AMERICAN
JOURNAL
OF
Vol. 231, No. 6, December
PHYSIOLOGY 1976.
Printed
in U.S.A.
Ion transport across isolated mucosa of the rabbit
antral
DAVID FROMM, JOHN H. SCHWARTZ, RUSSELL ROBERSTON, AND Department of Surgery, Harvard Medical School, and Beth Israel Hospital, Boston, Massachusetts 02215; and Department of Nephrolugy, Walter Reed Army Institute of Research, Washington, D.C. 20012
FROMM,DAVID,JOHN H. SCHWARTZ,RUSSELL ROBERTSON, AND ROBERT FUHRO. Ion transport across isolated antral mu.1976. cosa ofthe rabbit. Am. J. Physiol, 231(6): 17834789. Isotopic fluxes of Na, Cl, and K were measured across isolated antral mucosa under short-circuit conditions. HCO, fluxes were also measured with either isotopic and/or pH-stat methods. Net secretion of all four ions was observed. HCU, secretion is due to a transmural process requiring metabolic energy. Secretion of endogenous HCO, was not observed, and the unidirectional mucosal-to-serosal flux of HCO, was negligible. There appears to be a close relationship between HCO, secretion and the unidirectional mucosal-to-serosal Cl flux, but no relationships were observed between the unidirectional serosal-to-mucosal flux or either unidirectional Na flux. The bulk of HCO, secretion is independent of the unidirectional Cl fluxes, but there is a fraction of HCO, transport that is dependent on unidirectional Cl transport, However, HCO, transport is not measurably influenced by inhibition of net Cl (and Na) transport per se. stomach, antrum; HC@ secretion; acetylcholine; norepinephrine
ouabain;
ethacrynic
acid;
MAJORITY OF INVESTIGATIONS dealing with gastric antrum have been confined to factors relating to the release of gastrin. Ion transport processes of the antrum have been dealt with by comparatively few studies. Most of these have been concerned with permeability of the mucosa (2, 4, 8, 9, 12, 25) and few have been involved with active transport processes (6, 9). Active transport of Na, Cl, and HCO, by amphibian antrum have been described only recently (6), but there is little information for mammalian antrum. This study examines Na, K, Cl, and HCO, transport across isolated antral mucosa of the rabbit. THE
New Zealand white rabbits (l-3 kg), which had been allowed free access to a standard diet and water, were killed by a sharp blow to the neck. The antrum was immediately excised and rinsed with a Ringer solution. The serosa and muscularis were sharply stripped away, and the mucosa with its thin layer of muscularis mucosa was clamped between half chambers perfused with gas-lift circulating systems maintained at 37.5”C, In the majority of experiments both sides of the mucosa
FUHRO
were initially bathed with an identical Ringer solution that was bubbled with 100% O2 and contained the following in millimoles per liter: Na, 136; K, 5; Ca, 0.5; Mg, 1.2; Cl, 144.4; and glucose, 10. All tissues were short-circuited. Short-circuit current (I,,) and tissue electrical resistance (R) were determined as described previously (10). In a few experiments, the mucosa was bathed in Na isethionate, 144 mM. When NaHC03 was added either to the mucosal or serosal bathing solutions, an equimolar concentration of mannitol was added to the opposite bathing solution. Unless stated to the contrary, the HCO:, concentration was 25 mM. In the majority of instances the solution containing HCO, was bubbled with 95% O,-5% C02. Steady-state unidirectional mucosal-to-serosal (mto-s) and serosal-to-mucosal (s-to-m) fluxes of Na were either measured simultaneously across the antrum by labeling the reservoir on one side of the tissue with 22Na and the other side with 2”Na or measured across adjacent segments of stomach by labeling opposite sides of the tissue with 22Na. Unidirectional fluxes of Cl were measured simultaneously by labeling opposite sides of adjacent, mucosal segments with “YX 22Na was also added to alternate sides of these paired tissues so that the 22Na and 36C1were always present on the same side of the tissue. The fluxes were measured over 3% to 45min intervals beginning at least lo-15 min after the addition of the isotopes. In some experiments two 35- to 45-min flux determinations were made. When a given agent was added to one bathing solution, an equimolar concentration of mannitol was added to the opposite bathing solution. Duplicate samples were removed from both the m and s reservoirs at the beginning and end of each flux measurement, or l-ml samples were removed
at lo-min
initially METHODS
ROBERT
intervals
from
the
reservoir
not
labeled with isotope and were replaced with
isotope-free
solution,
24Na and 22Na were assayed in a gamma spectrometer. 22Na was assayed 2 wk after the assay of z4Na. The sum of the 22Na and 36C1was assayed in a liquid scintillation spectrometer. The activity of z6C1was obtained by subtracting the activity of 22Na, which was determined
by gamma
counting
and corrected
for efficiency
between the spectrometers. In triple-labeled experiments (24Na, 22Na, and 36C1),the unidirectional fluxes of Na were determined for each tissue and then averaged in order to give a single value for the tissue pair.
