Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells
Steven Coon,1
James Kim,2
Guohong Shao,2 and
Uma Sundaram1
2Section of Digestive Diseases, Department of Medicine, The Ohio State University, Columbus, Ohio; and 1West Virginia University Medical Center, Morgantown, West Virginia
Submitted 21 March 2005
; accepted in final form 30 April 2005
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ABSTRACT
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Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with NG-nitro-L-arginine methyl ester (L-NAME) resulted in the inhibition of Na-stimulated 3H-O-methyl-D-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The L-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with L-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from L-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.
mucosal blood flow; glucose absorption; sodium absorption; intestinal absorption; regulation of intestinal glucose; amino acid absorption
NITRIC OXIDE (NO) has been demonstrated to alter gastrointestinal functions in normal and pathophysiological states. In the normal gastrointestinal tract, NO has been demonstrated to alter motility, mucosal blood flow, mucus secretion, and intestinal electrolyte and fluid transport (18, 26). We have previously demonstrated that inhibition of constitutive nitric oxide (cNO) synthase with NG-nitro-L-arginine methyl ester (L-NAME) resulted in the unique regulation of the three functionally different rabbit enterocyte brush border membrane (BBM) anion/HCO3 exchangers. It did not affect villus cell BBM Cl/HCO3 exchange; stimulated crypt cell BBM Cl/HCO3 exchange; and inhibited villus cell BBM short-chain fatty acid (SCFA)/HCO3 exchange. Furthermore, kinetic studies demonstrated that the mechanism of inhibition of crypt cell BBM Cl/HCO3 exchange is secondary to a decrease in the maximal rate of uptake of Cl, without an alteration in the affinity of the transporter for Cl. However, in contrast, the mechanism of stimulation of villus cell BBM SCFA/HCO3 exchange is secondary to an increase in the affinity of the transporter for SCFA without an alteration in the maximal rate of uptake. These results indicated that cNO uniquely regulates the three BBM anion/HCO3 transporters in the rabbit small intestine (4). However, the effect of cNO on an important class of mammalian small intestinal mucosal absorptive systems, specifically Na-solute cotransport processes, is unknown.
Na-solute cotransport processes are not only important for the assimilation of essential solutes such as adenosine, bile acids, and nutrients, but also for the absorption of Na (6). Na-glucose (SGLT-1) and Na-amino acid cotransporters (NAcT) are two of the more important Na-nutrient cotransport processes in the mammalian small intestine (6, 12, 14). For example, alanine is transported into villus cells by one of these Na-driven amino acid transporters specifically by the Na-neutral amino acid cotransporter system B0 (Asct1)(3) and will be used to measure the effects of cNO on NAcT. Both cotransporters have been localized to the BBM of the absorptive villus but not the secretory crypt cells in the mammalian small intestine including rabbits (23, 24). The favorable transcellular Na gradient for these secondary active transport processes is provided by Na-K-ATPase on the basolateral membrane (BLM) of villus cells (16). Thus an agent such as NO may regulate these cotransporters at the level of the cotransporter and/or at Na-K-ATPase on the BLM.
A variety of hormones and immune-inflammatory mediators have been demonstrated to alter the SGLT-1 (1, 5, 13, 27). SGLT-1 is unaffected in secretory diarrheal diseases such as cholera (12). However, it has been demonstrated the SGLT-1 is inhibited during chronic small intestinal inflammation (24). A variety of hormones has also been demonstrated to alter NAcT in the mammalian small intestine (8). Although NAcT is inhibited in the chronically inflamed small intestine, it is of note that the mechanism of inhibition of this cotransport process is uniquely different from that of SGLT-1 during chronic ileitis (23). For example, during chronic ileitis, it has been demonstrated that whereas SGLT-1 inhibition is during chronic ileitis secondary to a decrease in cotransporter numbers, NAcT inhibition is secondary to a decrease in the affinity of the cotransporter for the amino acid without change in cotransporter numbers (23, 24). These findings indicate that these cotransporters lend themselves to unique regulation.
Given this background, the first aim of this study was to determine whether cNO might in fact regulate Na-nutrient cotransport processes in the normal mammalian small intestine. The second aim was to determine whether this regulation might be unique amongst different types of Na-nutrient cotransport processes. The final aim was to decipher the mechanism of this regulation by cNO in the mammalian small intestine.
