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Membrane Resistances and Electromotive Forces during Stimulation of Chloride Secretion in the Canine Tracheal Epithelium
to match that across the apical membrane. Whether secretagogues directly augment Na-K pump activity and/ or NaCl co-transport across the basolateral membrane is not certain at the present time, but the secondary increase in basolateral membrane K permeability which accompanies their action has two effects: first, it provides for increased diffusional K exit from the cell to match the increase in Na-K pump rate so that cell composition is not markedly affected by large changes in transepithelial transport rate. Second, the resulting repolarization of the cell contributes, in an essential fashion, to the driving force for Cl exit from the cell across the apical membrane.
1 Olver RE, Davis B, Marin MG, Nadel JA. Active transport of Na and Cl across the canine tracheal epithelium in vitro. Am Rev Respir Dis 1975; 112:811 2 Widdicombe JH, Welsh MJ. Ion transport by dog tracheal epithelium. Federation Proc. 1980; 39:3062 3 Frizzell RA, Field M, Schultz SC. Sodium-coupled choride transport by epithelial tissues. Am J Physiol 1979; 238:5Fl 4 Al-Bazzaz F., Yadava VP, Westenfelder C. Modification of Na and Cl transport in canine tracheal mucosa by prostaglandins. Am J Physiol 1981; 240:Fl01 5 Smith PL, Frizzell RA, Welsh MJ. Chloride secretion by canine tracheal epithelium. I. Effects of agents which mediate secretion on cAMP content. J Membrane Bioi (in press) 6 Welsh MJ, Frizzell RA, Smith PL. Chloride secretion by canine tracheal epithelium. II. The cellular electrical potential profile. J Membrane Bioi (in press)
1n
Michael/. Welsh, M.D.; Philip L . Smith, M.D.; and Raymond A. Frizzell, M.D.
A
}though the transport properties of Na-absorbing epithelia have been modeled as equivalent electrical circuits since the time of Ussing, these techniques and theories have not, for the most part, been applied to the study of electrogenic chloride secretion. The purpose of this study was to examine the mechanism of ion transport and stimulation of Cl secretion by the canine tracheal epithelium using intracellular microelectrode techniques and an equivalent electrical circuit analysis to obtain values for individual membrane resistances and electromotive forces. •Department of Physiology, University of Texas Medical School at Houston, and Department of Internal Medicine, University of Iowa Hospitals, Iowa City. Supported by NIH grants AM26702, HL07159, and AM05973.
CHEST, 81: 5, MAY, 1982 SUPPLEMENT
Cell
Submucosal Solution
+ ...._______,t+.__ _ _ _ ___.t -,a -,b , l
REFERENCEs
Membrane Resistances and Electromotive Forces during Stimulation of Chloride Secretion the Canine Tracheal Epithelium*
Mucosal Solution
T
-,,,R,
FIGURE 1. An equivalent electrical circuit model for describing electrogenic ion transport in the canine tracheal epithelium. The shaded areas indicate the epithelial cells. See text for details. Figure I depicts the equivalent electrical circuit model that we used. Isc refers to the short ciruit, .; refers to the measured electrical potential difference, R refers to an electrical resistance, E refers to an electromotive force, and the subscripts a, b, p, and t refer to apical, basolateral, paracellular, and transepithelial values, respectively. At each barrier to ion movement the ionic permeabilities are represented as electrical resistances, the electrochemical driving forces or Nernst equilibrium potentials are represented as electromotive forces, and the flow of ions is represented as an electrical current. Tissues were treated with indomethacin ( 10-e M) to reduce the basal rate of Cl secretion and then Cl secretion was stimulated by the addition of epinephrine ( IQ-e M) . The method of determining the equivalent circuit parameters was similar to that used by Reuss and Finn (J Gen Physiol 1974; 64:1-25) and Fromter and GebTable 1-EfJecr of Epinephrine on Elecrrical
Properlie• and Equi.,alent Circuit Parameler•
Isc (pA•cm-2 )
R, (O•cm2 )
,Ya (mV)
fa
29±4
502±142
-60±3
0.82±0.05
221 ± 48 Rb (O•cm2 )
-50±7
E.