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1784
FROMM,
The net flux of each tissue or tissue pair is the arithmetic difference between the unidirectional m-to-s and s-to-m fluxes. The rate of alkali appearance in the mucosal or serosal bathing solution was titrated at pH 7 by a pH stat with HCl, 0.01 N, as the titrant. Unidirectional mto-s or s-to-m fluxes of H14C03 were also measured. The isotope was added to the bathing solution containing HC03, 25 mM, and both sides of the mucosa were bubbled with 100% 0, that was passed through a KOH, 3 M, trap. The rate of alkali appearance in the opposite bathing solution was continuously titrated with a pH stat to maintain the pH at 6,O during these H14C0, flux measurements. Preliminary experiments indicated that there was no significant difference in the rate of alkali appearance titrated at pH 7 and pH 6. The lower pH was used during these experiments to minimize 14C trapping in the bathing solution. The gas phase of the sampling reservoir was separately collected in vials containing Hyamine 10X over a 30- to 40-min interval. When H14C03 flux measurements were made in the absence of a HCO, gradient across the mucosa (no titration was performed during these experiments), samples from the bathing solutions were added to their respective Hyamine 10X traps. 14C was analyzed in a liquid scintillation spectrometer. The individual rates of CO, appearance on the mucosal and serosal sides of the antrum were measured by a conductometric method (lo), and lactate appearance was measured by a lactate dehydrogenase method (10).
The rates of spontaneous Na, Cl, and K transport across antral mucosa bathed in HCO,-free Ringer solution are shown in Table 1. Antral mucosa secretes Na and Cl. The net flux of Cl is approximately 3 times greater than that observed for Na. The sum of the net Na and Cl fluxes does not differ significantly (P > 0.5) from the I,,, suggesting the absence of a significant residual flux (J:&. However, in a separate group of tissues a net secretory flux of K (P < 0,001) was observed, but the rate of K secretion is negligible with respect to the rates of Na and Cl secretion. Na and Cl secretion also occur when both sides of antral mucosa are bathed in a Ringer solution containing HCO, (Table 1). The rates of Na and Cl transport do not appear to differ substantially from those obtained in the absence of HCO,. However, in contrast to 1. Unidirectional n
JYa
and net Na, CL, and K fluxes across JNa sm
ms
JNa
J”’
F’ ms
nel
33X3-free 45
2.4 + 0.1
3.7 2 0.1
20
0.12 -+ 0.01 JN” ms
0.24 4 0.03
2.1 2 0.2
3.6 t 0.3
-1.3
-t 0.1
-0.12
net
Values are means * 1 SE. Measurement Negative value for Jnet indicates secretion.
-1.5
J”’
sm
Ringer
2 0.2
7.3 t 0.3
JC’ms
JC’ Em
nei
I SC
JL
-4.0
k 0.3
2.8 k 0.1
0.1 4 0.3
2.3 -+ 0.1
3.5 + 0.3
Ringer
FUHRO
R, slcm2
PD, mV
170.2 k 8.1
12.8 2 3.3
155.8 k 6.7
9.6 2 2.8
solution
3.3 2 0.2
HCO, 10
AND
antrum
5 0.03 JNa
J%
ROBERTSON,
flux measurements made in the absence of HC03, a significant (P < 0.025) residual flux was observed in the presence of HC03, suggesting the presence of HC03 secretion. When both sides of the mucosa are bathed in HCO,free Ringer solution bubbled with 100% 02, appearance of titratable acid may occur in the mucosal bathing solution. The rate of acid appearance did not exceed 0.26 peq/h cm* and was observed for approximately 43% of the tissue studied. UJhen mucosal appearance of acid occurred, it remained steady for at least 1 h and was not influenced by bubbling both sides of the mucosa with 100% N, (9). Serosal alkalinization was not observed. Bubbling the serosal side with 5% CO,-95% 0, resulted in the transient appearance of acid on the mucosal side (lo), but subsequently neither acid nor alkali appeared. The rate of CO, appearance on the mucosal side of eight tissues, 8,3 t 0.6 PM/h cm2 (means 2 1 SE), was not significantly different (P > 0.4) from that appearing on the serosal side, 7.8 t 0.7 PM/h cm? The luminal rate of lactate appearance for 16 tissues was 0.14 t 0.01 FM/h cm2 in the mucosal bathing solution and 0.18 * 0.01 PM/h cm2 in the serosal bathing solution. The rate of pyruvate appearance in the mucosal bathing solution of antrum has been shown to be negligible with respect to the rate of lactate appearance (12). Thus, it appears that isolated antral mucosa of the rabbit does not actively secrete acid, and when acid appears in the mucosal bathing solution it most likely is due to the release of lactic acid by the tissue. Occasionally, the spontaneous appearance of alkali in the mucosal or serosal bathing solution occurs, but this disappears within approximately 10 min after mounting the tissue in vitro. Addition of HCO, to the serosal bathing solution is followed by the appearance of alkali in the mucosal bathing solution. The mean rate of alkali appearance in the mucosal bathing solution of 50 tissues was 0.99 t 0.08 peq/h cm? In 15 experiments, the rate of alkali appearance in the mucosal bathing solution was not measurably influenced (P > 0.4) by changing the gas phase of the serosal bathing solution from 100% 0, to 95% 02-5% COZ, which changed the pH from approximately 8.1 to 7.4. Furthermore, in 10 experiments the rate of alkali appearance in the mucosal bathing solution was not significantly influenced (P > 0.4) by reducing the HC03 concentration from 25 to 15 mM, which changed the pH from approximately 7.4 to 7.1. In contrast to the addition of HCO, to the serosal
RESULTS
TABLE
SCHWARTZ,
JC1 net
solution
6.5 k 0.4
-3.0
of fluxes is in peqlh crn2. n, number of rabbits. J,,, unidirectional I,,, short-circuit current 12 JFe:,,,I,, - (“Jr,$ t “J$). R, electrical
2 0.4
2.6 k 0.1
1.1 t 0.4
mucosal-to-serosal flux. J,,, unidirectional resistance, PD, potential difference.