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METHODS
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Drug treatment.
A dose range from 0.1 to 1.3 mM of NG-nitro-L-arginine methyl ester (L-NAME) is necessary in vivo to inhibit cNO synthase (1, 5, 11, 13). In our model, we used a dose range from 0.01 to 0.5 mM per day. L-NAME was dissolved in 1 ml of sterile water given intramuscularly to New Zealand White male rabbits (2.52.7 kg) for 13 days. Sterile water was used as control. The lowest dose that produced a reproducible and near-maximal effect on both the inhibition of cNO production and an effect on transporter activity (e.g., SGLT-1) was then subsequently used in all studies. Thus we used a dose of 0.1 mM (75 mg), clearly a dose that is at the low end of what has been used in vivo in the literature. Alternatively rabbits were treated with the inactive analog NG-nitro-D-arginine methyl ester (D-NAME; 75 mg/day im for 2 days) or with a more selective inhibitor of inducible NO (iNO) synthase N6-(1-imonoethyl)-L-lysine dihydrochloride (L-NIL; 0.1 mg/day im for 2 days). (Animal assurance welfare no. A-3292-01.)
Cell isolation.
Villus and crypt cells were isolated from the rabbit intestine by a calcium chelation technique as previously described (21, 22). Previously established criteria were used to validate good separation of villus and crypt cells. Some of these criteria included 1) marker enzymes (e.g., alkaline phosphatase), 2) transporter specificity (e.g., Na/H on the BBM of villus, but not crypt cells), and 3) morphological differences (e.g., villus cells are larger and with better-developed BBM compared with crypt cells). Previously established criteria were also used to study cells with good viability and to exclude cells that showed evidence of poor viability: 1) trypan blue exclusion and 2) the demonstration of Na/H and Cl/HCO3 exchange activities (21, 22).
BBM vesicle preparation.
BBM vesicles (BBMV) from rabbit intestinal villus cells were prepared by CaCl2 precipitation, and differential centrifugation as previously reported (23, 24). BBMV were resuspended in a medium appropriate to each experiment. BBMV purity was assured with marker enzyme enrichment (e.g., alkaline phosphatase).
Uptake studies in villus cells.
Uptake studies were performed in villus cells by the rapid filtration technique as previously described (23, 24). Villus cells (100 mg wet wt) were washed and resuspended in HEPES buffer containing (in mM) 1.25 3-O-methyl-D-glucose (3-OMG) or L-alanine, 4.5 KC1, 1.2 KH2PO4, 1.0 MgSO4, 1.25 CaC12, 20 HEPES, and either 130 NaCl or choline chloride and were gassed with 100% O2 (pH 7.4 at 37°C). 3H-OMG or 3H-alanine (10 µCi; Amersham) was added to 1 ml cell suspension in the HEPES buffer, and 100-µl aliquots were removed at desired time intervals. The uptake was arrested by mixing with 1 ml ice-cold stop solution (choline-HEPES buffer). The mixture was filtered on 0.65-µm Millipore (HAWP) filters. After two washes with ice-cold stop solution, the filter was dissolved in 5 ml Ecoscint, and the radioactivity was determined.
Uptake studies in BBMV.
Vesicle uptake studies were performed by the rapid-filtration technique as previously described (23, 24). In brief, 5 µl of BBMV resuspended in 100 mM choline chloride, 0.10 mM MgSO4, 50 mM HEPES-Tris (pH 7.5), 50 mM mannitol, and 75 mM KCl were incubated in 95 µl of reaction medium that contained 50 mM HEPES-Tris buffer (pH 7.5), 0.1 mM 3-OMG or 0.2 mM L-alanine, 20 µM 3H-OMG or 3H-alanine, 0.10 mM MgSO4, 75 mM KC1, 50 mM mannitol, 100 mM of either NaCl or choline chloride, 10 µM valinomycin, and 100 µM carbonyl cyanide p-(tri-flouromethoxy)phenyl-hydrazone (FCCP). At desired times, uptake was arrested by mixing with ice-cold stop solution (50 mM HEPES-Tris buffer, 0.10 mM MgSO4, 75 mM KC1, and 100 mM choline chloride, pH 7.5). The mixture was filtered on a 0.45-µm Millipore (HAWP) filter and washed two times with 5 ml ice-cold stop solution. Filters with BBMV were dissolved in Ecoscint, and radioactivity was determined.
