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Modulation of chloride secretion in the rat colon by intracellular bicarbonate
GASTROENTEROLOGY
1992:103:120-127
Modulation of Chloride Secretion in the Rat Colon by Intracellular Bicarbonate PIERRE C. DAGHER, LEAH BALSAM, J. TODD WEBER, RICHARD W. EGNOR, and ALAN N. CHARNEY Nephrology Section, Department of Veterans Affairs Medical Center, and New York University School of Medicine New York, New York
Extracellular HCO,- stimulates colonic net Cl- absorption in part by inhibiting basal Cl- secretion. This inhibition was investigated by measuring serosal-to-mucosal Cl- flux across short-circuited colonic segments from Sprague-Dawley rats. Mucosal intracellular pH and bicarbonate were estimated using the pH-sensitive dye BCECF. When extracellular [HCO,-] ([HCO,-I,) was increased from 0 to 39 mmol/L at Pco, 33 mm Hg, mucosal intracellular [HCO,-] ([HCO,-1,) increased to 25.3 mmol/L and serosal-to-mucosal Cl- flux decreased from 13.0to 7.1pEq - cm-‘. h-‘. When Pco, was increased to 72 mm Hg at [HCO,-1, 39 mmol/L, [HCO,-]i increased to 29.8 mmol/L and serosal-to-mucosal Clflux decreased to 5.9 pEq* cm-’ - h-‘. In Ringer’s solution containing 21 mmol/L HCO,- and 20 mmol/L Cl- (but not 100 mmol/L Cl-), increasing Pcoz from 21 to 70 mm Hg increased [HCO,-]I to 22.6 mmol/L and decreased serosal-to-mucosal Cl- flux from 3.0 to 1.7 pEq - crnp2 - h-l. Overall, serosal-to-mucosal Cl- flux was inversely related to [HCO,-]I on either side of an [HCO,-1, plateau of 9-18 mmol/L at which flux was stable. These data suggest that [HCO,-lI is an important modulator of basal Cl- secretion in rat distal colon. e previously reported that colonic chloride absorption and bicarbonate secretion correlated with blood bicarbonate concentration in the rat.’ In this in vivo perfusion study, such changes suggested that alterations in a mucosal chloride-absorptive/bicarbonate-secretory exchange process accounted for the effects of blood bicarbonate. Therefore, it was of interest that subsequent in vitro isotope flux measurements showed that this bicarbonate effect was mediated through changes in both serosal-to-mucosal chloride flux (Jth) and mucosal to serosal flux (Jz:).’ Inasmuch as changes in J:A could represent changes in active secretion, we hypothesized that bicarbonate modulated an ongoing chloride-secretory process.
W
The idea that net chloride transport in the intestine reflects both active absorption and secretion is not new.3 Two oppositely directed chloride pathways have been described under a variety of experimental conditions. Whereas absorption occurs primarily in surface epithelial cells, secretion was originally thought to be confined to the crypts.4 Recently, chloride secretion has also been shown in surface cells.5*6 Similarly, the temporal separation of these two processes, while evident during stimulated secretion, is less obvious in resting epithelium. Data from different species and various intestinal segments including human jejunum, rabbit ileum, and teleost small intestine support the presence of an ongoing basal chloride secretory process that could modulate net absorption.7-g The present study was designed to investigate systematically the relationship between extracellular bicarbonate concentration ([HCO,-I,) and J:A in the rat colon. We also measured intracellular bicarbonate ([HCO,-I,) in the colonic epithelium because the strong correlation between extracellular and intracellular concentrations of this ionlo suggested that [HCO,-1, may mediate the effects of [HCO,-I,. The experimental conditions were designed to vary [HCO,-1, and [HCO,-1, independently and to correlate these changes with J:k. The results suggest that net chloride absorption in the rat colon is modulated by [HCO,-Ii through an effect on an active, ongoing chloride-secretory process. Materials and Methods Male Sprague-Dawley rats weighing 250-350 g were maintained on a standard diet with free access to water, While rats were under pentobarbital sodium anesthesia (5 mg/lOO g body wt), the distal 10 cm of descending colon was removed, rinsed with 0.9% saline, and stripped of serosa. This is a U.S. government work. There are no restrictions on its use. 0016-5065/92/$0.00
July
[HCO,-Ii MODULATION OF Cl- SECRETION
1992
Electrical and Radioisotope Measurements
Flux
Details of the method of flux measurement have been described previously.’ Briefly, tissue pairs were mounted in modified Ussing half-chambers exposing 1.12 cm* surface area. The transepithelial potential difference (PD) was expressed as serosal side positive with respect to the mucosa. Tissues were studied under short-circuit conditions except for l-second intervals every 100 seconds, during which bipolar pulses of 0.5 mV yielded electrical current values used to calculate tissue conductance (G). The short-circuit current (1s~) was given the same polarity as PD. Tissues were paired for ion flux studies when differences in G were no greater than 25%. Unidirectional fluxes of Na+ and Cl- were measured by adding 2 FCi of “Na+ and 1 pCi of 36C1m(100 Ci/g sp act; New England Nuclear, Boston, MA) to the mucosal side of one member of each tissue pair and to the serosal side of the other. A 20-minute equilibration period was allowed after each change of condition, and then unidirectional flux was measured over a 45-minute period. The residual flux (JR)was calculated as Isc - (JF’,a, - Jz:,).
Fluorescence
Measurements
A segment of stripped distal colon (described above) was mounted as a flat sheet over a l-cm’ hollowring assembly consisting of two close-fitting concentric plastic rings. The tissue was thereafter handled in the solution designated to be tested first in the experimental protocol. First, the tissue was incubated in 1 mmol/L dldithiothreitol (DTT) (Sigma Chemical Co., St. Louis, MO), a mucolytic agent, for 10 minutes. It was then bathed for 30 minutes in a 7 pmol/L solution of the pH-sensitive dye 2’,7’-bis-(2-carboxyethyl)-5(and-6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, OR). The mounted tissue was washed twice in fresh test solution to remove extracellular dye. A Perkin-Elmer (South Plainfield, NJ) LS-5B spectrofluorometer equipped with a thermoregulated sample chamber maintained at 37°C was used. Data were collected using Fura software supplied by Perkin-Elmer and adapted for intracellular pH (pH,) measurement. The mounted tissue was placed in a fixed position at the bottom of a 4-mL cuvette (Markson Science, Phoenix, AZ) with the mucosal surface facing the excitation beam at a 45” angle. All readings were done after the tissue was exposed to the test solution for 10 minutes, during which three exchanges of the solution with fresh aliquots added from sealed syringes were performed. These measurements represent steady-state values because in preliminary experiments they were not found to change for up to 23 minutes. At all times the cuvette was tightly closed with a plastic cap to prevent CO, leakage. Fluorescence measurements were performed in triplicate using excitation wavelengths of 440 and 500 nm in sequence. The emission was measured at 530 nm. Excitation and emission slits were adjusted to 3-nm bandwidths. An average fluorescence ratio (500 nm/440 nm) was computed from the triplicate readings. Tissue autofluores-
cence was 5%-10% of the total fluorescence
121
and was auto-
matically subtracted from the readings. In preliminary experiments, we found that the plastic ring showed no autofluorescence and that dye leakage and photobleaching were minimal during the course of the experimental protocols. Tissue viability was documented for periods up to 40 minutes using trypan blue-exclusion criteria. The intracellular site and responsiveness of the fluorescent signal were verified by the ammonium-pulse technique” (Figure 1). The location and distribution of the dye within surface and crypt cells were determined by fluorescence microscopy of frozen sections of BCECF-exposed tissues. As shown in Figure 2, homogeneous uptake of the dye is evident in surface epithelial cells and to a lesser degree in crypt cells. Fluorescence is totally absent in the crypt spaces. DTT, used exclusively in the pH, studies, nevertheless had no effect on the electrical properties of the tissue or on its response to CO, in the Ussing flux chamber. At the end of each experiment, the mounted tissue was exposed to HEPES-buffered solution (described below) containing 140 mmol/L KC1 and 10 pg/mL nigericin (Sigma Chemical Co.) for pH, calibration.” The medium was titrated with NaOH to four different pH values. A calibration curve was thus constructed and used to convert the observed fluorescence ratios to pH, values. The calibration curves were linear over the pH range of 6.5-7.8 in accordance with the known properties of the dye.13 The [HCO,-1, was computed using the HendersonHasselbalch equation and the measured pH,. Intracellular PCO, was assumed to be equal to the medium Pco,, and the pK’ and CO, solubility were 6.115and 0.0306, respectively. Bathing solution pH and Pco, were measured using a Radiometer BMS 3 Mk 2 system with a PHM 73 acidbase analyzer (The London Company, Cleveland, OH).
z z P
7.6
7.0 I
I 5
I 10
I 15
I
I
20
25
TIME (min) Figure 1.A
typical experiment showing the pH, response of a whole mucosal tissue mount in HEPES-Ringer’s at pH 7.38 to a 20 mmol/L NH&l pulse. A, Control pH,; B, rapid entry of NH,; C, slower entry of NH,+; D, rapid exit of NH,; E, recovery.
122 DAGHER ET AL.
GASTROENTEROLOGY Vol. 103,No. 1
Figure 2. Fluorescence photomicrograph (original magnification X1000) of a stripped segment of colonic mucosa incubated in a 7 pmol/L solution of BCECF-AM for 30 minutes. Frozen sections examined by fluorescence microscopy showed homogeneous distribution of the dye in surface cells (S) and to a lesser extent in crypt cells (C).
[HCO,-1, was computed equation as above.
using the Henderson-Hasselbalch
Solutions and Acid-Base Conditions All solutions were maintained at 37°C. The HCO, Ringers contained (mmol/L) glucose, 10;NaCl, 96;KCl, 4; Na,HPO,, 2.4; NaH,PO,, 0.4; CaSO,, 1; MgSO,, 1.2; NaHCO,, 21; and Na+ gluconate, 18.The [HCO,J was adjusted to 5,11,21, and 39 mmol/L by reciprocal alterations in Na+ gluconate to keep the osmolality constant. These solutions were gassed with 3% CO,/97% 0,, 5% CO,/95% O,, or 11% CO,/89% 0, to obtain various pH and PCO, values. In HEPES Ringer’s, 21 mmol/L HEPES, Na+ salt, was substituted for NaHCO, and the medium was gassed with 100% 0,. In the low-Cl_ solution, gluconate replaced Cl- in equimolar amounts. The acid-base variables were otherwise identical to the 100 mmol/L Cl- solution. The pHi calibration solution contained (mmol/L): HEPES, 5; KCl, 140; MgCl,, 1; CaCl,, 1; and glucose, 10. In some studies, bumetanide (Sigma Chemical Co.) was added to the serosal reservoir at a final concentration of 10m4mol/L.
