in humans is rare, but when it occurs leads to pellagra, a disease characterized by inflammation of mucous membranes, skin lesions, and diarrhea (5, 31). Nicotinic acid deficiency and suboptimal levels have been reported in patients with Hartnup's disease and in alcoholics (15, 20, 23, 31). Patients with Hartnup disease have mutations in the gene that encodes the membrane transporter of the amino acid tryptophan, which is an endogenous precursor of nicotinic acid.

Humans obtain nicotinic acid from two sources: an exogenous source where the vitamin is obtained from the diet via intestinal absorption and an endogenous source where the vitamin is produced (when the exogenous supply of the vitamin is low) via the metabolic conversion of tryptophan (1, 14). Nicotinic acid is distributed to different body tissues via the circulation, where the vitamin exists in plasma at levels of 0.1–1 µM (9, 29). The liver plays an important role in the metabolism of exogenous nicotinic acid (3), and both in vivo and in vitro studies have shown significant uptake of administered vitamin by hepatocytes (3, 16). Little, however, is known about the mechanism(s) by which liver cells transport negatively charged (pKa = 4.9) nicotinic acid. A low-affinity and nonspecific nicotinic acid membrane transport system(s) has been proposed to function in a number of tissues, including the intestine (2, 6, 7, 22, 28–30), but the importance of such a system(s) in transporting physiological concentrations of the vitamin is not clear. A recent study (22) has reported the existence of a high-affinity system for nicotinic acid uptake in human intestinal epithelial cells. This system was found to function at the physiological (micromolar) concentration range of the vitamin and is specific for nicotinic acid. In addition to providing a challenge to the previous belief that the intestinal nicotinic acid uptake process involves a nonspecific low-affinity uptake system (7, 22, 28, 30), these findings have also raised the possibility that a similar uptake process may function in other cell types if the proper conditions are used to study the vitamin uptake event. Our aim in the present study was, therefore, to to test this possibility in human hepatocytes using both human-derived liver HepG2 cells and human primary hepatocytes as models. The results showed, for the first time, that human heptocytes do indeed have a specific high-affinity carrier system for nicotinic acid uptake, which most likely is the system responsible for the uptake of physiological concentrations of the vitamin. The results also showed the system to be pH dependent and appears to be under the regulation of an intracellular Ca2+/calmodulin-mediated pathway.

MATERIALS AND METHODS

[3H]nicotinic acid (specific activity: 50 Ci/mmol, radiochemical purity: 97%) was obtained from American Radiolabeled Chemicals (St. Louis, MO). Culture medium (DMEM) and other cell culture ingredients were obtained from Sigma (St. Louis, MO). All other chemicals and reagents used in this investigation were of analytical grade and were obtained from commercial sources.

The human-derived liver HepG2 cells (passages 10–20, American Type Culture Collection, Manassas, VA) used in this investigation were grown to confluence in DMEM growth medium containing 10% FBS. Uptake experiments were performed on monolayers 3–5 days after confluence. When human primary hepatocytes were used, they were obtained in 24 cell plates (collagen coated/Matrigel overlay) from CellzDirect (Austin, TX), cultured for 24 h in DMEM containing 10% FBS, and then used for uptake investigations. Human primary hepatocytes were obtained from a male (73 yr) Caucasian donor and a female (62 yr) Caucasian donor. Use of these cells is exempt by our local Institutional Review Board Committee. Uptake of [3H]nicotinic acid by both HepG2 cells and human primary hepatocytes was examined by incubating cells in Krebs-Ringer buffer [containing (in mM) 133 NaCl, 4.93 KCl, 1.23 MgSO4, 0.85 CaCl2, 5 glucose, 5 glutamine, 5 MES, and 5 HEPES] at 37°C (unless otherwise stated) in the presence of labeled and unlabeled nicotinic acid. Uptake was terminated at the appropriate time point by the addition of ice-cold buffer (x2) followed by immediate aspiration and rinsing. Cells were then digested with 1 ml of 1 N NaOH, neutralized with HCl, and then counted for radioactivity. A Bio-Rad kit (Richmond, VA) was used to measure the protein content of cell digests.