174.1 t 10.2 s-to-m
flux.
12.2 A 3.8 J,,, = J,,
- J,,.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
ION
TRANSPORT
ACROSS
ANTRUM
1785
bathing solution, addition of HCO, to the mucosal bathing solution resulted in the titratable appearance of alkali on the serosal side of only 0.07 -+ 0.05 peq/h cm2 (n, 10, P > 0.2). In 10 experiments HCO, was placed in the serosal bathing solution and a unidirectional flux of H14C0, was measured simultaneously with titration of alkali appearance in the mucosal bathing solution. The rate of isotopically measured HCO, secretion was 1.18 t 0.20 peq/h cm2 and the titratable appearance of alkali was 0.80 t 0.15 peq/h cm2. The difference between these two values, 0.38 t 0.10 peq/h cm2, is statistically significant (P < 0.01). In five experiments, the opposite flux was measured. Bicarbonate was placed in the mucosal bathing solution and a unidirectional m-to-s H14C0, flux was measured simultaneously with titration of the serosal bathing solution. The unidirectional m-to-s H14C0, flux was 0.38 t 0.2 peq/h cm2, whereas the titratable appearance of serosal alkali was 0.07 k 0.05 peq/h cm2. Therefore, it appears that the difference between the isotopically determined flux and that measured by titration probably is due to conversion of HCO, to CO,. In part, this may be due to some titration by release of organic acid, (e.g., lactic acid) as well as due to equilibration of HCO, with 14C02 in solution. In five experiments the rate of titratable HCO, secretion,l 1.01 L 0.14 peq/h cm2, was reduced to zero when the gas bubbling both sides of the tissues was changed from 100% 0, to 100% N,. Rebubbling with 100% 0, lo- to 15-min later resulted in the return of HCO, secretion in four of five tissues and was not significantly different (P > 0.2) from that observed prior to gassing with N,. The electrical resistance increased by 52.2 t 4.8 ficm2 in the presence of Ne, and after rebubbling with 02, the resistance was not significantly different from that observed prior to starting N2 (P > 0.2). In an additional five experiments, antral mucosa was bathed in a Ringer solution (7) containing HCO, on both sides of the tissue that was short-circuited during measurement of a unidirectional s-to-m H14C0, flux, Under these conditions the mean flux was 1.15 t 0.14 peq/h cm2. A unidirectional m-to-s H14C03 flux was also measured across two tissues, and the rates of transport were 0.21 and 0.35 peq/h cm? Although no obvious differences in the magnitudes of the unidirectional or net Na and Cl fluxes are apparent when antral mucosa is bathed in the absence or presence of HC03, a separate series of experiments were performed in which the effects of HCO, secretion on the unidirectional fluxes of Na and Cl were individually measured. A unidirectional flux of either 22Na or 36C1 was initially measured over a 30-min interval for mucosae bathed in HC03-free Ringer solution. Subsequently, HCO, was added to the serosal bathing solution, which was then bubbled with 95% O,-5% C02, and the isotopic flux measurement was continued lo-15 min later. Analysis of flux measurements made every
10 min (n, 3 for each unidirectional flux of Na and Cl) before and after HCO, addition indicated the presence of steady-state rates of isotopic transfer. Titration of the luminal bathing solution was performed during both flux measurements in order to maintain the pH at 7. The addition of HCO, to 12 tissues was followed by a significant increase in the unidirectional m-to-s Cl flux (Table 2). A statistical correlation existed between the rate of HCO, secretion and the increase in unidirectional m-to-s flux (Fig. 1). In contrast, the unidirectional s-to-m Cl flux or either unidirectional Na flux was not significantly altered after the addition of HC03 (Table Z), These data imply that HCO, secretion is associated with a decrease in net Cl secretion2 The I,, does not change significantly (-0.39 -t 0.24 peq/h cm2; P > 0.1, n, 46) after HCO, is added to the serosal bathing solution. Furthermore, the tissue electrical resistance also does not change significantly under these conditions (4.6 -+ 5.2 flcm2, P > 0.4, n, 46). No significant changes in I,, or tissue electrical resistance were observed when HCOz, 25 mM, was added to the mucosal bathing solution (P > 0.5, for each, n, 10). In a separate series of experiments, both sides of antral mucosa were bathed in Na isethionate solution, and the mucosal side was titrated with HZS04, 0.01 N. In seven tissues no alkali appeared on the mucosal side. However, after the addition of HCO, to the serosal side an alkali secretory rate of 1,21 t 0.09 r.Leg/h cm2 was observed. The rate of HCO, secretion did not appear to be measurably