J. Physiol. (1977), 268, pp. 353-369 With 7 text-figures Printed in Great Britain
353
SHORT-CIRCUIT CURRENT AND TOTAL CONDUCTANCE MEASUREMENTS ON RABBIT ILEUM
BY C. HENRIQUES DE JESUS From the Centro de Biologia, Instituto Gulbenkian de Ciencia, Oeiras, Portugal
(Received 13 September 1976) SUMMARY
1. Short-circuit current (SCC) and total conductance (at) measurements were made in in vitro preparations of rabbit terminal ileum. 2. Successive additions of D-glucose increased the SCC and Gt in a stepwise way and both effects were seen to correlate linearly. 3. Measurements of SCC and Gt were also made after replacing Na in the Krebs solution by K or sucrose. The SCC decreased to nearly zero in both cases but the decrease in Ot was larger when sucrose was used. 4. SCC and Gt were monitored after the addition of ouabain. SCC decreased to nearly zero but Gt remained constant. 5. On successive additions of sucrose after the preparation was mounted in 1/4 Na Krebs without osmolal compensation, SCC decreased and Gt increased, a linear relationship being found between both changes. 6. Successive additions of 2-deoxy-D-glucose caused a stepwise decrease of SCC and an increase in Gt. These effects seemed independent of the presence of glucose. 7. A model in which a resistance in series is added to the basic model by Ussing & Windhager (1964), modified by Rose & Schultz (1971), is proposed to account for these findings. INTRODUCTION
In 1964 Schultz & Zalusky presented a paper in which an exhaustive analysis of the behaviour of rabbit ileum in vitro and in short-circuit conditions was made (Schultz & Zalusky, 1964a). The short-circuit current (SCC) was seen to equate net Na transport both in the presence and in the absence of glucose. A cellular model was suggested in which the mucosal brush-border membrane was seen as the limiting barrier for diffusion of Na to the cell and the Na pump was located in the basal membrane.
C. HENRIQUES DE JESUS 354 Rose & Schultz (1971) presented the most recent electrical model for intestine. This model is based on morphological as well as electrical considerations involving micro-electrode measurements. The main elements of the networks are split into mucosal and serosal sections, series and parallel conductances with two electromotive forces, and an extracellular shunt pathway. This is elegant but it is difficult to relate the electrical elements to the measurable variables. These models are all elaborations over Ussing & Windhager's (1964) model for the frog skin and can be simply described as a conductance in series with the pump (Gm) and a parallel conductance (Gp). Em
Gm
Gt = Gp+ mA SCC = Em.Gm, GP
where Gt is the total conductance and Em the electromotive force. It was noted previously (Henriques de Jesus, 1974) in pig intestine that conductance changes are positively correlated with SCC when actively transported sugars are added to the mucosal side of the preparation. The object of the present work was to see initially whether a similar relation existed between SC( and conductance in the rabbit ileum. This being established further work was then undertaken to determine the nature of this interaction. Subsequent evidence suggested the need to consider a modified Ussing-Windhager model incorporating a conductance in series (GC') to describe the over-all electrical characteristics of the in vitro preparation. For such a model the following relationship between the parameters should be considered: Ga
sac
=
G'8 (G'm + G'p) + G's+G,
CmG, Gm' EMC+GIp+G,
A|r
.
G'
It can be shown that a single modification in any one of the elements of the first model will lead to the following results: (1) a modification in the electromotive force (Em) will be seen as a modification of the shortcircuit current (SCC) since SCC = Em . Gm, but not in total conductance (Ct) of the tissue since Ct = Cm + Gp; (2) a modification in Cm will be seen as a modification of SCC and of Ct; (3) a modification in Gp will only be detected as a modification on the conductance.