In examining the metabolic form of the transported substrate following incubation of HepG2 cells with [3H]nicotinic acid (18 nM), Silica-precoated thin-layer chromatography plates and a solvent system of 4:4:2 (vol/vol/vol) ratio of n-butanol, acetic acid, and water were used. When the effect of substrate level in the growth medium on the initial rate of [3H]nicotinic acid uptake was examined, HepG2 cells were maintained for 48 h in the absence or presence of nicotinic acid or nicotinimide (each at 1 mM) and then used for uptake investigations.

Data presentation and statistical analysis. Transport data are means ± SE of multiple separate uptake determinations and were expressed in terms of femtomoles or picomoles per milligram protein per unit time. Variability in the absolute amount of nicotinic acid taken up by HepG2 cells and human primary hepatocytes was observed in different batches of HepG2 cells and different individual human donors. For this reason, appropriate controls were run simultaneously with each set of experiments for comparison. Transport of nicotinic acid by the carrier-mediated process was calculated by subtracting the passive diffusion component (determined from the slope of the uptake line between 10 mM nicotinic acid and the point of origin, i.e., multiplication of the slope by the individual concentration) from total uptake at each concentration examined. Kinetic parameters of the saturable folic acid uptake process were calculated using a computerized model as previously described (25). Statistical analysis was performed using Student's t-test and ANOVA, with statistical significance being set at 0.05 (P < 0.05).

RESULTS

Basic characteristics of nicotinic acid transport in HepG2 cells. The initial rate (3 min, see below) of nicotinic acid (6 nM) transport into HepG2 cells was temperature dependent, as a significant (P < 0.01) decrease in transport was found upon lowering the incubation temperature from 37 to 22°C (389 ± 29 and 105.8 ± 5.8 fmol·mg protein–1·3 min–1, respectively). Also, transport of the vitamin into HepG2 cells was energy dependent, as significant (P < 0.01 for both) inhibition in the initial rate of nicotinic acid (6 nM) transport was observed following pretreatment (for 30 min) of cells with the metabolic inhibitors iodoacetate (0.5 mM) and 2,4-dinitrophenol (1 mM) (436 ± 24, 293.7 ± 13.7, and 236 ± 10 fmol·mg protein–1·3 min–1 for control and following pretreatment with iodoacetate and 2,4-dinitrophenol, respectively). Furthermore, uptake of nicotinic acid by HepG2 cells proceeded without metabolic alterations in the transported substrate (92.3% and 91.2% of the transported radioactivity following 7- and 10-min incubation of cells with 18 nM [3H]nicotinic acid, respectively) and was found to be in the form of nicotinic acid (see MATERIALS AND METHODS). Moreover, transport of low (0.1 µM) and high (10 µM) concentrations of nicotinic acid into HepG2 cells was linear with time for up to 10 min of incubation (Fig. 1), which was the reason behind our selection of a 3-min period as the standard incubation time in all experiments.


Figure 1
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 1. Uptake of nicotinic acid by HepG2 cells as a function of time. Confluent monolayers were incubated at 37°C in Krebs-Ringer buffer (pH 5.0) for different time intervals. Labeled and unlabeled nicotinic acid [0.1 µM (A) or 10 µM (B)] were added to the incubation medium at the start of uptake. Each data point represents the mean ± SE of 4–6 separate uptake determinations. When not shown, error bars are smaller than the symbol. A: y = 1.337x + 0.897, R = 0.992; B: y = 22.806x + 41.123, R = 0.999.

 
Role of H+ and Na+ in the transport of nicotinic acid in HepG2 cells and human primary hepatocytes. The role of extracellular H+ in the transport of nicotinic acid into HepG2 cells was studied by examining the effect of varying incubation buffer pH over the range of 8.0 to 5.0 on the initial rate of vitamin uptake. A sharp increase in nicotinic acid (6 nM) uptake was observed upon decreasing the incubation buffer pH from 8.0 to 5.0 (Fig. 2). Uptake of nicotinic acid at buffer pH 5.0 was ~28-fold higher than uptake at buffer pH 8.0. In a related investigation, we examined the effect of pretreatment of (for 30 min, buffer pH 5.0) HepG2 cells with the protonophore FCCP (50 µM) on the initial rate of uptake of 6 nM nicotinic acid. FCCP significantly (P < 0.01) inhibited nicotinic acid uptake (407 ± 13.5 and 182.8 ± 14.8 fmol·mg protein–1·3 min–1 for control and FCCP-pretreated cells, respectively).


