Ammonia blockade of intestinal epithelial
K+ conductance
Bruce J.
Hrnjez,
Jaekyung C.
Song,
Madhu
Prasad,
Julio M.
Mayol, and
Jeffrey B.
Matthews
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard
Medical School, and the Harvard Digestive Diseases Center, Boston,
Massachusetts 02215
 |
ABSTRACT |
Ammonia profoundly inhibits cAMP-dependent
Cl
secretion in model T84
human intestinal crypt epithelia. Because colonic lumen concentrations
of ammonia are high (10-70 mM), ammonia may be a novel regulator
of secretory diarrheal responsiveness. We defined the target of ammonia
action by structure-function analysis with a series of primary amines
(ammonia, methylamine, ethylamine, propylamine, butylamine,
pentylamine, hexylamine, heptylamine, and octylamine) that vary
principally in size and lipid solubilities. The amine concentrations
required for 50% inhibition of
Cl
secretion in intact
monolayers and 50% inhibition of outward K+ current
(IK) in
apically permeabilized monolayers vs. the logs of the respective amine
partition coefficients give two plots that are strikingly similar in
character. Half-maximal inhibition of short-circuit current
(Isc) by
ammonia was seen at 6 mM and for
IK at 4 mM;
half-maximal inhibition for octylamine was 0.24 mM and 0.19 mM for
Isc and
IK, respectively.
The preferentially water-soluble hydrophilic amines (ammonia,
methylamine, ethylamine) increase in blocking ability with decreasing
size and lipophilicity. Conversely, the preferentially lipid-soluble
hydrophobic (propylamine, butylamine, pentylamine, hexylamine,
heptylamine, octylamine) amines increase in blocking ability with
increasing size and lipophilicity. Ammonia does not affect isolated
apical Cl
conductance;
amine-induced changes in cytosolic and endosomal pH do not correlate
with secretory inhibition. We propose that ammonia in its protonated
ammonium form (NH+4) inhibits
cAMP-dependent Cl
secretion
in T84 monolayers by blocking basolateral
K+ channels.
ammonia; chloride; diarrhea; T84 cells; pH
 |
INTRODUCTION |
Ammonia and related amines are normal products of cellular
transamination and deamination
reactions.1
The renal medulla contains a high level of ammonia, which is ultimately
eliminated in the urine in the form of urea, the chief final product of
nitrogen metabolism in mammals. The concentration of ammonia in most
other healthy organs is quite low; levels exceeding 1 mM are usually
toxic to mammalian cells. It is well known, however, that the lumen of
the lower gastrointestinal tract is the setting for bacterial action on
ingested protein and that the colon consequently experiences
concentrations of the protein degradation product ammonia that may
reach 100 mM. Little is known about the impact of such levels of
ammonia on normal intestinal epithelial cell function (1, 3, 7, 13, 22,
38). We recently reported that ammonia markedly inhibits cyclic
nucleotide-dependent Cl
secretion in the T84 human intestinal cell model of secondary active
epithelial Cl
transport
(22, 38) and proposed that luminal ammonia may be an endogenous
negative regulator of colonic secretion, serving to dampen epithelial
responsiveness to potentially diarrheagenic stimuli in the
bacteria-rich lumen. We were unable at that time to identify the
specific target of ammonia action but concluded that it is distal to
cAMP generation. Indirect evidence indicated that ammonia action is
independent of cytosolic alkalinization.
Here, we report that this inhibition results from
NH+4 block of the basolateral membrane
K+ conductance. This observation
runs counter to the conventional attribution of ammonia's biological
action to the ability to alter cytosolic or endosomal pH (1, 3, 7, 13).
It also runs counter to the well-known ability of many
K+ channels and transport pathways
to support NH+4 movement (25). Recognition
that NH+4 can block K+ channels may well be important
to understanding regulated ion transport in organ systems exposed to
high levels of ammonia under normal circumstances, in disease states
such as cirrhosis (32) and chronic renal insufficiency (11, 35, 49) and
during infections with urealytic organisms such as
Helicobacter pylori (6).
In accord with observations (12) on the action of ammonia and several
lysosomotropic amine weak bases on Dictyostelium
discoideum, preliminary experiments in our laboratory
showed that the same amines also suppress
Cl
secretion in T84 cells
and that the ability to do so may increase with increasing amine
lipophilicity. Such compounds have been observed in many instances to
perturb plasma membrane recycling, an effect that is widely assumed to
be due to their ability to accumulate within and raise the pH of acidic
endosomal compartments (13). Furthermore, recent evidence (4, 5, 39)
suggests that cAMP-dependent regulation of transepithelial
Cl
secretion may involve
rapid insertion or retrieval of membrane vesicles containing ion
channels such as cystic fibrosis transmembrane conductance regulator.
This finding initially led us to speculate that the effect of ammonia
on T84 cell secretion could be due to perturbation of endosomal pH and,
consequently, the regulated insertion or retrieval of key transport
proteins involved in transepithelial Cl
transport. However, the
variations in structural, functional, and chemical properties of the
amines we chose to examine made it difficult to draw conclusions about
a common mode of action. Moreover, we recently noted (33) some
similarities in ammonia's effect on epithelial transport to that of
Ba2+, a well-known
K+ channel blocker, and could not
easily reconcile this finding with the membrane-recycling hypothesis.
To define more clearly the target of ammonia action, we examined the
effect of structural variation about ammonia's nitrogen center with a
series of more closely related amines that have widely varying lipid
solubilities (Table 1). We assumed the
importance of nitrogen functionality to inhibitory ability and chose
amines whose structures, functionalities, and chemical properties vary in a well-defined manner. They comprise the basis set of a classical "structure-activity" study intended to disclose a common mode of
inhibitory action. Specifically, we chose these amines to evaluate the
relative importance of size, basicity, and lipophilicity to inhibitory
ability and then measured their effects on
Cl
secretion,
agonist-regulated apical membrane
Cl
and basolateral membrane
K+ conductance, cytosolic pH,
endosomal pH, and fluid-phase endocytosis in the T84 cell model.
 |
EXPERIMENTAL PROCEDURES |
Cell culture and buffers.
T84 cells were obtained from the American Type Culture Collection and
K. Barrett (Univ. of California, San Diego) and grown to confluence at
pH 7.40 in 150-ml flasks with DMEM-Ham's F-12 nutrient (1:1) and 6%
(vol/vol) fetal bovine serum, supplemented with HEPES (1.50 × 10
2 M),
NaHCO3 (1.43 × 10
2 M), penicillin G sodium
salt (1.70 × 10
4 M),
amphotericin B (2.70 × 10
7 M), and streptomycin
sulfate (6.86 × 10
5
M). The monolayers were maintained in culture with weekly passage by
trypsinization in Ca2+- and
Mg2+-free phosphate-buffered
solution at a surface ratio of 1:2.