influenced by changing the gas phase on the serosal side from 100% O2 to 95% O,5% CO,. In five of these tissues the isethionate solution was replaced with a standard Ringer solution containing HC03 on the serosal side. In the remaining two tissues the isethionate solution was maintained, but choline Cl, 100 mM, was added to the serosal side. In each instance there was an increase in HCO, secretion, the mean of which for these seven tissues was 0.57 * 0.07 peq/h cm2 (P < 0.001). In three experiments, changing the gas phase to 100% N, resulted in cessation of HC03 secretion by mucosa bathed in isethionate solution. In order to d etermi ne if ion transport by antral’mucosa is altered by ouabain, ethacrynic acid, acetylcholine, and norepinephrine, as has been reported for other tissues, the effects of these agents on the&, were initial1 -Y measured. Each of these agents causes a decrease in .Z,,. Figure 2 shows the effects of that concentration of agent added to the serosal side” which caused a maximal decrease in I,, of antrum bathed in HCO,free Ringer solution. Lower concentrations of ethacrynic acid, lo-” to 10B4M, acetylcholine, 10s4to 10m5M, and norepinephrine, 10m4to lo+ M, caused lesser declines in Z,,. The effect of acetylcholine was inhibited by pretreatment with atropine (Fig. Z), but atropine did not prevent the effects of ouabain, ethacrynic acid, or norepinephrine.
1 “HC03 secretion” is used without distinguishing between alterations in OH and H fluxes that could bring about alkali secretion. Furthermore, no distinction is made between transmural flux of HCO, and extrusion into the lumen.
2 The most likely reason that statistically significant differences were not noted for J$,\ and JE& of Table 1 measured in the absence and presence of HCOs is that these were unpaired experiments. 3 The fact that these agents are effective on the serosal side does not a priori determine the locus or orientation of the secretory pumps (23).
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1786
FROMM,
Measurement of spontaneous Na and Cl fluxes over two 30- to 40-min intervals indicated that the fluxes, Isc, and R did not change significantly (Table 3). In a separate group of tissues, ouabain, 10s6 M, was added to the serosal bathing solution after base-line fluxes were measured. Ouabain inhibited the net fluxes of Na and Cl without causing measurable alterations in the unidirectional m-to-s fluxes of Na and Cl, JEet, or R (Table 3). Since there was a relatively prolonged delay before the I,, reached a plateau after the addition of ethacrynic acid, acetylcholine, or norepinephrine (Fig. 2), each tissue was not used as its own control for Na and Cl flux measurements, Antral mucosa from the same TABLE 2. Effects of HCO, secretion unidirectional Cl and Na fluxes
SCHWARTZ,
ROBERTSON,
AND
FUHRO
rabbit could not be used for measurement of control fluxes, since rabbit antrum is small and only two segments per rabbit could be used. Therefore, control flux measurements were made on tissues from a separate group of animals, but were begun at a time interval approximating that at 0*5
0
n, number of rabbits. AJ, difference (mean 2 1 SE) between flux measured before and after HC@ addition to serosal bathing solution. J,,, unidirectional mucosal-to-serosal flux. J,,, unidirectional s-to-m flux. PAJ, P value for AJ (Student t test for paired variates). Measurement of fluxes is in peq/h cm?
I
2 JHC03 SM
4
3
,A Eq/hr cm2
FIG. 1. Increase in unidirectional m-to-s Cl flux (AJ$,\) as a function of HCO, secretion. Line drawn by method of least squares. Significance of r is 0.2) was detected in the presence of ethacrynic acid. Norepinephrine also inhibited net Na and Cl secretion, but in contrast to ethacrynic acid and acetylcholine, the unidirectional fluxes of Na and Cl were at least half of those observed for control tissues. Furthermore, the electrical resistance in the presence of norepinephrine was strikingly greater than that observed for control tissues, A similar increase in electrical resistance. was noted in five of the tissues shown in Fig. 2 in which this measurement was made. The experiments shown in Tables 3 and 4 were repeated with HCO, present on both sides of the mucosa. The effects of ouabain (12, 6), ethacrynic acid (n, 5), acetylcholine (n, 5), and norepinephrine (n, 8) on Na and Cl transport were the same as those shown in the tables. However, statistically significant residual ion TABLE Period
of uuabain
3. Effects J""
ms
JN"
on ion transport
sm
JNa net
1.9 1*7 -0.2 4 0.1 >0.05
3.2 2.8 -0.4 k 0.3 >O.Ol
-1.3 -1.1 0*2 * 0.3 BO.5 Ouabain
I II A P
2.2 2.0 -0.2 f 0.1 >0.05
3.6 2.0 -1.6 k 0.2
JOURNAL
OF
Vol. 231, No. 6, December
PHYSIOLOGY 1976.