(mV)
0.58±0.07 Eb (mV)
2785±562 562± 98
+19±14
+77±3
Epinephrine 238 ± 63 164 ± 34
-31 ± 6
+64±5
Control
Epinephrine 85±9 R. (O•cm2 ) Control
n=4 tissues. All values represent steady-state measurements. Indomethacin (10-• M) was present in mucosal solution during both conditions. Isc refers to the short circuit current; R,, R., and Rb to the transepithelial, apical membrane, and basolateral membrane resistances; !{Ia to the electrical potential difference across the apical membrane; fa to the fractional resistance of the apical membrane ( = R./R. + Rb); and E. and Eb refer to the electromotive forces at the apical and basolateral cell membrane. Paracellular resistance was 686 ± 244 O•cm2•
LUNG FLUIDS AND SECRETIONS 3S
ler (Pflugers Arch 1977; 371:99-108) and is based on the assumption that the initial effect (&rst 5-10 sec) of epinephrine is to decrease the apical cell membrane resistance (Ra). This assumption was based on our previous observations (Welsh et al, J Gen Physiol1980; 76:27a) and has been confirmed with independent methods (unpublished observation) . Table 1 shows the steady-state values of the electrical properties for 4 tissues during the control condition and during stimulation of Cl secretion with epinephrine. In agreement with previous studies, the short circuit current ( Isc) increased, reflecting an increased rate of electrogenic Cl secretion, and the transepithelial resistance ( Rt) decreased. These changes were accompanied by a significantly depolarization of the intracellular electrical potential and a decrease in the fractional resistance of the apical membrane (where fR Ra - - - - ) . The decrease in transepithelial resistance and R. + Rb fR can be explained by a fall in both R. and Rb, the apical and basolateral membrane resistances. The tenfold decreases in R., together with the increased rate of Cl secretion, indicates that Cl movement at the apical membrane is an electrically conductive process. There was also an inverse correlation between Isc and Ra indicating that the decrease in Ra is due to an increased Cl permeability. This conclusion is further supported by our previous observation (Welsh et al, J Gen Phys 1930; 76:27a) that substitution of Cl with sulfate or gluconate in the mucosal bathing solution generates a diffusion potential across the apical membrane and increases its fractional resistance. Finally, the inverse relationship between transport rate and Ra and the time course of the change in Ra ( R. decreases in 20-30 sec following the onset of stimulation) suggest that
=
secretagogues may mediate the rate of secretion by
regulating R•. During control conditions the electromotive force at the apical membrane (E.) varied over a wide range (+59 mV to -8 mV) but decreased in every case, to an average value of -31 m V with a range one-third the size of that in the control condition. Furthermore, we observed a direct relationship between Ea and R., suggesting that Ea is a membrane diffusion potential due to both Cl and Na concentration gradient across the apical membrane. The significance of this relationship can be appreciated from an examination of the following equation:
where E" and E"• represent the Nemst equilibrium potentials for Na and Cl across the apical membrane and t•' and t"• refer to the transference numbers for Cl and Na across the apical membrane. Thus, when secretion is stimulated, the apical membrane resistance to Cl movement will decrease, t'' will increase, and t"• decrease, (since t" + E"" = 1) and E" will be the pre-
4S 24TH ASPEN LUNG CONFERENCE
dominant factor determining Ea. On the other hand, when secretion is minimized by the addition of indomethacin during the control period, the resistance to Cl movement may be in the same range as, or greater than the resistance to Na movement. Therefore, t•' will be approximately equal to or less than t"•, and Ea will be determined by both E•' and E"•. We can draw two conclusions from these findings. First, since during stimulated conditions Ea will represent the Nernst equilibrium for Cl, we can calculate an intracellular Cl concentration of 37 mM, twice the value expected for an equilibrium distribution across the apical membrane. Since apical membrane Cl