Lumen-to-surface pH gradients in opossum and rabbit esophagi: role of submucosal glands

Solange Abdulnour-Nakhoul, Nazih L. Nakhoul, and Roy C. Orlando

Departments of Medicine and Physiology, Tulane University School of Medicine, and Veterans Administration Medical Center, New Orleans, Louisiana 70112-2699


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The opossum esophagus, like that of humans, contains a network of submucosal glands with the capacity to secrete bicarbonate ions into the esophageal lumen. To evaluate the role of these glands in protecting the epithelial surface from acid insult, we measured the lumen-to-surface pH gradient in opossum esophagus at different luminal pH and compared it to that of rabbit esophagus, an organ devoid of submucosal glands. Sections of opossum and rabbit esophageal epithelium were mounted luminal side up in a modified Ussing chamber. pH-sensitive microelectrodes, positioned within 5 µm of the epithelial cell surface, were used to monitor surface pH during perfusion with solutions of different pH. At luminal pH 7.5, the pHs of both opossum and rabbit were similar (pHs = 7.5). Lowering luminal pH from 7.5 to 3.5 in opossum decreased pHs to 4.2 ± 0.16, a value significantly higher than pH of perfusate, whereas in rabbit this maneuver decreased pHs to 3.69 ± 0.08, a value not significantly different from pH of perfusate. In opossum but not in rabbit, addition of carbachol to the serosal solution increased basal pHs to 7.8 ± 0.1 and significantly blunted the decline in pHs on perfusion with acidic Ringer solution (pH 3.5), with pHs falling to 5.6 ± 0.45. The effect of carbachol on surface buffering was inhibited by prior treatment with atropine. Luminal acidification to pH 2.0 in opossum (as in rabbit) abolished the lumen-to-surface pH gradient even after addition of serosal carbachol. We conclude that the presence of submucosal glands in esophagus contributes through bicarbonate secretion to creation of a lumen-to-surface pH gradient. Although this gradient can be modulated by carbachol, its capacity to buffer (and therefore to protect) the epithelial surface against back-diffusing H+ is limited and dissipated at pH 2.0.

carbachol; alkaline secretion; microelectrodes; acid reflux


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ONE OF THE DEFENSES that upper gastrointestinal epithelia use for protection against injury by high concentrations of luminal acid is the establishment and maintenance of a lumen-to-surface pH gradient. This gradient protects by limiting the degree of acidity that comes in contact with surface epithelial cells. Multiple factors determine the magnitude and resilience of the gradient among species and among organs within a species, but the most important factors appear to be the capacity of the tissue to secrete mucus and bicarbonate. It is therefore not surprising that the stomach and duodenum, with both mucus and bicarbonate-secreting surface epithelial cells, have been shown in both animals and humans to maintain a sizable lumen-to-surface pH gradient in the presence of high luminal acidity (1, 6, 7, 12, 17, 18, 20). In contrast to stomach and duodenum, the moist stratified squamous epithelium lining the esophagus has neither mucus nor bicarbonate-secreting cells and as such appears to be disadvantaged in its capacity to produce or maintain a sizable lumen-to-surface pH gradient. However, humans and some animal species like opossum have submucosal glands (SMGs) within the esophageal wall (2, 8), which are reported to secrete both mucin and bicarbonate into the esophageal lumen (4, 9, 11, 15, 16). However, the role of the secretions from these glandular structures to create and support a lumen-to-surface pH gradient in esophagus has not been evaluated yet. We therefore characterized the magnitude of the lumen-to-surface pH gradient in the opossum, a species with a rich network of submucosal glands, and compared its capacity for protection against luminal acidity to that of the rabbit, a species whose esophagus is devoid of such glands. The results of this comparison demonstrate that the presence of SMGs, through bicarbonate secretion, contributes to the ability of the esophagus to limit back-diffusing luminal acid from reaching the epithelial surface; however, this capacity is limited and can be overwhelmed at low pH.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal and tissue preparation. American opossums (Northeastern Wildlife, South Plymouth, NY) and New Zealand White rabbits were killed by administration of an intravenous overdose of pentobarbital sodium (60 mg/ml). The esophagus was excised, opened longitudinally, and pinned mucosal side down in a paraffin tray containing ice-cold oxygenated Ringer. The muscle layers were lifted up with forceps, and the underlying mucosa was dissected free with a scalpel. The sheet of mucosa obtained was cut, and a section was mounted apical side up, horizontally in a modified Ussing chamber with an aperture of 1.13 cm2. The chamber allowed continuous and independent perfusion of the apical and the serosal sides of the tissue. The fluid for the perfusion of the tissue was delivered by gravity, and all solutions were placed at the same height from the chamber. The perfusion solutions could be switched quickly and with minimal dead space by means of a combination of six-way rotary valves and four-way pneumatically activated slider valves (Rainin, Emeryville, CA). In this arrangement, one of six experimental solutions could flow to each side of the chamber. The volume of fluid in the upper and in the lower sides of the chamber was ~0.25 ml. The rate of flow of the solution to the upper chamber was 2 ml/min, which yielded a turnover rate (i.e., time for a solution to be washed out completely from the chamber) of ~8 s. Perfusion fluid to the lower part of the chamber was delivered at 2 ml/min; however, only part of this fluid (~1 ml/min) entered the chamber, the rest leaving through a drain that was kept at a fixed level. The turnover rate in the lower side of the chamber was ~15 s. The solutions were prewarmed and delivered to the chamber at 37°C.

