Proc. Nat. Acad. Sci. USA
Vol. 72, No. 9, pp. 3731-3734, September 1975 Physiology
Titration of sodium channels in canine gastric mucosa (gastric mucosal permeability/oxyntic and pyloric mucosas/protein shedding)
GORDON L. KAUFFMAN, JR. AND MICHAEL R. THOMPSON Department of Physiology, The University of Michigan, Ann Arbor, Mich. 48104; and The Center for Ulcer Research and Education, VA Wadsworth Hospital Center, Los Angeles, California 90073
Communicated by Horace W. Davenport*, June 19, 1975
Net Na+ flux from mucosa to lumen, potenABSTRACT tial difference, and volume and plasma protein outputs were measured in vagally denervated, separated pouches of the dog's oxyntic or pyloric glandular mucosa when the pouches were irrigated with Na+-free solutions whose pH ranged from 1.5 to 12.2. The apparent permeability to Na+(P'Na) was calculated. P'Na is lowest when the mucosa is bathed with acid and increases 2- to 3-fold when the pH is raised to 10. In the range of pH 10.0-11.2 P'Na is greater by an order of magnitude, but volume output is small, and no plasma proteins are shed. When the pH is above 11.2 there is an abrupt increase in P'Na, and the mtcosa sheds a large volume of fluid containing plasma proteins. The change effected by raising the pH to the range of 10.0-11.2 occurs within 10 sec, and it is reversible. The change effected by raising the pH above 11.2 also occurs within 10 sec, and it is partly reversible.
The normal gastric mucosa is only very slightly permeable HI and Na+ (1, 2) and its hydraulic coefficient is low (3). Permeability to H+ and Na+ can be increased by treating the mucosa with detergents (4) or mercurials (5, 6) in neutral solution. In this state flux of Na+ from mucosal interstitial fluid to lumen is large, but little fluid and no plasma proteins are shed. If the mucosa is treated with dithiothreitol (6) or with acetylcholine (7) mucosal permeability is enormously increased. Flux of Na+ into the lumen is very large, and a large volume of fluid containing plasma proteins is rapidly filtered into the lumen. In the normal state the permeability of both the oxyntic and pyloric glandular mucosas to Na+ is affected by the [H+] of the fluid bathing the mucosa (1). When this fluid is acid, mucosal permeability to Na+ is least, and as the pH of the fluid is raised, permeability to Na+ increases. We can assume that in this state Na+ moves down its electrochemical gradient from mucosal interstitial fluid to lumen through small channels lined with ionizable groups. If the [H+] of the fluid in contact with the mucosa is manipulated, the degree of ionization of the groups is altered, and permeability to Na+ changes. Moreno and Diamond (8) have developed this idea of the influence of [H+] upon ionic selectivity of a membrane in their study of the cation conductance of the gallbladder. They found that the apparent pKIa of the group which governs the Na+ conductance of the rabbit gallbladder is 4.55. When the pH of the fluid bathing the gallbladder is below 4.55, Na+ conductance falls to a minimum. Raising the pH above 6 increases Na+ conductance many times. In work based upon similar considerations Hille (9) found that Na+ channels in nerve appear to be dominated by a single group having a pKIa of 5.2. We have measured the net Na+ flux from mucosa to to
Abbreviation: PD, potential difference. To whom reprint requests should be addressed at Department of Physiology, The University of Michigan Medical Center, Ann Arbor, Mich. 48104.