Experiments were carried out in HEPES-phosphate-buffered Ringer
solution (HPBR) containing NaCl (1.35 × 10
1 M), KCl (5.0 × 10
3 M),
NaH2PO4
(3.33 × 10
3 M),
Na2HPO4
(8.30 × 10
4 M),
CaCl2 (1.0 × 10
3 M),
MgCl2 (1.0 × 10
3 M), glucose (1.0 × 10
2 M), and HEPES
(5.0 × 10
3 M).
Solutions containing amines (Aldrich Chemical, Milwaukee, WI) were
prepared by addition of the hydrochloride salt or the free amine to
HPBR without adjustment for variation in osmolarity or
Cl
concentration. Control
experiments with
N-methyl-D-glucamine (NMG) demonstrated that hyperosmolar apical or basolateral solutions slightly suppressed secretion only at high concentrations (>40 mM).
All solutions were used at room temperature and adjusted to a pH of
7.40 (with HCl or NaOH) unless otherwise specified.
Transepithelial Cl
transport.
Dose-response curves for suppression of
Cl
secretion with the
amines in Table 1 were obtained with T84 cells seeded and grown to
confluence on collagen-coated permeable supports with an area of 0.33 cm2 (24-well inserts, Costar,
Cambridge, MA). Monolayers were fed every 2-3 days and the night
before the experiment and were used between 7 and 14 days after they
were plated. Transepithelial potential difference and short-circuit
current (Isc)
were measured with a dual voltage-current clamp (model 616C-2, Univ. of
Iowa, Iowa City, IA) with apical and basolateral Ag-AgCl and calomel electrodes connected via "chopstick" KCl-agar bridges
(38). This setup facilitated the performance of large
numbers of transport studies that would have been substantially more
cumbersome in traditional Ussing chamber models. Forskolin-stimulated
Isc at room
temperature is approximately equivalent to that obtained at 37°C in
this model, and the effects of ammonia and amines were similar (not
shown). Typically, electrical measurements were made at 0 min on
monolayers in HPBR after they were washed free of medium, after a
30-min equilibration in HPBR at room temperature, after a 30-min
equilibration with or without amine, and 15 and 45 min after
basolateral stimulation with forskolin (1.0 × 10
5 M; Calbiochem, La
Jolla, CA). Each reported value for transepithelial resistance and
Isc is the
average of n = 4 measurements.
Permeabilized system.
The outward K+ current
(IK) was
measured, and dose responses were constructed for basolaterally applied
amines 1-9 to monolayers apically
permeabilized by nystatin (23, 28, 52). T84 cells grown to confluent
monolayers (resistance of >900
· cm2) on
collagen-coated Snapwell inserts (0.40-mm pore size, 12 mm diameter,
Costar) were washed with a low
K+-low
Na+ gluconate solution (1.0 × 10
2 M sodium
gluconate, 1.35 × 10
1 M NMG-gluconate, 5.0 × 10
2 M potassium gluconate,
1.0 × 10
3 M
MgSO4, 1.0 × 10
3 M calcium gluconate,
1.0 × 10
2 M glucose,
and 1.0 × 10
2 M HEPES
at pH 7.40). The monolayers were mounted in an Ussing-type diffusion
chamber system (model DCCSYS, Precision Instrument Design, Tahoe City,
CA) interfaced with computer-based data acquisition software (MacLab
System Chart v3.5.4) and a multichannel voltage-current clamp (model
VCC MC6 revision A, Precision Instrument Design). Apical and
basolateral chambers contained the wash solution described above during
15-min monolayer equilibration at 37°C. Current as a function of
time was measured with the voltage clamped at 0 mV. Voltage pulses of
1-s duration and 10-mV magnitude were passed across the monolayer at
10-s intervals to allow determination of transepithelial resistance.
The apical chamber solution was replaced with a high
K+-low
Na+ gluconate solution (1.0 × 10
2 M sodium
gluconate, 1.4 × 10
1
M potassium gluconate, 1.0 × 10
3 M
MgSO4, 1.0 × 10
3 M calcium gluconate,
1.0 × 10
2 M glucose,
and 1.0 × 10
2 M HEPES
at pH 7.40) to apply an apical-to-basolateral
K+ gradient across the monolayer.
Nystatin (500 U/ml; Sigma, St. Louis, MO) was added to the apical
solution to permeabilize the apical membrane to small monovalent ions.
Cells were treated with ouabain (1.0 × 10
4 M, Sigma) to inhibit
the basolateral
3Na+-2K+-ATPase
and prevent its contribution to the
IK. The resultant IK through
basolateral K+ channels was
thereby isolated and measured. Small aliquots of a concentrated
solution of the appropriate amine from Table 1 were added to the
basolateral solution, and the effects on
IK were observed
and recorded. In some cases, carbachol (1.0 × 10
4 M, Sigma) was added to
determine amine effects on
Ca2+-sensitive
K+ channels when the
Ca2+-independent or
"resting"
IK reached zero
after amine treatment. These apical permeabilization experiments were
conducted in Cl
-free
solutions to prevent swelling-induced opening of
K+ channels in the cell
basolateral membrane.
The outward Cl
current
(ICl) through
apical Cl
channels was
measured under similar conditions after the basolateral membrane was
permeabilized with nystatin. T84 cells grown to confluency on Snapwell
inserts were washed with a low
Cl
-high
K+ gluconate solution (1.0 × 10
2 M sodium gluconate, 1.4 × 10
1 M potassium
gluconate, 1.0 × 10
3
M calcium gluconate, 1.0 × 10
3 M
MgSO4, 1.0 × 10
2 M glucose, and 1.0 × 10
2 M HEPES at pH
7.40). Monolayers were mounted in the same diffusion chamber system as
described above for
IK measurement.
The basolateral chamber contents were replaced with a high
Cl
-high
K+ solution (1.0 × 10
2 M sodium gluconate, 1.4 × 10
1 M KCl, 1.0 × 10
3 M
MgSO4, 1.0 × 10
3 M
CaCl2, and 1.0 × 10
2 M HEPES at pH 7.40) to
apply a basolateral-to-apical
Cl
concentration gradient
across the monolayer. Nystatin (500 U/ml) was added to the basolateral
solution to permeabilize the basolateral membrane to small monovalent
ions. Forskolin (1.0 × 10
5 M) was added to the
apical solution to stimulate the
Cl
secretion through apical
Cl
channels, and the
isolated cAMP-stimulated
ICl was measured
and recorded. Several experiments with amines
1-9 were performed with the appropriate amine
added to the basolateral and/or apical solutions to test for direct
effects on apical Cl
conductance.