Electrodes. Transepithelial potential difference (VTE) was measured as the voltage difference between a free-flowing KCl electrode (tip <10 µm) placed in the bath fluid of the serosal side and a similar electrode placed in the bath fluid of the apical side. Both electrodes were fitted with an Ag-AgCl wire, and the leads were connected to the amplifier of a voltage clamp (Physiologic Instruments, San Diego, CA). The voltage clamp was also used to deliver a direct-current pulse of 5-15 µA via platinum wires located in each side of the chamber. This allowed us to determine the transepithelial resistance (RTE) from the voltage deflection (Delta VTE) as follows
<IT>R</IT><SUB>TE</SUB> = &Dgr;<IT>V</IT><SUB>TE</SUB>/<IT>I</IT>
where I is current. Given the use of free-flowing KCl electrodes to measure VTE, junction potentials produced during exposure to solutions of varying composition, including pH 2.0 Ringer solution, were negligible and therefore were not corrected for.

The pH-sensitive microelectrodes were of the liquid ion exchanger type. Alumino-silicate glass tubes (1.2 mm OD × 0.86 mm ID; Frederick Haer, Brunswick, MD) were pulled on a vertical microelectrode puller (David Kopf, Tujunga ,CA) to a tip of ~1-2 µm and dried in an oven at 200°C for 2 h. The electrodes were exposed in a closed vessel to tri-n-butyl-chlorosilane fumes for 2 min, after which the silane fumes were vented, and the electrodes were left in the oven for an additional 30 min. The exchanger (Fluka, Hydrogen Ionophore II, cocktail A) was then introduced into the tip of the electrodes by means of a very fine glass capillary. The electrodes were then backfilled with a buffer solution containing 0.04 M KH2PO4, 0.023 M NaOH, and 0.015 M NaCl, pH 7.0 (3) and calibrated in standard buffer solutions of pH 2, 4, and 7. The average slope was 57.8 ± 0.4 mV/pH unit (n = 15).

For measurements of pH at the epithelial surface (pHs), the pH-sensitive electrode was advanced using a motorized micromanipulator (Merhauzer, Fine Science Tools) and under microscopic control at ×140 (Stereozoom7, Leica) to a position immediately above, but without touching, the tissue. The pH electrode was then further advanced using the micromanipulator until the electrode was observed microscopically to touch the apical membrane of a surface cell, an event that was accompanied by an increase in the magnitude of the direct- current pulses. The tip size of the microelectrode was big enough not to allow intracellular impalements of the epithelial cells. The pH electrode was then withdrawn in steps of 1-2 µm until the direct-current pulse returned to that observed in free solution (12). This permitted localization of the pH electrode within 5 µm of the epithelial cell surface, a position that was maintained throughout the course of the experiments. All electrical and pH readings were recorded on a three-channel strip chart recorder (Kipp & Zonen, Bohemia, NY).