Figure 2
View larger version (7K):
[in this window]
[in a new window]

  		Fig. 2. Effect of incubation buffer pH on nicotinic acid uptake by HepG2 cells. Confluent monolayers were used. [3H]niacin (6 nM) was added to the incubation medium (Krebs-Ringer buffer of varying pH) at the onset of a 3-min incubation (i.e., initial rate). Values are means ± SE of 3–6 separate uptake determinations. When not shown, error bars are smaller than the symbol.

 
When the effect of incubation buffer pH on [3H]nicotinic acid (12 nM) transport into human primary hepatocytes was examined, a significantly (P < 0.01, 7.2-fold) higher vitamin uptake was again observed at buffer pH 5.0 compared with 7.4 (5.3 ± 0.16 and 0.74 ± 0.05 pmol·mg protein–1·3 min–1, respectively). Pretreatment (for 30 min, buffer pH 5.0) with FCCP (50 µM) also caused a significant (P < 0.01) inhibition in the initial rate of nicotinic acid (12 nM) uptake by human primary hepatocytes (1.35 ± 0.05 and 0.18 ± 0.01 pmol·mg protein–1·3 min–1 for control and FCCP-pretreated cells, respectively). As demonstrated by these results, the absolute amount of nicotinic acid uptake by human primary hepatocytes was markedly higher than that by HepG2 cells. Because of this, buffer pH 5.0 was used in all our investigations.

The role of extracellular Na+ in driving the initial rate of uptake of nicotinic acid by HepG2 cells was examined by testing the effect of replacement of Na+ with an eqimolar concentration of the monovalent cations K+ and Li+ or with mannitol on the vitamin (6 nM) uptake by process. Similar nicotinic acid uptake was found in the presence and absence of Na+ (379.3 ± 14.7, 371.3 ± 19.6, 375.5 ± 39, and 365 ± 26 fmol·mg protein–1·3 min–1 in the presence of Na+, K+, Li+, and mannitol, respectively). In a related experiment, we examined the effect of ouabain (1 mM), an inhibitor of Na+-K+-ATPase, on the initial rate of nicotinic acid (6 nM) uptake. The results showed a lack of significant effect of ouabain on vitamin uptake (435.8 ± 24 and 416.7 ± 19 fmol·mg protein–1·3 min–1 for control and ouabain-pretreated cells, respectively).

Involvement of a carrier-mediated system for nicotinic acid transport in HepG2 cells and human primary hepatocytes. The initial rate of nicotinic acid transport into HepG2 cells was examined as a function of concentration over a wide range of substrate concentrations spanning nanomolar and micromolar ranges (6 nM–10 µM), i.e., spanning the entire physiological range. We observed saturation in the initial rate of nicotinic acid uptake in the micromolar range only (Fig. 3). Kinetic parameters of the saturable process were determined as described in MATERIALS AND METHODS and found to be 0.73 ± 0.16 µM and 25.02 ± 1.45 pmol·mg protein–1·3 min–1 for the apparent Km and Vmax, respectively. These findings suggest the involvement of a functional carrier-mediated process in nicotinic acid transport into HepG2 cells.


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Uptake of nicotinic acid by HepG2 cells as a function of concentration in the nanomolar (A) and micromolar (B) ranges. Confluent monolayers of HepG2 cells were incubated at 37°C in Krebs-Ringer buffer (pH 5.0) in the presence of different concentrations of nicotinic acid. Uptake was measured after a 3-min incubation (i.e., initial rate). Values are means ± SE of 4–6 separate uptake determinations. When not shown, error bars are smaller than the symbol. A: y = 0.029x + 0.167, R = 0.991.