Cytosolic pH.
Polarized T84 cells grown to confluence on Anocell inserts (surface
area = 0.33 cm2; Whatman,
Maidstone, UK) were used to fluorometrically (Spex DM3000, Spex
Industries, Edison, NJ) measure cytosolic pH and its response to
separate apical and basolateral application of the
amines 1-9 in Table 1 (42). Cell
monolayers were loaded with the acetoxymethyl ester of
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM, 2.0 × 10
7
M in HPBR with glucose; Molecular Probes, Eugene, OR) for 1 h at room
temperature and then mounted in a modified spectroscopic quartz cuvette
that afforded separate access to the isolated apical and basolateral
aspects of the confluent monolayers. Small aliquots of a concentrated
solution of the appropriate amines
1-9 were added to the apical and/or basolateral
solution, and the effects on intracellular pH were observed and
recorded as a function of time.
The quartz cuvette was modified with a clear, colorless, and flat piece
of Lucite (thickness = 1.0 mm) that was cut (width = 10.0 mm,
height = 65.0 mm) so as to fit snugly a quartz fluorescence cuvette of
standard internal dimensions (width = length = 10.0 mm, height = 43.0 mm). A single hole (diameter = 3.0 mm, centered at height = 13.0 mm),
intended to coincide with the incident excitation light beam, was
drilled in the cut Lucite. When inserted into the cuvette, the plane of
the flat Lucite is perpendicular to the sides, parallel to the front
and rear faces, and bisects the distance between the front and rear
faces, affording a cuvette with equal volume front (apical) and rear
(basolateral) compartments. The seal between the edges of the Lucite
insert and the inner walls of the cuvette was accomplished with
fingertip rope caulk (Frost King, Paterson, NJ). Sample, in the form of
a T84 monolayer loaded with dye on an irregular flat fragment of an
optically clear and rigid Anocell-permeable support, was placed in the
cuvette, cell side up, on the Lucite insert so as to cover completely
the 3-mm-diameter drilled hole. The seal between the bottom surface of
the permeable support and the Lucite surface surrounding the hole was
made with a thin film of high-vacuum silicone grease (Dow Corning,
Midland, MI). The cuvette was consistently inserted in the fluorometer
sample compartment so that the excitation beam initially struck the
apical rather than the basolateral aspect of the monolayer. The
monolayer surface was perpendicular to the excitation beam, and
fluorescence was collected at an angle 45° to the incident beam
(front-face mode). The sample was excited at 505 and 439 nm. Separate
emission intensities were collected at 535 nm as a function of time
(data point interval = 4 s) and ratioed (ratio of 505-nm to 439-nm
excitation) after data collection. Excitation and emission
slits were set at 0.5 mm, corresponding to a bandpass of 1.8 nm.
The emission ratio at 535 nm was correlated with pH via intracellular
calibration. The sodium salt of the polyether nigericin (0.1 ml of a
stock solution containing 10 mg in 1.0 ml dimethylformamide and 0.3 ml
ethanol) in high-K+ buffer (40 ml,
the same as the aforementioned HPBR except the concentrations of
Na+ and
K+ are reversed) at 2.6 × 10
5 M was used to
bilaterally permeabilize the cell membrane to the ions
H3O+
and K+. Monolayers loaded with dye
and mounted normally were sequentially and bilaterally exposed to the
high-K+ nigericin buffer at five
different pH values between 6.60 and 8.00, and the respective emission
ratios were measured after an ~15-min equilibration of the
intracellular and extracellular K+
and
H3O+
concentrations. The results from several different monolayers were
averaged, and a plot of pH vs. emission ratio
(y = 3.06 + 0.58x;
r = 0.98) afforded a calibration curve.
Endosomal pH.
The average pH of a broad population of endosomal compartments was also
measured fluorometrically (Spex DM3000). T84 cells grown to confluence
in a 163-cm2 flask containing the
cell culture medium (30 ml) described above were loaded with
fluorescein isothiocyanate-dextran (FITC-dextran, molecular weight of
12,000, with 0.73 mol fluorescein/mol dextran, 15.0 mg/ml; Sigma) at
37°C for 24 h (37). The cells were then washed (3 × 30 ml at
pH 7.40 and 37°C) with PBS (8.0 × 10
3 M
Na2HPO4,
1.5 × 10
3 M
KH2PO4,
1.4 × 10
1 M NaCl, 2.7 × 10
3 M KCl),
trypsinized, quenched with medium (30 ml), and centrifuged (1,200 rpm,
5 min at room temperature). The resultant cells were washed with HPBR
(3 × 30 ml) and then finally suspended at pH 7.40 in HPBR (10 ml). Aliquots (1 ml) of this dye-loaded cell suspension were diluted to
3 ml with HPBR in a standard quartz fluorescence cuvette and
magnetically stirred at room temperature during data collection. Small
aliquots of a concentrated solution of the appropriate
amines 1-9 were added to the
stirred cell suspension, and the effects on endosomal pH were observed
and recorded.
Fluorescence was collected at an angle of 45° to the incident beam
(front-face mode). The sample was excited at 495 and 450 nm. Separate
emission intensities were collected at 519 nm as a function of time
(data point interval = 4 s) and ratioed (ratio of 495-nm to 450-nm
excitation) after data collection. Excitation and emission
slits were set at 0.5 mm, corresponding to a bandpass of 1.8 nm.
The emission ratio at 519 nm was correlated with pH by in vitro
extracellular calibration. FITC-dextran (3.0 mg,
~10
7 mol fluorescein) was
dissolved in HPBR (1.0 liters). Fifteen aliquots of this solution were
adjusted to different pH values between 5.18 and 6.87, and the
respective emission ratios were measured. A plot of pH vs. emission
ratio afforded a calibration curve that was linear from pH 5.18 to 6.37 (y =
5.14 + 1.44x; r = 0.99) but nonlinear in the region
from 6.47 to 6.87. This nonlinear region was approximated effectively
as a line (y = 0.037 + 0.52x;
r = 0.96).
Fluid-phase endocytosis.