Solutions. The composition of the solutions is shown in Table 1. The chemicals were obtained from Sigma.

                              
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Table 1.   Composition of solutions

Statistical analysis. The results are presented as means ± SE. Data were analyzed using the two-tailed paired Student's t-test unless otherwise indicated; n is the number of observations. The initial rates of pHs change were determined from the slope of a linear regression fit of pHs vs. time. Only the initial portion of pHs change was taken, which amounted to 30-40 s, during which the data could be tightly fit to a straight line.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Both the opossum and rabbit esophagi are lined by a moist stratified squamous epithelium; however, the opossum esophagus contains an extensive network of tubuloacinar SMGs (8, 13, 14), whereas that of the rabbit is completely devoid of SMGs (Fig. 1). Using pH-selective microelectrodes positioned within 5 µm of the esophageal epithelium, we measured pHs in both rabbit and opossum esophagi and monitored the changes during luminal perfusion with acidic Ringer solutions of pH 3.5 or 2.0 (Table 1). RTE and VTE were monitored as well.


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Fig. 1.   Light micrographs (×100) of opossum (A) and rabbit (B) esophageal mucosa. Although both esophagi possess stratified squamous epithelia (uppermost layer), only the opossum esophagus shows an extensive network of submucosal glands (arrows).

Resting condition. Esophageal tissues obtained from six opossums and five rabbits were perfused with control Ringer (pH 7.5) in the lumen and in the serosal bath. Under basal conditions in opossum, pHs was 7.5 ± 0.03, VTE was -7.6 ± 0.94 mV, and RTE was 1,616 ± 214 Omega  · cm2 (n = 13, 1-3 tissues were used from each animal). In the rabbit, pHs was 7.5 ± 0.04, VTE was -12.0 ± 0.94 mV, and RTE was 2,001 ± 157 Omega  · cm2 (n = 7). Both RTE and VTE were significantly higher in the rabbit than in the opossum (P < 0.004, unpaired t-test).

Effect of luminal acidification to pH 3.5. After baseline recordings, luminal pH was lowered by switching the luminal solution to acidic Ringer, pH 3.5. In the opossum, this maneuver resulted in a small depolarization of VTE (1.9 ± 0.4 mV, P < 0.001) but no significant change in RTE. However, acidification of the lumen resulted in a progressive decline in pHs, over 7.4 ± 1.14 min, to a new pHs of 4.2 ± 0.16 (Fig. 2, segment ab). The initial rate of decline in pHs during perfusion with pH 3.5 was 1.78 ± 0.48 pH/min, and Delta pHs was 3.3 ± 0.15 pH units. Thus pHs at the new steady state was 0.64 ± 0.16 pH units higher than that of the luminal perfusate pH 3.5 (n = 13, P < 0.002). When the luminal bathing solution was switched back to bicarbonate-Ringer solution, pH 7.5, the changes were reversible and pHs returned to its previous baseline value (Fig. 2, segment bc).


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Fig. 2.   Tracing of an experiment showing the changes in surface pH (pHs) (top trace) and transepithelial potential difference (VTE) (bottom trace) of the opossum esophagus on exposure of the lumen to a Ringer solution of pH 3.5 in the absence (segment ab) and in the presence of carbachol (segment ef) and in the presence of carbachol and atropine (segment hi). The addition of carbachol to the serosal bath caused an increase in pHs (segment de). Also, in the presence of carbachol, the decrease in pHs and the rate of acidification were smaller than in control. In the presence of atropine, the effect of carbachol is partially inhibited. The break in the tracing is ~20 min.