*
3731
lumen and the potential difference (PD) in pouches of the dog's oxyntic and pyloric glandular mucosa when the pouches were irrigated with Na+-free solutions whose pH ranged from 1.5 to 12.2. We have calculated the apparent Na+ permeability coefficient from the data obtained at each pH. METHODS AND CALCULATIONS Three dogs with separated, vagally denervated (Heidenhain) pouches of the oxyntic glandular area of the stomach and two dogs with separated, vagally denervated pouches of the pyloric glandular mucosa were used. The preparation and care of the animals, the methods for measuring the net Na+ flux, volume and plasma protein outputs, and the potential difference have been described (2, 6, 7, 10). Solutions used to irrigate the mucosa were HCl at pH 1.5 or solutions buffered with tris(hydroxymethyl)aminomethane, the buffers described by Good et al. (11), lysine, or arginine, all titrated to the desired pH with KOH. All solutions were made isotonic with mannitol. Control experiments showed that the [K+] of the solutions did not influence the results. All solutions were Na+-free when placed in the pouches. The pH of the solutions did not change significantly during the period of irrigation. The apparent permeability coefficient was calculated by means of the equation
XN; [Na+],
exp(-FE/2RT) - [Na+],
exp(FE/2RT) [1]
derived from the integrated Nernst-Planck equation by Jacquez and Schultz (12, 13). JNa is the measured net flux of Na+ from mucosa to lumen in geq min-. [Na+]p is the plasma concentration of Na+ in geq ml-l, and [Na+]lf is that in the luminal fluid. The other symbols have their usual meanings. RESULTS Effect of pH upon the oxyntic glandular mucosa The effect of irrigating the pouch of the oxyntic glandular mucosa of one dog with solutions of different pH's is shown in Fig. 1. Net Na+ output rose and PD fell only slightly as the pH was raised from 1.5 to 10.0. In the range from 10.0 to approximately 11.5 Na+ output rose steeply, and the PD fell. There was a small output of fluid from the mucosa, but this fluid did not contain plasma proteins. Above pH 11.5 there was a large output of fluid containing plasma proteins, and Na+ output rose to high values. Moreno and Diamond (8) measured Na+ conductance of the rabbit gallbladder as a function of pH, and they were able to fit their data with a titration curve based on the assumption that a single ionizable group governs the Na+ conductance. The pKa of the group was calculated to be 4.55.
3732
Physiology: Kauffman and Thompson
Proc. Nat. Acad. Sci. USA 72 (1975)
~2000
P
-
4
710 0)
1000 -
2
CL~ zo .500500-
~~p 1 15
19
.
FIG2.NtN+otpt30E n iescesie3 1.5
11.1
1.5
11.9
e
1.5
pH
pH FIG. 1. Net Na+ output (0) and potential difference (0) (PD) in a pouch of a dog's oxyntic glandular mucosa irrigated for 30 min periods with Na+-free solutions of the pH indicated on the abscissa. The solid line is the calculated titration curve of a single acidic group having a pK, of 11.05. The broken line was drawn by eye. Total height of the bars indicates fluid output in the 30 min irrigation period, and the solid part of the bars indicates output of plasma.
Our cdata do not permit a similar calculation, for the transition from the state of intermediate permeability to the protein-losing state robs us of the upper half of the titration curve. Nevertheless, we have fitted the data with a titration curve having a pKIa of 11.05, and this curve is drawn as the solid line in the figure. Its purpose is merely to show that the data below pH 11.5 might be fitted with part of a titration curve. Data obtained using pouches of the oxyntic glandular area in two other dogs have similar results. Those data might be fitted with curves having pKIa values of about 10.5.
Reversibility The net Na+ output and the PD were measured in a pouch of the oxyntic glandular area when the pouch was irrigated for five successive 30 min periods with solutions in the following sequence: pH 1.5, pH 11.1, pH 1.5, pH 11.9, and pH 1.5. The series of observations was repeated on the same dog on different days for a total of three times. The results are shown in Fig. 2. When the solution of pH 1.5 was replaced with a solution of higher pH the PD was observed to fall to essentially its final value in 10 sec. When the solution of higher pH was replaced by one of pH 1.5 the PD climbed to nearly its final value in 15 min. There were no statistically significant differences between the Na+ outputs during irrigation with a solution of pH 1.5 before and after irrigation with one of pH 11.1. The permeability change effected by raising the pH from 1.5 to 11.1 is almost entirely reversible. The results obtained when the solution of pH 11.9 was replaced with one of pH 1.5 show that the change effected by a solution which induces a very large Na+ output accompanied by plasmashedding is partially reversible. Effect of pH upon the pyloric glandular mucosa The results obtained by irrigating the pouch of the pyloric glandular area of one dog with solutions of different pH are shown in Fig. 3. As the pH of the irrigation fluid was raised
FIG. 2. Net Na+ outputs 4- SEM in five successive 30 min periods in which a pouch of a dog's oxyntic glandular mucosa was irrigated with Na+-free solutions of the pH indicated. The circles with vertical bars are the mean PD's i SEM during the 30 min periods. Between the 2nd and 3rd periods and between the 4th and 5th periods the pouch was filled with 100 mN HCl, and the PD was read at 3 min intervals. These readings are shown as filled circles connected with a line. The series of observations was repeated three times on different days.