The apical or basolateral medium solution of T84 cells grown to
confluence on collagen-coated permeable supports (area = 4.70 cm2, 3.0-µm pore size) was
replaced with medium containing FITC-dextran (molecular weight of
12,000, with 0.73 mol fluorescein/mol dextran, 15.0 mg/ml, Sigma) and
incubated for 6 min at 37°C. Immediately after incubation, each
monolayer was immersed in a large volume (250 ml) of HPBR at 4°C
and then washed twice more by immersion for 30 s in fresh HPBR at
4°C. Residual liquid was aspirated from the edges of the filter
membrane, which was then excised from its plastic support and inserted
into an Eppendorf tube (1.5 ml capacity) containing distilled water
(0.43 ml). The contents of the tube were sonicated (model 550 sonic
dismembrator, setting 3, Fisher Scientific). The porous membrane was
removed, and the remaining suspension was freed of solid cellular
debris by centrifugation (2 × 5 min at 14,000 g). The resultant clear solution was
analyzed fluorometrically (excitation wavelength = 495 nm, emission
wavelength = 565 nm) for its relative FITC content. Complete
suppression of FITC-dextran uptake at 4°C supports this technique
as a general measure of pinocytotic behavior.
Rubidium ion efflux.
T84 cells grown to confluence on collagen-coated permeable supports
(area = 0.33 cm2) were washed
free of medium and placed in HPBR. Cells were loaded basolaterally with
86Rb+
(1.5 mCi/ml in HPBR) at 37°C for 2 h and then washed free of radioisotope-containing solution with HPBR. Baseline efflux was measured for 5 min, and then monolayers were simultaneously and basolaterally exposed to forskolin
(10
5 M) alone or forskolin
plus ammonia (amine 1; 4.0 × 10
3 M), methylamine
(amine 2; 1.0 × 10
2 M), or octylamine
(amine 9; 7.0 × 10
4 M). Data points were
collected as aliquots from the basolateral bath solution every 2 min
and counted in Atomlight (Packard, Meriden, CT) scintillation fluid
with a liquid scintillation analyzer (Packard model 1600 TR).
Statistical analyses.
Data are reported as means ± SE. Analysis included the Student's
t-test for paired or unpaired variates
and two-way ANOVA when appropriate, with
P < 0.05 considered significant.
 |
RESULTS |
Characteristics of amines.
The amines listed in Table 1 share the presence of a single formally
sp3 hybridized nitrogen. Entries
2 through
9 are straight-chain primary alkyl
amines that vary according to the number of methylene units (
CH2
) in the single alkyl
group attached to the primary amine (
NH2) functionality. Together,
they represent a tool designed to elucidate the means by which ammonia
suppresses Cl
secretion in
T84 monolayers. They vary most obviously in size, that is, length, of
the lipophilic alkyl tail bonded to the nitrogen that they hold in
common with ammonia (amine 1). As
evidenced by the relatively invariant
pKa values of
their conjugate acids (pKaH+)
in water (50), successive addition of methylene groups for
2 through
9 has little effect on their relative
basicities. This is because the electron-donating character of
higher-order alkyl chains is about the same as that of methyl (amine 2). Electronically, series
2-9 (average
pKaH+ = 10.68) is uniform in its difference from the parent unsubstituted ammonia
(pKaH+ = 9.24) with respect to the nitrogen center. Likewise, the
aqueous nucleophilicity of the unprotonated amines is
approximately invariant in amines
2-9. Nucleophilicity often parallels basicity, and
the steric environment in the immediate vicinity of the active nitrogen
center changes little from methylamine (amine
2) through
n-octylamine (amine 9). This uniformity in electronic and steric
character at nitrogen in primary amines
2-9 further suggests that the intermolecular interactions between solvent water molecules and the
NH2 functionality are similarly
uniform. Amines 2-9 therefore
present a uniform nitrogen "head" but nonuniform alkyl
"tails" to the target inhibitory site (8, 30).
Alkyl amines suppress Cl
secretion.
We measured the
Isc responses to
cAMP in the presence or absence of amines
1-9 over a range of concentrations.
Representatively, Fig. 1 depicts a small
portion of the data for the two apparent extremes in the amine series.
Basolaterally applied ammonia (amine 1) in Fig. 1A
gives the previously reported diminution in forskolin-stimulated Isc (a measure of
Cl
secretion in epithelial
monolayers) relative to control monolayers (22). Basolaterally applied
octylamine (amine 9) in Fig.
1B similarly inhibits
Isc but requires
considerably less of this lipophilic amine to almost completely
suppress secretion. Pretreatment with amine has no effect on baseline
Isc before
forskolin stimulation. Figure 2 illustrates
that the concomitant sharp drop in transepithelial resistance with
administration of forskolin is markedly blunted by
amines 1 and
9, and amine pretreatment also has no
effect on baseline transepithelial resistance.

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Fig. 1.
Ammonia and primary alkyl amines inhibit
Cl secretion in T84 cell
monolayers. A: representative
short-circuit current
(Isc) response
of ammonia (amine 1)-treated ( )
monolayers relative to control ( ) as a function of time.
B: representative
Isc response of
octylamine (amine 9)-treated ( )
monolayers relative to control ( ) as a function of time. f,
Forskolin treatment at time (t) = 60 min. Dose responses for the basolateral and apical application of
amines 1-9 were similarly
acquired. In each case, the amine sample
Isc response
normalized to control
Isc response at
t = 75 min was the time point
reported.
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Fig. 2.
Ammonia and primary alkyl amines blunt the forskolin-induced decrease
in transepithelial resistance (R) in T84 cell monolayers.
A: representative transepithelial
resistance response of ammonia-treated ( ) monolayers relative to
control ( ) as a function of time.
B: representative transepithelial
resistance response of octylamine-treated ( ) monolayers relative to
control ( ) as a function of time. Data for the basolateral and
apical application of amines 1-9
were similarly acquired.
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|
Ammonia and alkyl amines suppress basolateral
K+ conductance.
Transepithelial Cl
transport by T84 and many other secretory epithelial cells requires the
integrated activity of four membrane-bound systems: apical
Cl
channels and a
basolateral machinery consisting of the
Na+-K+-2Cl
cotransporter, the
3Na+-2K+-ATPase,
and K+ channels. We previously
noted a parallel between the effect of NH+4
and the effect of Ba2+, a
well-known K+ channel blocker, on
cAMP- and Ca2+-regulated
transepithelial Cl
secretion and speculated that the basolateral
K+ channel could be a target of
action (33). The monovalent cations NH+4 and
K+ are of similar size; their
Pauling ionic radii are 1.48 and 1.33 Å, respectively (9).