Printed
in U.S.A.
Ion transport across isolated mucosa of the rabbit
antral
DAVID FROMM, JOHN H. SCHWARTZ, RUSSELL ROBERSTON, AND Department of Surgery, Harvard Medical School, and Beth Israel Hospital, Boston, Massachusetts 02215; and Department of Nephrolugy, Walter Reed Army Institute of Research, Washington, D.C. 20012
FROMM,DAVID,JOHN H. SCHWARTZ,RUSSELL ROBERTSON, AND ROBERT FUHRO. Ion transport across isolated antral mu.1976. cosa ofthe rabbit. Am. J. Physiol, 231(6): 17834789. Isotopic fluxes of Na, Cl, and K were measured across isolated antral mucosa under short-circuit conditions. HCO, fluxes were also measured with either isotopic and/or pH-stat methods. Net secretion of all four ions was observed. HCU, secretion is due to a transmural process requiring metabolic energy. Secretion of endogenous HCO, was not observed, and the unidirectional mucosal-to-serosal flux of HCO, was negligible. There appears to be a close relationship between HCO, secretion and the unidirectional mucosal-to-serosal Cl flux, but no relationships were observed between the unidirectional serosal-to-mucosal flux or either unidirectional Na flux. The bulk of HCO, secretion is independent of the unidirectional Cl fluxes, but there is a fraction of HCO, transport that is dependent on unidirectional Cl transport, However, HCO, transport is not measurably influenced by inhibition of net Cl (and Na) transport per se. stomach, antrum; HC@ secretion; acetylcholine; norepinephrine
ouabain;
ethacrynic
acid;
MAJORITY OF INVESTIGATIONS dealing with gastric antrum have been confined to factors relating to the release of gastrin. Ion transport processes of the antrum have been dealt with by comparatively few studies. Most of these have been concerned with permeability of the mucosa (2, 4, 8, 9, 12, 25) and few have been involved with active transport processes (6, 9). Active transport of Na, Cl, and HCO, by amphibian antrum have been described only recently (6), but there is little information for mammalian antrum. This study examines Na, K, Cl, and HCO, transport across isolated antral mucosa of the rabbit. THE
New Zealand white rabbits (l-3 kg), which had been allowed free access to a standard diet and water, were killed by a sharp blow to the neck. The antrum was immediately excised and rinsed with a Ringer solution. The serosa and muscularis were sharply stripped away, and the mucosa with its thin layer of muscularis mucosa was clamped between half chambers perfused with gas-lift circulating systems maintained at 37.5”C, In the majority of experiments both sides of the mucosa
FUHRO
were initially bathed with an identical Ringer solution that was bubbled with 100% O2 and contained the following in millimoles per liter: Na, 136; K, 5; Ca, 0.5; Mg, 1.2; Cl, 144.4; and glucose, 10. All tissues were short-circuited. Short-circuit current (I,,) and tissue electrical resistance (R) were determined as described previously (10). In a few experiments, the mucosa was bathed in Na isethionate, 144 mM. When NaHC03 was added either to the mucosal or serosal bathing solutions, an equimolar concentration of mannitol was added to the opposite bathing solution. Unless stated to the contrary, the HCO:, concentration was 25 mM. In the majority of instances the solution containing HCO, was bubbled with 95% O,-5% C02. Steady-state unidirectional mucosal-to-serosal (mto-s) and serosal-to-mucosal (s-to-m) fluxes of Na were either measured simultaneously across the antrum by labeling the reservoir on one side of the tissue with 22Na and the other side with 2”Na or measured across adjacent segments of stomach by labeling opposite sides of the tissue with 22Na. Unidirectional fluxes of Cl were measured simultaneously by labeling opposite sides of adjacent, mucosal segments with “YX 22Na was also added to alternate sides of these paired tissues so that the 22Na and 36C1were always present on the same side of the tissue. The fluxes were measured over 3% to 45min intervals beginning at least lo-15 min after the addition of the isotopes. In some experiments two 35- to 45-min flux determinations were made. When a given agent was added to one bathing solution, an equimolar concentration of mannitol was added to the opposite bathing solution. Duplicate samples were removed from both the m and s reservoirs at the beginning and end of each flux measurement, or l-ml samples were removed
at lo-min
initially METHODS
ROBERT
intervals
from
the
reservoir
not
labeled with isotope and were replaced with
isotope-free
solution,
24Na and 22Na were assayed in a gamma spectrometer. 22Na was assayed 2 wk after the assay of z4Na. The sum of the 22Na and 36C1was assayed in a liquid scintillation spectrometer. The activity of z6C1was obtained by subtracting the activity of 22Na, which was determined
by gamma
counting
and corrected
for efficiency
between the spectrometers. In triple-labeled experiments (24Na, 22Na, and 36C1),the unidirectional fluxes of Na were determined for each tissue and then averaged in order to give a single value for the tissue pair.
Downloaded from www.physiology.org/journal/ajplegacy by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 16, 2019.