from 8.6 to 12.2 the PD fell, and the Na+ output rose. At pH 11 volume output was greater than at lower pH's, and above pH 11.9 plasma-shedding occurred. Results obtained with the other pyloric glandular pouch were almost identical. A series of observations similar to those made on the oxyntic glandular mucosa was performed to test reversibility. A pouch of the pyloric glandular mucosa was irrigated for five successive 30 min periods with solutions in the following sequence: pH 1.5, pH 11.5-12.0, pH 1.5, pH 12.1, and pH 1.5. The results were qualitatively identical with those obtained on the oxyntic glandular mucosa. The transition occurring when the pH is raised to an intermediate value is almost instantaneous and almost completely reversible. The transition effected by raising the pH to a high value is just as rapid and is partially reversible. Apparent Na+ permeability coefficient One dog with a pouch of the oxyntic glandular area was killed with an overdose of anesthetic, and the mucosa of her
.c 1000*oo 0 C,, cr
50
0
I
40
4) 750
k soo i 2500
0'
;
20
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ol
R10
i
30
* *
2 5'g
n
, 7Qn n
n ffl;
13
pH
FIG. 3. Net Na+ output (-) and potential difference (0) (PD) in a pouch of a dog's pyloric glandular mucosa irrigated for 30 min periods with Na+-free solutions of the pH indicated in the abscissa. The lines are drawn by eye. Total height of the bars indicates fluid output in the 30 min periods, and the solid part of the bars indicates output of plasma.
Proc. Nat. Acad. Sci. USA 72 (1975)
Kauffman and Thompson
Physiology: 700
0
0
600 o PYLORIC
to0 x
500
*
0
* OXYNTIC
0
400 _
C
.E E
z
a-
300 _ 200 _
f
100 _ 0 8 0
0 0 0
00
2
4
8
6
10
12
14
pH
FIG. 4. Apparent permeability to sodium of the oxyntic glandular mucosa (0) of one dog and of the pyloric glandular mucosa (0) of another dog in cm min1 plotted against the pH of the solutions used to irrigate the mucosa.
pouch was dissected out and pinned to a cork board with minimal stretching. Its area was measured by planimetry. A dog with a pouch of the pyloric glandular mucosa was killed in the same way, and the surface area of the mucosa of her pouch was measured. This allowed calculation of permeability to be related to surface area of the mucosa, and the units of P'Na as calculated with Eq. are therefore cm min1. The data are plotted in Fig. 4. Because the actual surface area of the gastric mucosa is some 13 times the gross area (14) and because we have no evidence what fraction of the surface contains the Na+ channels, the numerical values have little meaning. The data show that in the pH range of 1.5-10 the permeability of the pyloric glandular mucosa to sodium is greater than that of the oxyntic glandular mucosa. This confirms the observations of Dyck et al. (15) made on the dog and of Spenney et al. (16, 17) on Necturus. At pH's above 10 the permeability of each mucosa increases abruptly and there is no difference between them. DISCUSSION These data are consistent with the idea that the channels through which Na+ diffuses from gastric interstitial fluid into the lumen are dominated by ionizable groups having an apparent pKa in the range 10.5-11.5. When the pH of the fluid bathing the mucosa is below 9 these groups maximally impede the passage of Na+. There is competition between Na+ and H+ for permeation of the gastric mucosa (1), and it is likely that the channels through which Na+ moves are the same as those through which H+ diffuses back into the mucosa. The same forces which oppose Na+ keep back-diffusion of H+ to a minimum. Thus, the high apparent pKa is a teleological necessity if the mucosa is to contain within the lumen the acid it secretes. In the pH range of 9-11 the charges of the channels are affected in such a way as to increase Na+ permeability. The effect is rapid and reversible. H+ or OH- diffuses quickly to and from the groups whose ionization it affects. The change in ionization may occur without any alteration in the dimensions of the channels. Fluid output from the mucosa in this pH range is slightly greater than at lower pH. It is possible that the increase in P'Na is accompanied by an increase in the mucosa's hydraulic coefficient, but this remains to be determined. When the pH is raised above about 11.2 there is a major qualitative change in the properties of the gastric mucosa:
3733
numbers of very large channels appear. These allow rapid filtration of interstitial fluid into the lumen, and because gastric interstitial fluid contains a high concentration of all plasma proteins (18), these too appear in the lumen. When a similar protein-losing state is induced by treating the mucosa with dithiothreitol, plasma proteins leak through tight junctions joining surface epithelial cells (19). Perhaps these junctions are also opened by high pH. Permeation of Na+ through the frog gallbladder is governed by acidic groups having an apparent pKa of 4.4 (8), and the Na+ permeability of this tissue can be almost completely blocked by 2,4,6-triaminopyrimidine (20). The Na+ channels in nerve are dominated by a single acidic group having a pKa of 5.2 (9), and these channels are blocked by tetrodotoxin (21). In each case blockade is effected by the combination of the blocking agent with charged groups in the channels and by the appropriate steric configuration of the agent. The apparent pKa of the groups controlling the Na+ channels through the gastric mucosa is some 5 pH units higher than the pKa's of the corresponding groups in gallbladder and nerve, and consequently a compound which might be expected to block the gastric channels must have properties very different from those of 2,4,6-triaminopyrimidine and tetrodotoxin. Such a compound can be expected to block the back-diffusion of H+ through the gastric mucosa as well as the passage of Na+ in the other direction. The authors are deeply grateful to Dr. Jared Diamond for advice and criticism and to Dr. John A. Jacquez for help with the permeability calculations. This investigation was supported by Grant AM17328 and Grant AM-08716 from the U.S. Public Health Service. G.L.K. is Academic Trainee in General Surgery, Section of General Surgery, the University of Michigan. M.R.T. was Harkness Fellow of the Commonwealth Fund of New York, 1973-1975.
Code, C. F., Higgins, J. A., Moll, J. C., Orivs, A. L. & Scholer, J. F. (1963) "The influence of acid on the gastric absorption of water, sodium and potassium," J. Physiol. (London) 166, 110-119. 2. Davenport, H. W., Warner, H. A. & Code, C. F. (1964) "Functional significance of gastric mucosal barrier to sodium," Gastroenterology 47, 142-152. 3. Durbin, R. P. & Moody, F. G. (1965) "Water movement through a transporting epithelial membrane: the gastric mucosa," Symp. Soc. Exp. Biol. 19, 229-306. 4. Davenport, H. W. (1968) "Destruction of the gastric mucosal barrier by detergents and urea," Gastroenterology 54, 1751.
181. M. & Hunt, J. N. (1956) "The effect of sodium fluoride on the output of some electrolytes from the gastric mucosa of cats," J. Physiol. (London) 133, 317-329. 6. Davenport, H. W. (1971) "Protein-losing gastropathy produced by sulfhydryl reagents," Gastroenterology 60, 8705.
Bond, A.
879. 7.
8. 9. 10. 11.
Davenport, H. W. & Kauffman, G. L., Jr. (1975) "Plasma shedding by the canine oxyntic and pyloric glandular mucosa induced by topical acetylcholine; Effect of atropine and physostigmine," Gastroenterology 70, in press. Moreno, J. H. & Diamond, J. M. (1974) "Discrimination of monovalent inorganic cations by 'tight' junctions of gallbladder epithelium," J. Membr. Biol. 15, 277-318. Hille, B (1968) "Charges and potentials at the nerve surface. Divalent ions and pH," J. Gen. Physiol. 51, 221-236. Davenport, H. W. (1966) "Fluid produced by the gastric mucosa during damage by acetic and salicylic acids," Gastroenterology 50, 487-499. Good, N. E., Winget, G. D., Winter, W., Connolly, T. N.,
Izawa, S. & Singh, M. M. (1966) "Hydrogen ion buffers for biological research," Biochemistry 5, 465-477.