In the rabbit esophagus, perfusion of the lumen with acidic Ringer, pH 3.5 (solution 2), produced no change in VTE (-12.8 ± 1.25 mV) or RTE (1,928 ± 180 Omega  · cm2; n = 7, P > 0.05). However, pHs declined at an initial rate of 2.58 ± 0.78 pH/min, by 3.81 ± 0.07 units (n = 6) to reach a plateau value of 3.69 ± 0.08 (Fig. 3, segment ab). This latter pHs value was not significantly different from pH 3.5 of perfusate (plateau reached within 5.1 ± 1.01 min). These changes were reversible on switching back the luminal solution to a control Ringer solution (Fig. 3, segment bc).


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Fig. 3.   Tracing of an experiment showing the changes in pHs (top trace) and VTE (bottom trace) in the rabbit esophagus on exposure of the lumen to a solution of pH 3.5 in the absence (segment ab) and the presence of carbachol (segment ef). In the presence of carbachol, both the decrease in pHs and the rate of acidification are not significantly different from control. This is in contrast to the findings in the opossum (see Fig. 2).

Effect of carbachol on pHs. Carbachol, a cholinergic agonist, has been reported to stimulate bicarbonate secretion in the opossum esophagus (10). Therefore, the effect of serosal carbachol on pHs was studied in the opossum and, for comparison, in the rabbit. Perfusing the serosal side of opossum esophagus with a Ringer solution containing carbachol (10-7 M) increased pHs by 0.3 ± 0.11 pH units (Fig. 2, segment de), decreased RTE by 236 ± 49 Omega  · cm2, and had no effect on VTE (n = 9, P < 0.01). These data are summarized in Fig. 4. In contrast to the opossum, the addition of carbachol to the serosal bath of rabbit esophagus had no effect on pHs (pHs remaining 7.5 ± 0.1; Fig. 3, segment de), RTE (2,043 ± 125 Omega  · cm2), or VTE (-12 ± 0.95 mV; n = 9, P > 0.05). These data are summarized in Fig. 4.



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Fig. 4.   Effect of carbachol on steady-state pHs (A), VTE (B), and transepithelial resistance (RTE; C) in the opossum and rabbit esophagi. In the opossum but not in the rabbit, addition of carbachol (10-7 M) to the serosal bath increased pHs and decreased RTE. VTE remained unchanged. * Significantly different from control (P < 0.05, paired data); § significantly different from opossum (P < 0.05, unpaired t-test).

Effect of luminal acidification to pH 3.5 in the presence of carbachol. When tissues perfused with carbachol in opossum were subsequently exposed to acidic Ringer, pH 3.5, pHs dropped only by 2.2 ± 0.36 pH units, a value significantly smaller than the decline in pHs of 3.27 ± 0.23 (P < 0.01, n = 8) observed in the absence of carbachol (Fig. 2, segment ef, and Fig. 5). Hence, at luminal pH 3.5, pHs in opossum was 5.6 ± 0.45 vs. 4.2 ± 0.24 in the absence of carbachol (n = 8, P < 0.01). Also, in the presence of carbachol, the initial rate of acidification was slower, at 0.58 ± 0.2 pH/min, than in its absence (1.78 ± 0.48 pH/min), and the time it took to reach the new steady-state value for pHs was longer (11.5 ± 0.5 min vs. 7.4 ± 1.14 min) than in the absence of carbachol (n = 8, P < 0.03 for each comparison). pHs values at defined time points of 1, 3, and 5 min after acidification are shown in Table 2. Luminal perfusion with pH 3.5 in the presence or absence of carbachol resulted similarly in a depolarization of VTE of 2.32 ± 0.77 mV and no change in RTE.


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Fig. 5.   Changes in pHs (Delta pHs, A) and initial rate of acidification (B) of the opossum and rabbit esophagi on exposure to acidic Ringer of pH 3.5 in the absence or the presence of carbachol. In the opossum, in the presence of carbachol, the decline in pHs was significantly smaller than the decline in pHs observed in the absence of carbachol. Also, in the presence of carbachol, initial rate of acidification was slower than in its absence. In the rabbit, decrease in pHs was not significantly different from that observed in the absence of carbachol. Also, in the rabbit, there was no difference in the initial rate of decline of pHs at luminal pH 3.5 in the presence of carbachol compared with its absence. * Significantly different from control (P < 0.05, paired data); § significantly different from opossum (P < 0.05, unpaired t-test).