Statistics Results are expressed as means + SE. Data were analyzed by paired or unpaired Student’s t test, one-way analysis of variance, and simple linear regression.14 Results
Table 1 shows the effect of [HCO,-1, on coionic electrolyte fluxes under short-circuit conditions. In a CO,-free, HEPES-buffered Ringer’s, minimal net Naf and Cl- absorption were observed. In HCO,- Ringer’s at a Pco, of approximately 33 mm Hg, net Na+ and Cl- absorption and a residual flux were present. As the Ringer’s [HCO,-] was increased, an interesting pattern emerged: over the en-
tire range of HCO,- concentrations from 0 to 39 mmol/L, JFAvaried inversely with [HCO,-I,. Jzj progressively decreased almost 50% from 13.0 + 0.9 pEq - cm-‘- h-’ in HEPES-buffered Ringer’s to 7.1 -t 0.3 pEq * cm-’ - h-’ at a [HCO,-1, of 39 mmol/L and PCO, of 33.7 mm Hg. G tended to decrease in parallel with J:A. No relationship was found between Na.No changes in J,, and Jnetfor either [HCO,-I, and J,, Naf or Cl- were statistically significant. Furthermore, changes in J,, ” did not correlate with either extracellular pH (pH,) or Pco,. To examine the role of [HCO,-1, in mediating these changes in JzA, we measured colonic mucosal pHi and calculated [HCO,-1, in another group of tissues prepared identically. The measurements were made over a broad range of HCO,- concentrations at two CO, tensions: 32 and 72 mm Hg. As shown in Figure 3, there were strong correlations between [HCO,-1, and both mucosal [HCO,-1, and pH, at each CO, tension (r = 0.99 at Pco, 32 mm Hg, P < 0.001; r = 0.99 at Pco, 72 mm Hg, P < 0.001). The slopes of these correlations were similar so that at both CO, tensions, [HCO,-1, increased 0.6 mmol/L for each 1 mmol/L increase in [HCO,-I,. However, at each [HCO,-I,, mucosal pH, was lower and [HCO,-1, was higher at Pco, 72 mm Hg than at Pco, 32 mm Hg. For example, at [HCO,-1, = 39 mmol/L pH, was 0.3 U lower and [HCO,-1, was 4.4 mmol/L higher at PCO, 72 mm Hg than at Pco, 32 mm Hg. Similar changes were noted at 21 mmol/L [HCO,-I,, but there was no effect of Pco, on [HCO,-Ii at [HCO,-1, = 5 and 11 mmol/L, despite a marked effect on pHi. Because Pco, and [HCO,-1, were additive in their effects on [HCO,-I,, we investigated whether an increase in CO, tension could cause a further decrease in Jzk in colonic tissue exposed to a [HCO,-1, of 39 mmol/L. As shown in Table 1, JFAwas reduced from 7.1 + 0.3 FEq - cm-’ - h-l at Pco, 33.7 mm Hg to 5.9 + 0.3 PEq - cm-‘. h-’ at Pco, 71.2 mm Hg (P < 0.05). This reduction was reversible by decreasing Pco,. There was no significant change in Jr:. G decreased significantly from 6.8 * 0.5 to 4.6 + 0.3 mS/cm’ (P < Cl The effects of PCO, to in0.05) in parallel with J,,. crease J,, for both Na+ and Cl- and decrease PD and Isc were similar to our previous observations in the rat distal colon.’ We then examined the effects of changing mucosal [HCO,-Ii by altering the Ringer’s [Cl-]. A decrease in medium [Cl-] inhibits or reverses apical membrane Cl-/HCO,- e xc h ange and thus increases [HC03-li.15 These experiments were performed at an [HCO,-1, of 21 mmol/L at Pco, 21, 34, and 69 mm Hg. As shown in Figure 4, at each CO, tension [HCO,-1, was 4 mmol/L greater and pH, was 0.1 U greater at a [Cl-], of 20 mmol/L than at 100 mmol/L. Figure 4 also shows that this effect of [Cl-], did not
[HCO,-1,
July 1992
Table I. Effect of [HCO,], and CO, on Colonic Electrolyte
IN” bEq.
@Eq. cm-‘.
PCO,
(mmol/L)
(mm Hg)
cm-‘.h-‘]
(mS/cm’)
WI
123
IC’(_uEq~cm~‘~h~‘)
G
PD
h-‘)
PH.
OF Cl- SECRETION
Transport
kc
IHCO;IF
MOlJULATION
ms
sm
net
ms
sm
net
bEq.
lR cm ~‘. h-‘)
7.39 + 0.04
2.0 f 0.2
7.0 i 0.6
7.9 + 0.3
6.1 * 0.8
4.9 i 0.2
1 0 k 0.7
12.3 i 1.4
13.0 * 0.9
k 0.8
0.3 k 0.6
11
34.3 i; 0.6
7.20 I? 0.01
1.1 + 0.1
4.4 * 0.7
7.1 t 0.8
9.1 i 0.8
4.9 f 0.5
4.2 i- 0 9
13.6 i 1.0
8.5 * 0.4
5 2 k 0.8
2.0 k 0.6
21
32.1 k 1.0
7.39 * 0.03
1.6 + 0.2
5.8 + 0.3
7.2 * 0.4
7.3 * 0.8
4.2 + 0.3
3.1 + 0 7
12.1 + 0.6
7.9 * 0.4
4.1 i- 0.5
2.6 -r 0.4
39
33.7 * 0.9b
7.67 + 0.02”
1.1 + 0.1”
4.6 f 0.7O
6.8 k 0.5
5.7 k 0.6
3.9 * 0.4
1.9 + 0.7
10.2 2 0.3
7.1 k 0.3”
3.2 t 0 5h
2.4 + 0.7
39
71.2 i- 2.5’
7.35 2 0.02
0.5 + O.lC
2.8 k 0.5c
4.6 + 0.3
8.7 zk 1.1‘
3.5 + 0.4
5.2 2 1.4‘
13.0 k 0.7’
5.9 + 0.3’
7.1 k 0.8’
2.4 i 1.1
0
0
NOTE. [HCO;]. and
Values
are means
of 0 (HEPES)
21 mmol/L),
f SE. Data were
is 5. at [HCO;].
or 5% COJ95X
obtained
in four separate
11 is 8. at [HCO;]. 0, and
11% CO,/89%
([HCO;],
the four groups
‘P < 0.05 when
mmol/L. PCO, 71.2 mm Hg was compared
39
obscure
the effect of
creases
in Pco,
PCO,
compared
0,
“P < 0.05. ‘P < 0.001 when [HCO,],
were
groups
of tissue
21 is 17, and at [HCO;], by one-way
pairs
in 100 mmol/L solutions
Cl- Ringer’s WOT~ gassed
and various wth
[HCO;
100% 0, ([HCO;],,
]._ Number
of tissue
= 0). 5% CO,/95%
pairs
studted
at an
0, ([HCO;],
= II
= 39 mmol/I.). analysis with
of variance.
[HCO;].
39 mmol/L.
on [HCO,~], and pHi. In21 and 69 mm Hg caused progressive increases in [HCO,-1, and decreases in pHi of similar magnitude at [Cl-], of 20 and 100 mmol/L. Thus, the lowest [HCO,-1, (11.3 + 0.8 mmol/L) was measured at PCO, 21 mm Hg at a [Cl-], of 100 mmol/L, and the highest [HCO,-1, (22.5 + 1.2 mmol/L) was measured at PCO, 69 mm Hg at a [Cl-], of 20 mmol/L. This additive effect of [Cl-], and Pco, was evident whether [Cl-], was altered at one CO, tension or whether Pco, was altered at one [Cl-],. Table 2 shows the effect of PCO, on ion fluxes at [Cl-], of 100 and 20 mmol/L. Because the larger part of any flux represents passive paracellular diffusion and is directly proportional to the concentration of the particular ion in the medium, the absolute magnitude of Cl- fluxes at [Cl-], of 20 and 100 mmol/L cannot be compared directly. However, changes in ion flux at these [Cl-], values can still be evaluated qualitatively. As shown in Table 2, there was no significant change in JFA (and G) at a [Cl-], of 100 mmol/L when Pco, was increased. At a [Cl-], of 20 mmol/L, however, increases in Pco, caused Jzk to decrease from 3.0 + 0.3 to 1.7 * 0.2 PEq. cm-‘- h-’ (P < 0.001). A concomitant and significant decrease in G also was noted. There was no effect of Pco, on Jr: at either [Cl-],. The changes in Isc, PD, and J,, and Jnetfor both Na+ and Cl- in response to increasing PCO, were similar to our previous observations.’ The overall relationship between mucosal [HCO,-Ii and JzAis shown in Figure 5. The data were obtained from the ion-flux studies in which the [Cl-],, PCO, were altered, and the correPCO3-L. f and/or sponding [HCO,-1, for each condition. Colonic JzA was highest (13.0 pEq* cm-‘- h-l) in CO,-free, HEPES-buffered Ringer’s in which [HCO,-1, was assumed to be negligible. JzA was much lower in Ringer’s containing HCO, and CO,, In Ringer’s containing a [Cl-], of 100 mmol/L, J:A was 8.3 pEq - crnm2. h-’ at an [HCO,-1, of 9 mmol/L. Thereafter, up to an [HCO,-1, of 18 mmol/L, JfA was relatively stable. J$.. then decreased at [HCO,-1, >18 between
bathed
39 is 7. The Ringer’s
-0.7
PCO, 33.7 mm
Hg by paired
Student’s
t test.