Because the positively charged NH+4 is likely
to compete with other positively charged species for electron-rich
sites, we reasoned that the cationic NH+4 may
interfere with the secretory machinery at a site with high affinity for
cations of similar size and electronegativity (24).
Accordingly, we used apically permeabilized, ouabain-inhibited T84
monolayers to isolate and measure the basolateral
IK and its
dose-response to amines 1-9.
Representatively, Fig. 3 depicts block of
resting IK by
basolaterally applied ammonia and octylamine, respectively. In our
hands, this resting
IK, and its block
by amines, is insensitive to the cAMP agonist forskolin, and results were similar in the presence or absence of a prior cAMP stimulus. For
clarity, we show only portions of the curves subsequent to application
of the apical-to-basolateral K+
gradient, treatment with ouabain, and permeabilization of the apical
membrane with nystatin. The
IK typically
remained stable after nystatin permeabilization without substantial
rundown over the course of a 20-to 30-min experiment. The
transepithelial voltage pulses from 0 to 10 mV for calculation of
transepithelial resistance as a function of time also are omitted for
clarity. These amines and the remaining entries
(2-8) block outward
IK on the same
order of magnitude as they block
Isc, suggesting
that they may share a common target. Ammonia and selected alkyl amines
also inhibited basolateral
86Rb+
efflux in intact monolayers. As shown in Fig.
4, efflux was suppressed by >50%
relative to control under forskolin-stimulated conditions in the
presence of basolateral ammonia (amine
1; 8 mM), methylamine (amine
2; 10 mM), and octylamine (amine
9; 0.8 mM).

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Fig. 3.
Ammonia and primary alkyl amines inhibit basolateral
K+ current
(IK) in the
apically permeabilized system. A:
representative IK
response to 2 separate ammonia doses (6 mM) as a function of time.
B: representative
IK response to 2 separate octylamine doses (0.4 mM) as a function of time. Complete dose
responses for the basolateral and apical application of
amines 1-9 were similarly
acquired but with smaller amine doses. These traces depict responses to
relatively large doses to unambiguously demonstrate immediate
IK block.
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Fig. 4.
Amines suppress
86Rb+
efflux. Ammonia (amine 1, 4 mM, ),
methylamine (amine 2, 10 mM, ), and
octylamine (amine 9, 0.7 mM, )
suppress forskolin-stimulated
86Rb+
efflux relative to control ( ).
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Ammonia does not alter cAMP-regulated apical
Cl
conductance.
Basolateral permeabilization of monolayers with nystatin allows us to
isolate and study apical Cl
conductance in the same way that apical permeabilization with nystatin
allows isolation and study of basolateral
K+ conductance. We determined that
basolaterally applied ammonia does not affect forskolin-stimulated
(cAMP-mediated) apical Cl
conductance in this permeabilized system. For example, a representative monolayer (of n = 4) gave a baseline
ICl of 9 µA
with a basolateral-to-apical Cl
concentration gradient
of 140:5 mM. Forskolin stimulation sharply increased
ICl to a stable
96.5 µA. Basolateral application of 30 mM
NH4Cl left
ICl virtually
unchanged at 97.5 µA. Ammonia interference with the cAMP-regulated
component of the apical Cl
conductance is therefore unlikely. We did not examine whether amines
also inhibited the baseline
ICl of
nystatin-permeabilized monolayers and thus cannot determine whether
cell swelling-induced Cl
currents are affected.
Lipophilicity is the key variable relating inhibition of
Cl
secretion and basolateral
K+ conductance.
The similarity of
Isc and
IK responses to
lipophilic amines 4-9 is
underscored by the summarized linear regions of the natural logarithms
of the dose-response curves for
Isc and
IK in Fig. 5, A and
B, respectively. In both cases, the
amines show an orderly progression in their increasing ability to block
Isc and
IK from the least
lipophilic, propylamine (amine 4),
to the most lipophilic, octylamine (amine
9). The slopes within the
Isc subset (Fig.
5A) are similar (average slope =
0.28 ± 0.02) as are those within the
IK subset
(average slope =
0.42 ± 0.03). Control of amine probe
variables allows us to focus on the change in lipophilicity within
series 1-9. This variable
approximates solubility within the lipid bilayer of plasma and
vesicular membrane environments and is given by the partition
coefficient (P) between
n-octanol and water (14, 27),
conveniently represented as its logarithm (log
P). A plot of the amine
concentrations at 50% inhibition of
Isc
(50Isc)
vs. log P (Fig.
6A)
reveals that series 1-9 is
organized into two groups, those that are preferentially water soluble
(amines 1-3) and those that are
preferentially lipid-soluble (amines
4-9). The preferentially water-soluble amines
increase in ability to block secretion with
decreasing size and lipophilicity. Conversely, the
preferentially lipid-soluble amines increase in ability to block
secretion with increasing size and lipophilicity. This dichotomous behavior reflects a departure from strict adherence to the
Meyer-Overton rule (18, 34) for membrane diffusivity. Inhibitory
potency does not, therefore, reflect the ability to traverse membranes to reach a cytosolic target or to enter acidic intracellular
compartments. The dichotomy is resolved, however, if these two amine
subtypes (lipophobic and lipophilic) have essentially the same target
but reach it principally by two different routes and interact with the
same target in two slightly different modes. This target could be a
membrane-bound protein that has a functionally relevant lipophobic domain, such as an aqueous pore or transport site. Thus these data are
consistent with a membrane-bound ion transport site such as a
K+ channel as the amine target.

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Fig. 5.
Linear regions of normalized dose responses for lipophilic amines.
A: normalized
Isc response with
respect to natural logarithm of amine
1-9 concentrations.
B: normalized
IK response with
respect to natural logarithm of amine
1-9 concentrations.
I0 is the
IK or
Isc in the
absence of amine inhibitor.
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Fig. 6.
Intact secretory apparatus response bears striking resemblance to
isolated K+ current response.
A: amine concentrations at 50%
inhibition of Isc
(50Isc)
as a function of the log of the partition coefficient for basolateral
application of amines 1-9 to T84
cell monolayers. B: amine
concentrations at 50% inhibition of
IK
(50IK)
as a function of the log of the partition coefficient for basolateral
application of amines 1-9 to
monolayers apically permeabilized by nystatin.
