Am J Physiol Gastrointest Liver Physiol 285: G1084–G1090, 2003;
10.1152/ajpgi.00013.2003.
Unique regulation of anion/HCO3⫺ exchangers by
constitutive nitric oxide in rabbit small intestine
Steven Coon and Uma Sundaram
Digestive Diseases Unit, Department of Medicine, The University
of Rochester Medical Center, Rochester, New York 14642
Submitted 13 January 2003; accepted in final form 14 July 2003
brush border; intestinal absorption; short-chain fatty acid
transport; secretion
IN THE RABBIT SMALL INTESTINE, three different brushborder membrane (BBM) anion/HCO3⫺ exchangers
have been demonstrated, which have unique functions:
1) Cl/HCO3⫺ exchange on the BBM of villus cells, which
coupled by intracellular pH with Na/H exchange, is
responsible for coupled NaCl absorption (25); 2) Cl/
HCO3⫺ exchange on the BBM of crypt cells possibly
involved in HCO3⫺ secretion (25); and 3) short-chain
fatty acid (SCFA)/HCO3⫺ exchange on the BBM of villus
cells (11). Although the molecular identity of these
three anion exchangers has yet to be elucidated, it has
been well established that in the normal intestine and
in intestinal pathophysiology, these three exchangers
Address for reprint requests and other correspondence: U.
Sundaram, Digestive Diseases Unit, The Univ. of Rochester Medical
Center, 601 Elmwood Ave., Box 646, Rochester, NY 14642.
G1084
are uniquely regulated. For example, in the normal
rabbit intestine, a secretogogue such as serotonin has
been demonstrated to inhibit coupled NaCl absorption
by inhibiting Cl/HCO3⫺ but not Na/H exchange on the
BBM of villus cells (26). In contrast, serotonin stimulates Cl/HCO3⫺ exchange on the BBM of crypt cells,
which may result in HCO3⫺ secretion by these cells (26).
Similarly, during chronic intestinal inflammation all
three of these anion exchangers are again uniquely
regulated as well (4, 7, 27). For example, although both
villus cell BBM Cl/HCO3⫺ and SCFA/HCO3⫺ exchange
are inhibited, the mechanism of inhibition of each
transporter is unique (4, 7). Cl/HCO3⫺ exchange is inhibited secondary to an alteration in the affinity of the
transporter for Cl without a change in transporter
numbers (4). In contrast, SCFA/HCO3⫺ exchange is
inhibited secondary to a decrease in transporter numbers without an change in the affinity of the transporter (7). Unlike both of these villus cell BBM anion
exchangers, the crypt cell BBM Cl/HCO3⫺ exchange is
secondarily stimulated to secrete HCO3⫺ during chronic
intestinal inflammation (27). Thus studies clearly indicate that there are at least three functionally different anion/HCO3⫺ exchangers on the BBM of villus and
crypt cells in the rabbit intestine.
NO has been demonstrated to be produced by multiple cell types in the intestine. Nitric oxide (NO) produced by constitutive nitric oxide (cNO) synthase in
the normal intestine and by inducible NO (iNO) synthase in pathophysiological states has been demonstrated to regulate gastrointestinal functions (2, 39). In
general, the small quantities of NO produced by cNO
synthase in the normal mammalian intestine has generally been thought to be beneficial (2, 3, 7, 14, 19, 21,
36, 39). In contrast, during pathophysiological states
(e.g., Crohn’s disease and ulcerative colitis), the larger
quantities of NO produced by iNO are thought to be
deleterious (2, 5, 7, 8, 22, 39).