3734
Physiology: Kauffman and Thompson
12. Jacquez, J. A. (1971) "A generalization of the Goldman equation including the effect of electrogenic pumps." Math. Biosci. 12, 185-196. 13. Jacquez, J. A. & Schultz, S. G. (1974) "A general relation between membrane potential, ion activities, and pump fluxes for symmetric cells in a steady state," Math. Biosci. 20, 10-25. 14. Canosa, C. A. & Rehm, W. S. (1958) "Microscopic dimensions of the pit region of the dog's gastric mucosa," Castroenterology 35, 292-297. 15. Dyck, W. P., Werther, J. L., Rudick, J. & Janowitz, H. D. (1969) "Electrolyte movement across canine antral and fundic mucosa," Gastroenterology 56, 488-495. 16. Spenney, J. G., Shoemaker, R. L. & Sachs, G. (1974) "Microelectrode studies of fundic gastric mucosa: Cellular coupling
Proc. Nat. Acad. Sci. USA 72 (1975) and shunt conductance," J. Membr. Biol. 19, 105-128. 17. Spenney, J. G., Flenstrom, G., Shoemaker, R. L. & Sachs, G. (1975) "Quantitation of conductance pathways in antral gastric mucosa," J. Gen. Physiol. 65, 645-662. 18. Bruggeman, T. M. (1975) "Plasma proteins in canine gastric lymph," Gastroenterology 35, 1204-1210. 19. Munro, D. R. (1974) "Route of protein loss during a model protein-losing gastropathy in dogs," Gastroenterology 66, 960-972. 20. Moreno, J. H. (1974) "Blockage of cation permeability across the tight junctions of gallbladder and other leaky epithelia," Nature 251, 150-151. 21. Hille, B. (1975) "An essential ionized acid group in sodium channels," Fed. Proc. 34, 1318-1321.
Vol. 72, No. 9, pp. 3731-3734, September 1975 Physiology
Titration of sodium channels in canine gastric mucosa (gastric mucosal permeability/oxyntic and pyloric mucosas/protein shedding)
GORDON L. KAUFFMAN, JR. AND MICHAEL R. THOMPSON Department of Physiology, The University of Michigan, Ann Arbor, Mich. 48104; and The Center for Ulcer Research and Education, VA Wadsworth Hospital Center, Los Angeles, California 90073
Communicated by Horace W. Davenport*, June 19, 1975
Net Na+ flux from mucosa to lumen, potenABSTRACT tial difference, and volume and plasma protein outputs were measured in vagally denervated, separated pouches of the dog's oxyntic or pyloric glandular mucosa when the pouches were irrigated with Na+-free solutions whose pH ranged from 1.5 to 12.2. The apparent permeability to Na+(P'Na) was calculated. P'Na is lowest when the mucosa is bathed with acid and increases 2- to 3-fold when the pH is raised to 10. In the range of pH 10.0-11.2 P'Na is greater by an order of magnitude, but volume output is small, and no plasma proteins are shed. When the pH is above 11.2 there is an abrupt increase in P'Na, and the mtcosa sheds a large volume of fluid containing plasma proteins. The change effected by raising the pH to the range of 10.0-11.2 occurs within 10 sec, and it is reversible. The change effected by raising the pH above 11.2 also occurs within 10 sec, and it is partly reversible.
The normal gastric mucosa is only very slightly permeable HI and Na+ (1, 2) and its hydraulic coefficient is low (3). Permeability to H+ and Na+ can be increased by treating the mucosa with detergents (4) or mercurials (5, 6) in neutral solution. In this state flux of Na+ from mucosal interstitial fluid to lumen is large, but little fluid and no plasma proteins are shed. If the mucosa is treated with dithiothreitol (6) or with acetylcholine (7) mucosal permeability is enormously increased. Flux of Na+ into the lumen is very large, and a large volume of fluid containing plasma proteins is rapidly filtered into the lumen. In the normal state the permeability of both the oxyntic and pyloric glandular mucosas to Na+ is affected by the [H+] of the fluid bathing the mucosa (1). When this fluid is acid, mucosal permeability to Na+ is least, and as the pH of the fluid is raised, permeability to Na+ increases. We can assume that in this state Na+ moves down its electrochemical gradient from mucosal interstitial fluid to lumen through small channels lined with ionizable groups. If the [H+] of the fluid in contact with the mucosa is manipulated, the degree of ionization of the groups is altered, and permeability to Na+ changes. Moreno and Diamond (8) have developed this idea of the influence of [H+] upon ionic selectivity of a membrane in their study of the cation conductance of the gallbladder. They found that the apparent pKIa of the group which governs the Na+ conductance of the rabbit gallbladder is 4.55. When the pH of the fluid bathing the gallbladder is below 4.55, Na+ conductance falls to a minimum. Raising the pH above 6 increases Na+ conductance many times. In work based upon similar considerations Hille (9) found that Na+ channels in nerve appear to be dominated by a single group having a pKIa of 5.2. We have measured the net Na+ flux from mucosa to to
Abbreviation: PD, potential difference. To whom reprint requests should be addressed at Department of Physiology, The University of Michigan Medical Center, Ann Arbor, Mich. 48104.