 
To confirm the existence of a carrier system for nicotinic acid transport in HepG2 cells, we examined the effect of addition of unlabeled nicotinic acid (50 µM) to the incubation buffer on the initial rate of [3H]nicotinic acid (6 nM) transport. Significant (P < 0.01) cis-inhibition in [3H]nicotinic acid transport into cells was observed by the unlabeled substrate (416.9 ± 18.6 and 132.7 ± 6.7 fmol·mg protein–1·3 min–1 for control and in the presence of unlabeled nicotinic acid, respectively). We also examined the effect of addition of unlabeled nicotinic acid to the incubation buffer on the rate of efflux of [3H]nicotinic acid from preloaded HepG2 cells (to test for the existence of trans-stimulation). In this experiment, cells were first preloaded with [3H]nicotinic acid (by incubating them with 20 nM of [3H]nicotinic acid for 10 min), followed by removal of the incubation buffer, washing of cells with ice-cold buffer, and then incubating cells in buffer in the presence and absence of 1 mM unlabeled nicotinic acid. The results showed the cell content of 3H radioactivity to be significantly (P < 0.01) lower in cells incubated in the presence of unlabeled nicotinic acid in the incubation buffer compared with those incubated in buffer alone (271.9 ± 1.7 and 715.7 ± 16.4 fmol·mg protein–1·7-min incubation–1, respectively). This finding demonstrates trans-stimulation in vitamin transport across the HepG2 cell membrane.

We also examined the effect of unlabeled nicotinic acid (500 µM) in the incubation medium on [3H]nicotinic acid (12 nM) transport into human primary hepatocyes. The results showed significant (P < 0.01) inhibition in [3H]nicotinic acid transport by unlabeled nicotinic acid (5.3 ± 0.16 and 0.52 ± 0.05 pmol·mg protein–1·3-min incubation–1 for control and in the presence of unlabeled nicotinic acid, respectively). These results validate the findings with HepG2 cells with regard to the involvement of a carrier-mediated system for vitamin transport.

Specificity of the hepatic nicotinic acid uptake system of human liver cells and effect of unrelated organic anions and transport inhibitors. The effect of the nicotinic acid structural analogs nicotinamide, isonicotinic acid, nicotinyl alcohol, and nicotinuric acid (all at 50 µM) on the initial rate of [3H]nicotinic acid (6 nM) uptake into HepG2 cells was examined. The results showed that among the tested structural analogs, only nicotinamide significantly (P < 0.1) inhibited [3H]nicotinic acid uptake (Table 1). Nicotinamide (50 µM) also inhibited the transport of [3H]nicotinic acid (12 nM) into human primary hepatocytes (5.3 ± 0.165 and 2.4 ± 0.14 pmol·mg protein–1·3 min–1 for control and in the presence of nicotinamide, respectively), thus validating the data with HepG2 cells. In addition, 5-methyl-1H-pyrazole-3-carboxylic acid, a high-affinity ligand for the nicotinic acid HM74A receptor (28), showed no effect on [3H]nicotinic acid (6 nM) uptake by HepG2 cells (427 ± 18.6 and 420 ± 18.8 fmol·mg protein–1·3 min–1 for control and in the presence of 5-methyl-1H-pyrazole-3-carboxylic acid, respectively).


View this table:
[in this window]
[in a new window]

  		Table 1. Effect of nicotinic acid structural analogs on the intial rate of uptake of [3H]nicotinic acid by HepG2 cells

 
Since nicotinic acid is negatively charged at physiological pH (pKa = 4.9), we also tested the effect of the unrelated carboxylic anions lactate, acetate, butyrate, succinate, citrate, and valproate (all at 100 µM) on the initial rate of [3H]nicotinic acid (6 nM) uptake by HepG2 cells. The results showed that none of the tested compounds cause significant inhibition in nicotinic acid uptake (Table 2). Similarly, the anions acetate and lactate (100 µM) failed to affect the transport of [3H]nicotinic acid (12 nM) into human primary hepatocytes (1.44 ± 0.17, 1.49 ± 1.22, and 1.60 ± 0.08 pmol·mg protein–1·3 min–1 for control and in the presence of acetate and lactate, respectively).