The role of NO on intestinal electrolyte transport in
general is poorly understood (9). Whether NO has no
effect, promotes absorption, or secretion appears to
depend, at least in part, on 1) whether the condition of
the intestine is physiological or pathophysiological, 2)
the part of the intestinal tract studied, and 3) the
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Coon, Steven, and Uma Sundaram. Unique regulation
of anion/HCO3⫺ exchangers by constitutive nitric oxide in
rabbit small intestine. Am J Physiol Gastrointest Liver Physiol
285: G1084–G1090, 2003; 10.1152/ajpgi.00013.2003.—In the
rabbit small intestine, there are three functionally different
brush-border membrane (BBM) anion/HCO3⫺ exchangers: 1)
Cl/HCO3⫺ exchange on the BBM of villus cells responsible for
coupled NaCl absorption; 2) Cl/HCO3⫺ exchange on the BBM
of crypt cells possibly involved in HCO3⫺ secretion; and 3)
short-chain fatty acid (SCFA)/HCO3⫺ exchange on the BBM of
villus cells, which facilitates SCFA absorption. Although
constitutive nitric oxide (cNO) has been postulated to alter
many gastrointestinal tract functions, how cNO may specifically alter these three transporters is unknown. Inhibition
of cNO synthase with N G-nitro-L-arginine methyl ester
(L-NAME) 1) did not affect villus cell BBM Cl/HCO3⫺ exchange, 2) stimulated crypt cell BBM Cl/HCO3⫺ exchange,
and 3) inhibited villus cell BBM SCFA/HCO3⫺ exchange.
D-NAME, an inactive analog of L-NAME, and L-N6-(1-iminoethyl)lysine, a more selective inhibitor of inducible NO, did
not affect these transport processes. Kinetic studies demonstrated that 1) 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, and 2) 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 of SCFA. These
results indicate that cNO uniquely regulates the three BBM
anion/HCO3⫺ transporters in the rabbit small intestine.
NO REGULATION OF ANION EXCHANGERS
METHODS
Drug treatment. New Zealand male, white rabbits (2–2.5
kg) were treated with NG-nitro-L-arginine methyl ester (LNAME; 75 mg/day im for 2 days) or with the inactive analog
NG-nitro-D-arginine methyl ester (D-NAME; 75 mg/day im for
2 days) or with L-N6-(1-iminoethyl)lysine (L-NIL, 0.1 mg/day
im for 2 days). L-NAME has been well demonstrated previously (11, 16, 23, 30–33) to inhibit cNOS and to reduce NO
production in intestinal tissue.
Cell isolation. Villus and crypt cells were isolated from the
rabbit intestine by a calcium chelation technique as previously described (27, 29). Previously established criteria were
used to validate good separation of villus and crypt cells.
Some of these criteria included 1) marker enzymes (alkaline
phosphatase), 2) transporter specificity (SGLT-1 on the BBM
of villus, but not crypt cells), and 3) morphological differences
(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 such as 1) Trypan blue exclusion
and 2) the demonstration of Na/H and C1/HCO3⫺ exchange
activities (27, 29).
BBM vesicle preparation. BBM vesicles (BBMV) from rabbit intestinal villus and crypt cells were prepared by CaCl2
precipitation and differential centrifugation as previously
reported (25, 28). 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 cell BBMV. BBMV uptake studies
were performed by the rapid-filtration technique as previously described (25, 28). For Cl/HCO3⫺ exchange experiments,
BBMV were resuspended and incubated for 2 h in 105 mM
NMG gluconate, 50 mM HEPES-Tris, (pH 7.7) and either 50
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mM KHCO3⫺ gassed with 5% CO2-95% N2 or 50 mM K
gluconate gassed with 100% N2. The reaction was started by
adding 5 ␮l of vesicles to 95 ␮l reaction mix containing 5 mM
N-methyl-D-glucamine (NMG)36Cl, 149.7 mM K gluconate,
50 mM MES-Tris, and 0.3 mM KHCO3⫺, pH 5.5 gassed with
5% CO2-95% N2 or 150 mM K gluconate (pH 5.5) gassed with
100% N2. DIDS (1 mM) was added to the reaction mix when
relevant. For SCFA/HCO3⫺ experiments, BBMV were resuspended in 100 mM NMG gluconate, 0.10 mM MgSO4, 50 mM
HEPES-Tris (pH 7.5) and 50 mM KHCO3⫺ or K gluconate.