SCC AND Gt MEASUREMENTS ON RABBIT ILEUM 355 In the second model any modification of a single parameter with the exception of the electromotive force will be reflected in both measurable variables, the SCC and the Gt. It was this type of change which was generally seen to take place in the rabbit ileum subjected to different experimental treatments. METHODS
Rabbits weighing about 2 kg were starved for 18 hr before the experiment. The animals were killed with a blow on the head. The terminal portion of the ileum (5 cm proximal to the ileo-caecal junction) was dissected in bicarbonate saline (Krebs & Henseleit, 1932) and used immediately. The tissue was mounted between two halves of a leucite chamber Ussing-type. This chamber was designed to optimize different parameters known to be important in in vitro preparations, specially mammalian intestines. Fig. I shows the diagram of this chamber and a full description of it can be found in Henriques de Jesus (1974). The area of exposed tissue is 2-4 cm2. SCC was measured using an automatic system of voltage clamp (Henriques de Jesus & Smith, 1974). The resistance of the medium between the two tips of agar bridges was of the order of 5-10 % of the total resistance measured in the presence of the tissue. Corrections were made for this extra resistance. A square-wave generator with variable pulse duration (time used 21 sec) was connected so that a pulse from 0 to + 2 mV could be applied across the tissue, serosal side positive. A circuit equivalent to the electrical properties of the intestine was also constructed with an internal conductance which could be varied from 5 to 60 m-mho and with an open-circuit voltage similar to that found in the living tissue. This equivalent circuit was connected to the input terminals of the first amplifier so that the scale of the recorder could be calibrated for different known conductances. Addition of drugs was made with a micropipette, always after steady-state conditions were achieved. Added volume never surpassed 200 #ul. in a volume of 6 ml. for each half-chamber. The electrolyte composition of the Krebs solution was checked regularly. Mea8urement of adenine nucleotides. Mucosal scrapings from rabbit ileum (0-5 g) were homogenized in 3 ml. 5 % (w/v) trichloroacetic acid and the resulting suspension centrifuged at 3000 g for 5 min. All operations were carried out in the cold, the time from killing the animal to putting the scrapings in trichloroacetic acid varying from 5 to 7 min. Trichloroacetic acid was removed from the supernatant by ether extraction (three extractions with three volumes of ether), ether in the aqueous phase then being removed by a nitrogen stream. The resulting solution was added to a small column of Dowex 1 (Sigma Chemical Co) and AMP, ADP and ATP eluted consecutively by the stepwise addition of 0-01 N-HCl containing 0-02 N-NH4Cl and 1 N-HCl. Samples were scanned for absorbance using a Perkin Elmer series 402 spectrophotometer and the peak of absorbance at 259 nm compared with that obtained with standard solutions of ATP (Bronk & Leese, 1973). RESULTS
The first observation made is that the electrical characteristics of these preparations vary over a wide range, even if taken from the same animal and immediately adjacent to one another. For this reason it was decided
rn _7 z r s 7 U. HENR1V4UENS DPI JPjNUA _.