*
3731
lumen and the potential difference (PD) in pouches of the dog's oxyntic and pyloric glandular mucosa when the pouches were irrigated with Na+-free solutions whose pH ranged from 1.5 to 12.2. We have calculated the apparent Na+ permeability coefficient from the data obtained at each pH. METHODS AND CALCULATIONS Three dogs with separated, vagally denervated (Heidenhain) pouches of the oxyntic glandular area of the stomach and two dogs with separated, vagally denervated pouches of the pyloric glandular mucosa were used. The preparation and care of the animals, the methods for measuring the net Na+ flux, volume and plasma protein outputs, and the potential difference have been described (2, 6, 7, 10). Solutions used to irrigate the mucosa were HCl at pH 1.5 or solutions buffered with tris(hydroxymethyl)aminomethane, the buffers described by Good et al. (11), lysine, or arginine, all titrated to the desired pH with KOH. All solutions were made isotonic with mannitol. Control experiments showed that the [K+] of the solutions did not influence the results. All solutions were Na+-free when placed in the pouches. The pH of the solutions did not change significantly during the period of irrigation. The apparent permeability coefficient was calculated by means of the equation
XN; [Na+],
exp(-FE/2RT) - [Na+],
exp(FE/2RT) [1]
derived from the integrated Nernst-Planck equation by Jacquez and Schultz (12, 13). JNa is the measured net flux of Na+ from mucosa to lumen in geq min-. [Na+]p is the plasma concentration of Na+ in geq ml-l, and [Na+]lf is that in the luminal fluid. The other symbols have their usual meanings. RESULTS Effect of pH upon the oxyntic glandular mucosa The effect of irrigating the pouch of the oxyntic glandular mucosa of one dog with solutions of different pH's is shown in Fig. 1. Net Na+ output rose and PD fell only slightly as the pH was raised from 1.5 to 10.0. In the range from 10.0 to approximately 11.5 Na+ output rose steeply, and the PD fell. There was a small output of fluid from the mucosa, but this fluid did not contain plasma proteins. Above pH 11.5 there was a large output of fluid containing plasma proteins, and Na+ output rose to high values. Moreno and Diamond (8) measured Na+ conductance of the rabbit gallbladder as a function of pH, and they were able to fit their data with a titration curve based on the assumption that a single ionizable group governs the Na+ conductance. The pKa of the group was calculated to be 4.55.
3732
Physiology: Kauffman and Thompson
Proc. Nat. Acad. Sci. USA 72 (1975)
~2000
P
-
4
710 0)
1000 -
2
CL~ zo .500500-
~~p 1 15
19
.
FIG2.NtN+otpt30E n iescesie3 1.5
11.1
1.5
11.9
e
1.5
pH
pH FIG. 1. Net Na+ output (0) and potential difference (0) (PD) in a pouch of a dog's oxyntic glandular mucosa irrigated for 30 min periods with Na+-free solutions of the pH indicated on the abscissa. The solid line is the calculated titration curve of a single acidic group having a pK, of 11.05. The broken line was drawn by eye. Total height of the bars indicates fluid output in the 30 min irrigation period, and the solid part of the bars indicates output of plasma.
Our cdata do not permit a similar calculation, for the transition from the state of intermediate permeability to the protein-losing state robs us of the upper half of the titration curve. Nevertheless, we have fitted the data with a titration curve having a pKIa of 11.05, and this curve is drawn as the solid line in the figure. Its purpose is merely to show that the data below pH 11.5 might be fitted with part of a titration curve. Data obtained using pouches of the oxyntic glandular area in two other dogs have similar results. Those data might be fitted with curves having pKIa values of about 10.5.