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A plot of the amine concentrations at 50% inhibition of
IK
(50IK)
vs. log P (Fig. 6B) bears a striking
resemblance to the
50Isc
functional dependence on amine lipophilicity shown in Fig. 6A. That is, the isolated
K+ current portion of the
secretory apparatus responds with the same character and magnitude as
does the intact Cl
secretory apparatus. In particular, ammonia gives a
50Isc
of 6.2 mM and a
50IK
of 3.9 mM and octylamine gives a
50Isc
of 0.24 mM and a
50IK
of 0.19 mM.
Dose-response curve fits.
Each dose-response curve for the normalized current amplitude
(Isc/I0
or
IK/I0)
as a function of amine 1-9
concentration was fit to a Langmuir adsorption isotherm (10, 36) of the
form
where
[a] is the amine concentration,
K1/2 is the
apparent equilibrium constant for binding of the amine to the discrete target site, n is the Hill
coefficient, IX
is IK or
Isc, and
I0 is the
IK or
Isc in the
absence of amine inhibitor. When
K1/2 = [a], the expression
IX/I0
is one-half and [a] corresponds to the dose required for
50% suppression of current. Indeed, data fits to the above equation
gave our reported
50Isc
and
50IK
values. Fits (see Table 2) for
Isc are
consistent with the functional form describing a one-to-one interaction
between the amine inhibitor and its target site. Fits for
IK suggest the
possibility of positive cooperativity. However, any apparent
cooperativity could potentially be explained by a tendency to
overestimate inhibitory potency of amines in the
IK experiments
due to the cumulative dose-response protocol used to obtain these
values. This confounding factor was not the case for
Isc experiments,
in which single amine concentrations were used with each monolayer.
Asymmetry and symmetry of apical and basolateral application of
amines correlates with amine size and lipophilicity.
We previously noted that the inhibitory ability of basolaterally
applied ammonia was ~10-fold greater than apically applied ammonia
(38). That is, the apical ammonia
50Isc
is 50 mM and the basolateral ammonia
50Isc
is 5 mM. It was therefore of interest to examine the "sidedness" of the
50Isc
values for amines 2-9. In data
not shown, apical treatment of T84 monolayers with lipophilic
amines 4-9 gives the same
50Isc
values as does the basolateral treatment shown in Fig.
6A, but apical treatment with
lipophobic amines 1-3 gives
50Isc
values fivefold higher than those obtained by basolateral treatment.
Thus the sidedness of lipophilic amines
4-9 is symmetrical, but the sidedness of
lipophobic amines 1-3 is
asymmetrical. Because the compounds were added to only one chamber,
this may lead to an underestimation by as much as one-half of the
inhibitory constant for the lipophilic subset of amines
for both the Isc
and IK experiments.
This correlation of symmetry with lipophilicity is supported by
experiments with the highly lipophilic amines
10-13 (Table 1) that lie outside the well-ordered
series given by amines 1-9. Amines 10-13 gave about the same
50Isc
values for both apical and basolateral application (mean = 27.4, 12.7, 45.3, and 3.3 mM, respectively).
Ammonia and alkyl amine effects on cytosolic pH do not correlate
with inhibitory action.
When confronted with the observation that ammonia interferes with any
aspect of physiological function, the most obvious conclusion is that
ammonia is exerting its effect via an ammonia-induced pH change of the
cytosolic medium or that of intracellular compartments. Although the
foregoing data strongly support the notion that
NH+4 blocks
K+ channels and thereby blocks
Cl
secretion, it is also
necessary to determine if the ammonia-induced pH change of the
cytosolic medium or that of intracellular compartments is related to
the block of Cl
secretion.
We therefore directly examined the effect of basolateral or apical
application of ammonia on cytosolic pH with the knowledge that 10-fold
more apical ammonia (50 mM) than basolateral ammonia (5 mM) is needed
to suppress Cl
secretion
(38). The cytosolic pH responses to apically and basolaterally applied
ammonia (30 mM) differ markedly, probably reflecting the permeability
differences between the species
NH3 and
NH+4 (Fig. 7).
Apical application gives an immediate cytosolic pH increase from 7.19 to 7.90 followed by a slow decline to 7.70 after 20 min of continued
apical exposure. Basolateral application gives an immediate but
tempered increase and relatively rapid recovery to the initial
cytosolic pH and below. Apical application likely gives persistent
alkalinization because there are limited apical pathways for
NH+4 transmembrane movement. The cytosolic pH
response to basolateral ammonia (blunted peak alkalinization followed
by rapid recovery) resembles that of renal epithelia, in which
competitive influx of the protonated NH+4 via
the
Na+-K+-2Cl
cotransporter and the
3Na+-2K+-ATPase
has been documented (25).

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Fig. 7.
Cytosolic pH responses to apical and basolateral application of ammonia
markedly differ. Initial alkalinization in response to apical ammonia
(30 mM, ) relaxes slowly, whereas blunted initial alkalinization in
response to basolateral ammonia (30 mM, ) relaxes rapidly.
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Inhibition of Cl
secretion
does not correlate with these changes in bulk cytosolic pH. Figure
8 depicts the cytosolic pH responses of
BCECF-loaded monolayers superimposed on the forskolin-stimulated Isc responses to
apically (Fig. 8A) and basolaterally
(Fig. 8B) applied 50 mM (apical
50Isc)
and 5 mM (basolateral
50Isc)
NH4Cl, respectively. The apically
applied 50 mM NH4Cl reduces Isc by 50% and
substantially alkalinizes the cytosol (7.25 to 7.55 after 38-min
exposure). However, basolaterally applied 5 mM
NH4Cl also reduces
Isc by 50% but
only slightly alkalinizes the cytosol (7.15 to 7.25 after 38-min
exposure). This observation is underscored by related observations with
the higher-order amines of our series. Figure
9 depicts the cytosolic pH response of
BCECF-loaded monolayers to basolaterally applied octylamine
(amine 9) and propylamine (amine 4). Cytosolic pH is increased
only slightly (
pH = +0.2 units) by enough amine
9 (1 mM) to suppress
Cl
secretion by 85%,
whereas cytosolic pH is substantially increased (
pH = +0.7 units) by
a high amine 4 concentration (50 mM)
that can suppress secretion by only 50%. Hence, the ability to
alkalinize the bulk cytosolic environment does not correlate with the
ability to suppress secretion. It is important to acknowledge that the BCECF method we utilized does not allow us to rule out the possibility that the pH microclimate around the relevant transport sites could be
altered in a functionally meaningful way by ammonia; because NH+4 can move via various basolateral
K+ transport pathways, the pH
microenvirnoment in the vicinity of basolateral
K+ channels may indeed be
different from bulk pH.