The reactions were started by adding 5 ␮l vesicles to 95 ␮l
reaction mix containing 100 mM NMG gluconate, 0.1 mM
MgSO4, 10 ␮M valinomycin, 50 ␮M [14C]butyrate, 50 mM
HEPES-Tris (pH 7.5 or pH 6.0), and 50 mM KHCO3⫺ or K
gluconate. At desired times, uptake was arrested by mixing
with ice-cold stop solution [50 mM HEPES-Tris buffer (pH
7.5), 0.10 mM MgSO4, 50 mM K gluconate, and 100 mM
NMG gluconate]. The mixture was filtered on 0.45 ␮m Millipore (HAWP) filters and washed twice with 5 ml ice-cold
stop solution. Filters with BBMV were dissolved in Liquiscint, and radioactivity was determined.
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.
RESULTS
Villus cell BBM Cl/HCO3⫺ exchange. Figure 1 demonstrates the effect of inhibition of cNO production
with L-NAME on Cl/HCO3⫺ exchange in villus cells.
L-NAME treatment did not affect the DIDS-sensitive
and HCO3⫺ gradient-stimulated 36Cl uptake (e.g., Cl/
HCO3⫺ exchange) in villus cell BBMV. These data indicated that cNO did not affect villus cell BBM Cl/
HCO3⫺ exchange.
Crypt cell BBM Cl/HCO3⫺ exchange. To determine
the effect of cNO on crypt cell BBM Cl/HCO3⫺ exchange,
we then looked at DIDS-sensitive and HCO3⫺ gradientstimulated 36Cl uptake in crypt cell BBMV prepared
from control and L-NAME-treated rabbit small intes-
Fig. 1. Effect of inhibition of constitutive nitric oxide (cNO) production with NG-nitro-L-arginine methyl ester (L-NAME) on villus cell
brush-border membrane (BBM) Cl/HCO3⫺ exchange. L-NAME did not
alter HCO3⫺-dependent and DIDS-sensitive Cl uptake in villus cell
BBM vesicles (BBMV). These data indicated that cNO did not affect
villus cell BBM Cl/HCO3⫺ exchange.
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species studied (1, 10, 11, 21, 34, 40). Further inhibition of NO vs. stimulation of NO production has not
necessarily yielded the expected corollary response on
electrolyte absorption and secretion from study to
study (16, 23, 38). At least some of the conflicting
information pertaining to the effect of NO on electrolyte transport in the intestine may be owing to the
inability to reproduce the NO levels similar to cNO of
the normal mammalian small intestine in these studies (1, 11, 21).
Another potential concern with intact tissue studies
that were used to determine the role of NO on electrolyte transport is that the epithelium is composed of
functionally different villus and crypt cells (25). The
former is absorptive, and the latter is secretory, thus
any observed effect will be a compilation of these two
properties and therefore is less likely to be mechanistic. So, it is important to separate these two cell types
to best determine the effect of an agent on electrolyte
transport in the intestine, as was done in this study.
Also in studies to date, whether cNO can uniquely
regulate a related family of BBM transporters and
between absorptive villus and secretory crypt cells has
not been deciphered. Thus, given this background, the
aims of this study were to determine whether cNO may
in fact regulate anion/HCO3⫺ exchangers and to determine the mechanism of this regulation by cNO in the
mammalian small intestine.
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NO REGULATION OF ANION EXCHANGERS
tine. As shown on Fig. 2, 36Cl uptake was significantly
reduced in L-NAME-treated intestine at all early time
points measured. These data indicated that cNO stimulates crypt cell BBM Cl/HCO3⫺ exchange under physiological conditions.
To ensure that the effect of L-NAME is indeed specific to diminishing the production of cNO, the effect of
the inactive analog D-NAME was then studied.
⫺
D-NAME had no effect on DIDS-sensitive and HCO3
36
gradient-stimulated Cl uptake in crypt cell BBMV
(Fig. 3). Similarly, whereas one would not expect a role
for iNO in the normal mammalian intestine, the effect
of L-NIL, a more specific inhibitor of iNO synthase was
nevertheless studied. As demonstrated in Fig. 4, L-NIL
also had no effect on Cl/HCO3⫺ exchange in crypt cell
BBMV. These data indicate that the L-NAME treatment inhibits crypt cell BBM Cl/HCO3⫺ exchange and
that the effect is specific to its ability to reduce cNO.