Reversibility The net Na+ output and the PD were measured in a pouch of the oxyntic glandular area when the pouch was irrigated for five successive 30 min periods with solutions in the following sequence: pH 1.5, pH 11.1, pH 1.5, pH 11.9, and pH 1.5. The series of observations was repeated on the same dog on different days for a total of three times. The results are shown in Fig. 2. When the solution of pH 1.5 was replaced with a solution of higher pH the PD was observed to fall to essentially its final value in 10 sec. When the solution of higher pH was replaced by one of pH 1.5 the PD climbed to nearly its final value in 15 min. There were no statistically significant differences between the Na+ outputs during irrigation with a solution of pH 1.5 before and after irrigation with one of pH 11.1. The permeability change effected by raising the pH from 1.5 to 11.1 is almost entirely reversible. The results obtained when the solution of pH 11.9 was replaced with one of pH 1.5 show that the change effected by a solution which induces a very large Na+ output accompanied by plasmashedding is partially reversible. Effect of pH upon the pyloric glandular mucosa The results obtained by irrigating the pouch of the pyloric glandular area of one dog with solutions of different pH are shown in Fig. 3. As the pH of the irrigation fluid was raised
FIG. 2. Net Na+ outputs 4- SEM in five successive 30 min periods in which a pouch of a dog's oxyntic glandular mucosa was irrigated with Na+-free solutions of the pH indicated. The circles with vertical bars are the mean PD's i SEM during the 30 min periods. Between the 2nd and 3rd periods and between the 4th and 5th periods the pouch was filled with 100 mN HCl, and the PD was read at 3 min intervals. These readings are shown as filled circles connected with a line. The series of observations was repeated three times on different days.
from 8.6 to 12.2 the PD fell, and the Na+ output rose. At pH 11 volume output was greater than at lower pH's, and above pH 11.9 plasma-shedding occurred. Results obtained with the other pyloric glandular pouch were almost identical. A series of observations similar to those made on the oxyntic glandular mucosa was performed to test reversibility. A pouch of the pyloric glandular mucosa was irrigated for five successive 30 min periods with solutions in the following sequence: pH 1.5, pH 11.5-12.0, pH 1.5, pH 12.1, and pH 1.5. The results were qualitatively identical with those obtained on the oxyntic glandular mucosa. The transition occurring when the pH is raised to an intermediate value is almost instantaneous and almost completely reversible. The transition effected by raising the pH to a high value is just as rapid and is partially reversible. Apparent Na+ permeability coefficient One dog with a pouch of the oxyntic glandular area was killed with an overdose of anesthetic, and the mucosa of her
.c 1000*oo 0 C,, cr
50
0
I
40
4) 750
k soo i 2500
0'
;
20
LoaJo
ol
R10
i
30
* *
2 5'g
n
, 7Qn n
n ffl;
13
pH
FIG. 3. Net Na+ output (-) and potential difference (0) (PD) in a pouch of a dog's pyloric glandular mucosa irrigated for 30 min periods with Na+-free solutions of the pH indicated in the abscissa. The lines are drawn by eye. Total height of the bars indicates fluid output in the 30 min periods, and the solid part of the bars indicates output of plasma.
Proc. Nat. Acad. Sci. USA 72 (1975)
Kauffman and Thompson
Physiology: 700
0
0
600 o PYLORIC
to0 x
500
*
0
* OXYNTIC
0
400 _
C
.E E
z
a-
300 _ 200 _
f
100 _ 0 8 0
0 0 0
00
2
4
8
6
10
12
14
pH
FIG. 4. Apparent permeability to sodium of the oxyntic glandular mucosa (0) of one dog and of the pyloric glandular mucosa (0) of another dog in cm min1 plotted against the pH of the solutions used to irrigate the mucosa.