mmol/L. This reduction in J:A as [HCO,-1, was increased above 18 mmol/L was seen also in a Ringer’s solution containing a [Cl-], of 20 mmol/L, although as noted above the absolute flux rate was lower than at a [Cl-], of 100 mmol/L. Next, the modulation of J$Aby [HCO,-1, was examined in the presence of 10m4mmol/L serosal bumetanide, a known inhibitor of Cl- secretion. The rationale was as follows: if the Cl--secretory pathway modulated by [HCO,-1, was inhibited by this drug, a subsequent alteration in [HCO,-1, should have minimal or no effect on J,,. ” As shown in Table 3, bumetanide caused significant reductions in J:A and G at an [HCO,-1, of 21 mmol/L. No effect on Jr: was seen. In Ringer’s solution containing 39 mmol/L HCO,- at a Pco, of 72 mm Hg, J:j was 5.1 + 0.6 pEq - cm-‘. h-’ and was not further reduced by bumetanide. In addition, the increase in J$, noted upon reduction of Pco, from 72 to 34 mm Hg (1.2 FEq - cm-‘- h-‘: Table 1) was totally inhibited by bumetanide (-1.0 PEq. cm-2. h-‘; Table 3). Discussion Whereas stimulated intestinal Cl- secretion has been studied extensively,3 much less attention has been given to the basal Cl--secretory process apparent in various epithelia in the resting state.7-g This stems from the direct relevance of the former process to the pathophysiology of diarrhea1 disease. Although the clinical significance of the basal secretory process is unknown, the unraveling of its physiology and regulation is nevertheless essential for a more complete understanding of overall epithelial Cl- homeostasis. Furthermore, the simultaneous presence of basal secretion and absorption creates a new dimension of complexity in the interpretation of in vivo transport data measuring only net Cl- fluxes. The present study was designed to examine the basal Cl--secretory process in the distal colon of the rat. In previous studies, we found electrolyte transport across this epithelium responsive to alterations in the acid-base variables.‘~2~10~‘6These studies were
124 DAGHER ET AL.
GASTROENTEROLOGY Vol. 103, No. I
30 0
25
PC02 =22mmHg
PC02 = 72mmHg
[HCO3]0mM 5
11
21
6.7
6.9
functional in this absorbing epithelium. In addition, it suggested that this transport process was regulated by changes in the intracellular as well as the extracellular acid-base milieu. In the present study, we measured J:k under a variety of experimental conditions. Part of this flux (and in particular changes in this flux) appeared to reflect an underlying active secretory process. This was suggested by our finding that the maneuvers used to alter JFA, namely changing [HCO,-1, and Pco, or adding bumetanide, are not known to affect passive Clmovement through the paracellular shunt pathway. Furthermore, changes in J:A in our study were always accompanied by parallel changes in G. Jr:, on the other hand, remained unchanged under all conditions. Inasmuch as Jyz is a measure of paracellular ion flux, the observed variations in J:A and G are likely to represent changes in active transcellular Cl- secretion. The correlations between colonic mucosal [HCO,-1, and ion flux were justified largely because these measurements were made in identical tissue preparations. Despite its histological diversity, a colonic
g 7.1 25 0
7.3
[Cl]=lOOmM [Cl) =20mM
T
20 7.5
E ‘;; 15
Figure 3. Effect of [HCO,J, and Pco, on [HCO,Ji and pH,. Tissues were bathed consecutively in 5, 11, 21, and 39 mmol/L HCO,- Ringer’s at Pco, 32 mm Hg (n = 5) or 72 mm Hg (n = 5). At each Pco,, both [HCO,J, and pH, increased as [HCO,J, was increased (one-way analysis of variance, P < 0.001). *P < 0.05 when [HCO,-Ii or pH, at Pco, 32 mm Hg was compared with Pco, 72 mm Hg at each [HCO,J, by unpaired Student’s t test. Values are means k SE.
P 10
0 PCO3(mmHg) 21.3 to.0
34.5 iO.6
68.8 2 3.2
7.0
performed in vivo over a physiological range of systemic acid-base conditions, and in vitro where active and passive components of ion flux, pHi, and putative transport inhibitors could be examined. We found that J,, and Jnet for Na+ and Cl- were stimulated by an increase in ambient Pco,.’ This was apparently due to stimulation of apical membrane Na+/H+ and Cl-/HCO,- exchangers after intracellular hydration of CO, and subsequent generation of hydrogen and bicarbonate ions.2~10 We also found that changes in net Cl- absorption in the colon correlated with the systemic [HCO,-I.’ This effect was primarily caused by changes in J::.” This finding suggested that J:A reflected in part an active, basal secretory process that was present and
7.2 % 7.4
7.6 L Figure 4. Effect of [Cl-], and PCO, on [HCO,Ji and pH,. Five tissues were bathed consecutively in 21 mmol/L HCO,- Ringer’s containing [Cl-]. of 100 and 20 mmol/L. At each [Cl-]., the PCO, was varied between 21.3, 34.5, and 68.8 mm Hg. At each Pco,, [HCO,Ji and pH, were higher at a [Cl-], of 20 mmol/L than at 100 mmol/L (paired Student’s t test, P < 0.05). At each [Cl-]., [HCO,J, was higher and pH, was lower as Pco, increased (oneway analysis of variance, P c 0.02). Values are means + SE.
[HCO,-1,
July 1992
Table 2. Effect of CO, and [Cl-], on Colonic Electrolyte
100 mmol/L
(mmol/L)
(mm Hg)
Cl
20 mmol/L
NOTE.
PCO,
JN” @Eq.
Cl
Values
HCO;.Ringer’s
PD
(/IEq.cm-‘. h-‘)
PH.
cm-‘.
sm
ms
net
7.62 + 0.01
1.2 * 0 2
3.7 -t 0.6
7.8 f 1 1
9.0 k 0.9
70.1 i 3.2b
7.12 + 0.02b
0.6 * 0.20
2.5 t 0.7”
6.6 f 0.5
13.2 + 1.8”
21
2, 3 k 1.0
7.61 + 0.01
1.2 i 0.1
5.4 + 0.5
6.2 i 0.5
6.3 k 0.4
4.4 + 0.6
21
69.8 k 3.9*
7.13 + 0.02b
0.7 t 0.10
3.8 + 0.9’
5.1 + 0.Y
8.7 + 0.6b
4.1 k 0.6
gassed
were with
obtained
in two
tw” different
separate
CO, tensions.
groups
125
cm-‘.h-‘) JR
(mS/cm’)
WI
20.4 + 0.7
f SE. Data
Jc’ @Eq.
h-‘)
21
are means
Cl- SECRETION
G
21
consecutively
OF
Transport
kc WW.
MODUI.ATION
of tissue
The second
group
pairs.
The
consisted
net
@Eq . cm-‘.
he’)
5.0 + 0.5
3.9 k 0.8
13.5 + 1.4
8.3 k 0.3
5.2 k 1.6
2.5 + 1.0
4.6-r
8.7 i; 2.1’
16.9+
2.3
8.4 + 0.5
8.5 + 2.4’
0.5 + 0.5
2.0 k 0.7
3.9 -t 0.3
3.0 + 0.3
0.9 i 0.3
0.1 + 0.6
4.7 f O.Sb
6.6 f 0.3b
1.7 k 0.2h
4.9 + 0.4b
0.9 * 0.7
first group ofsix
sm
Ins
tissue
0.4
consisted pairs
of five tissue
treated
as described
pairs above
bathed
in 100 mmol/L
Cl-,
that 20 mmol/L
Cl- Ringer’s
except
21 mmol/L was
used. “P < 0.05. hP < 0.001 when
values
at Pco,
20 and
70 mm Hg at each
Ringer’s
[Cl-]
were
compared
on Colonic Electrolyte
Student’s
t test.
its distribution among the various cell types are not exchange process, yet known. ‘O The Cl-/HCO,which likely mediated the increases in pH, and [HCO,-Ii caused by a reduction in [Cl-],,I5 is definitely present along the apical membrane of colonic surface cell? and has been shown in crypt cells in other intestinal segmentsz2 The results of the present study strongly suggest that [HCO,-1, is the specific acid-base variable affecting Jcl . This relationship is supported by the findings thar[HCO,-1, correlated with [HCO,-1, (Figure 3) and that J,, ” decreased when [HCO,-1, was increased regardless of the prevailing Pco, and the direction of change in pH, (Table 1).A direct role for [HCO,-1, was ruled out by the finding that [HCO,-1, and Jzh were affected by changes in Pco, in the absence of changes in [HCO,-1, at an [HCO,-1, of 39 mmol/L (Table 1) or a [Cl-], of 20 mmol/L (Table 2). The additive effects of low [Cl-],, [HCO,-I,, and Pco, on JzAcan most easily be interpreted as due to a common effect of these variables on [HCO,-I,. Of note, JEL,did not incre ase despite significant increments in [HCO,-1, when [HCO,-1, was increased. This finding would appear to contradict our previous in vivo studies’ in which a significant stimulatory effect of [HCO,-1, on JzA,was seen. However, this can be explained by the symmetrical increase in bathing-solution [HCO;-] used in the flux chamber and the blood-to-lumen gradient for [HCO,-] present in vivo. In vitro, the increased mucosal bathing solution [HCO,-] decreased JgS by inhibiting mucosal
segment stripped of serosal elements is considered one functional unit when studied in the flux chamber. Fluxes are appropriately labeled “mucoSal” without further specifications because all (or most) epithelial cells undoubtedly contribute to the measured ion fluxes. Furthermore, the original separation of surface and crypt ceils as sites of absorption and secretion, respectively, is emerging as somewhat artificial if not incorrect. Recent studies have shown the existence of functional apical Cl- channels and active Cl- secretion in surface cells of rat colon,’ necturus small intestine,6 and hen intestine.17 In this regard, the tissue preparation used to measure intracellular acid-base variables was particularly suitable for comparison with the flux studies because the fluorescence was localized to the epithelial elements of the tissue (the surface cells contributing the larger part of the fluorescence). The qualitative responses of mucosal pH, and [HCO,-Ii to changes in Pco,, [HCO,-I,, and [Cl-], further support the epithelial origin of these measurements. Thus, the response of these acid-base variables to alterations in Pco, is most prominent in carbonic anhydrase-rich cells.1~‘7~‘8This enzyme has been localized to both the surface and crypt epithelium of the colon but not to nonepithelial colonic elements.” The increase in [HCO,-Ii caused by increasing [HCO,-1, is most likely caused by basolateral Nafdependent HCO,- entry into the cell. This process has been identified in rat colonic epithelium, although the exact nature of this transport process and
Table 3. Effect of Bumetanide
by paired
Transport J”‘(IIEq~cm~‘.h~‘)
kc [HCO,].
PCO,
(_uEq cm-‘.