Villus cell BBM SCFA/HCO3⫺ exchange. Because
cNO had no effect on villus cell BBM Cl/HCO3⫺ exchange, to determine whether a different villus cell
BBM anion/HCO3⫺ exchanger may be affected differently by cNO, we looked at SCFA/HCO3⫺ exchange.
This exchanger is known to be present only on the
BBM of villus cells in the rabbit small intestine (12).
We studied the effect of inhibition of cNO production
Fig. 3. The effect of NG-nitro-D-arginine methyl ester (D-NAME), an
inactive analog of L-NAME, on Cl/HCO3⫺ exchange in crypt cell
BBMV. D-NAME treatment had no effect on Cl/HCO3⫺ exchange.
These data indicated that the effect of L-NAME on crypt cell Cl/
HCO3⫺ exchange is specific for this agent.
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with L-NAME on pH and HCO3⫺ gradient-stimulated
[14C]butyrate uptake in villus cell BBMV prepared
from control and L-NAME-treated rabbit small intestine. As shown in Fig. 5, butyrate uptake was significantly enhanced in villus cell BBM from L-NAMEtreated intestine at all early time points measured.
These data indicated that cNO inhibits villus cell BBM
SCFA/HCO3⫺ exchange under physiological conditions.
To determine whether the effects of L-NAME on
SCFA/HCO3⫺ exchange is indeed specific to diminishing the production of cNO, the effect of the inactive
analog D-NAME was then studied. D-NAME had no
effect on pH and HCO3⫺ gradient-stimulated [14C]butyrate uptake in villus cell BBMV (Fig. 6). Also,
whereas one would not expect any substantial amount
of iNO in the normal mammalian intestine, the effect
of L-NIL, a more specific inhibitor of iNO synthase, was
nevertheless studied on SCFA/HCO3⫺ exchange. As
demonstrated in Fig. 7, L-NIL also had no effect on
SCFA/HCO3⫺ in villus cell BBMV. These data indicate
that the L-NAME treatment stimulates villus cell
SCFA/HCO3⫺ exchange and that the effect is specific to
its ability to reduce cNO.
Kinetic studies. To determine the mechanism of diminished cNO-mediated inhibition of crypt cell BBM
Cl/HCO3⫺ exchange, kinetic studies were performed.
Fig. 5. Effect of inhibition of cNO production with L-NAME on
short-chain fatty acid (SCFA)/HCO3⫺ in villus cell BBMV. L-NAME
treatment stimulates HCO3⫺-dependent butyrate uptake in villus cell
BBMV. These data indicated that cNO inhibits villus cell BBM
SCFA/HCO3⫺ exchange.
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Fig. 2. Effect of inhibition of cNO production with L-NAME on crypt
cell BBM Cl/HCO3⫺ exchange. L-NAME treatment resulted in the
reduction of HCO3⫺-dependent and DIDS-sensitive Cl uptake in crypt
cell BBMV. These data indicated that cNO stimulates crypt cell BBM
Cl/HCO3⫺ exchange.
Fig. 4. Effect of L-N6-(1-iminoethyl)lysine (L-NIL), a more selective
inhibitor of inducible NOS on crypt cell Cl/HCO3⫺ exchange. L-NIL
had no effect on Cl/HCO3⫺ exchange in crypt cell BBMV. These data
indicated that L-NAME-mediated inhibition of Cl/HCO3⫺ exchange in
crypt cell BBM is via its effects on cNO.
NO REGULATION OF ANION EXCHANGERS
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L-NAME treated, n ⫽ 4; P ⬍ 0.05). These data indicated that inhibition of cNO production stimulates
SCFA/HCO3⫺ exchange in villus cells by likely increasing the affinity of the transporter for butyrate.
