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Microelectrode and short-circuiting techniques for the study of ion transport in the lens
Exp. Eye Res. (1973) 15, 219-223
Microelectrode and Short-Circuiting Ttxlm@es for the Study of Ion Tramp-t in the Lens OSCAR
A.
CANDIA
Department of Ophthalmology, Mount Sinai School of Medicine City University of New York, New York 10029, U.S.A.
of
the
Ion transport was studied by considering the lens as a composite asymmetrical system rather than a closed system. When the toad lens is isolated in a chamber, the anterior surface of the lens is 30 mV positive with respect to the posterior surface. ‘Iranslentioular unidirectional fluxes of Na, c1, and K were measured under short-circuit conditions. It was found that only Na is actively transported across the lens; the transport being in a posterior-anterior direction. A discrepancy between net Na flux and short-circuit-current is interpreted on the basis of Na being gained by the lens across its posterior surface and K being lost across its anterior surface. With the aid of a glass microelectrode the potential difference across the epithelial layer under the anterior capsule was determined and a larger short-circuit-current obtained. It is concluded that in the amphibian lens the movement of K appears to be secondary to electrical gradients created by an electrogenic Na pump.
1. Introduction Active ionic transport is of great relevance to most biological membranes including ocular membranes such as the cornea and the ciliary body. In the crystalline lens, although the maintenance of a proper ionic composition seems to be necessary for the control of a normal degree of hydration and transparency, no direct relationship has been established. One of the reasons is, perhaps, that techniques widely used on other transport systems to characterize ionic transport have not been applied in the lens, until very recently. I am referring in particular to the short-circuiting technique and the measurement of transepithelial unidirectional fluxes. The movement of ions across membranes depends basically on two factors: the driving forces and the permeability of the membrane. Depending upon the nature of the driving forces, movement of ions can be separated into two categories: passive and active. Passive driving forces are, for example, the electrical potential difference (PD) and the chemical gradient. Active forces usually involve metabolic energy coupled to the transport system by resistance cross-coefficients. Depending upon their geometrical characteristics, transport systems can be classified into two main categories : (1) closed or symmetrical systems ; and (2) open or asymmetrical systems. An example of a closed system is the red cell where the membrane symmetrically surrounds the red cell compartment. Examples of open asymmetrical systems are the classical models: isolated frog skin and toad urinary bladder. In closed systems, in a steady state, opposite fluxes are equal and there is no net flux across the membrane. Nevertheless, concentration gradients and PD are found across the cell membrane. Metabolic energy is expended to maintain this electrochemical gradient even though no net %ux normally occurs across the membrane. In asymmetrical open systems a net transfer of ions between compartments usually occurs. Because of the accessibility to the compartments, transport properties of open systems are generally easier to characterize. F
219
220
0.
A.
CAKDIA
A superficial look at the lens geometry and ionic gradients may suggest its classification as a closed symmetrical system. Indeed, the lens has been compared by many to a giant cell (Harris, 1960; Bonting, 1970). However, both anatomical and functional asymmetrical characteristics are immediately apparent, due to the presence of a layer of epithelial cells only under the anterior capsule. The lens should really be classified as a composite asymmetrical system. Because of this we decided to adapt to the lens two of the most profitable techniques for studying ion transport, namely short-circuiting and measurements of unidirectional fluxes. The basic consideration in applying the short-circuiting technique is that regardless of the complexity of the membrane (that can be considered as a black box), passive unidirectional fluxes should be the samein opposite directions if the electrochemical gradient between the two compartments is zero. This is commonly accomplished by using identical solutions on both sides of the membrane and cancelling any PD created by the membrane by means of an external battery. On the other hand, a net ionic flux occurring in this condition is compelling evidence of an active transport of that ion. Equality between the short-circuit current (SCC) and the net flux of the ion is further confirmation of the transport. In studying ion transport in the lens, two general approaches were commonly used: one was to measure ionic uptake and washouts in various experimental conditions, and the other was to introduce a microelectrode into the lens and measure the PD in reference to the outside bathing medium in which ionic composition was modified. 2. Results and Discussion About three years ago we decided to investigate the possibility that a PD existed acrossthe entire lens. We isolated the lens of the toad in a chamber (seeFig. 1) and found a translenticular PD of 30 mV in the anterior-posterior direction, the anterior side being positive. We examined this PD in a variety of bathing solutions in which the ionic composition was altered. Complete removal of Cl from either/or both bathing solutions had little effect on the PD. Removal of K from both bathing solutions increased the translenticular PD by 5 mV, while removal of Na reduced it by 12 mV. These results pointed to a possibletransport of Na, but were inconclusive
tiole
fia. 1. Glass chamber solution.