movement is an electrically conductive process and there is a favorable electrochemical gradient, Cl may leave the cell passively via a process that needs to be no more complicated than simple diffusion. Second, these findings (plus our observation that amiloride increases Ra in the tracheal epithelium) indicate that the same cell contains the transport processes and ion permeabilities required for both Na absorption and Cl secretion. Thus, the direction of net salt and water movement across the epithelium may be mediated by factors that regulate the ratio of Na and Cl permeabilities in the apical cell membrane. Stimulation of secretion decreased the basolateral membrane resistance to one third the value observed during non-secreting conditions. There was also an inverse correlation between Isc and Rb. These changes indicate an increase in the passive ion permeabilities at the basolateral membrane with an increase in the transepithelial transport rate. However, despite a wide range of values of Rb there were only minimal changes in the electromotive force at the basolateral membrane (Eb). Although the transport processes at the basolateral membrane are more complex than those at the apical membrane (since they include passive ionic permeabilities in parallel with the Na pump), these observations allow us to draw some inferences about the mechanisms involved. The decrease in Rb, together with minimal changes in Eb, indicate an increase in ionic permeability with minimal changes in ion concentration. The decrease in Rb is most likely due to an increase in K permeability based on four lines of evidence: first, we have previously found that there is no appreciable Cl conductance in the basolateral membrane; second, a significant Na conductive pathway is very unlikely based on findings in Na absorbing epithelia; third, Stutts and Gatzy (Physiologist 1980; 23 :62) found that increased concentrations of K in the submucosal bathing solution inhibit transport; and fourth, Smith and Frizzell (these proceedings) found that increasing the K concentration of the submucosal bathing solution depolarized the basolateral membrane potential during both control and stimulated conditions. Thus, as the transport rate increases, K entry into the cell via the Na-K-ATPase will increase. The increased K permeability (decreased Rb) will allow K recycling across the basolateral membrane and prevent an increase in intracellular K concentration (thus, Eb will not increa~e).
CHEST, 81: 5, MAY, 1982 SUPPLEMENT
1 Olver RE, Davis B, Marin MG, Nadel JA. Active transport of Na and Cl across the canine tracheal epithelium in vitro. Am Rev Respir Dis 1975; 112:811 2 Widdicombe JH, Welsh MJ. Ion transport by dog tracheal epithelium. Federation Proc. 1980; 39:3062 3 Frizzell RA, Field M, Schultz SC. Sodium-coupled choride transport by epithelial tissues. Am J Physiol 1979; 238:5Fl 4 Al-Bazzaz F., Yadava VP, Westenfelder C. Modification of Na and Cl transport in canine tracheal mucosa by prostaglandins. Am J Physiol 1981; 240:Fl01 5 Smith PL, Frizzell RA, Welsh MJ. Chloride secretion by canine tracheal epithelium. I. Effects of agents which mediate secretion on cAMP content. J Membrane Bioi (in press) 6 Welsh MJ, Frizzell RA, Smith PL. Chloride secretion by canine tracheal epithelium. II. The cellular electrical potential profile. J Membrane Bioi (in press)
1n
Michael/. Welsh, M.D.; Philip L . Smith, M.D.; and Raymond A. Frizzell, M.D.
A
}though the transport properties of Na-absorbing epithelia have been modeled as equivalent electrical circuits since the time of Ussing, these techniques and theories have not, for the most part, been applied to the study of electrogenic chloride secretion. The purpose of this study was to examine the mechanism of ion transport and stimulation of Cl secretion by the canine tracheal epithelium using intracellular microelectrode techniques and an equivalent electrical circuit analysis to obtain values for individual membrane resistances and electromotive forces. •Department of Physiology, University of Texas Medical School at Houston, and Department of Internal Medicine, University of Iowa Hospitals, Iowa City. Supported by NIH grants AM26702, HL07159, and AM05973.