1784
FROMM,
The net flux of each tissue or tissue pair is the arithmetic difference between the unidirectional m-to-s and s-to-m fluxes. The rate of alkali appearance in the mucosal or serosal bathing solution was titrated at pH 7 by a pH stat with HCl, 0.01 N, as the titrant. Unidirectional mto-s or s-to-m fluxes of H14C03 were also measured. The isotope was added to the bathing solution containing HC03, 25 mM, and both sides of the mucosa were bubbled with 100% 0, that was passed through a KOH, 3 M, trap. The rate of alkali appearance in the opposite bathing solution was continuously titrated with a pH stat to maintain the pH at 6,O during these H14C0, flux measurements. Preliminary experiments indicated that there was no significant difference in the rate of alkali appearance titrated at pH 7 and pH 6. The lower pH was used during these experiments to minimize 14C trapping in the bathing solution. The gas phase of the sampling reservoir was separately collected in vials containing Hyamine 10X over a 30- to 40-min interval. When H14C03 flux measurements were made in the absence of a HCO, gradient across the mucosa (no titration was performed during these experiments), samples from the bathing solutions were added to their respective Hyamine 10X traps. 14C was analyzed in a liquid scintillation spectrometer. The individual rates of CO, appearance on the mucosal and serosal sides of the antrum were measured by a conductometric method (lo), and lactate appearance was measured by a lactate dehydrogenase method (10).
The rates of spontaneous Na, Cl, and K transport across antral mucosa bathed in HCO,-free Ringer solution are shown in Table 1. Antral mucosa secretes Na and Cl. The net flux of Cl is approximately 3 times greater than that observed for Na. The sum of the net Na and Cl fluxes does not differ significantly (P > 0.5) from the I,,, suggesting the absence of a significant residual flux (J:&. However, in a separate group of tissues a net secretory flux of K (P < 0,001) was observed, but the rate of K secretion is negligible with respect to the rates of Na and Cl secretion. Na and Cl secretion also occur when both sides of antral mucosa are bathed in a Ringer solution containing HCO, (Table 1). The rates of Na and Cl transport do not appear to differ substantially from those obtained in the absence of HCO,. However, in contrast to 1. Unidirectional n
JYa
and net Na, CL, and K fluxes across JNa sm
ms
JNa
J”’
F’ ms
nel
33X3-free 45
2.4 + 0.1
3.7 2 0.1
20
0.12 -+ 0.01 JN” ms
0.24 4 0.03
2.1 2 0.2
3.6 t 0.3
-1.3
-t 0.1
-0.12
net
Values are means * 1 SE. Measurement Negative value for Jnet indicates secretion.
-1.5
J”’
sm
Ringer
2 0.2
7.3 t 0.3
JC’ms
JC’ Em
nei
I SC
JL
-4.0
k 0.3
2.8 k 0.1
0.1 4 0.3
2.3 -+ 0.1
3.5 + 0.3
Ringer
FUHRO
R, slcm2
PD, mV
170.2 k 8.1
12.8 2 3.3
155.8 k 6.7
9.6 2 2.8
solution
3.3 2 0.2
HCO, 10
AND
antrum
5 0.03 JNa
J%
ROBERTSON,
flux measurements made in the absence of HC03, a significant (P < 0.025) residual flux was observed in the presence of HC03, suggesting the presence of HC03 secretion. When both sides of the mucosa are bathed in HCO,free Ringer solution bubbled with 100% 02, appearance of titratable acid may occur in the mucosal bathing solution. The rate of acid appearance did not exceed 0.26 peq/h cm* and was observed for approximately 43% of the tissue studied. UJhen mucosal appearance of acid occurred, it remained steady for at least 1 h and was not influenced by bubbling both sides of the mucosa with 100% N, (9). Serosal alkalinization was not observed. Bubbling the serosal side with 5% CO,-95% 0, resulted in the transient appearance of acid on the mucosal side (lo), but subsequently neither acid nor alkali appeared. The rate of CO, appearance on the mucosal side of eight tissues, 8,3 t 0.6 PM/h cm2 (means 2 1 SE), was not significantly different (P > 0.4) from that appearing on the serosal side, 7.8 t 0.7 PM/h cm? The luminal rate of lactate appearance for 16 tissues was 0.14 t 0.01 FM/h cm2 in the mucosal bathing solution and 0.18 * 0.01 PM/h cm2 in the serosal bathing solution. The rate of pyruvate appearance in the mucosal bathing solution of antrum has been shown to be negligible with respect to the rate of lactate appearance (12). Thus, it appears that isolated antral mucosa of the rabbit does not actively secrete acid, and when acid appears in the mucosal bathing solution it most likely is due to the release of lactic acid by the tissue. Occasionally, the spontaneous appearance of alkali in the mucosal or serosal bathing solution occurs, but this disappears within approximately 10 min after mounting the tissue in vitro. Addition of HCO, to the serosal bathing solution is followed by the appearance of alkali in the mucosal bathing solution. The mean rate of alkali appearance in the mucosal bathing solution of 50 tissues was 0.99 t 0.08 peq/h cm? In 15 experiments, the rate of alkali appearance in the mucosal bathing solution was not measurably influenced (P > 0.4) by changing the gas phase of the serosal bathing solution from 100% 0, to 95% 02-5% COZ, which changed the pH from approximately 8.1 to 7.4. Furthermore, in 10 experiments the rate of alkali appearance in the mucosal bathing solution was not significantly influenced (P > 0.4) by reducing the HC03 concentration from 25 to 15 mM, which changed the pH from approximately 7.4 to 7.1. In contrast to the addition of HCO, to the serosal
RESULTS
TABLE
SCHWARTZ,
JC1 net
solution
6.5 k 0.4
-3.0
of fluxes is in peqlh crn2. n, number of rabbits. J,,, unidirectional I,,, short-circuit current 12 JFe:,,,I,, - (“Jr,$ t “J$). R, electrical
2 0.4
2.6 k 0.1
1.1 t 0.4
mucosal-to-serosal flux. J,,, unidirectional resistance, PD, potential difference.