(mmol/L)
(mm Hg)
PH,
h-‘)
(mS/cm’)
WI
CO”trOl
21
34.2 i 0.9
7.40 + 0.01
2.3 f 0.3
Bumetanide
21
34 2 t 0.9
7.40 e 0.01
1.4 * 0.2b
7.7 * 1.5
Control
39
71.5 + 0.7
7.33 * 0.02
1.5 + 0.3
8.1 i 2.2
Bumetanide
39
71.5 t 0.7
7.33 i 0.02
1.2 * 0.3
8.7 k 2.4
Bumetanide
39
33.9 + 0.2
7.66 zk 0.01
0.9 k 0.3
6.6 + 2.6
4.5 f 0.3
NOTE. group
Values consisted
reservoirs
are means
-+ SE. Data were
of five tissue
pairs
at a final concentration
“P < 0.01, hP < 0.05 compared
bathed
obtained
in two separate
in 39 mmol/L
10.3 k 0.5
groups
HCO;-Ringer’s
the preceeding
condition
by paired
ms
net
sm
net
@Eq . cm-‘.
h-’
6.1 k 0.5
7.3 k 1.6
2.8 f 0.2
4.6 + 1.7
13.7 k 1.7
7.4 k 0.4
6.3 + 1.9
4.0 k 0.9
5.2 + 0.3b
7 9 i 1.5
2.6 + 0.5
5.3 k 1.9
14.3 + 1.9
6.0 k 0.4”
6.3 k 2.10
4.4 + 0.8
5.3 * 0.4
10.7 ? 1.9
2.3 k 0.3
8.4 + 1.9
15.4 +- 1.9
5.1 k 0.6
10.2 + 2.1
3.4 k 0.6
4.5 + 0.3O
10.1 + 1.4
2.2 i- 0.5
7.9 + 1.8
13.8 zk 1.2
5.7 + 1.2
8.0 k 1.6
1.3 + 1.1
8.6 k 1.7
2.2 k 0.4
6.5 + 1.6
11.8 + 2.0
4.7 It 0.7
7.1 + 1.9
1.6 + 1.6
of tissue
pairs.
The first group
and consecutively Student’s
sm
Ins
of lo-’ mol/L. with
JR
G
PD
t test.
gassed
with
consisted two different
of four tissue CO, tensions.
pairs
bathed
In both
in 21 mmol/L groups,
bumetanide
HCO;.Ringer’s was added
The second to the semsal
I
126
DAGHER ET AL.
GASTROENTEROLOGY
Cl-/HCO,e xc h ange, and this inhibition offset the effect of decreasing JzA on J$. We also considered the possibility that colonic mucosal pH, modulated Jz$,. Intracellular acidosis is known to inhibit basolateral membrane K+ channels, depolarize the cell, and thus diminish the driving force for Cl- secretion.‘5*23 However, in the present study JzAdecreased both when pH, increased (Table 1; Figure 3) and when pH, decreased (Tables 1 and 2; Figures 3 and 4). The quantitative effect of [HCO,-Ii on JzAalso was not affected by the direction of change in pH,. Nevertheless, as noted above, under certain physiological conditions not examined in this study, alterations in colonic mucosal pHi could influence
JCl . ““The relationship between [HCO,-1, and J,Cb,shows a pattern common to many physiological systems. J:’ is most sensitive to changes in [HCO,-1, above and below a range that encompasses the “normal” [HCO,-1, .I0 Th is range or plateau that extends from approximately 9 mmol/L to 18 mmol/L is associated with a stable JzA of 8.3 ~Eq.cm-2.h-’ when measured in a Ringer’s solution containing 21 mmol/L HCO,- and 100 mmol/L Cl- (Figure 5). The presence of this plateau may explain why moderate increases in Pco, or [HCO,-1, do not affect J,“b,when colonic fluxes are studied in Ringer’s of similar composition.
Vol. 103, No. 1
The nature of this colonic Cl--secretory pathway has not been systematically examined. However, by analogy with other epithelia active Cl- secretion in the colon occurs by Cl- entry via a basolateral membrane Na+-K+-2Cll cotransport process and Cl- exits via an apical membrane anion conductance.3’24 The lack of effect of [HCO,-1, on J:j in the presence of serosal bumetanide suggests that it is indeed this transport pathway that is modulated by [HCO,-Ii. The exact mechanism by which [HCO,-1, affects this Cl--secretory pathway remains uncertain. Clchannels in various epithelia may be quite permeable to HC03-.25,26 Thus, it is conceivable that changes in [HCO,-1, inversely affect Cl- flow through these channels. A second possibility is that alterations in [HCO,-1, change the proportion of mucosal membrane anion exchangers operating as Cl-/ Cl- self-exchange and Cl-/HCO,heteroexchange. This phenomenon has been demonstrated in the intercalated cells of the mammalian collecting tubulez7 but has not been studied in the colonic epithelium. No direct effect of [HCO,-1, on the basolateral membrane Na+-K+-2Cll cotransporter can be postulated. This cotransporter has no known sensitivity to pH or base gradients.28 Of course, [HCO,-1, could have an indirect effect at either the apical or the basolateral membrane. In this regard, [HCO,-1, modulation of intermediates such as cellular calcium or protein phosphorylation needs further investigation. In summary, we have described the modulation of an active, basal Cl--secretory process in the rat colon by mucosal [HCO,-Ii. This secretory process is part of the J,, measured under short-circuit conditions in The relationship between the Ussing chamber. [HCO,-1, and Jfk is triphasic; these variables are inversely related on either side of a plateau that encompasses the normal colonic mucosal [HCO,-1,. The shape of the [HC03-li/J2A relationship may account for the presence of intestinal Cl- preservation during clinical metabolic alkalosis and intestinal Cllosses during metabolic acidosis.‘~2*‘0*‘6 References 1. Charney
2.
L
1
I
I
I
I
0
5
IO
15
20
25
I
30
3.
[HCOs]i mM Figure 5. Effect of [HCO,-1, on JzA. Data for JzA were obtained from Tables 1 and z and plotted against the corresponding [HCO,-]I obtained from Figures 3 and 4. In CO,-free, HEPES-buffered Ringer’s, [HCO,-1, was assumed to be negligible. The relationship between [HCO,-Ii and JzA is triphasic. Values are means + SE.
4.
5.
AN, Haskell LP. Relative effects of systemic pH, PCO,, and bicarbonate concentration on colonic ion transport. Am J Physiol 1984;246:G159-G165, Goldfarb DS, Egnor RW, Charney AN. Effects of acid-base variables on ion transport in rat colon. J Clin Invest 1988;81:19031910. Binder HJ, Sandle GI. Electrolyte absorption and secretion in the mammalian colon. In: Johnson LR, ed. Physiology of the Gastrointestinal Tract. New York: Raven, 1987:1389-1417. Welsh MJ, Smith PL, Fromm M, Frizzell RA. Crypts are the site of intestinal fluid and electrolyte secretion. Science 1982;218:1219-1221. Diener M, Rummel W, Mestres P, Lindemann B. Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat. J Membrane Biol 1989;108:21-30.
July 1992
[HCO,-1, MODULATION
6. Giraldez F, Sepulveda
19.
1988;395:597-623. 7. Charney AN, Donowitz
20.
FV, Sheppard DN. A chloride conductance activated by adenosine 3’,5’-cyclic monophosphate in the apical membrane of Necturus enterocytes. J Physiol M. Functional significance of intesin vivo ouabain inhibition. Am J Physiol
8.
9.
10.
11.
12.
13.
14. 15.
16.
tinal Na-K-ATPase: 1978;234:E629-E636. Davis GR, Santa Ana CA, Morawski S, Fordtran JS. Active chloride secretion in the normal human jejunum. J Clin Invest 1980;66:1326-1333, O’Grady SM, Wolters PJ. Evidence for chloride secretion in the intestine of the winter flounder. Am J Physiol 1990;258:C243-C247. Wagner JD, Kurtin P, Charney AN. Effect of systemic acidbase disorders on colonic intracellular pH and ion transport. Am J Physiol 1985;249:G39-G47. Ganz MB, Boyarsky G, Boron WF, Sterzel RB. Effects of angiotensin II on intracellular pH of glomerular mesangial cells. Am J Physiol 1988;254:F787-F794, Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehlrich Ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 1979;18:2210-2218. Graber ML, DiLillo DC, Friedman BL, Pastoriza-Munoz E. Characteristics of fluoroprobes for measuring intracellular pH. Anal Biochem 1986;156:202-212. Snedecor GW, Cochran WG. Statistical methods. Ames, IA: Iowa State University, 1987. Duffey ME, Devor DC. Intracellular pH and membrane potassium conductance in rabbit distal colon. Am J Physiol 1990;258:C336-C343. Charney AN, Feldman GM. Systemic acid-base disorders and intestinal electrolyte transport. Am J Physiol 1984;247:Gl-
G12. 17. Holtug K, Shipley A, Dantzer V, Sten-Knudsen
0, Skadhauge E. Localization of sodium absorption and chloride secretion in an intestinal epithelium. J Membrane Biol 1991;122:215-229, 18. Charney AN, Wagner JD, Birnbaum GJ, Johnstone JN. Functional role of carbonic anhydrase in intestinal electrolyte transport. Am J Physiol 1986;251:G159-G165.
21.
22.
23.
24.
25.
26.
27.
28.
OF Cl- SECRETION
127
Lonnerholm G, Midtvedt T, Schenholm M, Wistrand PG. Carbonic anhydrase isoenzymes in the caecum and colon of normal and germ-free rats. Acta Physiol Stand 1988;132:159-166. Feldman GM, Koethe JD, Stephenson RL. Base secretion in rat distal colon: ionic requirements. Am J Physiol 1990;258: G825-G832. Binder HJ, Foster EF, Budinger ME, Hayslett JP. Mechanism of sodium chloride absorption in distal colon of the rat. Gastroenterology 1987;93:449-455, Knickelbein RG, Aronson PS, Dobbins JW. Membrane distribution of sodium-hydrogen and chloride-bicarbonate exchangers in crypt and villus cell membranes from rabbit ileum. J Clin Invest 1988;82:2158-2163. Christensen 0, Zeuthen T. Maxi K channels in leaky epithelia are regulated by intracellular Ca’+, pH and membrane potential. Pflugers Arch 1987;408:249-259. Dharmasathaphorn K, Mandel KG, Masui H, McRoberts JA. Vasoactive intestinal polypeptide-induced secretion by a coionic epithelial cell line. Direct participation of a basolaterally-localized Na, K, Cl cotransport system. J Clin Invest 1985;77:348-354. Kunzelmann K, Gerlach L, Frobe U, Greger R. Bicarbonate permeability of epithelial chloride channels. Pflugers Arch 1991;417:616-621. Tabcharani JA, Jensen TJ, Riordan JR, Hanrahan JW. Bicarbonate permeability of the outwardly rectifying anion channel. J Membrane Biol 1989;112:109-122. Schuster VL, Stokes JB. Chloride transport by the cortical and outer medullary collecting duct. Am J Physiol1987;253:F203F212. Haas M. Properties and diversity of (Na-K-Cl) cotransporters. Annu Rev Physiol 1989:51:443-457.
Received August 23, 1991. Accepted January 7, 1992. Address requests for reprints to: Alan N. Charney, M.D., Nephrology Section, Department of Veterans Affairs Medical Center, 423 East 23rd Street, New York, New York 10010. Supported by the General Medical Research Program of the Department of Veterans Affairs.