Enzyme measurement.
Na-K-ATPase was measured as Pi liberated in cellular homogenates from the same amount of cell as previously described (7). Enzyme specific activity was expressed as nanomoles of Pi released per milligram protein per minute.
Northern blot studies.
Total RNA was extracted from rabbit ileal villus cells by the guanidinium isothiocyanate-cesium chloride method (2). After denaturation, total RNA was electrophoresed on 1% agarose-formaldehyde gel, transferred to nylon membrane (Schleicher and Schuell, Keene, NH), and incubated with prehybridization solution. Membranes were hybridized with 32P-labeled SGLT-1 cDNA. The cDNA was random labeled with [32P]CTP with Klenow polymerase.
-Actin was used to ensure equal loading of total RNA onto the electrophoresis gels. Hybridized membrane was exposed to autoradiography film (New England Nuclear, Boston, MA). Dr. E. M. Wright generously provided the SGLT-1 cDNA.
Western blot studies.
BBMV (4 ug) were diluted in SDS buffer, boiled, and electrophoresed on a 12% SDS-PAGE gel. The gel was electroblotted onto a polyvinylidene difluoride membrane and blocked for 2 h in 5% BSA at room temperature. The membrane was incubated at room temperature with 1:3,000 anti-rabbit SGLT-1 antiserum followed by goat anti-rabbit IgG coupled to horseradish peroxidase (1:10,000, Pierce, Rockford, IL). After each incubation, the membrane was washed extensively with PBS-0.2% Tween 20. The signal was developed with the chemiluminescence Western blot kit (NEN). Desitometric analysis of the Western blots was done using Pharmacia LKB Ultrascan SX with Alpha Imager 2000.
Data presentation.
When data are averaged, means ± SE are shown, except when error bars are inclusive within the symbol. All uptakes were done in triplicate. The number (n) for any set of experiments refers to vesicle or isolated cell preparations from different animals. Preparations in which cell viability was >85% were excluded from analysis. Student's t-test was used for statistical analysis.
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RESULTS
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Na-nutrient cotransport in villus cells.
Inhibition of cNO with L-NAME resulted in a reduction in Na-stimulated glucose uptake (3-OMG uptake in the presence of Na minus 3-OMG uptake in the absence of Na) in intact villus cells (Fig. 1). These data indicated that SGLT-1 is inhibited when L-NAME inhibits cNO production in the normal rabbit intestine.

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Fig. 1. Effect of inhibition of constitutive nitric oxide (cNO) production with NG-nitro-L-arginine methyl ester (L-NAME) on Na-nutrient cotransport processes in intact villus cells. L-NAME treatment inhibited Na-stimulated glucose uptake.
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Because Na-K-ATPase on the basolateral membrane provides the favorable Na gradient for SGLT-1 on the BBM of villus cells, the effect of inhibition of cNO production on this enzyme was determined. As demonstrated in Fig. 2, L-NAME treatment did not diminish Na-K-ATPase levels, which would have explained the decrease in SGLT-1 on intact cells. If anything, Na-K-ATPase activity was stimulated by L-NAME treatment. Thus these data indicate that the effect of cNO on SGLT-1 is most likely at the level of the cotransporter itself.

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Fig. 2. Effect of inhibition of cNO production with L-NAME on Na-K-ATPase in villus cells. Na-K-ATPase activity was stimulated in villus cells from the L-NAME-treated rabbit small intestine (11.9 ± 2.2 nmol·mg protein1·min1 in control and 24.3 ± 4.1 nmol·mg protein1·min1 in L-NAME-treated cells, n = 7, P < 0.05).
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SGLT-1 in BBMV.
To determine whether L-NAME treatment had an effect at the level of the cotransporter itself, 3-OMG uptake was determined in villus cell BBMV prepared from control and L-NAME-treated rabbit intestine. In rabbits treated with L-NAME, Na-stimulated 3-OMG uptake was significantly reduced in villus cell BBMV (Fig. 3). These data demonstrated that inhibition of cNO production inhibits SGLT-1 at the level of the cotransporter on the BBM in the rabbit intestine.