pouch was dissected out and pinned to a cork board with minimal stretching. Its area was measured by planimetry. A dog with a pouch of the pyloric glandular mucosa was killed in the same way, and the surface area of the mucosa of her pouch was measured. This allowed calculation of permeability to be related to surface area of the mucosa, and the units of P'Na as calculated with Eq. are therefore cm min1. The data are plotted in Fig. 4. Because the actual surface area of the gastric mucosa is some 13 times the gross area (14) and because we have no evidence what fraction of the surface contains the Na+ channels, the numerical values have little meaning. The data show that in the pH range of 1.5-10 the permeability of the pyloric glandular mucosa to sodium is greater than that of the oxyntic glandular mucosa. This confirms the observations of Dyck et al. (15) made on the dog and of Spenney et al. (16, 17) on Necturus. At pH's above 10 the permeability of each mucosa increases abruptly and there is no difference between them. DISCUSSION These data are consistent with the idea that the channels through which Na+ diffuses from gastric interstitial fluid into the lumen are dominated by ionizable groups having an apparent pKa in the range 10.5-11.5. When the pH of the fluid bathing the mucosa is below 9 these groups maximally impede the passage of Na+. There is competition between Na+ and H+ for permeation of the gastric mucosa (1), and it is likely that the channels through which Na+ moves are the same as those through which H+ diffuses back into the mucosa. The same forces which oppose Na+ keep back-diffusion of H+ to a minimum. Thus, the high apparent pKa is a teleological necessity if the mucosa is to contain within the lumen the acid it secretes. In the pH range of 9-11 the charges of the channels are affected in such a way as to increase Na+ permeability. The effect is rapid and reversible. H+ or OH- diffuses quickly to and from the groups whose ionization it affects. The change in ionization may occur without any alteration in the dimensions of the channels. Fluid output from the mucosa in this pH range is slightly greater than at lower pH. It is possible that the increase in P'Na is accompanied by an increase in the mucosa's hydraulic coefficient, but this remains to be determined. When the pH is raised above about 11.2 there is a major qualitative change in the properties of the gastric mucosa:
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numbers of very large channels appear. These allow rapid filtration of interstitial fluid into the lumen, and because gastric interstitial fluid contains a high concentration of all plasma proteins (18), these too appear in the lumen. When a similar protein-losing state is induced by treating the mucosa with dithiothreitol, plasma proteins leak through tight junctions joining surface epithelial cells (19). Perhaps these junctions are also opened by high pH. Permeation of Na+ through the frog gallbladder is governed by acidic groups having an apparent pKa of 4.4 (8), and the Na+ permeability of this tissue can be almost completely blocked by 2,4,6-triaminopyrimidine (20). The Na+ channels in nerve are dominated by a single acidic group having a pKa of 5.2 (9), and these channels are blocked by tetrodotoxin (21). In each case blockade is effected by the combination of the blocking agent with charged groups in the channels and by the appropriate steric configuration of the agent. The apparent pKa of the groups controlling the Na+ channels through the gastric mucosa is some 5 pH units higher than the pKa's of the corresponding groups in gallbladder and nerve, and consequently a compound which might be expected to block the gastric channels must have properties very different from those of 2,4,6-triaminopyrimidine and tetrodotoxin. Such a compound can be expected to block the back-diffusion of H+ through the gastric mucosa as well as the passage of Na+ in the other direction. The authors are deeply grateful to Dr. Jared Diamond for advice and criticism and to Dr. John A. Jacquez for help with the permeability calculations. This investigation was supported by Grant AM17328 and Grant AM-08716 from the U.S. Public Health Service. G.L.K. is Academic Trainee in General Surgery, Section of General Surgery, the University of Michigan. M.R.T. was Harkness Fellow of the Commonwealth Fund of New York, 1973-1975.
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12. Jacquez, J. A. (1971) "A generalization of the Goldman equation including the effect of electrogenic pumps." Math. Biosci. 12, 185-196. 13. Jacquez, J. A. & Schultz, S. G. (1974) "A general relation between membrane potential, ion activities, and pump fluxes for symmetric cells in a steady state," Math. Biosci. 20, 10-25. 14. Canosa, C. A. & Rehm, W. S. (1958) "Microscopic dimensions of the pit region of the dog's gastric mucosa," Castroenterology 35, 292-297. 15. Dyck, W. P., Werther, J. L., Rudick, J. & Janowitz, H. D. (1969) "Electrolyte movement across canine antral and fundic mucosa," Gastroenterology 56, 488-495. 16. Spenney, J. G., Shoemaker, R. L. & Sachs, G. (1974) "Microelectrode studies of fundic gastric mucosa: Cellular coupling
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