View this table:
[in this window]
[in a new window]

  		Table 2. Effect of unrelated carboxylic acids on the initial rate of nicotinic acid uptake by HepG2 cells

 
The effect of the membrane transport inhibitors phloretin, {alpha}-cyano-4-hydroxycinnamate, SITS, DIDS, probenecid, and furosemide (all at 1 mM) on the initial rate of nicotinic acid (6 nM) uptake by HepG2 cells was also examined. None of the tested compounds were found to affect nicotinic acid uptake (Table 3).


View this table:
[in this window]
[in a new window]

  		Table 3. Effect of membrane transport inhibitors on the initial rate of uptake of nicotinic acid by HepG2 cells

 
Possible regulation of the nicotinic acid uptake process of human liver cells. Possible regulation of the nicotinic acid uptake process of human liver cells by intracellular and extracellular factors was investigated. The effect of intracellular factors was examined by testing the effect of pretreatment of HepG2 cells with specific modulators of the PKA-, PKC-, protein tyrosine kinase (PTK)-, and Ca2+/calmodulin-mediated regulatory pathways on the initial rate of uptake of nicotinic acid (6 nM). The results showed that all the PKC-mediated pathway modulators used had no effect on nicotinic acid (6 nM) uptake (Table 4). Similarly, modulators of the PKA-mediated pathway [8-bromo-cAMP (1 mM), forskolin (0.1 mM), IBMX (1 mM), and H89 (1 mM)] did not affect nicotinic acid (6 nM) uptake (Table 5). Modulators of the PTK pathway also failed to affect nicotinic acid uptake by HepG2 cells (data not shown). On the other hand, modulators of the Ca2+/calmodulin-mediated pathway (trifluoperazine and calmidazolium) were found to cause a concentration-dependent and significant (P < 0.01) inhibition in vitamin uptake (Table 2). When the effect of calmidazolium (100 µM) pretreatment on nicotinic acid (12 nM) uptake was examined in human primary hepatocytes, significant (P < 0.01) inhibition was also observed (3.16 ± 0.1 and 1.32 ± 0.08 pmol·mg protein–1·3 min–1 for control and in the presence of calmidazolium, respectively). These results suggest the possible involvement of a Ca2+/calmodulin-mediated pathway in the regulation of nicotinic acid uptake by human liver cells.


View this table:
[in this window]
[in a new window]

  		Table 4. Effect of pretreatment of HepG2 cells with modulators of the PKC-mediated pathway on the uptake of nicotinic acid

 

View this table:
[in this window]
[in a new window]

  		Table 5. Effect of pretreatment of HepG2 cells with modulators of the PKA-mediated pathway on the uptake of nicotinic acid

 
The effect of the extracellular nicotinate level on vitamin uptake by HepG2 cells was also investigated. HepG2 cells were maintained for 48 h in growth medium oversupplemented with either 1 mM nicotinic acid or with 1 mM nicotinamide followed by a test of the initial rate of [3H]nicotinic acid (6 nM) uptake. The results showed a similar rate of [3H]nicotinic acid uptake under all conditions (412 ± 14, 405 ± 20, and 401 ± 13 fmol·mg protein–1·3 min–1 for cells maintained in control growth medium and those maintained in growth media supplemented with nicotinic acid and nicotinamide, respectively).

DISCUSSION

We investigated the mechanism of transport of nicotinic acid into human liver cells using two cell models: human-derived liver HepG2 cells and human primary hepatocytes obtained from organ donors. Use of a cell line together with primary cells of the tissue of origin provides physiological relevance to the findings with the cell line model. Our results showed that the initial rate of the transport process in HepG2 cells is temperature and energy dependent and occurs with minimal metabolic alterations in the transported substrate. The latter suggests that metabolic trapping does not influence the initial rate of nicotinic acid uptake in these cells. Transport of nicotinic acid in both HepG2 cells and primary hepatocytes was highly dependent on extracellular pH with a significantly higher uptake of the vitamin at acidic compared with neutral or alkaline buffer pHs. Pretreatment of HepG2 cells and primary hepatocytes with the protonphore FCCP (which leads to a collapse in the trans-membrane pH gradient) led to a significant reduction in the initial rate of nicotinic acid uptake. These findings suggest the involvement of a nicotinic acid-H+ cotransport (and/or nicotinic acid/OH– exchange) process in the vitamin transport process in human liver cells. The source of H+ needed for driving nicotinic acid uptake into liver cells could be provided by the activity of Na+/H+ exchangers, which are abundantly expressed in hepatocytes (4, 11, 21). In contrast to the role of incubation buffer pH (i.e., H+), no role for extracellular Na+ in nicotinic acid uptake by HepG2 cells was found, as isoosmotically replacing this monovalent cation with an equimolar concentration of other monovalent cations or with mannitol failed to affect the initial rate of uptake of this vitamin. This conclusion was further supported by the finding of a lack of an effect of pretreatment of HepG2 cells with ouabain (an inhibitor of Na+-K+-ATPase) on the initial rate of nicotinic acid uptake.