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Fig. 8.
Ammonia inhibition of
Isc is
independent of ammonia-induced changes in cytosolic pH. Representative
curves in A and
B labeled
Isc depict
transepithelial Cl current
as a function of time with the voltage clamped at 0 mV and pulsed from
0 to 10 mV at 1-min intervals to provide a record of monolayer
resistance change over the course of the experiment. The 2 separate
monolayers in A and
B were basolaterally stimulated with
forskolin (10 mM) at the arrows designated
a (t = 14 min), resulting in stimulation of
Isc from a
resting value of ~5 µA to ~55 µA within 8 min.
A: apical treatment with 50 mM
NH4Cl at arrow designated
b (t = 22 min) results in a 50% decrease in
Isc within 28 min. Apical treatment with 50 mM
NH4Cl at arrow designated
b (t = 22 min) immediately results in a large increase in cytosolic pH that
slowly declines with continued apical exposure.
B: basolateral treatment with 5 mM
NH4Cl at arrow designated
b (t = 22 min) results in a 50% decrease in
Isc within 16 min. In separate experiments in which polarized and confluent
monolayers were mounted to afford isolated apical and basolateral
access in a quartz cuvette for fluorescence spectroscopy,
representative heavy curves depict the cytosolic pH as a function of
time according to the
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
method. Basolateral treatment with 5 mM
NH4Cl at arrow designated
b (t = 22 min) results in a relatively slow and small increase in cytosolic
pH that persists with continued basolateral exposure.
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Fig. 9.
Cytosolic pH responses to basolateral octylamine and propylamine do not
correlate with
Isc suppression.
Basolaterally applied octylamine (amine
9, 1 mM, 85% suppression of
Isc, ) affects
pH only slightly but greatly suppresses
Isc.
Basolaterally applied propylamine (amine
4, 50 mM, 50% suppression of
Isc, ) has a
large effect on cytosolic pH but suppresses
Isc 35% less
than does the comparatively small dose of octylamine.
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Ammonia and alkyl amine effects on endosomal pH or membrane
recycling do not correlate with inhibitory action.
The ability to alter endosomal pH also does not correlate with the
ability to inhibit secretion. Figure 10,
A-C,
depicts the change in the H+
concentration
(
[H+]) of a broad
population of endosomal compartments in suspended T84 cells as a
function of time after acute treatment with ammonia (1), propylamine
(4), and octylamine
(9), respectively. The three suspensions were treated separately with the concentrations of amines 1,
4, and
9 required to suppress
Cl
secretion by 50%
(50Isc),
but the endosomal [H+]
responses differ dramatically. All amines initially alkalinize endosomes, but the initial
[H+] for ammonia
(1) and octylamine
(9) is about
0.3 mM and that for propylamine (4) is about
0.9 mM. Furthermore, prolonged incubation with
amine 4 alkalinizes endosomal
compartments, whereas prolonged incubation with amine
9 acidifies endosomal compartments. Maximal effects of
the amines on Cl
secretion
are typically reached within 10 min of basolateral exposure and persist
for at least 24 h. Because our dose-response measurements reflect a
routine 30-min preincubation with amine, our
50Isc
for amines 1,
4, and
9 correspond to a time point at which these three amines exert quite divergent effects on endosomal pH
(
[H+] at 30 min = 0.0,
0.2, and +0.3 µM, respectively). Thus neither endosomal
nor cytosolic pH correlates with
Isc change.

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Fig. 10.
Comparative endosomal H+
concentration ([H+])
response to amine
50Isc
concentration as a function of time.
A: endosomal
[H+] response to
ammonia
50Isc
(5 mM). B: endosomal
[H+] response to
propylamine
50Isc
(42 mM). C: endosomal
[H+] response to
octylamine
50Isc
(0.3 mM).
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Finally, fluid-phase endocytosis was estimated by 6-min uptake of
FITC-dextran by T84 monolayers grown on plastic and permeable supports.
In our hands, the cAMP agent forskolin slightly inhibits endocytosis
(70 ± 10% relative to control in the absence of forskolin; n = 6) in monolayers grown on plastic,
a result similar to that obtained by others (4, 5, 39). Forskolin
slightly inhibits apical (80 ± 10%) but not basolateral (110 ± 16%) endocytosis in filter-grown cells
(n = 12 for each). Ammonia (10 mM) has
no effect (96 ± 10% relative to control in the absence of ammonia; n = 3) on the cAMP-dependent slight
inhibition of FITC-dextran uptake in monolayers grown on plastic.
Examination of apical and basolateral endocytosis using monolayers
grown on permeable supports showed that preincubation (30 min) with
ammonia (10 mM) has little or no effect (85 ± 10% and 92 ± 8%, respectively, relative to control in the absence of ammonia) on
FITC-dextran uptake used as a method to estimate pinocytotic uptake.
 |
DISCUSSION |
The assumption that biological effects of ammonia and alkyl amines are
exerted via effects on cytosolic and/or endosomal pH is firmly
entrenched and only occasionally questioned. Indeed, these agents are
widely used as experimental probes for just this purpose. Our
experiments clearly indicate that the mechanism by which ammonia
inhibits transepithelial Cl
secretion is independent of its ability to alkalinize the bulk cytoplasm or raise the pH of acidic endosomal compartments.
Structure-function and kinetic analyses using a coherent series of
amine probes provide direct evidence to support our recent speculation
that the target of ammonia action is instead the basolateral
K+ conductance that is required
for K+ recycling and maintenance
of the driving force for electrogenic apical
Cl
exit. Our data are
inconsistent with a mechanism involving inhibition of cAMP-regulated
apical Cl
conductance or
interference with membrane recycling.
The importance of outward
IK at the
basolateral membrane for transepithelial
Cl
secretion is well
recognized (43, 44, 51), and basolateral K+ channels are thought to carry
five-sixths of the current (48) through the membrane during
electrogenic Cl
secretion.
At least two pharmacologically distinct but molecularly uncharacterized
K+ conductances coexist in the T84
basolateral membrane (31). One is sensitive to the well-known
K+ channel blocker
Ba2+, whereas the other is
Ca2+ dependent but insensitive to
Ba2+. These
K+ channels are targets of drug
development against secretory diarrheal disorders. Indeed, clotrimazole
(41) may act by a mechanism similar to the one we propose here. In data
not shown, we found that the small hydrophilic amines
1-3 do not block
Ca2+-regulated
(carbachol-stimulated and
Ba2+-insensitive) basolateral
K+ conductance in the apically
permeabilized system or transepithelial Cl
secretion in intact
monolayers within our dose ranges. The lipophilic amines 4-9, however, do block
these Ca2+-regulated processes at
concentrations effective in blocking resting K+ conductance, and inhibitory
ability increases with lipophilicity.