1992:103:120-127
Modulation of Chloride Secretion in the Rat Colon by Intracellular Bicarbonate PIERRE C. DAGHER, LEAH BALSAM, J. TODD WEBER, RICHARD W. EGNOR, and ALAN N. CHARNEY Nephrology Section, Department of Veterans Affairs Medical Center, and New York University School of Medicine New York, New York
Extracellular HCO,- stimulates colonic net Cl- absorption in part by inhibiting basal Cl- secretion. This inhibition was investigated by measuring serosal-to-mucosal Cl- flux across short-circuited colonic segments from Sprague-Dawley rats. Mucosal intracellular pH and bicarbonate were estimated using the pH-sensitive dye BCECF. When extracellular [HCO,-] ([HCO,-I,) was increased from 0 to 39 mmol/L at Pco, 33 mm Hg, mucosal intracellular [HCO,-] ([HCO,-1,) increased to 25.3 mmol/L and serosal-to-mucosal Cl- flux decreased from 13.0to 7.1pEq - cm-‘. h-‘. When Pco, was increased to 72 mm Hg at [HCO,-1, 39 mmol/L, [HCO,-]i increased to 29.8 mmol/L and serosal-to-mucosal Clflux decreased to 5.9 pEq* cm-’ - h-‘. In Ringer’s solution containing 21 mmol/L HCO,- and 20 mmol/L Cl- (but not 100 mmol/L Cl-), increasing Pcoz from 21 to 70 mm Hg increased [HCO,-]I to 22.6 mmol/L and decreased serosal-to-mucosal Cl- flux from 3.0 to 1.7 pEq - crnp2 - h-l. Overall, serosal-to-mucosal Cl- flux was inversely related to [HCO,-]I on either side of an [HCO,-1, plateau of 9-18 mmol/L at which flux was stable. These data suggest that [HCO,-lI is an important modulator of basal Cl- secretion in rat distal colon. e previously reported that colonic chloride absorption and bicarbonate secretion correlated with blood bicarbonate concentration in the rat.’ In this in vivo perfusion study, such changes suggested that alterations in a mucosal chloride-absorptive/bicarbonate-secretory exchange process accounted for the effects of blood bicarbonate. Therefore, it was of interest that subsequent in vitro isotope flux measurements showed that this bicarbonate effect was mediated through changes in both serosal-to-mucosal chloride flux (Jth) and mucosal to serosal flux (Jz:).’ Inasmuch as changes in J:A could represent changes in active secretion, we hypothesized that bicarbonate modulated an ongoing chloride-secretory process.
W
The idea that net chloride transport in the intestine reflects both active absorption and secretion is not new.3 Two oppositely directed chloride pathways have been described under a variety of experimental conditions. Whereas absorption occurs primarily in surface epithelial cells, secretion was originally thought to be confined to the crypts.4 Recently, chloride secretion has also been shown in surface cells.5*6 Similarly, the temporal separation of these two processes, while evident during stimulated secretion, is less obvious in resting epithelium. Data from different species and various intestinal segments including human jejunum, rabbit ileum, and teleost small intestine support the presence of an ongoing basal chloride secretory process that could modulate net absorption.7-g The present study was designed to investigate systematically the relationship between extracellular bicarbonate concentration ([HCO,-I,) and J:A in the rat colon. We also measured intracellular bicarbonate ([HCO,-I,) in the colonic epithelium because the strong correlation between extracellular and intracellular concentrations of this ionlo suggested that [HCO,-1, may mediate the effects of [HCO,-I,. The experimental conditions were designed to vary [HCO,-1, and [HCO,-1, independently and to correlate these changes with J:k. The results suggest that net chloride absorption in the rat colon is modulated by [HCO,-Ii through an effect on an active, ongoing chloride-secretory process. Materials and Methods Male Sprague-Dawley rats weighing 250-350 g were maintained on a standard diet with free access to water, While rats were under pentobarbital sodium anesthesia (5 mg/lOO g body wt), the distal 10 cm of descending colon was removed, rinsed with 0.9% saline, and stripped of serosa. This is a U.S. government work. There are no restrictions on its use. 0016-5065/92/$0.00
July
[HCO,-Ii MODULATION OF Cl- SECRETION
1992
Electrical and Radioisotope Measurements
Flux
Details of the method of flux measurement have been described previously.’ Briefly, tissue pairs were mounted in modified Ussing half-chambers exposing 1.12 cm* surface area. The transepithelial potential difference (PD) was expressed as serosal side positive with respect to the mucosa. Tissues were studied under short-circuit conditions except for l-second intervals every 100 seconds, during which bipolar pulses of 0.5 mV yielded electrical current values used to calculate tissue conductance (G). The short-circuit current (1s~) was given the same polarity as PD. Tissues were paired for ion flux studies when differences in G were no greater than 25%. Unidirectional fluxes of Na+ and Cl- were measured by adding 2 FCi of “Na+ and 1 pCi of 36C1m(100 Ci/g sp act; New England Nuclear, Boston, MA) to the mucosal side of one member of each tissue pair and to the serosal side of the other. A 20-minute equilibration period was allowed after each change of condition, and then unidirectional flux was measured over a 45-minute period. The residual flux (JR)was calculated as Isc - (JF’,a, - Jz:,).
Fluorescence
Measurements
A segment of stripped distal colon (described above) was mounted as a flat sheet over a l-cm’ hollowring assembly consisting of two close-fitting concentric plastic rings. The tissue was thereafter handled in the solution designated to be tested first in the experimental protocol. First, the tissue was incubated in 1 mmol/L dldithiothreitol (DTT) (Sigma Chemical Co., St. Louis, MO), a mucolytic agent, for 10 minutes. It was then bathed for 30 minutes in a 7 pmol/L solution of the pH-sensitive dye 2’,7’-bis-(2-carboxyethyl)-5(and-6)-carboxyfluoresceinacetoxymethyl ester (BCECF-AM) (Molecular Probes, Eugene, OR). The mounted tissue was washed twice in fresh test solution to remove extracellular dye. A Perkin-Elmer (South Plainfield, NJ) LS-5B spectrofluorometer equipped with a thermoregulated sample chamber maintained at 37°C was used. Data were collected using Fura software supplied by Perkin-Elmer and adapted for intracellular pH (pH,) measurement. The mounted tissue was placed in a fixed position at the bottom of a 4-mL cuvette (Markson Science, Phoenix, AZ) with the mucosal surface facing the excitation beam at a 45” angle. All readings were done after the tissue was exposed to the test solution for 10 minutes, during which three exchanges of the solution with fresh aliquots added from sealed syringes were performed. These measurements represent steady-state values because in preliminary experiments they were not found to change for up to 23 minutes. At all times the cuvette was tightly closed with a plastic cap to prevent CO, leakage. Fluorescence measurements were performed in triplicate using excitation wavelengths of 440 and 500 nm in sequence. The emission was measured at 530 nm. Excitation and emission slits were adjusted to 3-nm bandwidths. An average fluorescence ratio (500 nm/440 nm) was computed from the triplicate readings. Tissue autofluores-
cence was 5%-10% of the total fluorescence
121
and was auto-
matically subtracted from the readings. In preliminary experiments, we found that the plastic ring showed no autofluorescence and that dye leakage and photobleaching were minimal during the course of the experimental protocols. Tissue viability was documented for periods up to 40 minutes using trypan blue-exclusion criteria. The intracellular site and responsiveness of the fluorescent signal were verified by the ammonium-pulse technique” (Figure 1). The location and distribution of the dye within surface and crypt cells were determined by fluorescence microscopy of frozen sections of BCECF-exposed tissues. As shown in Figure 2, homogeneous uptake of the dye is evident in surface epithelial cells and to a lesser degree in crypt cells. Fluorescence is totally absent in the crypt spaces. DTT, used exclusively in the pH, studies, nevertheless had no effect on the electrical properties of the tissue or on its response to CO, in the Ussing flux chamber. At the end of each experiment, the mounted tissue was exposed to HEPES-buffered solution (described below) containing 140 mmol/L KC1 and 10 pg/mL nigericin (Sigma Chemical Co.) for pH, calibration.” The medium was titrated with NaOH to four different pH values. A calibration curve was thus constructed and used to convert the observed fluorescence ratios to pH, values. The calibration curves were linear over the pH range of 6.5-7.8 in accordance with the known properties of the dye.13 The [HCO,-1, was computed using the HendersonHasselbalch equation and the measured pH,. Intracellular PCO, was assumed to be equal to the medium Pco,, and the pK’ and CO, solubility were 6.115and 0.0306, respectively. Bathing solution pH and Pco, were measured using a Radiometer BMS 3 Mk 2 system with a PHM 73 acidbase analyzer (The London Company, Cleveland, OH).
z z P
7.6
7.0 I
I 5
I 10
I 15
I
I
20
25
TIME (min) Figure 1.A
typical experiment showing the pH, response of a whole mucosal tissue mount in HEPES-Ringer’s at pH 7.38 to a 20 mmol/L NH&l pulse. A, Control pH,; B, rapid entry of NH,; C, slower entry of NH,+; D, rapid exit of NH,; E, recovery.
122 DAGHER ET AL.
GASTROENTEROLOGY Vol. 103,No. 1
Figure 2. Fluorescence photomicrograph (original magnification X1000) of a stripped segment of colonic mucosa incubated in a 7 pmol/L solution of BCECF-AM for 30 minutes. Frozen sections examined by fluorescence microscopy showed homogeneous distribution of the dye in surface cells (S) and to a lesser extent in crypt cells (C).
[HCO,-1, was computed equation as above.
using the Henderson-Hasselbalch
Solutions and Acid-Base Conditions All solutions were maintained at 37°C. The HCO, Ringers contained (mmol/L) glucose, 10;NaCl, 96;KCl, 4; Na,HPO,, 2.4; NaH,PO,, 0.4; CaSO,, 1; MgSO,, 1.2; NaHCO,, 21; and Na+ gluconate, 18.The [HCO,J was adjusted to 5,11,21, and 39 mmol/L by reciprocal alterations in Na+ gluconate to keep the osmolality constant. These solutions were gassed with 3% CO,/97% 0,, 5% CO,/95% O,, or 11% CO,/89% 0, to obtain various pH and PCO, values. In HEPES Ringer’s, 21 mmol/L HEPES, Na+ salt, was substituted for NaHCO, and the medium was gassed with 100% 0,. In the low-Cl_ solution, gluconate replaced Cl- in equimolar amounts. The acid-base variables were otherwise identical to the 100 mmol/L Cl- solution. The pHi calibration solution contained (mmol/L): HEPES, 5; KCl, 140; MgCl,, 1; CaCl,, 1; and glucose, 10. In some studies, bumetanide (Sigma Chemical Co.) was added to the serosal reservoir at a final concentration of 10m4mol/L.