Our investigations provided evidence that the transport of nicotinic acid into HepG2 cells and human primary hepatocytes involves a carrier-mediated process. This conclusion is based on the findings of saturation in the initial rate of nicotinic acid uptake as a function of concentration, the ability of unlabeled nicotinic acid to cis-inhibit the initial rate of [3H]nicotinic acid uptake, and the trans-stimulation in [3H]nicotinic acid efflux from preloaded cells by extracellular unlabeled vitamin. The apparent Km of the saturable component was determined to be 0.73 ± 0.16 µM, which suggests a role for this system in the transport of physiological concentrations of the vitamin that exist in human plasma at a range of 0.1–1 µM (9, 29).

The nicotinic acid membrane transport system of HepG2 cells and human primary hepatocytes was found to be specific for the vitamin, as only biologically active nicotinamide was able to inhibit the initial rate of [3H]nicotinic acid uptake. The inability of 5-methyl-1H-pyrazole-3-carboxylic acid [a high-affinity ligand for the nicotinic acid receptor (32)] to inhibit nicotinic acid uptake by HepG2 cells is in line with recently published data showing lack of expression of the nicotinic acid receptor in liver tissue (32), and together they clearly suggest that this receptor plays no role in liver uptake of the vitamin. We also tested the effect of a host of unrelated carboxylic acids on the initial rate of negatively charged nicotinic acid uptake, but none were found to cause a significant effect, further establishing the specific nature of the process. A number of the tested organic anions were substrates or inhibitors (phloretin and {alpha}-cyano-4-hydroxycinnamate) for monocarboxylate transporter (MCT) systems (9, 12). Thus, because these compounds did not affect nicotinic acid uptake and since affinity of MCT systems is low [i.e., in the millimolar range (9, 12)], it is reasonable to assume that MCT systems are not involved in the uptake of physiological concentrations of nicotinic acids by human liver cells.

Following functional identification of a nicotinic acid transport system in human liver cells, we tested the possible regulation of the transport process by intracellular and extracellular factors. The effect of intracellular factors was tested by examining the effect of pretreatment of HepG2 cells with modulators of the PKA-, PKC-, PTK-, and Ca2+/calmodulin-mediated pathways on the initial rate of nicotinic acid uptake. The results showed that while the PKA-, PKC-, and PTK-mediated pathways had no role in the regulation of nicotinic acid uptake by HepG2 cells, a possible role for the Ca2+/calmodulin-mediated pathway was observed, as inhibitors of this pathway inhibited nicotinic acid uptake by these cells. This inhibition in nicotinic acid uptake was also observed when human primary hepatocytes were used. The molecular mechanism(s) involved in the Ca2+/calmodulin mediated effect on nicotinic acid uptake by human liver cells is not clear and requires further investigation. Whether the nicotinic acid uptake process of HepG2 cells is adaptively regulated by the substrate level in the extracellular medium was also examined by testing the effect of maintenance of cells in the presence of a high concentration of nicotinic acid or its metabolically active derivative nicotinamide on the initial rate of [3H]nicotinic acid uptake. The results, however, showed no such regulation in the vitamin uptake process in HepG2 cells, a finding that is in contrast to the adaptive regulation that occurs in the uptake of other water-soluble vitamins (e.g., folate, riboflavin, and pyridoxine) by their levels in the extracellular environment (24–26). Whether this lack of adaptive regulation in nicotinic acid uptake is related to the ability of hepatocytes to synthesize some nicotinic acid (compared with their inability to synthesize other vitamins) is not clear.