356
_
-
--
A
Fig. 1. Ussing-type chamber used for experiments with rabbit ileum. A, half chamber. B, compartment for Krebs solution. C, temperature controlled bath. D, clamping point. d, pin-holes. E, current bridge. F, potential bridge. G and G', water inlet and outlet. H, drainage tubing. I, aeration chimney. JI, nemnbrane window. Scale in cm,
SCC AND Gt MEASUREMENTS ON RABBIT ILEUM 357 to present results of individual experiments, statistical treatment of the data being tried only when possible. The second finding is that in these preparations the normal decay of the SCC is much smaller than previously reported: stability for periods over 1 hr, followed by slow decay of about 0-2 ,uA/min in our case, compared with decays of 1 #uA/min in the literature (Schultz & Zalusky, 1964a). A specific role has recently been suggested by Moreno (1974) for 2,4,6triaminopyrimidinium. This drug is supposed to decrease the part of passive conductance related with tight junctions. If this would prove correct for intestine, it must be expected to affect Gp alone in model (1) (see Introduction) or G'p (model (2)) and this would prove most useful in defining the model. For this reason 2,4,6-triaminopyrimidinium was tried in the present preparation. SCC was slightly inhibited with successive additions of this drug but the effects were very small and changes in conductance did not appear to have a consistent pattern. It was known that glucose and other actively transported sugars affected SCC (Schultz & Zalusky, 1964b; Goldner, Schultz & Curran, 1969; Riklis & Quastel, 1958) and in preliminary experiments it also affected conductance. These effects were rapid and reproducible and could be analysed precisely. The amounts of glucose added were chosen after measuring the apparent half-maximal stimulation on the Na-sugar interaction. This was done by assessing the effects on the SCC on different concentrations of glucose with Na at the concentration of 140 mm. The value found was 2X2 mm, which is smaller than the value found by Schultz & Zalusky (4.0 mM). Fig. 2 shows the effect on the SCC and Gt of successive additions of D-glucose in thirteen experiments. The intercepts of the dotted lines with the ordinate show the value of Gt that would represent either the value of Gp if model (1) is considered or the value of G'p/(G'p +G'8) if model (2) represents the behaviour of the preparation. The slopes would be, respectively, 1/Em or (1/Em) . G's/(G'8 + G'p). In three of the thirteen experiments it was possible to discriminate five additions of glucose, giving to the value of Gt for SCC = 0 a good degree of precision. Initial points (control) in each preparation are quite different, variations being larger for the initial Gt than for SCC. The slopes of individual preparations are very similar. On the other hand, the values of Gt for SCC = 0 are different for each preparation. This would mean that, whatever the chosen material, preparations varied more on the value of the shunt parallel conductance than on the value of the respective electromotive forces. The values of the ordinate for SCC = 0 and the slopes would allow for the calculations of the electrical basic parameters of the preparation if model (1) would be a good description of the isolated ileum,
358 C. HENRIQUES DE JESUS but they are not sufficient if, as it will be argued, model (2) is a better description of the ileum in the present conditions. It was decided to investigate further the relative importance of the two paths, the series and the parallel one. Experiments were designed 201-
C/
. '"' ' . '. '
15
E
CC
353
SHORT-CIRCUIT CURRENT AND TOTAL CONDUCTANCE MEASUREMENTS ON RABBIT ILEUM
BY C. HENRIQUES DE JESUS From the Centro de Biologia, Instituto Gulbenkian de Ciencia, Oeiras, Portugal
(Received 13 September 1976) SUMMARY
1. Short-circuit current (SCC) and total conductance (at) measurements were made in in vitro preparations of rabbit terminal ileum. 2. Successive additions of D-glucose increased the SCC and Gt in a stepwise way and both effects were seen to correlate linearly. 3. Measurements of SCC and Gt were also made after replacing Na in the Krebs solution by K or sucrose. The SCC decreased to nearly zero in both cases but the decrease in Ot was larger when sucrose was used. 4. SCC and Gt were monitored after the addition of ouabain. SCC decreased to nearly zero but Gt remained constant. 5. On successive additions of sucrose after the preparation was mounted in 1/4 Na Krebs without osmolal compensation, SCC decreased and Gt increased, a linear relationship being found between both changes. 6. Successive additions of 2-deoxy-D-glucose caused a stepwise decrease of SCC and an increase in Gt. These effects seemed independent of the presence of glucose. 7. A model in which a resistance in series is added to the basic model by Ussing & Windhager (1964), modified by Rose & Schultz (1971), is proposed to account for these findings. INTRODUCTION
In 1964 Schultz & Zalusky presented a paper in which an exhaustive analysis of the behaviour of rabbit ileum in vitro and in short-circuit conditions was made (Schultz & Zalusky, 1964a). The short-circuit current (SCC) was seen to equate net Na transport both in the presence and in the absence of glucose. A cellular model was suggested in which the mucosal brush-border membrane was seen as the limiting barrier for diffusion of Na to the cell and the Na pump was located in the basal membrane.