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Fig. 3. Na-dependent uptake of 3-O-methyl-D-glucose (3-OMG) in villus brush-border membrane vesicles (BBMV) as a function of time from control and L-NAME-treated rabbit small intestine. Na-dependent glucose uptake was significantly reduced in villus cell BBMV from the L-NAME-treated intestine.
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To determine whether cNO affected SGLT-1 uniquely or broadly affects other Na-nutrient cotransport processes as well, we investigated its effects on Na-neutral amino acid cotransport. This cotransport process previously was shown to have the same distribution as SGLT-1 (villus cell BBM) and to have the same secondary active transport properties as SGLT-1 (e.g., BLM Na-K-ATPase dependent) in the rabbit small intestine (8, 27). As shown in Fig. 4, in contrast to SGLT-1, NAcT uptake was unaffected when cNO production was inhibited with L-NAME. These data indicated that SGLT-1 is regulated by cNO in the normal rabbit intestine, whereas NAcT is unaffected.

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Fig. 4. Effect of L-NAME treatment on Na-amino acid cotransport (NAcT). Na-dependent alanine uptake was unaffected in villus cell BBMV from the L-NAME-treated intestine.
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To ensure that the effects of L-NAME are indeed specific to diminishing the production of cNO, the effect of the inactive analog, D-NAME, was investigated. D-NAME had no effect on Na-dependent glucose uptake in villus cells (Fig. 5A). Because L-NAME can block both cNO and iNO the effects of L-NIL, a specific inhibitor of iNO was used to block the effects of NO. However, under normal physiological conditions, one would not expect any significant amount of iNO produced. As demonstrated in Fig. 5B, L-NIL also had no effect on SGLT-1 in villus cells. These data indicate that the L-NAME inhibits SGLT-1 and that the effect is specific to its ability to reduce the production cNO.

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Fig. 5. Effect of NG-nitro-D-arginine methyl ester (D-NAME) and N6-(1-imonoethyl)-L-lysine dihydrochloride (L-NIL) on SGLT-1 in villus cell BBMV. D-NAME, an inactive analog of L-NAME, did not alter SGLT-1 in villus cells. L-NIL, a more selective inhibitor of inducible NO synthase also had no effect in SGLT-1 in villus cells from the normal rabbit small intestine.
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Kinetic studies.
To determine the mechanism of diminished cNO-mediated inhibition of the SGLT-1 in the small intestine, kinetic studies were performed. Uptake for all the concentrations was carried out at 6 s because the initial uptake studies for Na-dependent glucose uptake in BBMV were linear for at least 10 s. Figure 6 demonstrates the kinetics of glucose uptake in villus cells BBMV from the rabbit small intestine. Figure 6A shows the uptake of Na-dependent 3-OMG as a function of varying concentration of extravesicular glucose. As the concentration of extravesicular glucose was increased, the uptake of Na-dependent 3-OMG was simulated and subsequently became saturated in all conditions. With the use of Enzfitter, kinetic parameters derived from this data demonstrated that the affinity [1/Michaelis constant (Km)] for 3-OMG uptake was significantly diminished by the inhibition of cNO production (Fig. 6B; Km for 3-OMG uptake in BBMV was 4.5 ± 0.2 mM in control and 9.3 ± 1.1 in L-NAME-treated intestine, n = 4, P < 0.01). However, the maximal rate of uptake (Vmax) of 3-OMG was not altered by L-NAME treatment (Fig. 6B; Vmax for 3-OMG uptake in BBMV was 4.6 ± 0.3 nmol·mg protein1·6 s1 in control and 5.3 ± 0.4 nmol·mg protein1·6 s1in L-NAME-treated intestine). These data demonstrated that inhibition of cNO production inhibits SGLT-1 by decreasing the affinity of the cotransporter for glucose.

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Fig. 6. Kinetics of glucose uptake in villus cell BBMV from control and L-NAME-treated intestine. A: Na-dependent uptake of [3H]OMG is shown as a function of varying concentrations of extravesicular D-glucose. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations was determined at 6 s. As the concentration of extravesicular glucose was increased, uptake of glucose was stimulated and subsequently became saturated in villus cell BBMV in all conditions. B: analysis of these data with a Lineweaver Burk plot yielded kinetic parameters. The maximal rate of uptake of 3-OMG was not affected by L-NAME. However, the affinity for 3-OMG uptake was markedly reduced in the L-NAME-treated intestine.