DISCUSSION
Uptake for all the concentrations was carried out at 6 s,
because the initial uptake studies for pH and HCO3⫺
gradient-stimulated 36Cl uptake in crypt cell BBMV
were linear for at least 10 s. Figure 8 demonstrates the
kinetics of 36Cl uptake in crypt cell BBMV from the
rabbit small intestine. Figure 8 shows the uptake of
36
Cl as a function of varying concentration of extravesicular Cl. As the concentration of extravesicular Cl
was increased, the uptake of 36Cl was simulated and
subsequently became saturated in all conditions. With
the use of Enzfitter, kinetic parameters derived from
these data demonstrated that Vmax was markedly reduced by the inhibition of cNO production (Fig. 8A;
Vmax was 54.6 ⫾ 0.3 nmol 䡠 mg protein⫺1 䡠 6 s⫺1 in control and 25.8 ⫾ 1.0 nmol 䡠 mg protein⫺1 䡠 6 s⫺1 in LNAME treated, n ⫽ 4; P ⬍ 0.05). However, the affinity
(1/Km) for Cl uptake was not altered by L-NAME treatment (Fig. 8B; Km was 6.6 ⫾ 0.2 mM in control and
6.04 ⫾ 0.1 mM in L-NAME treated, n ⫽ 4). These data
demonstrated that inhibition of cNO production inhibits Cl/HCO3⫺ exchange in crypt cells by likely reducing
transporter numbers.
Next, to determine the mechanism of reduced cNO
production-mediated stimulation of villus cell SCFA/
HCO3⫺ exchange, kinetic studies were performed. Uptake for all of the concentrations were carried out at
3 s, because the initial uptake studies for pH and
HCO3⫺ gradient-stimulated [14C]butyrate uptake in villus cell BBMV were linear for at least 6 s. Figure 9
demonstrates the kinetics of [14C]butyrate uptake in
villus cell BBMV from the rabbit small intestine. Figure 9 shows the uptake of [14C]butyrate as a function of
varying concentration of extravesicular butyrate. As
the concentration of extravesicular butyrate was increased, the uptake of [14C]butyrate was simulated and
subsequently became saturated in all conditions. With
the use of Enzfitter, kinetic parameters derived from
these data demonstrated that Vmax was not affected by
the inhibition of cNO production (Fig. 9A; Vmax was
15.1 ⫾ 0.2 nmol 䡠 mg protein⫺1 䡠 3 s⫺1 in control and
14.5 ⫾ 0.3 nmol 䡠 mg protein⫺1 䡠 3 s⫺1 in L-NAME
treated, n ⫽ 4). However, Km for butyrate uptake was
markedly reduced by L-NAME treatment (Fig. 9B; Km
was 39.6 ⫾ 0.8 mM in control and 12.4 ⫾ 0.4 mM in
AJP-Gastrointest Liver Physiol • VOL
Fig. 7. Effect of L-NIL, a more selective inhibitor of inducible NOS on
villus cell SCFA/HCO3⫺ exchange. L-NIL had no effect on SCFA/
HCO3⫺ exchange in villus cell BBMV. These data indicated that
⫺
L-NAME-mediated stimulation of SCFA/HCO3 exchange in villus
cell BBM is via its effects on cNO.
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Fig. 6. The effect of D-NAME, the inactive analog of L-NAME on
SCFA/HCO3⫺ exchange in villus cell BBMV. D-NAME treatment had
no effect on SCFA/HCO3⫺ exchange. These data indicated that the
effect of L-NAME on villus cell SCFA/HCO3⫺ exchange is specific for
this agent.
This study, for the first time, demonstrates directly
that cNO uniquely regulates the three functionally
different anion/HCO3⫺ exchangers on the BBM mammalian small intestinal villus and crypt cells. On the
villus cell BBM, Cl/HCO3⫺ exchange, thought to be
important for coupled NaCl absorption, is not altered
by cNO. However, Cl/HCO3⫺ exchange on the BBM of
crypt cells, thought to be involved in intestinal HCO3⫺
secretion, is inhibited when cNO production is diminished. This indicates that in physiological states, cNO
exerts a positive tone on HCO3⫺ secretion in the small
intestine. Unlike these two anion/HCO3⫺ exchangers,
SCFA/HCO3⫺, which is only found on the BBM of villus
cells, is stimulated when cNO production is reduced.