C. HENRIQUES DE JESUS 354 Rose & Schultz (1971) presented the most recent electrical model for intestine. This model is based on morphological as well as electrical considerations involving micro-electrode measurements. The main elements of the networks are split into mucosal and serosal sections, series and parallel conductances with two electromotive forces, and an extracellular shunt pathway. This is elegant but it is difficult to relate the electrical elements to the measurable variables. These models are all elaborations over Ussing & Windhager's (1964) model for the frog skin and can be simply described as a conductance in series with the pump (Gm) and a parallel conductance (Gp). Em
Gm
Gt = Gp+ mA SCC = Em.Gm, GP
where Gt is the total conductance and Em the electromotive force. It was noted previously (Henriques de Jesus, 1974) in pig intestine that conductance changes are positively correlated with SCC when actively transported sugars are added to the mucosal side of the preparation. The object of the present work was to see initially whether a similar relation existed between SC( and conductance in the rabbit ileum. This being established further work was then undertaken to determine the nature of this interaction. Subsequent evidence suggested the need to consider a modified Ussing-Windhager model incorporating a conductance in series (GC') to describe the over-all electrical characteristics of the in vitro preparation. For such a model the following relationship between the parameters should be considered: Ga
sac
=
G'8 (G'm + G'p) + G's+G,
CmG, Gm' EMC+GIp+G,
A|r
.
G'
It can be shown that a single modification in any one of the elements of the first model will lead to the following results: (1) a modification in the electromotive force (Em) will be seen as a modification of the shortcircuit current (SCC) since SCC = Em . Gm, but not in total conductance (Ct) of the tissue since Ct = Cm + Gp; (2) a modification in Cm will be seen as a modification of SCC and of Ct; (3) a modification in Gp will only be detected as a modification on the conductance.
SCC AND Gt MEASUREMENTS ON RABBIT ILEUM 355 In the second model any modification of a single parameter with the exception of the electromotive force will be reflected in both measurable variables, the SCC and the Gt. It was this type of change which was generally seen to take place in the rabbit ileum subjected to different experimental treatments. METHODS
Rabbits weighing about 2 kg were starved for 18 hr before the experiment. The animals were killed with a blow on the head. The terminal portion of the ileum (5 cm proximal to the ileo-caecal junction) was dissected in bicarbonate saline (Krebs & Henseleit, 1932) and used immediately. The tissue was mounted between two halves of a leucite chamber Ussing-type. This chamber was designed to optimize different parameters known to be important in in vitro preparations, specially mammalian intestines. Fig. I shows the diagram of this chamber and a full description of it can be found in Henriques de Jesus (1974). The area of exposed tissue is 2-4 cm2. SCC was measured using an automatic system of voltage clamp (Henriques de Jesus & Smith, 1974). The resistance of the medium between the two tips of agar bridges was of the order of 5-10 % of the total resistance measured in the presence of the tissue. Corrections were made for this extra resistance. A square-wave generator with variable pulse duration (time used 21 sec) was connected so that a pulse from 0 to + 2 mV could be applied across the tissue, serosal side positive. A circuit equivalent to the electrical properties of the intestine was also constructed with an internal conductance which could be varied from 5 to 60 m-mho and with an open-circuit voltage similar to that found in the living tissue. This equivalent circuit was connected to the input terminals of the first amplifier so that the scale of the recorder could be calibrated for different known conductances. Addition of drugs was made with a micropipette, always after steady-state conditions were achieved. Added volume never surpassed 200 #ul. in a volume of 6 ml. for each half-chamber. The electrolyte composition of the Krebs solution was checked regularly. Mea8urement of adenine nucleotides. Mucosal scrapings from rabbit ileum (0-5 g) were homogenized in 3 ml. 5 % (w/v) trichloroacetic acid and the resulting suspension centrifuged at 3000 g for 5 min. All operations were carried out in the cold, the time from killing the animal to putting the scrapings in trichloroacetic acid varying from 5 to 7 min. Trichloroacetic acid was removed from the supernatant by ether extraction (three extractions with three volumes of ether), ether in the aqueous phase then being removed by a nitrogen stream. The resulting solution was added to a small column of Dowex 1 (Sigma Chemical Co) and AMP, ADP and ATP eluted consecutively by the stepwise addition of 0-01 N-HCl containing 0-02 N-NH4Cl and 1 N-HCl. Samples were scanned for absorbance using a Perkin Elmer series 402 spectrophotometer and the peak of absorbance at 259 nm compared with that obtained with standard solutions of ATP (Bronk & Leese, 1973). RESULTS
The first observation made is that the electrical characteristics of these preparations vary over a wide range, even if taken from the same animal and immediately adjacent to one another. For this reason it was decided
rn _7 z r s 7 U. HENR1V4UENS DPI JPjNUA _.