Statistics Results are expressed as means + SE. Data were analyzed by paired or unpaired Student’s t test, one-way analysis of variance, and simple linear regression.14 Results
Table 1 shows the effect of [HCO,-1, on coionic electrolyte fluxes under short-circuit conditions. In a CO,-free, HEPES-buffered Ringer’s, minimal net Naf and Cl- absorption were observed. In HCO,- Ringer’s at a Pco, of approximately 33 mm Hg, net Na+ and Cl- absorption and a residual flux were present. As the Ringer’s [HCO,-] was increased, an interesting pattern emerged: over the en-
tire range of HCO,- concentrations from 0 to 39 mmol/L, JFAvaried inversely with [HCO,-I,. Jzj progressively decreased almost 50% from 13.0 + 0.9 pEq - cm-‘- h-’ in HEPES-buffered Ringer’s to 7.1 -t 0.3 pEq * cm-’ - h-’ at a [HCO,-1, of 39 mmol/L and PCO, of 33.7 mm Hg. G tended to decrease in parallel with J:A. No relationship was found between Na.No changes in J,, and Jnetfor either [HCO,-I, and J,, Naf or Cl- were statistically significant. Furthermore, changes in J,, ” did not correlate with either extracellular pH (pH,) or Pco,. To examine the role of [HCO,-1, in mediating these changes in JzA, we measured colonic mucosal pHi and calculated [HCO,-1, in another group of tissues prepared identically. The measurements were made over a broad range of HCO,- concentrations at two CO, tensions: 32 and 72 mm Hg. As shown in Figure 3, there were strong correlations between [HCO,-1, and both mucosal [HCO,-1, and pH, at each CO, tension (r = 0.99 at Pco, 32 mm Hg, P < 0.001; r = 0.99 at Pco, 72 mm Hg, P < 0.001). The slopes of these correlations were similar so that at both CO, tensions, [HCO,-1, increased 0.6 mmol/L for each 1 mmol/L increase in [HCO,-I,. However, at each [HCO,-I,, mucosal pH, was lower and [HCO,-1, was higher at Pco, 72 mm Hg than at Pco, 32 mm Hg. For example, at [HCO,-1, = 39 mmol/L pH, was 0.3 U lower and [HCO,-1, was 4.4 mmol/L higher at PCO, 72 mm Hg than at Pco, 32 mm Hg. Similar changes were noted at 21 mmol/L [HCO,-I,, but there was no effect of Pco, on [HCO,-Ii at [HCO,-1, = 5 and 11 mmol/L, despite a marked effect on pHi. Because Pco, and [HCO,-1, were additive in their effects on [HCO,-I,, we investigated whether an increase in CO, tension could cause a further decrease in Jzk in colonic tissue exposed to a [HCO,-1, of 39 mmol/L. As shown in Table 1, JFAwas reduced from 7.1 + 0.3 FEq - cm-’ - h-l at Pco, 33.7 mm Hg to 5.9 + 0.3 PEq - cm-‘. h-’ at Pco, 71.2 mm Hg (P < 0.05). This reduction was reversible by decreasing Pco,. There was no significant change in Jr:. G decreased significantly from 6.8 * 0.5 to 4.6 + 0.3 mS/cm’ (P < Cl The effects of PCO, to in0.05) in parallel with J,,. crease J,, for both Na+ and Cl- and decrease PD and Isc were similar to our previous observations in the rat distal colon.’ We then examined the effects of changing mucosal [HCO,-Ii by altering the Ringer’s [Cl-]. A decrease in medium [Cl-] inhibits or reverses apical membrane Cl-/HCO,- e xc h ange and thus increases [HC03-li.15 These experiments were performed at an [HCO,-1, of 21 mmol/L at Pco, 21, 34, and 69 mm Hg. As shown in Figure 4, at each CO, tension [HCO,-1, was 4 mmol/L greater and pH, was 0.1 U greater at a [Cl-], of 20 mmol/L than at 100 mmol/L. Figure 4 also shows that this effect of [Cl-], did not
[HCO,-1,
July 1992
Table I. Effect of [HCO,], and CO, on Colonic Electrolyte
IN” bEq.
@Eq. cm-‘.
PCO,
(mmol/L)
(mm Hg)
cm-‘.h-‘]
(mS/cm’)
WI
123
IC’(_uEq~cm~‘~h~‘)
G
PD
h-‘)
PH.
OF Cl- SECRETION
Transport
kc
IHCO;IF
MOlJULATION
ms
sm
net
ms
sm
net
bEq.
lR cm ~‘. h-‘)
7.39 + 0.04
2.0 f 0.2
7.0 i 0.6
7.9 + 0.3
6.1 * 0.8
4.9 i 0.2
1 0 k 0.7
12.3 i 1.4
13.0 * 0.9
k 0.8
0.3 k 0.6
11
34.3 i; 0.6
7.20 I? 0.01
1.1 + 0.1
4.4 * 0.7
7.1 t 0.8
9.1 i 0.8
4.9 f 0.5
4.2 i- 0 9
13.6 i 1.0
8.5 * 0.4
5 2 k 0.8
2.0 k 0.6
21
32.1 k 1.0
7.39 * 0.03
1.6 + 0.2
5.8 + 0.3
7.2 * 0.4
7.3 * 0.8
4.2 + 0.3
3.1 + 0 7
12.1 + 0.6
7.9 * 0.4
4.1 i- 0.5
2.6 -r 0.4
39
33.7 * 0.9b
7.67 + 0.02”
1.1 + 0.1”
4.6 f 0.7O
6.8 k 0.5
5.7 k 0.6
3.9 * 0.4
1.9 + 0.7
10.2 2 0.3
7.1 k 0.3”
3.2 t 0 5h
2.4 + 0.7
39
71.2 i- 2.5’
7.35 2 0.02
0.5 + O.lC
2.8 k 0.5c
4.6 + 0.3
8.7 zk 1.1‘
3.5 + 0.4
5.2 2 1.4‘
13.0 k 0.7’
5.9 + 0.3’
7.1 k 0.8’
2.4 i 1.1
0
0
NOTE. [HCO;]. and
Values
are means
of 0 (HEPES)
21 mmol/L),
f SE. Data were
is 5. at [HCO;].
or 5% COJ95X
obtained
in four separate
11 is 8. at [HCO;]. 0, and
11% CO,/89%
([HCO;],
the four groups
‘P < 0.05 when
mmol/L. PCO, 71.2 mm Hg was compared
39
obscure
the effect of
creases
in Pco,
PCO,
compared
0,
“P < 0.05. ‘P < 0.001 when [HCO,],
were
groups
of tissue
21 is 17, and at [HCO;], by one-way
pairs
in 100 mmol/L solutions
Cl- Ringer’s WOT~ gassed
and various wth
[HCO;
100% 0, ([HCO;],,
]._ Number
of tissue
= 0). 5% CO,/95%
pairs
studted
at an
0, ([HCO;],
= II
= 39 mmol/I.). analysis with
of variance.
[HCO;].
39 mmol/L.
on [HCO,~], and pHi. In21 and 69 mm Hg caused progressive increases in [HCO,-1, and decreases in pHi of similar magnitude at [Cl-], of 20 and 100 mmol/L. Thus, the lowest [HCO,-1, (11.3 + 0.8 mmol/L) was measured at PCO, 21 mm Hg at a [Cl-], of 100 mmol/L, and the highest [HCO,-1, (22.5 + 1.2 mmol/L) was measured at PCO, 69 mm Hg at a [Cl-], of 20 mmol/L. This additive effect of [Cl-], and Pco, was evident whether [Cl-], was altered at one CO, tension or whether Pco, was altered at one [Cl-],. Table 2 shows the effect of PCO, on ion fluxes at [Cl-], of 100 and 20 mmol/L. Because the larger part of any flux represents passive paracellular diffusion and is directly proportional to the concentration of the particular ion in the medium, the absolute magnitude of Cl- fluxes at [Cl-], of 20 and 100 mmol/L cannot be compared directly. However, changes in ion flux at these [Cl-], values can still be evaluated qualitatively. As shown in Table 2, there was no significant change in JFA (and G) at a [Cl-], of 100 mmol/L when Pco, was increased. At a [Cl-], of 20 mmol/L, however, increases in Pco, caused Jzk to decrease from 3.0 + 0.3 to 1.7 * 0.2 PEq. cm-‘- h-’ (P < 0.001). A concomitant and significant decrease in G also was noted. There was no effect of Pco, on Jr: at either [Cl-],. The changes in Isc, PD, and J,, and Jnetfor both Na+ and Cl- in response to increasing PCO, were similar to our previous observations.’ The overall relationship between mucosal [HCO,-Ii and JzAis shown in Figure 5. The data were obtained from the ion-flux studies in which the [Cl-],, PCO, were altered, and the correPCO3-L. f and/or sponding [HCO,-1, for each condition. Colonic JzA was highest (13.0 pEq* cm-‘- h-l) in CO,-free, HEPES-buffered Ringer’s in which [HCO,-1, was assumed to be negligible. JzA was much lower in Ringer’s containing HCO, and CO,, In Ringer’s containing a [Cl-], of 100 mmol/L, J:A was 8.3 pEq - crnm2. h-’ at an [HCO,-1, of 9 mmol/L. Thereafter, up to an [HCO,-1, of 18 mmol/L, JfA was relatively stable. J$.. then decreased at [HCO,-1, >18 between
bathed
39 is 7. The Ringer’s
-0.7
PCO, 33.7 mm
Hg by paired
Student’s
t test.