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Molecular biology studies.
To determine whether indeed the levels of the cotransporter are unaltered in villus cells after L-NAME treatment, we looked at the message for SGLT-1. Northern blot studies demonstrated that the message for SGLT-1 was indeed unaltered in villus cells from the L-NAME-treated animals (Fig. 7). Because steady-state mRNA levels may not directly correlate with functional protein levels on the BBM, immunoreactive SGLT-1 levels on the BBM were also determined (Fig. 8). Western blot analysis of villus cell BBM showed that the immunoreactive protein levels of SGLT-1 were also unchanged in animals treated with L-NAME. These molecular biology studies are consistent with the kinetic studies and indicate that the mechanism of inhibition of SGLT-1 when cNO production is inhibited is secondary to an alteration in the affinity of the cotransporter without a change in the number of cotransporters.

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Fig. 7. Northern blot analysis demonstrated that steady-state levels of SGLT-1 mRNA were unaffected in villus cells from the L-NAME-treated small intestine. Each blot is a representative of 4 experiments each with different animals. -Actin was used to ensure equal loading of the gel.
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Fig. 8. Western blot analysis demonstrated that the amount of immunoreactive SGLT-1 in BBMV from the L-NAME-treated small intestine. Results were also unchanged when compared with control. Each blot is a representative of 4 experiments each with different animals.
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DISCUSSION
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This study, for the first time, demonstrates that cNO uniquely regulates Na-nutrient cotransport processes in the mammalian small intestine. Inhibition of cNO production resulted in the inhibition of SGLT-1, but not NAcT. The mechanism of inhibition of SGLT-1 was secondary to an alteration in the affinity of the cotransporter for glucose without an alteration in the number of cotransporters. Thus cNO in the mammalian small intestine regulates SGLT-1 by altering the affinity of the cotransporter for the glucose.
In the mammalian small intestine, the two most important means of assimilation of Na are via coupled NaCl absorption and Na-nutrient cotransport processes (6). The latter class of transporters is also very important for the assimilation of critical nutrients such as glucose and amino acids. Glucose is transported via SGLT-1, whereas amino acids are mostly transported via sodium-driven amino acid transporters.
Both cotransport processes have been demonstrated to be on the BBM of the absorptive villus, but not secretory crypt cells in the mammalian small intestine. These two cotransport processes are not only important for the absorption of Na and nutrients in the normal intestine, but they may be even more important in intestinal pathophysiology. In fact, in a mammalian model of chronic small intestinal inflammation, both of these cotransport processes were inhibited during chronic enteritis. However, the mechanism of inhibition was markedly different. SGLT-1 was inhibited secondary to a decrease in a number of the cotransporters on the BBM of the villus cells, whereas NAcT was inhibited secondary to an alteration in the affinity of the cotransporter for the amino acid rather than a change in the number of cotransporters (23, 24).
Whereas these two Na-nutrient cotransporter processes may be altered in chronic diarrheal diseases, they are entirely preserved in certain severe acute diarrheal diseases such as cholera (9). Thus, whereas the significance of Na-nutrient cotransport processes in health and in diseased states is substantial, how these cotransport processes are altered by one of the more biologically active molecules, cNO, is at present unknown (11, 15, 17, 19, 20).
cNO has been demonstrated to influence a variety of gastrointestinal functions (18, 26). However, this study, for the first time, demonstrates that when cNO production was inhibited with L-NAME, SGLT-1 was inhibited in the rabbit small intestinal absorptive villus cells. This effect of cNO on SGLT-1 was unique for this cotransporter because inhibition of cNO production had no effect on NAcT also known to be present on the BBM of villus cells. Thus it is likely that in the normal intestine, the low levels of NO produced by cNO, while having an effect on SGLT-1, apparently do not have an effect on NAcT. However, because we have previously demonstrated that during chronic intestinal inflammation, SGLT-1 and NAcT are inhibited and it is known that iNOS is upregulated in the inflamed intestine, it is conceivable that higher quantities of NO produced by iNOS may 1) regulate SGLT-1 differently and/or 2) have an effect on NAcT.