In summary, these results show, for the first time, the existence of an acidic pH-dependent, high-affinity, and specific carrier-mediated system for nicotinic acid transport in human liver cells. This system operates in the physiological micromolar range and appears to be regulated by an intracellular Ca2+/calmodulin-mediated pathway.

GRANTS

This work was supported by a grant from the Department of Veterans Affairs and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-56061, DK-58057, and DK-64165.

FOOTNOTES


Address for reprint requests and other correspondence: H. M. Said, Veterans Affairs Medical Center 151, 5901 E. 7th St., Long Beach, CA 90822 (e-mail: hmsaid{at}uci.edu)

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.

* The laboratories of H. M. Said and M. L. Kashyap contributed equally to this work. Back

REFERENCES

1. Bechgaard H, Jespersen S. GI absorption of niacin in humans. J Pharm Sci 66: 871–872, 1977.[CrossRef][Web of Science][Medline]

2. Boumendil-Podevin EF, Podevin RA. Nicotinic acid transport by brush border membrane vesicles from rabbit kidney. Am J Physiol Renal Fluid Electrolyte Physiol 240: F185–F191, 1981.[Abstract/Free Full Text]

3. Collins PB, Chaykin S. The management of nicotinamide and nicotinic acid in the mouse. J Biol Chem 247: 778–783, 1972.[Abstract/Free Full Text]

4. Collins JF, Honda T, Knobel S, Bulus NM, Conary J, Du Bois R, Ghishan FK. Molecular cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger (NHE-2). Proc Natl Acad Sci USA 90: 3938–3942, 1993.[Abstract/Free Full Text]

5. DesGroseilliers JP, Shiffman NJ. Pellagra. Can Med Assoc J 115: 768–770, 1976.[Abstract]

6. Elbert J, Daniel H, Rehner G. Intestinal uptake of nicotinic acid as a function of microclimate-pH. Int J Vitam Nutr Res 56: 85–93, 1986.[Web of Science][Medline]

7. Fox KR, Adrian C, Hogben M. Nicotinic acid active transport by in vitro bullfrog small intestine. Biochim Biophys Acta 332: 336–340, 1974.

8. Ganji SH, Zhang L, Kamanna VS, Kahyap ML. Effect of niacin on lipoprotein and atherosclerosis. Future Lipidol 1: 549–557, 2006.[CrossRef]

9. Garcia CK, Brown MS, Pathak R, Goldstein JL. cDNA cloning of MCT2, a second moncarboxylate transporter expressed in different cells than MCT1. J Biol Chem 270: 1843–1849, 1995.[Abstract/Free Full Text]

10. Guilarte TR, Pravlik K. Radiometric-microbiologic assay of niacin using Kloeckera brevis: analysis of human blood and food. J Nutr 113: 2587–2594, 1983.[Abstract/Free Full Text]

11. Haddad P, Noel J, Vadnais J, Mathe L, Vallerand D. Modulation of liver cell membrane NHE-1, Na+-K+ ATPase, and GLUT-2 protein content after cold preservation and rewarming. Can J Physiol Pharmacol 77: 852–859, 1999.[CrossRef][Web of Science][Medline]

12. Halestrap AP, Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 343: 281–299, 1999.[CrossRef][Web of Science][Medline]

13. Ijichi H, Ichiyama A, 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: 3701–3707, 1966.[Abstract/Free Full Text]

14. Ikeda M, Tsuji H, Nakamura S, Ichiyama A, Nishizuka Y, 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: 1395–1401, 1965.[Free Full Text]

15. Ishii N, Nishihara Y. Pellagra among chronic alcoholics: clinical and pathological study of 20 necropsy cases. J Neurol Neurosurg Psychiatry 44: 209–215, 1981.[Abstract/Free Full Text]

16. Keller J, Liersch M, Grunicke H. Studies on the biosynthesis of NAD from nicotinamide and on the intracellular pyridine nucleotide cycle in isolated perfused rat liver. Eur J Biochem 22: 263–270, 1971.[Web of Science][Medline]