mmol/L. This reduction in J:A as [HCO,-1, was increased above 18 mmol/L was seen also in a Ringer’s solution containing a [Cl-], of 20 mmol/L, although as noted above the absolute flux rate was lower than at a [Cl-], of 100 mmol/L. Next, the modulation of J$Aby [HCO,-1, was examined in the presence of 10m4mmol/L serosal bumetanide, a known inhibitor of Cl- secretion. The rationale was as follows: if the Cl--secretory pathway modulated by [HCO,-1, was inhibited by this drug, a subsequent alteration in [HCO,-1, should have minimal or no effect on J,,. ” As shown in Table 3, bumetanide caused significant reductions in J:A and G at an [HCO,-1, of 21 mmol/L. No effect on Jr: was seen. In Ringer’s solution containing 39 mmol/L HCO,- at a Pco, of 72 mm Hg, J:j was 5.1 + 0.6 pEq - cm-‘. h-’ and was not further reduced by bumetanide. In addition, the increase in J$, noted upon reduction of Pco, from 72 to 34 mm Hg (1.2 FEq - cm-‘- h-‘: Table 1) was totally inhibited by bumetanide (-1.0 PEq. cm-2. h-‘; Table 3). Discussion Whereas stimulated intestinal Cl- secretion has been studied extensively,3 much less attention has been given to the basal Cl--secretory process apparent in various epithelia in the resting state.7-g This stems from the direct relevance of the former process to the pathophysiology of diarrhea1 disease. Although the clinical significance of the basal secretory process is unknown, the unraveling of its physiology and regulation is nevertheless essential for a more complete understanding of overall epithelial Cl- homeostasis. Furthermore, the simultaneous presence of basal secretion and absorption creates a new dimension of complexity in the interpretation of in vivo transport data measuring only net Cl- fluxes. The present study was designed to examine the basal Cl--secretory process in the distal colon of the rat. In previous studies, we found electrolyte transport across this epithelium responsive to alterations in the acid-base variables.‘~2~10~‘6These studies were
124 DAGHER ET AL.
GASTROENTEROLOGY Vol. 103, No. I
30 0
25
PC02 =22mmHg
PC02 = 72mmHg
[HCO3]0mM 5
11
21
6.7
6.9
functional in this absorbing epithelium. In addition, it suggested that this transport process was regulated by changes in the intracellular as well as the extracellular acid-base milieu. In the present study, we measured J:k under a variety of experimental conditions. Part of this flux (and in particular changes in this flux) appeared to reflect an underlying active secretory process. This was suggested by our finding that the maneuvers used to alter JFA, namely changing [HCO,-1, and Pco, or adding bumetanide, are not known to affect passive Clmovement through the paracellular shunt pathway. Furthermore, changes in J:A in our study were always accompanied by parallel changes in G. Jr:, on the other hand, remained unchanged under all conditions. Inasmuch as Jyz is a measure of paracellular ion flux, the observed variations in J:A and G are likely to represent changes in active transcellular Cl- secretion. The correlations between colonic mucosal [HCO,-1, and ion flux were justified largely because these measurements were made in identical tissue preparations. Despite its histological diversity, a colonic
g 7.1 25 0
7.3
[Cl]=lOOmM [Cl) =20mM
T
20 7.5
E ‘;; 15
Figure 3. Effect of [HCO,J, and Pco, on [HCO,Ji and pH,. Tissues were bathed consecutively in 5, 11, 21, and 39 mmol/L HCO,- Ringer’s at Pco, 32 mm Hg (n = 5) or 72 mm Hg (n = 5). At each Pco,, both [HCO,J, and pH, increased as [HCO,J, was increased (one-way analysis of variance, P < 0.001). *P < 0.05 when [HCO,-Ii or pH, at Pco, 32 mm Hg was compared with Pco, 72 mm Hg at each [HCO,J, by unpaired Student’s t test. Values are means k SE.
P 10
0 PCO3(mmHg) 21.3 to.0
34.5 iO.6
68.8 2 3.2
7.0
performed in vivo over a physiological range of systemic acid-base conditions, and in vitro where active and passive components of ion flux, pHi, and putative transport inhibitors could be examined. We found that J,, and Jnet for Na+ and Cl- were stimulated by an increase in ambient Pco,.’ This was apparently due to stimulation of apical membrane Na+/H+ and Cl-/HCO,- exchangers after intracellular hydration of CO, and subsequent generation of hydrogen and bicarbonate ions.2~10 We also found that changes in net Cl- absorption in the colon correlated with the systemic [HCO,-I.’ This effect was primarily caused by changes in J::.” This finding suggested that J:A reflected in part an active, basal secretory process that was present and
7.2 % 7.4
7.6 L Figure 4. Effect of [Cl-], and PCO, on [HCO,Ji and pH,. Five tissues were bathed consecutively in 21 mmol/L HCO,- Ringer’s containing [Cl-]. of 100 and 20 mmol/L. At each [Cl-]., the PCO, was varied between 21.3, 34.5, and 68.8 mm Hg. At each Pco,, [HCO,Ji and pH, were higher at a [Cl-], of 20 mmol/L than at 100 mmol/L (paired Student’s t test, P < 0.05). At each [Cl-]., [HCO,J, was higher and pH, was lower as Pco, increased (oneway analysis of variance, P c 0.02). Values are means + SE.
[HCO,-1,
July 1992
Table 2. Effect of CO, and [Cl-], on Colonic Electrolyte
100 mmol/L
(mmol/L)
(mm Hg)
Cl
20 mmol/L
NOTE.
PCO,
JN” @Eq.
Cl
Values
HCO;.Ringer’s
PD
(/IEq.cm-‘. h-‘)
PH.
cm-‘.
sm
ms
net
7.62 + 0.01
1.2 * 0 2
3.7 -t 0.6
7.8 f 1 1
9.0 k 0.9
70.1 i 3.2b
7.12 + 0.02b
0.6 * 0.20
2.5 t 0.7”
6.6 f 0.5
13.2 + 1.8”
21
2, 3 k 1.0
7.61 + 0.01
1.2 i 0.1
5.4 + 0.5
6.2 i 0.5
6.3 k 0.4
4.4 + 0.6
21
69.8 k 3.9*
7.13 + 0.02b
0.7 t 0.10
3.8 + 0.9’
5.1 + 0.Y
8.7 + 0.6b
4.1 k 0.6
gassed
were with
obtained
in two
tw” different
separate
CO, tensions.
groups
125
cm-‘.h-‘) JR
(mS/cm’)
WI
20.4 + 0.7
f SE. Data
Jc’ @Eq.
h-‘)
21
are means
Cl- SECRETION
G
21
consecutively
OF
Transport
kc WW.
MODUI.ATION
of tissue
The second
group
pairs.
The
consisted
net
@Eq . cm-‘.
he’)
5.0 + 0.5
3.9 k 0.8
13.5 + 1.4
8.3 k 0.3
5.2 k 1.6
2.5 + 1.0
4.6-r
8.7 i; 2.1’
16.9+
2.3
8.4 + 0.5
8.5 + 2.4’
0.5 + 0.5
2.0 k 0.7
3.9 -t 0.3
3.0 + 0.3
0.9 i 0.3
0.1 + 0.6
4.7 f O.Sb
6.6 f 0.3b
1.7 k 0.2h
4.9 + 0.4b
0.9 * 0.7
first group ofsix
sm
Ins
tissue
0.4
consisted pairs
of five tissue
treated
as described
pairs above
bathed
in 100 mmol/L
Cl-,
that 20 mmol/L
Cl- Ringer’s
except
21 mmol/L was
used. “P < 0.05. hP < 0.001 when
values
at Pco,
20 and
70 mm Hg at each
Ringer’s
[Cl-]
were
compared
on Colonic Electrolyte
Student’s
t test.
its distribution among the various cell types are not exchange process, yet known. ‘O The Cl-/HCO,which likely mediated the increases in pH, and [HCO,-Ii caused by a reduction in [Cl-],,I5 is definitely present along the apical membrane of colonic surface cell? and has been shown in crypt cells in other intestinal segmentsz2 The results of the present study strongly suggest that [HCO,-1, is the specific acid-base variable affecting Jcl . This relationship is supported by the findings thar[HCO,-1, correlated with [HCO,-1, (Figure 3) and that J,, ” decreased when [HCO,-1, was increased regardless of the prevailing Pco, and the direction of change in pH, (Table 1).A direct role for [HCO,-1, was ruled out by the finding that [HCO,-1, and Jzh were affected by changes in Pco, in the absence of changes in [HCO,-1, at an [HCO,-1, of 39 mmol/L (Table 1) or a [Cl-], of 20 mmol/L (Table 2). The additive effects of low [Cl-],, [HCO,-I,, and Pco, on JzAcan most easily be interpreted as due to a common effect of these variables on [HCO,-I,. Of note, JEL,did not incre ase despite significant increments in [HCO,-1, when [HCO,-1, was increased. This finding would appear to contradict our previous in vivo studies’ in which a significant stimulatory effect of [HCO,-1, on JzA,was seen. However, this can be explained by the symmetrical increase in bathing-solution [HCO;-] used in the flux chamber and the blood-to-lumen gradient for [HCO,-] present in vivo. In vitro, the increased mucosal bathing solution [HCO,-] decreased JgS by inhibiting mucosal
segment stripped of serosal elements is considered one functional unit when studied in the flux chamber. Fluxes are appropriately labeled “mucoSal” without further specifications because all (or most) epithelial cells undoubtedly contribute to the measured ion fluxes. Furthermore, the original separation of surface and crypt ceils as sites of absorption and secretion, respectively, is emerging as somewhat artificial if not incorrect. Recent studies have shown the existence of functional apical Cl- channels and active Cl- secretion in surface cells of rat colon,’ necturus small intestine,6 and hen intestine.17 In this regard, the tissue preparation used to measure intracellular acid-base variables was particularly suitable for comparison with the flux studies because the fluorescence was localized to the epithelial elements of the tissue (the surface cells contributing the larger part of the fluorescence). The qualitative responses of mucosal pH, and [HCO,-Ii to changes in Pco,, [HCO,-I,, and [Cl-], further support the epithelial origin of these measurements. Thus, the response of these acid-base variables to alterations in Pco, is most prominent in carbonic anhydrase-rich cells.1~‘7~‘8This enzyme has been localized to both the surface and crypt epithelium of the colon but not to nonepithelial colonic elements.” The increase in [HCO,-Ii caused by increasing [HCO,-1, is most likely caused by basolateral Nafdependent HCO,- entry into the cell. This process has been identified in rat colonic epithelium, although the exact nature of this transport process and
Table 3. Effect of Bumetanide
by paired
Transport J”‘(IIEq~cm~‘.h~‘)
kc [HCO,].
PCO,
(_uEq cm-‘.