This indicates that under physiological conditions,
cNO exerts a negative tone on SCFA/HCO3⫺ exchange.
Several anion exchanger families have been identified and cloned. Anion exchangers 1–4 (AE 1–4), downregulated adenoma (DRA), pendrin (PDS), and putative anion transporter (PAT 1) have all been proposed
as candidates for BBM anion/HCO3⫺ exchange activity
(13, 20, 24, 37). Melvin et al. (13) suggested that DRA
is the main candidate for Cl/HCO3⫺ exchange in these
cells. However, other studies would point to a member
of the AE family, specifically AE1–3, because all have
been proposed as the BBM Cl/HCO3⫺ exchanger by
some, whereas others have demonstrated them on the
BBM (13, 15, 20, 24, 37). Thus, whereas the molecular
identity of the three BBM anion/HCO3⫺ exchangers
studied here has yet to be resolved, they without a
doubt function uniquely in the normal intestine and
are also altered uniquely in the chronically inflamed
intestine. This suggests that they may indeed be different proteins and thus lend themselves to unique
regulation by an agent. Indeed, as demonstrated in
this study, NO, a 30-kDa biological mediator known to
be present in the intestine, uniquely regulates these
G1088
NO REGULATION OF ANION EXCHANGERS
three BBM aninon/HCO3⫺ exchangers. DIDS-sensitive
and HCO3⫺-dependent Cl uptake, defined as Cl/HCO3⫺
exchange, was not affected in the villus cell BBM,
however, it was significantly reduced when NO production was diminished in crypt cells. In contrast, HCO3⫺and pH-dependent butyrate uptake, defined as SCFA/
HCO3⫺ exchange, known only to be present on the BBM
of villus cells, was markedly enhanced when NO production was reduced. Whereas the molecular identity
of this anion exchange is also not known, monocarboxylate-type transport proteins (MCT1 and -2) are possible
candidates (15). In summary, these data indicate that
cNO, under normal physiological conditions, stimulates crypt cell Cl/HCO3⫺ exchange and inhibits villus
cell SCFA/HCO3⫺ exchange, whereas it has no effect on
villus cell Cl/HCO3⫺ exchange.
To date, specifically how NO may alter intestinal
electrolyte transport has been unclear, partly due to
species variation, segment of the gut studied, and the
condition of the intestine studied. This last is especially important, because it is known that there are
three NO synthases: neuronal or nNOS or NOS1; endothelial or eNOS or NOS3; and inducible or iNOS or
NOS2. NOS1 and 3 are considered constitutive and
produce nanomolar quantities of NO. On the other
hand, NOS2 predominantly occurs during pathophysiological conditions and produces micromolar quantities
of NO. Thus the inconclusive results seen to date regarding NO regulation of electrolyte and fluid transport in the intestine (9, 10, 14, 29, 35, 38) may be
related to the levels of NO generated in these studies.
Variable NO levels could have led to physiological
and/or pathophysiological responses in electrolyte
transport in these studies. To allay these concerns,
cNO was inhibited in vivo in these studies.
To determine the mechanism of inhibition of crypt
cell BBM Cl/HCO3⫺ exchange when NO production was
diminished with L-NAME, kinetic studies were then
Fig. 9. Kinetics of SCFA uptake in villus cell BBMV from control and L-NAME treated intestine. A: pH and HCO3⫺
gradient stimulated [14C]butyrate uptake of is shown as a function of varying concentrations of extravesicular
butyrate. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations
was determined at 3 s. As the concentration of extravesicular butyrate was increased, uptake of butyrate was
stimulated and subsequently became saturated in villus cell BBMV in all conditions. B: analysis of these data with
Lineweaver-Burk plot yielded kinetic parameters. The maximal rate of uptake of SCFA (Vmax) was unaffected.