                              
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Table 2.   Surface pH in the opossum at defined time points after luminal acidification to pH 3.5 in the absence and presence of carbachol

In contrast to the opossum, luminal exposure to pH 3.5 in the rabbit, in the presence of serosal carbachol, resulted in a decrease of pHs to 3.9 ± 0.18 (Fig. 3, segment ef), a value not significantly different from that observed in the absence of carbachol (pHs = 3.7 ± 0.1) or from that of the luminal perfusate (n = 5, P > 0.05 for both comparisons; Fig. 5). Also, in the rabbit, there was no difference in the initial rate of decline of pHs at luminal pH 3.5 (2.58 ± 0.78 pH/min) or time to plateau (4.9 ± 1.12 min) in the presence of carbachol compared with the values recorded in its absence (P > 0.05 for each comparison; Fig. 5).

Because serosal carbachol had a marked effect on the lumen-to-surface pH gradient in opossum, we assessed the effect of atropine, a cholinergic antagonist, on this parameter. This was done by simultaneously exposing the tissue to both serosal carbachol and atropine (10-6 M) and then acidifying the luminal bath with Ringer at pH 3.5. As shown in Fig. 2, the ability of carbachol alone to reduce the rate and degree of decline in pHs with luminal acidification (segment ef) was markedly reduced by the addition of atropine to the serosal solution (segment hi). Indeed, in the presence of atropine, the change of pHs and the rate of acidification in response to pH 3.5 in the lumen were similar to the changes obtained in control conditions. Thus, in tissues from four opossums when the lumen was acidified to pH 3.5, pHs decreased to 4.0 ± 0.09 in control, 6.1 ± 0.54 in the presence of carbachol (n = 4, P < 0.05), and 5.1 ± 0.6 for atropine plus carbachol. This is reflected in different Delta pHs shown in Fig. 6. Also observed is that the initial rate of acidification in the presence of atropine (1.1 ± 0.61 pH/min) was not significantly different from the rate of 1.3 ± 0.4 pH/min in the control period (Fig. 6).


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Fig. 6.   Changes in pHs (A) and the rate of acidification (B) of the opossum esophagus on exposure of the lumen to a solution of pH 3.5 in the absence or presence of carbachol and in the presence of carbachol and atropine. Although in the presence of carbachol Delta pHs and the rate of acidification were significantly smaller than control, the addition of atropine (1 µM) reversed, at least partially, the effect of carbachol. * Significantly different from control (P < 0.05, paired data).

Effect of luminal acidification to pH 2.0 in the absence and presence of carbachol. Because the opossum esophagus maintained a significant lumen-to-surface pH gradient during luminal perfusion at pH 3.5, we investigated its ability to handle an increased acid load by perfusing the lumen with an acidic Ringer at pH 2.0, in both the presence and absence of serosal carbachol. In the opossum, when the luminal perfusate was switched to an acidic Ringer at pH 2.0, there was a rapid decline in pHs, which reached a value similar to that of the perfusate (Delta pHs of 5.43 ± 0.15 units) over a period of 2.5 ± 0.67 min (Fig. 7, segment ab). Although the presence of carbachol was able to reduce the initial rate of surface acidification (1.70 ± 0.5 pH/min); compared with its absence (3.45 ± 0.9 pH/min; n = 4, P < 0.05), acidifying the luminal solution to pH 2.0 in the presence or absence of carbachol resulted in similarly substantial declines in pHs (5.45 ± 0.2 units vs. 5.6 ± 0.28 pH units, respectively; n = 4, P > 0.05) such that both reached new steady-state values that were not significantly different from the luminal perfusate of pH 2.0 (Fig. 7, segment ef). These data are summarized in Fig. 8. In these experiments, it was also noted that luminal perfusion at pH 2.0 decreased RTE significantly from 1,347 ± 244 to 1,239 ± 240 Omega  · cm2 (P < 0.03, n = 9) in the absence of carbachol and from 1,449 ± 302 to 1,166 ± 262 Omega  · cm2 (P < 0.03, n = 7) in the presence of carbachol.