356
_
-
--
A
Fig. 1. Ussing-type chamber used for experiments with rabbit ileum. A, half chamber. B, compartment for Krebs solution. C, temperature controlled bath. D, clamping point. d, pin-holes. E, current bridge. F, potential bridge. G and G', water inlet and outlet. H, drainage tubing. I, aeration chimney. JI, nemnbrane window. Scale in cm,
SCC AND Gt MEASUREMENTS ON RABBIT ILEUM 357 to present results of individual experiments, statistical treatment of the data being tried only when possible. The second finding is that in these preparations the normal decay of the SCC is much smaller than previously reported: stability for periods over 1 hr, followed by slow decay of about 0-2 ,uA/min in our case, compared with decays of 1 #uA/min in the literature (Schultz & Zalusky, 1964a). A specific role has recently been suggested by Moreno (1974) for 2,4,6triaminopyrimidinium. This drug is supposed to decrease the part of passive conductance related with tight junctions. If this would prove correct for intestine, it must be expected to affect Gp alone in model (1) (see Introduction) or G'p (model (2)) and this would prove most useful in defining the model. For this reason 2,4,6-triaminopyrimidinium was tried in the present preparation. SCC was slightly inhibited with successive additions of this drug but the effects were very small and changes in conductance did not appear to have a consistent pattern. It was known that glucose and other actively transported sugars affected SCC (Schultz & Zalusky, 1964b; Goldner, Schultz & Curran, 1969; Riklis & Quastel, 1958) and in preliminary experiments it also affected conductance. These effects were rapid and reproducible and could be analysed precisely. The amounts of glucose added were chosen after measuring the apparent half-maximal stimulation on the Na-sugar interaction. This was done by assessing the effects on the SCC on different concentrations of glucose with Na at the concentration of 140 mm. The value found was 2X2 mm, which is smaller than the value found by Schultz & Zalusky (4.0 mM). Fig. 2 shows the effect on the SCC and Gt of successive additions of D-glucose in thirteen experiments. The intercepts of the dotted lines with the ordinate show the value of Gt that would represent either the value of Gp if model (1) is considered or the value of G'p/(G'p +G'8) if model (2) represents the behaviour of the preparation. The slopes would be, respectively, 1/Em or (1/Em) . G's/(G'8 + G'p). In three of the thirteen experiments it was possible to discriminate five additions of glucose, giving to the value of Gt for SCC = 0 a good degree of precision. Initial points (control) in each preparation are quite different, variations being larger for the initial Gt than for SCC. The slopes of individual preparations are very similar. On the other hand, the values of Gt for SCC = 0 are different for each preparation. This would mean that, whatever the chosen material, preparations varied more on the value of the shunt parallel conductance than on the value of the respective electromotive forces. The values of the ordinate for SCC = 0 and the slopes would allow for the calculations of the electrical basic parameters of the preparation if model (1) would be a good description of the isolated ileum,
358 C. HENRIQUES DE JESUS but they are not sufficient if, as it will be argued, model (2) is a better description of the ileum in the present conditions. It was decided to investigate further the relative importance of the two paths, the series and the parallel one. Experiments were designed 201-
C/
. '"' ' . '. '
15
E
CC