17. Meyers CD, Kashyap ML. Management of the metabolic syndrome-nicotinic acid. Endocrinol Metab Clin North Am 33: 557–575, 2004.[CrossRef][Web of Science][Medline]

18. Meyers CD, Kamanna VS, Kashyap ML. Niacin therapy in atherosclerosis. Curr Opin Lipidol 15: 659–665, 2004.[CrossRef][Web of Science][Medline]

19. Nabokina SM, Kashyap ML, Said HM. Mechanism and regulation of human intestinal niacin uptake. Am J Physiol Cell Physiol 289: C97–C103, 2005.[Abstract/Free Full Text]

20. Oakley A, Wallace J. Hartnup disease presenting in an adult. Clin Exp Dermatol 19: 407–408, 1994.[CrossRef][Web of Science][Medline]

21. Rieder CV, Fliegel L. Developmental regulation of Na+/H+ exchanger expression in fetal and neonatal mice. Am J Physiol Heart Circ Physiol 283: H273–H283, 2002.[Abstract/Free Full Text]

22. Sadoogh-Abasian F, Evered DF. Absorption of nicotinic acid and nicotinamide from rat small intestine in vitro. Biochim Biophys Acta 598: 385–391, 1980.[Medline]

23. Sashidhar RB, Ramakrishna Y, Bhat RV. Pellagra, mycotoxins and tryptophan-niacin metabolism. Adv Exp Med Biol 294: 631–636, 1991.[Medline]

24. Said HM, Chatterjee N, Haq RU, Subramanian VS, Ortiz A, Matherly LH, Sirotnak FM, Halsted C, Rubin SA. Adaptive regulation of intestinal folate uptake: effect of dietary folate deficiency. Am J Physiol Cell Physiol 279: C1889–C1895, 2000.[Abstract/Free Full Text]

25. Said HM, Ortiz A, Vaziri ND. Mechanism and regulation of vitamin B6 uptake by renal tubular epithelia: studies with cultured OK cells. Am J Physiol Renal Physiol 282: F465–F471, 2002.[Abstract/Free Full Text]

26. Said HM, Wang S, Ma TY. Mechanism of riboflavin uptake by cultured human retinal pigment epithelial ARPE-19 cells: possible regulation by an intracellular Ca2+-calmodulin-mediated pathway. J Physiol 566: 369–377, 2005.[Abstract/Free Full Text]

27. Shimada A, Nakagawa Y, Morishige H, Yamamoto A, Fujita T. Functional characteristics of H-dependant nicotinate transport in primary cultures of astrocytes from rat cerebral cortex. Neurosci Lett 392: 207–212, 2006.[CrossRef][Web of Science][Medline]

28. Simanjuntak MT, Tamai I, Terasaki T, Tsuji A. Carrier-mediated uptake of nicotinic acid by rat intestinal brush-border membrane vesicles and relation to monocarboxylic acid transport. J Pharmacobiodyn 13: 301–309, 1990.[Medline]

29. Spector R. Niacin and niacinamide transport in the central nervous system: in vivo studies. J Neurochem 33: 895–904, 1979.[CrossRef][Web of Science][Medline]

30. Takanaga H, Maeda H, Yabuuchi H, Tamai I, Higashida H, Tsuji A. Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane. J Pharm Pharmacol 48: 1073–1077, 1996.[Web of Science][Medline]

31. Vannucchi H, Moreno FS. Interaction of niacin and zinc metabolism in patients with alcoholic pellagra. Am J Clin Nutr 50: 364–369, 1989.[Abstract/Free Full Text]

32. 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, Pike NB. Molecular identification of high and low affinity receptors for nicotinic acid. J Biol Chem 278: 9869–9874, 2003.[Abstract/Free Full Text]




This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
D. A. Rodionov, X. Li, I. A. Rodionova, C. Yang, L. Sorci, E. Dervyn, D. Martynowski, H. Zhang, M. S. Gelfand, and A. L. Osterman
Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon
Nucleic Acids Res., April 1, 2008; 36(6): 2032 - 2046.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/6/C1773    most recent
00409.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Said, H. M.
Right arrow Articles by Kashyap, M. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Said, H. M.
Right arrow Articles by Kashyap, M. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.