Although a number of quartenary ammonium compounds are known to inhibit
certain K+ channels (21), the
effects of ammonia and monoamines have not been defined in most
systems. Indeed, a number of K+
channels are known to conduct NH+4 quite
readily, usually with ~30% or less efficiency (21). None is known
explicitly to be competitively blocked by
NH+4, but there is a recent suggestion of
such NH+4 block in the thick ascending limb
of Henle's loop in rat kidney (2, 26, 42). The naturally occurring
cytosolic polyamines spermine, spermidine, putrescine, and cadaverine
may serve as physiological blockers of inward rectifier
K+ channels (15, 17, 19, 29).
The use of structure-function analysis with the well-defined amine set
allows us to speculate on the mechanism of ammonia and primary alkyl
amine K+ channel block. We propose
that the discontinuity at log P = 0 in
Figs. 6 and 7 reflects the two different routes by which the two
classes of amines reach their target
K+ channels (Fig.
11). The relatively small hydrophilic
amines 1-3 reach their targets by
the aqueous path in which they are preferentially solubilized.
Inhibitory ability of amines
1 > 2 > 3 shows that the smallest and most
water-soluble of the three (NH+4) has the
highest probability of occupying the channel site normally occupied by
K+. As ion size first increases by
the volume of a methyl group (2) and
then by an ethyl group (3), the
ability to occupy the site intended for
K+ decreases as does the ability
to block secretion. In contrast, the large hydrophobic
amines 4-9 reach their target by
the hydrophobic pathway in which they are preferentially soluble. The
concentration of unprotonated amine in the membrane is directly related
to its partition coefficient P.
Neutral amine diffuses randomly within the plasma membrane and collides
with target K+ channels with a
probability dictated by amine concentration in the membrane. In some of
these collisions, the
NH2 head
will protrude far enough into the aqueous channel such that nitrogen is
quickly protonated to give a new NH+3
(ammonium) head that is now hydrophilic. The newly protonated ammonium
projects into the aqueous pore so that it interferes with the ability
of the pore to accommodate K+, and
the hydrophobic alkyl tail is solubilized by the lipid membrane interior or by a hydrophobic region of the channel protein. The residence time in this blocking configuration is a function of ammonium
aqueous solubility (the same for amines
4-9) and of the lipid solubility of the alkyl
tail, which increases with chain length. Inhibitory ability increases
with membrane solubility and residence time at the target site, both of
which increase with increasingly positive log
P. This model is analogous to the Hille model (19, 20) for Na+
channel block by local anesthetics.

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Fig. 11.
Proposed model. Inhibition of K+
channels by amines 1-9 via the
aqueous (amines 1-3) and
nonaqueous (amines 4-9)
pathways is shown. Despite the depicted amine approach from the
extracellular direction, detailed spatial knowledge of inhibition
site(s) is unknown.
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Two additional observations support the aqueous and nonaqueous pathway
proposition. First, apical treatment of T84 monolayers with lipophilic
amines 4-9 gives the same
50Isc
values as does the basolateral treatment shown in Fig.
6A, but apical treatment with
lipophobic amines 1-3 gives
50Isc
values fivefold higher than those obtained by basolateral treatment.
Apical lipophilic amines 4-9
travel to their basolateral targets via the hydrocarbon environment of
the lipid bilayer with a facility about equal to that of the same
lipophilic amines applied basolaterally. Apical lipophobic
amines 1-3 are hampered by their low membrane permeabilities. Second, the dominance of the log P variable is underscored by our
basolateral
50Isc
results with the di- and trialkyl amines
10-13. These amines differ markedly from their
primary counterparts 2-9 in terms
of basicity and steric environment at the nitrogen center. A plot of
the
50Isc
values vs. log P for
amines 10-13 gives a curve that
is nearly superimposable on the curve given by
4-9 in Fig.
6A.
We have speculated on the potential role of ammonia as an endogenous
negative regulator of intestinal secretion based on evidence developed
in the T84 model, but its effect on secretion in native mammalian
intestine is as yet undefined. Solomon et al. (45, 46) recently
reported that ammonia inhibits cAMP-dependent secretion in the shark
rectal gland preparation. The effect of ammonia on epithelial
absorptive processes is to our knowledge largely unexplored. It is
interesting to note that in the central nervous system ammonia is known
to impair synaptic transmission and neurotransmitter release (16, 40,
47). Its impact on neurohormonal regulatory events in the gut is
unknown; the effects of ammonia on integrated intestinal function are
thus likely to be complex. Recognition that
NH+4 can block certain
K+ channels may well encourage
reevaluation of ammonia's role in other biological systems,
particularly in those instances in which it has been assumed that
ammonia's action is due to effects on cytoplasmic and endosomal pH.
 |
ACKNOWLEDGEMENTS |
We are grateful for the technical assistance of Imran Hassan and Jeremy
Smith. We thank Seth Alper and David Vandorf for comments on the manuscript.
 |
FOOTNOTES |
We gratefully acknowledge support from National Institute of Diabetes
and Digestive and Kidney Diseases Grants R29-DK-48010 and
1-R01-DK-51630-01 (J. B. Matthews), the George H. A. Clowes, Jr. MD,
FACS Memorial Research Career Development Award of the American College
of Surgeons (J. B. Matthews), and a Feasibilty Study Award from the
Harvard Digestive Diseases Center, National Institute of Diabetes and
Digestive and Kidney Diseases Grant 5-P30-DK-34854-12 (B. J. Hrnjez).
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. §1734 solely to indicate this fact.
1
Ammonia
(pHaH+ = 9.24)
is present at pH 7.40 as its protonated ammonium
(NH+4) form to the extent of 98.5%. It is
convenient to use the word "ammonia" to mean the equilibrium
total of unprotonated NH3 and
protonated NH+4 in aqueous solution,
although, practically speaking, ammonia is almost exclusively present
as NH+4 under physiological conditions.
Address for reprint requests and other correspondence: J. B. Matthews,
Division of General and Gastrointestinal Surgery, Beth Israel Deaconess
Medical Center, East Campus ST-928, 330 Brookline Ave., Boston,
MA 02215 (E-mail: jmatthew{at}caregroup.harvard.edu).
Received 20 April 1998; accepted in final form 24 May 1999.
 |
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