In intact villus cells, Na-K-ATPase provides the favorable transcellular Na gradient for SGLT-1. Our results demonstrated that the effect of cNO on SGLT-1 is not secondary to a decrease in the Na-extruding capacity of the intact villus cells. If anything, Na-K-ATPase levels were increased in response to the inhibition of cNO production. This interesting observation has previously not been reported and has potential implications for multiple BBM transport processes. In any event, this stimulation of Na-K-ATPase would be expected to optimize the Na gradient across the BBM even more and stimulate SGLT-1. But, our observations suggest that the direct effect on SGLT-1 by cNO is more prevailing.
Subsequent studies were performed to determine whether cNO had an effect on SGLT-1 at the level of the cotransporter on the BBM of villus cells. The results demonstrated that inhibition of cNO indeed inhibited Na-dependent glucose uptake in villus cells BBMV. These findings indicated that the effect of cNO was at the level of the cotransporter itself. Because only L-NAME, but not the inactive analog D-NAME, altered SGLT-1, the effect of L-NAME in terms of inhibition of cNO production appears to be specific. Furthermore, L-NIL, a more selective inhibitor of iNOS, also had no effect on SGLT-1 in the normal intestine. This observation is as expected because one would not expect much, if any, iNOS activity in the normal intestine. Thus inhibition of little to no iNOS would not be expected to affect NO levels, which, in turn, should also not have an effect on SGLT-1. From these results, it is evident that the observed results with L-NAME on SGLT-1 are due to its effect on cNO.
To determine the mechanism of inhibition of SGLT-1, kinetic studies were then performed. The studies demonstrated that although the affinity of the cotransporter was significantly reduced when cNO was inhibited, there was no effect on the number of cotransporters. Indeed subsequent molecular studies demonstrated that neither the message for SGLT-1 nor the immunoreactive protein levels of SGLT-1 on the BBM changed when cNO production was inhibited by L-NAME. The intracellular pathways of regulation of SGLT-1 by cNO have yet to be deciphered. It is possible that NO may mediate its effect on intestinal electrolyte and nutrient transport by one of several mechanisms in the enterocyte, directly, via cGMP, which can then affect PKG or PKA, via PKC, or via ADP-associated ribosylation (25).
Thus this study for the first time shows a direct and unique effect of cNO on Na-nutrient cotransport processes in villus cells and in villus cell BBM from the small intestine. A previous study (10) in intact tissue from rat small intestine has suggested that endothelin-3 may increase Na-stimulated glucose uptake possibly via intracellular NO and guanylate cyclase. This study did not directly study the effect of inhibiting cNO production on SGLT-1. No mechanistic details were provided. Furthermore, it is conceivable that species differences, use of intact tissue vs. isolated absorptive villus cells, directly inhibiting cNO production, and avoiding other second-messenger systems an agonist such as endothelin-3 may alter may all be possible explanations for the differences in these two reports.
Although cNO has been postulated to alter many gastrointestinal tract functions, its direct effect on Na-nutrient cotransport processes was previously unknown. It is also interesting to note that despite global properties that altering cNO levels may have on the organism, including altering blood flow, motility, and renal function, all of which can nonspecifically alter absorption and secretion in the intestine (18, 26), inhibition of cNO production appears to have a direct effect on one specific type of Na-solute cotransport process amongst many known to be in the intestine. This would suggest that SGLT-1, unlike other Na-nutrient cotransport processes, either directly or via an intracellular mediator that NO may effect, lends itself to this type of regulation.
In conclusion, this novel study demonstrates that cNO uniquely regulates Na-nutrient cotransport processes in the mammalian small intestine. Whereas SGLT-1 is regulated by cNO, it appears that NAcT is not affected by cNO in the mammalian small intestine. Inhibition of cNO production results in the inhibition of SGLT-1. The mechanism of inhibition of SGLT-1 is secondary to an alteration in the affinity of the cotransporter for glucose without a change in the number of cotransporters.
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GRANT
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45062 and DK-58034 (to U. Sundaram).
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FOOTNOTES
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Address for reprint requests and other correspondence: U. Sundaram, Section of Digestive Diseases, West Virginia Univ. Medical Center, Medical Center Dr., PO Box 9161, Morgantown, WV 26506 (e-mail: usundaram{at}hsc.wvu.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.
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