174.1 t 10.2 s-to-m
flux.
12.2 A 3.8 J,,, = J,,
- J,,.
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ION
TRANSPORT
ACROSS
ANTRUM
1785
bathing solution, addition of HCO, to the mucosal bathing solution resulted in the titratable appearance of alkali on the serosal side of only 0.07 -+ 0.05 peq/h cm2 (n, 10, P > 0.2). In 10 experiments HCO, was placed in the serosal bathing solution and a unidirectional flux of H14C0, was measured simultaneously with titration of alkali appearance in the mucosal bathing solution. The rate of isotopically measured HCO, secretion was 1.18 t 0.20 peq/h cm2 and the titratable appearance of alkali was 0.80 t 0.15 peq/h cm2. The difference between these two values, 0.38 t 0.10 peq/h cm2, is statistically significant (P < 0.01). In five experiments, the opposite flux was measured. Bicarbonate was placed in the mucosal bathing solution and a unidirectional m-to-s H14C0, flux was measured simultaneously with titration of the serosal bathing solution. The unidirectional m-to-s H14C0, flux was 0.38 t 0.2 peq/h cm2, whereas the titratable appearance of serosal alkali was 0.07 k 0.05 peq/h cm2. Therefore, it appears that the difference between the isotopically determined flux and that measured by titration probably is due to conversion of HCO, to CO,. In part, this may be due to some titration by release of organic acid, (e.g., lactic acid) as well as due to equilibration of HCO, with 14C02 in solution. In five experiments the rate of titratable HCO, secretion,l 1.01 L 0.14 peq/h cm2, was reduced to zero when the gas bubbling both sides of the tissues was changed from 100% 0, to 100% N,. Rebubbling with 100% 0, lo- to 15-min later resulted in the return of HCO, secretion in four of five tissues and was not significantly different (P > 0.2) from that observed prior to gassing with N,. The electrical resistance increased by 52.2 t 4.8 ficm2 in the presence of Ne, and after rebubbling with 02, the resistance was not significantly different from that observed prior to starting N2 (P > 0.2). In an additional five experiments, antral mucosa was bathed in a Ringer solution (7) containing HCO, on both sides of the tissue that was short-circuited during measurement of a unidirectional s-to-m H14C0, flux, Under these conditions the mean flux was 1.15 t 0.14 peq/h cm2. A unidirectional m-to-s H14C03 flux was also measured across two tissues, and the rates of transport were 0.21 and 0.35 peq/h cm? Although no obvious differences in the magnitudes of the unidirectional or net Na and Cl fluxes are apparent when antral mucosa is bathed in the absence or presence of HC03, a separate series of experiments were performed in which the effects of HCO, secretion on the unidirectional fluxes of Na and Cl were individually measured. A unidirectional flux of either 22Na or 36C1 was initially measured over a 30-min interval for mucosae bathed in HC03-free Ringer solution. Subsequently, HCO, was added to the serosal bathing solution, which was then bubbled with 95% O,-5% C02, and the isotopic flux measurement was continued lo-15 min later. Analysis of flux measurements made every
10 min (n, 3 for each unidirectional flux of Na and Cl) before and after HCO, addition indicated the presence of steady-state rates of isotopic transfer. Titration of the luminal bathing solution was performed during both flux measurements in order to maintain the pH at 7. The addition of HCO, to 12 tissues was followed by a significant increase in the unidirectional m-to-s Cl flux (Table 2). A statistical correlation existed between the rate of HCO, secretion and the increase in unidirectional m-to-s flux (Fig. 1). In contrast, the unidirectional s-to-m Cl flux or either unidirectional Na flux was not significantly altered after the addition of HC03 (Table Z), These data imply that HCO, secretion is associated with a decrease in net Cl secretion2 The I,, does not change significantly (-0.39 -t 0.24 peq/h cm2; P > 0.1, n, 46) after HCO, is added to the serosal bathing solution. Furthermore, the tissue electrical resistance also does not change significantly under these conditions (4.6 -+ 5.2 flcm2, P > 0.4, n, 46). No significant changes in I,, or tissue electrical resistance were observed when HCOz, 25 mM, was added to the mucosal bathing solution (P > 0.5, for each, n, 10). In a separate series of experiments, both sides of antral mucosa were bathed in Na isethionate solution, and the mucosal side was titrated with HZS04, 0.01 N. In seven tissues no alkali appeared on the mucosal side. However, after the addition of HCO, to the serosal side an alkali secretory rate of 1,21 t 0.09 r.Leg/h cm2 was observed. The rate of HCO, secretion did