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Fig. 7.   Tracing of an experiment showing the changes in pHs (top trace) and VTE (bottom trace) in the opossum esophagus on exposure of the lumen to a solution of pH 2.0 in the absence and in the presence of carbachol. When the luminal perfusate was switched to an acidic Ringer at pH 2.0, there was a rapid decline in pHs, which reached a value similar to that of the perfusate within ~3 min (segment ab). Although the presence of carbachol was able to reduce the initial rate of surface acidification compared with its absence, acidifying the luminal solution to pH 2.0 in the presence (segment ef) or absence of carbachol (segment ab) resulted in similarly substantial declines in pHs. Inset: superimposed tracings showing the initial decrease in pHs in response to luminal pH 2.0 in the absence and presence of carbachol.



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Fig. 8.   Changes in pHs (A) and the rate of acidification (B) of the opossum and rabbit esophagi on exposure to a solution of pH 2.0 in the absence and presence of carbachol. In the opossum, although the presence of carbachol was able to reduce the initial rate of surface acidification compared with its absence, acidifying the luminal solution to pH 2.0 in the presence or absence of carbachol resulted in similarly substantial declines in pHs within ~3 min from acidification. * Significantly different from control (P < 0.05, paired data).

In the rabbit, when the lumen was similarly perfused with acidic Ringer (pH 2.0), pHs declined to 1.94 ± 0.17 in 1.7 ± 0.5 min and at an initial rate of acidification of 3.43 ± 0.32 pH/min. Furthermore, these values in the rabbit were not altered by the presence of serosal carbachol: pHs declined to 2.3 ± 0.3 in 2.6 ± 1.3 min and at an initial rate of 4.13 ± 1.05 pH/min (n = 5, P > 0.05 for each comparison). These data are summarized in Fig. 8. In the rabbit, acidification of the lumen to pH 2.0 hyperpolarized VTE by -2.7 ± 0.32 mV, whereas RTE remained unchanged.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present investigation, pH-sensitive microelectrodes were used to measure pHs in two species, the opossum (submucosal glands bearing) and the rabbit (submucosal glands free), under two conditions: 1) in the presence of two different levels of luminal acidity (pH 3.5 and 2.0), and 2) in the presence of a known stimulant of bicarbonate secretion by the submucosal glands.

As shown during luminal acidification at pH 3.5 of the rabbit esophagus, the absence of SMGs results in little to no capacity to protect the epithelium from being accessed by luminal acid even at this relatively modest level of acidity. Thus luminal acidity at pH 3.5 resulted in the rapid decline in pHs until it reached a value similar to that of the luminal perfusate.

In contrast to rabbit esophagus, the opossum esophagus responded to luminal acid perfusion of pH 3.5 with a slower rate of acidification and a longer time to reach a new steady-state pHs value that was higher than that of the rabbit esophagus and significantly above that of the luminal perfusate by a little more than one-half a pH unit. Because neither the rabbit nor the opossum stratified squamous esophageal epithelium secretes mucus or bicarbonate, this indicates that the superior lumen-to-surface pH gradient maintained by the opossum esophagus is due either to secretion of bicarbonate (and/or mucus, see below) by the submucosal glands or to greater passive paracellular diffusion of bicarbonate from blood to lumen. This latter possibility cannot be ignored, given that the transepithelial electrical resistance of opossum esophageal epithelium is significantly lower than that of rabbit and that this parameter is dominated by the ion permeability across the paracellular route (19).