However, the Km was reduced significantly in the L-NAME-treated intestine. These data indicated that the
stimulation of SCFA/HCO3⫺ exchange by L-NAME in villus cell BBM is most likely secondary to an increase in the
affinity of the transporter for butyrate.
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Fig. 8. Kinetics of chloride uptake in crypt cell BBMV from control and L-NAME-treated intestine. A: DIDSsensitive HCO3⫺ gradient-stimulated 36Cl uptake is shown as a function of varying concentrations of extravesicular
chloride. Isosmolarity was maintained by adjusting the concentration of mannitol. Uptake for all concentrations
was determined at 6 s. As the concentration of extravesicular chloride was increased, uptake of chloride was
stimulated, and subsequently became saturated in crypt cell BBMV in all conditions. B: analysis of these data with
Lineweaver-Brock plot yielded kinetic parameters. The affinity (1/Km) did not change significantly. However,
maximal rate of uptake of 36Cl (Vmax) was reduced significantly by L-NAME. This date indicated that the inhibition
of Cl/HCO3⫺ exchange by L-NAME in crypt cell BBM is most likely secondary to a reduction in the number of
transporters.
NO REGULATION OF ANION EXCHANGERS
DISCLOSURES
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases research Grants DK-45062 and DK58034 (to U. Sundaram).
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performed. The studies demonstrated that although
the affinity of the transporter was not affected, there
was a substantial decrease in the Vmax for Cl/HCO3⫺
exchange. This indicated that the mechanism of inhibition of crypt cell Cl/HCO3⫺ exchange when cNO is
reduced is secondary to a decrease in transporter numbers. Kinetic studies were also performed to determine
the mechanism of stimulation of villus cell BBM SCFA/
HCO3⫺ exchange when NO production was diminished.
These studies demonstrated that although the Vmax
was not affected, there was a substantial decrease in
Km. This indicated that the mechanism of stimulation
of villus cell SCFA/HCO3⫺ exchange when cNO is reduced is secondary to an increase in the affinity of the
transporter for SCFA.
Determination of the effect of cNO on electrolyte
transport in the normal mammalian small intestine is
also complicated by the fact that in intact tissue studies, it is not possible to separate the effects of a given
agent on these functionally different villus cells and
crypt cells. Current evidence would suggest that villus
cells are primarily absorptive, because Na-dependent
solute cotransport processes, SCFA/HCO3⫺ exchange,
and coupled NaCl absorption, which occurs via the
dual operation of Na/H and Cl/HCO3⫺ exchange, are
localized to the BBM of villus cells. In contrast, the
crypt cells, at least functionally, only have Cl/HCO3⫺
exchange on the BBM, thus they are not capable of
NaCl, SCFA, or solute absorption. In view of this, it is
important to isolate relatively pure populations of viable villus and crypt cells to ideally study the effect of
the cNO on electrolyte transport. This was done in this
study, and as the results demonstrate, anion/HCO3⫺
exchangers are indeed uniquely regulated between villus and crypt cells.
In conclusion, this study, for the first time, demonstrates that cNO uniquely regulates anion/HCO3⫺ exchange in the mammalian small intestine. Inhibition of
cNO does not alter the villus cell BBM Cl/HCO3⫺ exchange; it inhibits crypt cell BBM Cl/HCO3⫺ exchange,
whereas villus cell BBM SCFA/HCO3⫺ exchange is
stimulated. Thus cNO imposes an excitatory tone on
crypt cell BBM Cl/HCO3⫺ exchange and an inhibitory
tone on villus cell BBM SCFA/HCO3⫺ exchange in the
normal mammalian small intestine. Finally, the mechanism of cNO-mediated alterations of anion/HCO3⫺ exchangers is also different. cNO regulates crypt cell
BBM Cl/HCO3⫺ exchange by altering transporter numbers, whereas it regulates villus cell BBM SCFA/HCO3⫺
exchange by altering the affinity of the transporter.
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30.
NO REGULATION OF ANION EXCHANGERS