CHEST, 81: 5, MAY, 1982 SUPPLEMENT
Cell
Submucosal Solution
+ ...._______,t+.__ _ _ _ ___.t -,a -,b , l
REFERENCEs
Membrane Resistances and Electromotive Forces during Stimulation of Chloride Secretion the Canine Tracheal Epithelium*
Mucosal Solution
T
-,,,R,
FIGURE 1. An equivalent electrical circuit model for describing electrogenic ion transport in the canine tracheal epithelium. The shaded areas indicate the epithelial cells. See text for details. Figure I depicts the equivalent electrical circuit model that we used. Isc refers to the short ciruit, .; refers to the measured electrical potential difference, R refers to an electrical resistance, E refers to an electromotive force, and the subscripts a, b, p, and t refer to apical, basolateral, paracellular, and transepithelial values, respectively. At each barrier to ion movement the ionic permeabilities are represented as electrical resistances, the electrochemical driving forces or Nernst equilibrium potentials are represented as electromotive forces, and the flow of ions is represented as an electrical current. Tissues were treated with indomethacin ( 10-e M) to reduce the basal rate of Cl secretion and then Cl secretion was stimulated by the addition of epinephrine ( IQ-e M) . The method of determining the equivalent circuit parameters was similar to that used by Reuss and Finn (J Gen Physiol 1974; 64:1-25) and Fromter and GebTable 1-EfJecr of Epinephrine on Elecrrical
Properlie• and Equi.,alent Circuit Parameler•
Isc (pA•cm-2 )
R, (O•cm2 )
,Ya (mV)
fa
29±4
502±142
-60±3
0.82±0.05
221 ± 48 Rb (O•cm2 )
-50±7
E.
(mV)
0.58±0.07 Eb (mV)
2785±562 562± 98
+19±14
+77±3
Epinephrine 238 ± 63 164 ± 34
-31 ± 6
+64±5
Control
Epinephrine 85±9 R. (O•cm2 ) Control
n=4 tissues. All values represent steady-state measurements. Indomethacin (10-• M) was present in mucosal solution during both conditions. Isc refers to the short circuit current; R,, R., and Rb to the transepithelial, apical membrane, and basolateral membrane resistances; !{Ia to the electrical potential difference across the apical membrane; fa to the fractional resistance of the apical membrane ( = R./R. + Rb); and E. and Eb refer to the electromotive forces at the apical and basolateral cell membrane. Paracellular resistance was 686 ± 244 O•cm2•
LUNG FLUIDS AND SECRETIONS 3S
ler (Pflugers Arch 1977; 371:99-108) and is based on the assumption that the initial effect (&rst 5-10 sec) of epinephrine is to decrease the apical cell membrane resistance (Ra). This assumption was based on our previous observations (Welsh et al, J Gen Physiol1980; 76:27a) and has been confirmed with independent methods (unpublished observation) . Table 1 shows the steady-state values of the electrical properties for 4 tissues during the control condition and during stimulation of Cl secretion with epinephrine. In agreement with previous studies, the short circuit current ( Isc) increased, reflecting an increased rate of electrogenic Cl secretion, and the transepithelial resistance ( Rt) decreased. These changes were accompanied by a significantly depolarization of the intracellular electrical potential and a decrease in the fractional resistance of the apical membrane (where fR Ra - - - - ) . The decrease in transepithelial resistance and R. + Rb fR can be explained by a fall in both R. and Rb, the apical and basolateral membrane resistances. The tenfold decreases in R., together with the increased rate of Cl secretion, indicates that Cl movement at the apical membrane is an electrically conductive process. There was also an inverse correlation between Isc and Ra indicating that the decrease in Ra is due to an increased Cl permeability. This conclusion is further supported by our previous observation (Welsh et al, J Gen Phys 1930; 76:27a) that substitution of Cl with sulfate or gluconate in the mucosal bathing solution generates a diffusion potential across the apical membrane and increases its fractional resistance. Finally, the inverse relationship between transport rate and Ra and the time course of the change in Ra ( R. decreases in 20-30 sec following the onset of stimulation) suggest that
=