(mmol/L)
(mm Hg)
PH,
h-‘)
(mS/cm’)
WI
CO”trOl
21
34.2 i 0.9
7.40 + 0.01
2.3 f 0.3
Bumetanide
21
34 2 t 0.9
7.40 e 0.01
1.4 * 0.2b
7.7 * 1.5
Control
39
71.5 + 0.7
7.33 * 0.02
1.5 + 0.3
8.1 i 2.2
Bumetanide
39
71.5 t 0.7
7.33 i 0.02
1.2 * 0.3
8.7 k 2.4
Bumetanide
39
33.9 + 0.2
7.66 zk 0.01
0.9 k 0.3
6.6 + 2.6
4.5 f 0.3
NOTE. group
Values consisted
reservoirs
are means
-+ SE. Data were
of five tissue
pairs
at a final concentration
“P < 0.01, hP < 0.05 compared
bathed
obtained
in two separate
in 39 mmol/L
10.3 k 0.5
groups
HCO;-Ringer’s
the preceeding
condition
by paired
ms
net
sm
net
@Eq . cm-‘.
h-’
6.1 k 0.5
7.3 k 1.6
2.8 f 0.2
4.6 + 1.7
13.7 k 1.7
7.4 k 0.4
6.3 + 1.9
4.0 k 0.9
5.2 + 0.3b
7 9 i 1.5
2.6 + 0.5
5.3 k 1.9
14.3 + 1.9
6.0 k 0.4”
6.3 k 2.10
4.4 + 0.8
5.3 * 0.4
10.7 ? 1.9
2.3 k 0.3
8.4 + 1.9
15.4 +- 1.9
5.1 k 0.6
10.2 + 2.1
3.4 k 0.6
4.5 + 0.3O
10.1 + 1.4
2.2 i- 0.5
7.9 + 1.8
13.8 zk 1.2
5.7 + 1.2
8.0 k 1.6
1.3 + 1.1
8.6 k 1.7
2.2 k 0.4
6.5 + 1.6
11.8 + 2.0
4.7 It 0.7
7.1 + 1.9
1.6 + 1.6
of tissue
pairs.
The first group
and consecutively Student’s
sm
Ins
of lo-’ mol/L. with
JR
G
PD
t test.
gassed
with
consisted two different
of four tissue CO, tensions.
pairs
bathed
In both
in 21 mmol/L groups,
bumetanide
HCO;.Ringer’s was added
The second to the semsal
I
126
DAGHER ET AL.
GASTROENTEROLOGY
Cl-/HCO,e xc h ange, and this inhibition offset the effect of decreasing JzA on J$. We also considered the possibility that colonic mucosal pH, modulated Jz$,. Intracellular acidosis is known to inhibit basolateral membrane K+ channels, depolarize the cell, and thus diminish the driving force for Cl- secretion.‘5*23 However, in the present study JzAdecreased both when pH, increased (Table 1; Figure 3) and when pH, decreased (Tables 1 and 2; Figures 3 and 4). The quantitative effect of [HCO,-Ii on JzAalso was not affected by the direction of change in pH,. Nevertheless, as noted above, under certain physiological conditions not examined in this study, alterations in colonic mucosal pHi could influence
JCl . ““The relationship between [HCO,-1, and J,Cb,shows a pattern common to many physiological systems. J:’ is most sensitive to changes in [HCO,-1, above and below a range that encompasses the “normal” [HCO,-1, .I0 Th is range or plateau that extends from approximately 9 mmol/L to 18 mmol/L is associated with a stable JzA of 8.3 ~Eq.cm-2.h-’ when measured in a Ringer’s solution containing 21 mmol/L HCO,- and 100 mmol/L Cl- (Figure 5). The presence of this plateau may explain why moderate increases in Pco, or [HCO,-1, do not affect J,“b,when colonic fluxes are studied in Ringer’s of similar composition.
Vol. 103, No. 1
The nature of this colonic Cl--secretory pathway has not been systematically examined. However, by analogy with other epithelia active Cl- secretion in the colon occurs by Cl- entry via a basolateral membrane Na+-K+-2Cll cotransport process and Cl- exits via an apical membrane anion conductance.3’24 The lack of effect of [HCO,-1, on J:j in the presence of serosal bumetanide suggests that it is indeed this transport pathway that is modulated by [HCO,-Ii. The exact mechanism by which [HCO,-1, affects this Cl--secretory pathway remains uncertain. Clchannels in various epithelia may be quite permeable to HC03-.25,26 Thus, it is conceivable that changes in [HCO,-1, inversely affect Cl- flow through these channels. A second possibility is that alterations in [HCO,-1, change the proportion of mucosal membrane anion exchangers operating as Cl-/ Cl- self-exchange and Cl-/HCO,heteroexchange. This phenomenon has been demonstrated in the intercalated cells of the mammalian collecting tubulez7 but has not been studied in the colonic epithelium. No direct effect of [HCO,-1, on the basolateral membrane Na+-K+-2Cll cotransporter can be postulated. This cotransporter has no known sensitivity to pH or base gradients.28 Of course, [HCO,-1, could have an indirect effect at either the apical or the basolateral membrane. In this regard, [HCO,-1, modulation of intermediates such as cellular calcium or protein phosphorylation needs further investigation. In summary, we have described the modulation of an active, basal Cl--secretory process in the rat colon by mucosal [HCO,-Ii. This secretory process is part of the J,, measured under short-circuit conditions in The relationship between the Ussing chamber. [HCO,-1, and Jfk is triphasic; these variables are inversely related on either side of a plateau that encompasses the normal colonic mucosal [HCO,-1,. The shape of the [HC03-li/J2A relationship may account for the presence of intestinal Cl- preservation during clinical metabolic alkalosis and intestinal Cllosses during metabolic acidosis.‘~2*‘0*‘6 References 1. Charney
2.
L
1
I
I
I
I
0
5
IO
15
20
25
I
30
3.
[HCOs]i mM Figure 5. Effect of [HCO,-1, on JzA. Data for JzA were obtained from Tables 1 and z and plotted against the corresponding [HCO,-]I obtained from Figures 3 and 4. In CO,-free, HEPES-buffered Ringer’s, [HCO,-1, was assumed to be negligible. The relationship between [HCO,-Ii and JzA is triphasic. Values are means + SE.
4.
5.
AN, Haskell LP. Relative effects of systemic pH, PCO,, and bicarbonate concentration on colonic ion transport. Am J Physiol 1984;246:G159-G165, Goldfarb DS, Egnor RW, Charney AN. Effects of acid-base variables on ion transport in rat colon. J Clin Invest 1988;81:19031910. Binder HJ, Sandle GI. Electrolyte absorption and secretion in the mammalian colon. In: Johnson LR, ed. Physiology of the Gastrointestinal Tract. New York: Raven, 1987:1389-1417. Welsh MJ, Smith PL, Fromm M, Frizzell RA. Crypts are the site of intestinal fluid and electrolyte secretion. Science 1982;218:1219-1221. Diener M, Rummel W, Mestres P, Lindemann B. Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat. J Membrane Biol 1989;108:21-30.
July 1992
[HCO,-1, MODULATION
6. Giraldez F, Sepulveda
19.
1988;395:597-623. 7. Charney AN, Donowitz
20.
FV, Sheppard DN. A chloride conductance activated by adenosine 3’,5’-cyclic monophosphate in the apical membrane of Necturus enterocytes. J Physiol M. Functional significance of intesin vivo ouabain inhibition. Am J Physiol
8.
9.
10.
11.
12.
13.
14. 15.
16.
tinal Na-K-ATPase: 1978;234:E629-E636. Davis GR, Santa Ana CA, Morawski S, Fordtran JS. Active chloride secretion in the normal human jejunum. J Clin Invest 1980;66:1326-1333, O’Grady SM, Wolters PJ. Evidence for chloride secretion in the intestine of the winter flounder. Am J Physiol 1990;258:C243-C247. Wagner JD, Kurtin P, Charney AN. Effect of systemic acidbase disorders on colonic intracellular pH and ion transport. Am J Physiol 1985;249:G39-G47. Ganz MB, Boyarsky G, Boron WF, Sterzel RB. Effects of angiotensin II on intracellular pH of glomerular mesangial cells. Am J Physiol 1988;254:F787-F794, Thomas JA, Buchsbaum RN, Zimniak A, Racker E. Intracellular pH measurements in Ehlrich Ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 1979;18:2210-2218. Graber ML, DiLillo DC, Friedman BL, Pastoriza-Munoz E. Characteristics of fluoroprobes for measuring intracellular pH. Anal Biochem 1986;156:202-212. Snedecor GW, Cochran WG. Statistical methods. Ames, IA: Iowa State University, 1987. Duffey ME, Devor DC. Intracellular pH and membrane potassium conductance in rabbit distal colon. Am J Physiol 1990;258:C336-C343. Charney AN, Feldman GM. Systemic acid-base disorders and intestinal electrolyte transport. Am J Physiol 1984;247:Gl-
G12. 17. Holtug K, Shipley A, Dantzer V, Sten-Knudsen
0, Skadhauge E. Localization of sodium absorption and chloride secretion in an intestinal epithelium. J Membrane Biol 1991;122:215-229, 18. Charney AN, Wagner JD, Birnbaum GJ, Johnstone JN. Functional role of carbonic anhydrase in intestinal electrolyte transport. Am J Physiol 1986;251:G159-G165.
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28.
OF Cl- SECRETION
127
Lonnerholm G, Midtvedt T, Schenholm M, Wistrand PG. Carbonic anhydrase isoenzymes in the caecum and colon of normal and germ-free rats. Acta Physiol Stand 1988;132:159-166. Feldman GM, Koethe JD, Stephenson RL. Base secretion in rat distal colon: ionic requirements. Am J Physiol 1990;258: G825-G832. Binder HJ, Foster EF, Budinger ME, Hayslett JP. Mechanism of sodium chloride absorption in distal colon of the rat. Gastroenterology 1987;93:449-455, Knickelbein RG, Aronson PS, Dobbins JW. Membrane distribution of sodium-hydrogen and chloride-bicarbonate exchangers in crypt and villus cell membranes from rabbit ileum. J Clin Invest 1988;82:2158-2163. Christensen 0, Zeuthen T. Maxi K channels in leaky epithelia are regulated by intracellular Ca’+, pH and membrane potential. Pflugers Arch 1987;408:249-259. Dharmasathaphorn K, Mandel KG, Masui H, McRoberts JA. Vasoactive intestinal polypeptide-induced secretion by a coionic epithelial cell line. Direct participation of a basolaterally-localized Na, K, Cl cotransport system. J Clin Invest 1985;77:348-354. Kunzelmann K, Gerlach L, Frobe U, Greger R. Bicarbonate permeability of epithelial chloride channels. Pflugers Arch 1991;417:616-621. Tabcharani JA, Jensen TJ, Riordan JR, Hanrahan JW. Bicarbonate permeability of the outwardly rectifying anion channel. J Membrane Biol 1989;112:109-122. Schuster VL, Stokes JB. Chloride transport by the cortical and outer medullary collecting duct. Am J Physiol1987;253:F203F212. Haas M. Properties and diversity of (Na-K-Cl) cotransporters. Annu Rev Physiol 1989:51:443-457.
Received August 23, 1991. Accepted January 7, 1992. Address requests for reprints to: Alan N. Charney, M.D., Nephrology Section, Department of Veterans Affairs Medical Center, 423 East 23rd Street, New York, New York 10010. Supported by the General Medical Research Program of the Department of Veterans Affairs.