not appear to be measurably influenced by changing the gas phase on the serosal side from 100% O2 to 95% O,5% CO,. In five of these tissues the isethionate solution was replaced with a standard Ringer solution containing HC03 on the serosal side. In the remaining two tissues the isethionate solution was maintained, but choline Cl, 100 mM, was added to the serosal side. In each instance there was an increase in HCO, secretion, the mean of which for these seven tissues was 0.57 * 0.07 peq/h cm2 (P < 0.001). In three experiments, changing the gas phase to 100% N, resulted in cessation of HC03 secretion by mucosa bathed in isethionate solution. In order to d etermi ne if ion transport by antral’mucosa is altered by ouabain, ethacrynic acid, acetylcholine, and norepinephrine, as has been reported for other tissues, the effects of these agents on the&, were initial1 -Y measured. Each of these agents causes a decrease in .Z,,. Figure 2 shows the effects of that concentration of agent added to the serosal side” which caused a maximal decrease in I,, of antrum bathed in HCO,free Ringer solution. Lower concentrations of ethacrynic acid, lo-” to 10B4M, acetylcholine, 10s4to 10m5M, and norepinephrine, 10m4to lo+ M, caused lesser declines in Z,,. The effect of acetylcholine was inhibited by pretreatment with atropine (Fig. Z), but atropine did not prevent the effects of ouabain, ethacrynic acid, or norepinephrine.
1 “HC03 secretion” is used without distinguishing between alterations in OH and H fluxes that could bring about alkali secretion. Furthermore, no distinction is made between transmural flux of HCO, and extrusion into the lumen.
2 The most likely reason that statistically significant differences were not noted for J$,\ and JE& of Table 1 measured in the absence and presence of HCOs is that these were unpaired experiments. 3 The fact that these agents are effective on the serosal side does not a priori determine the locus or orientation of the secretory pumps (23).
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1786
FROMM,
Measurement of spontaneous Na and Cl fluxes over two 30- to 40-min intervals indicated that the fluxes, Isc, and R did not change significantly (Table 3). In a separate group of tissues, ouabain, 10s6 M, was added to the serosal bathing solution after base-line fluxes were measured. Ouabain inhibited the net fluxes of Na and Cl without causing measurable alterations in the unidirectional m-to-s fluxes of Na and Cl, JEet, or R (Table 3). Since there was a relatively prolonged delay before the I,, reached a plateau after the addition of ethacrynic acid, acetylcholine, or norepinephrine (Fig. 2), each tissue was not used as its own control for Na and Cl flux measurements, Antral mucosa from the same TABLE 2. Effects of HCO, secretion unidirectional Cl and Na fluxes
SCHWARTZ,
ROBERTSON,
AND
FUHRO
rabbit could not be used for measurement of control fluxes, since rabbit antrum is small and only two segments per rabbit could be used. Therefore, control flux measurements were made on tissues from a separate group of animals, but were begun at a time interval approximating that at 0*5
0
n, number of rabbits. AJ, difference (mean 2 1 SE) between flux measured before and after HC@ addition to serosal bathing solution. J,,, unidirectional mucosal-to-serosal flux. J,,, unidirectional s-to-m flux. PAJ, P value for AJ (Student t test for paired variates). Measurement of fluxes is in peq/h cm?
I
2 JHC03 SM
4
3
,A Eq/hr cm2
FIG. 1. Increase in unidirectional m-to-s Cl flux (AJ$,\) as a function of HCO, secretion. Line drawn by method of least squares. Significance of r is 0.2) was detected in the presence of ethacrynic acid. Norepinephrine also inhibited net Na and Cl secretion, but in contrast to ethacrynic acid and acetylcholine, the unidirectional fluxes of Na and Cl were at least half of those observed for control tissues. Furthermore, the electrical resistance in the presence of norepinephrine was strikingly greater than that observed for control tissues, A similar increase in electrical resistance. was noted in five of the tissues shown in Fig. 2 in which this measurement was made. The experiments shown in Tables 3 and 4 were repeated with HCO, present on both sides of the mucosa. The effects of ouabain (12, 6), ethacrynic acid (n, 5), acetylcholine (n, 5), and norepinephrine (n, 8) on Na and Cl transport were the same as those shown in the tables. However, statistically significant residual ion TABLE Period
of uuabain
3. Effects J""
ms
JN"
on ion transport
sm
JNa net
1.9 1*7 -0.2 4 0.1 >0.05
3.2 2.8 -0.4 k 0.3 >O.Ol
-1.3 -1.1 0*2 * 0.3 BO.5 Ouabain
I II A P
2.2 2.0 -0.2 f 0.1 >0.05
3.6 2.0 -1.6 k 0.2