secretagogues may mediate the rate of secretion by
regulating R•. During control conditions the electromotive force at the apical membrane (E.) varied over a wide range (+59 mV to -8 mV) but decreased in every case, to an average value of -31 m V with a range one-third the size of that in the control condition. Furthermore, we observed a direct relationship between Ea and R., suggesting that Ea is a membrane diffusion potential due to both Cl and Na concentration gradient across the apical membrane. The significance of this relationship can be appreciated from an examination of the following equation:
where E" and E"• represent the Nemst equilibrium potentials for Na and Cl across the apical membrane and t•' and t"• refer to the transference numbers for Cl and Na across the apical membrane. Thus, when secretion is stimulated, the apical membrane resistance to Cl movement will decrease, t'' will increase, and t"• decrease, (since t" + E"" = 1) and E" will be the pre-
4S 24TH ASPEN LUNG CONFERENCE
dominant factor determining Ea. On the other hand, when secretion is minimized by the addition of indomethacin during the control period, the resistance to Cl movement may be in the same range as, or greater than the resistance to Na movement. Therefore, t•' will be approximately equal to or less than t"•, and Ea will be determined by both E•' and E"•. We can draw two conclusions from these findings. First, since during stimulated conditions Ea will represent the Nernst equilibrium for Cl, we can calculate an intracellular Cl concentration of 37 mM, twice the value expected for an equilibrium distribution across the apical membrane. Since apical membrane Cl movement is an electrically conductive process and there is a favorable electrochemical gradient, Cl may leave the cell passively via a process that needs to be no more complicated than simple diffusion. Second, these findings (plus our observation that amiloride increases Ra in the tracheal epithelium) indicate that the same cell contains the transport processes and ion permeabilities required for both Na absorption and Cl secretion. Thus, the direction of net salt and water movement across the epithelium may be mediated by factors that regulate the ratio of Na and Cl permeabilities in the apical cell membrane. Stimulation of secretion decreased the basolateral membrane resistance to one third the value observed during non-secreting conditions. There was also an inverse correlation between Isc and Rb. These changes indicate an increase in the passive ion permeabilities at the basolateral membrane with an increase in the transepithelial transport rate. However, despite a wide range of values of Rb there were only minimal changes in the electromotive force at the basolateral membrane (Eb). Although the transport processes at the basolateral membrane are more complex than those at the apical membrane (since they include passive ionic permeabilities in parallel with the Na pump), these observations allow us to draw some inferences about the mechanisms involved. The decrease in Rb, together with minimal changes in Eb, indicate an increase in ionic permeability with minimal changes in ion concentration. The decrease in Rb is most likely due to an increase in K permeability based on four lines of evidence: first, we have previously found that there is no appreciable Cl conductance in the basolateral membrane; second, a significant Na conductive pathway is very unlikely based on findings in Na absorbing epithelia; third, Stutts and Gatzy (Physiologist 1980; 23 :62) found that increased concentrations of K in the submucosal bathing solution inhibit transport; and fourth, Smith and Frizzell (these proceedings) found that increasing the K concentration of the submucosal bathing solution depolarized the basolateral membrane potential during both control and stimulated conditions. Thus, as the transport rate increases, K entry into the cell via the Na-K-ATPase will increase. The increased K permeability (decreased Rb) will allow K recycling across the basolateral membrane and prevent an increase in intracellular K concentration (thus, Eb will not increa~e).
CHEST, 81: 5, MAY, 1982 SUPPLEMENT