Documentation that secretion from the esophageal submucosal glands is a major contributor to the lumen-to-surface pH gradient (and so preepithelial defense) in opossum was established by serosal exposure to carbachol, a known stimulant of submucosal gland secretion. Notably, the addition of carbachol both raised the resting pHs at luminal pH 7.5 and markedly enhanced the magnitude of the lumen-to-surface pH gradient at luminal pH 3.5 in opossum esophagus. In contrast, carbachol neither raised the resting pHs at luminal pH 7.5 nor did it improve the ability of the rabbit esophagus to establish a lumen-to-surface pH gradient in the presence of luminal acid of pH 3.5. Carbachol has no effect on esophageal stratified squamous epithelium (5), and so these observations indicate that the source of the enhanced lumen-to-surface pH gradient in opossum but not rabbit is due to its ability to stimulate secretion (bicarbonate) by the SMGs. Furthermore, bicarbonate, not mucin, is the likely factor in the secretion to be responsible for these observations, since mucin secretion, by creating a larger diffusion barrier, might slow the rate of H+ diffusion but would not probably change the ultimate pHs reached on luminal acidification. In effect, the ability of carbachol to produce a new higher steady-state value for pHs over that of the perfusate indicates that the secretion of bicarbonate from the submucosal glands was the likely component accounting for protection. Indeed, from the change in pHs, the increase in buffer capacity produced by carbachol can be estimated at 1.75 times over basal levels. This increase in buffering power can be calculated using the formula
&bgr;<SUB>carb</SUB>/&bgr;<SUB>control</SUB> = &Dgr;pH<SUB>carb</SUB>/&Dgr;pH<SUB>control</SUB>
where beta carb and beta control indicate the buffering power of the surface layer in the presence of carbachol and in control respectively. Moreover, Hamilton and Orlando (9) have shown that carbachol stimulation increases the basal rate of bicarbonate secretion in the opossum esophagus from 0.39 ± 0.03 to 0.74 ± 0.07 µeq · h-1 · cm-2. Assuming that this increase results in a proportionate increase in bicarbonate concentration in the surface unstirred layer, on the basis of the Henderson-Hasselbach equation, this increase in secretory rate could account for an increase in pHs of ~0.27 pH units. Thus our experiments document that the presence of submucosal glands can contribute to and modulate the protective capacity of the preepithelial defense against luminal acid and that such modulation by cholinergic agonists is clearly absent in esophagi devoid of such glands. Moreover, because atropine could block the enhancement in the gradient produced by carbachol, it is evident that cholinergic muscarinic receptors in the tissue mediate the response to carbachol in vitro just as they have been documented to do in vivo (15).

The effect of exposing the rabbit and opossum esophagi to luminal pH 2.0 was also of interest. In the absence of carbachol, luminal pH 2.0 resulted in a similar pattern of acidification in the opossum as in the rabbit esophagus, i.e., there was rapid equilibration of the pHs to that of the luminal solution. When stimulated by carbachol, however, the opossum esophagus was able to reduce the initial rate of acidification but not stop the ultimate decline in pHs to a value similar to that of the luminal perfusate. It is conceivable that below pH 3.5 most of the secreted or diffused bicarbonate would be consumed by an overwhelming H+ influx, causing an abrupt decrease in pHs. Nonetheless, together, these data suggest that, under both basal and stimulated conditions, the SMG-bearing esophagus (opossum) has a limited capacity to resist acidification of its surface cells and that at luminal acidity in the range of pH 2.0, which occurs commonly during episodes of gastroesophageal reflux in humans, the protective capacity of the lumen-to-surface pH gradient is overwhelmed.

In summary, this study establishes that the presence of submucosal glands in the esophageal wall is an important contributor to the preepithelial (buffering) defense against luminal acid. This defense is generated in large measure through the ability of these glands to secrete bicarbonate, and so the capacity for protection against luminal acidity varies and is dependent in part on the degree of glandular secretion to protect the epithelial surface against back-diffusing H+. However, the capacity of the glandular secretion to maintain a lumen-to-surface pH gradient is limited. In humans, whose SMG-bearing esophagus is often exposed to luminal pH equal 2.0, additional intrinsic (epithelium proper) and extrinsic (acid clearance mechanisms) esophageal defenses are necessary for protection against gastroesophageal reflux disease.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-36013.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: S. Abdulnour-Nakhoul, Dept. of Medicine, Section of Gastroenterology, SL 35, 1430 Tulane Ave., New Orleans, LA 70112-2699 (E-mail: solange{at}mailhost.tcs.tulane.edu).

Received 24 June 1999; accepted in final form 6 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 278(1):G113-G120
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