for
microelectrode
iu which the lens is mounted separating
the anterior
from
the posterior
bathing
MICROELECTRODE
AND
ION
TRANSPORT
IN
LENS
221
about the participation of K. The next logical step was to short-circuit the translenticular PD and measure unidirectional fluxes across the entire lens. Chloride fluxes were equal in both directions and amounted to 0.33 pequiv/cm2-hr. K fluxes were also equal in both directions and amounted to only O~03~equiv/cm2-hr. Na fluxes were different, 0.38 pequiv/cm2-hr in the posterior-anterior direction and 0.17 pequiv/cms-hr in the anterior-posterior direction. The difference between Na fluxes expressed as an electrical current is equal to 5.6 pA/cm2. However, the average SCC measured during the Na flux determination was much larger, 25.4 pA/cm2. The equality of the two unidirectional K fluxes and the higher PD measured in Kfree solutions are two strong arguments against an active transport of K. If there was a K transport into the lens across the anterior surface, part of it would leak out across the posterior surface and a net flux would be observed. The fact that removal of Na reduces the PD only partially is in good agreement with the finding that the net Na transport is only about 25% of the SCC. We have been unsuccessful in determining which ion is responsible for the remaining PD and SCC. Because of that, we decided to consider the possibility that the lens compartment is not in a steady state situation when isolated in the chamber. It is well known that during prolonged incubation the lens gains Na and loses K. It is also known that the anterior face is more permeable to K than the posterior, while the opposite occurs for Na (Harris and Becker, 1965). A net transfer of positive charges would occur from the posterior to the anterior solution if the net loss of K across the anterior surface exceeded the loss across the posterior surface and if Na gain across the posterior surface was larger than across the anterior face. The SCC would be carried by sodium moving in across the posterior face and by K moving out across the anterior face. We have done some experiments with the frog lens isolated in a chamber in which the uptake of Na22 by either surface was measured simultaneously with the total K loss towards both bathing solutions. We found that in a period of 10 hr a lens of about 200 mg (7 mm in diameter) loses 6 pequiv of K when short-circuited in a chamber. Four pequiv are lost to the anterior bathing solution and 2 pequiv to the posterior bathing solution. We did not measure the total gain of Na, but the relative uptake at each surface. Toyofuku and Bentley (1970) found that toad lenses gain 1.5 times as much Na as they lose K. The difference is made up by a gain of Cl and other anions (Bentley, pers. comm.). From our data and those of Toyofuku and Bentley we estimate a net gain of Na of 6.5 pequiv across the posterior surface, and 2.5 pequiv across the anterior surface. A complete balance of the movement of K and Na is shown in Fig. 2. As a result, there is a net movement of positive charges in the post,erior-anterior direction equivalent to 3.5 pequiv/lens during a period of 10 hr. This represents 0.35 pequiv/hr per lens, or 0.83 pequivlhr-cma, which is surprisingly similar to the difference between the net translenticular Na flux and the SCC (0.74 pequiv/hr-cm2). Ouabain and iodoaeetate depress the SCC in the isolated lens, but it takes 4-10 hr before complete inhibition is achieved. This is also in agreement with our interpretation that part of the SCC is produced by ionic diffusion. The measurement of SCC and ionic fluxes across the entire lens can determine the amount and direction of ionic fluxes, but it does not provide information on the anatomic location of the pump. Previous studies have indicated that Na-K ATPase activity and ionic pumps are found in the epithelial cells of the anterior face. It was expected then, that the PD across the layer of epithelial cells in the anterior face would he larger than across the posterior capsule. To confirm this assumption we
0.
222
A.
CANDIA
introduced a fine glass pipette with a tip diameter of about 80 p into the lens fibers while the lens was isolated in the chamber. This pipette served as a third electrode to record the PD across either the anterior or posterior surface. The inside of the lens was found, as expected from previous work (Brindley, 1956; Andree, 1958; Duncan, 1969), to be negative with respect to either side. However, the PD across the anterior side was 61 mV and the PD across the posterior side was 32 mV. The electrical resistance of the anterior side was about half of the resistance across the posterior side. Because of this, when the PD across the entire lens was short-circuited, the inside of the lens remained about 48 mV negative. Consequently, the layer of epithelial cells where the pump is located is not really completely short-circuited when the translenticular PD is cancelled. When the PD across the epithelial cells is short-circuited the posterior solution becomes 107 mV positive with respect to the inside of the lens or the anterior solution. The SCC when the epithelial cells are short-circuited, is about 117 pA/cm2; an extraordinary amount compared to SCC found in other membranes. Anterior
Posterior
2~57-T‘\“~5 Na + 4-f
2---w-l
Fro.
2. Balance
of
ionic movement between
-72
K
t
I
anions
-
3.5
net
+
8 lens mounted in
a chamber
and bathing
solutions.
The anatomical complexity of the lens poses the following question: which is the real SCC, the one measured with the whole lens short-circuited or when the epithelial cells are short-circuited? The SCC measured with the entire lens short-circuited is affected by the extra load imposed by the resistance of the posterior capsule and lens fibers, and does not represent the full capability of the epithelial pump. However, when cells themselves are short-circuited they are not in the ideal condition of electrochemical neutrality. The PD is zero, but a chemical gradient exists across the cells because of the difference between the ionic composition of the lens and the anterior bathing solution. To solve this problem, the anterior face of the lens was bathed with a solution in which the concentration of Na, K, and Cl were 31, 75, 33 mV, respectively. These ionic concentrations were similar to those found in the toad lens (Candia et al., 1970). With this solution bathing the anterior face of the lens, the PD across the anterior cell layer was reduced from 61 mV to about 30 mV and the average SCC was 60 pA/cm2. We believe this value to represent the real short-circuit current capability of the pump in the epithelial cells. Under this condition, if the measurements of unidirectional Na fluxes across the anterior face were possible, we would expect the net Na flux to account for the total SCC. Our results and analysis suggest that the movement of K is secondary to electrochemical gradients created by an electrogenic Na pump located at the epithelial cells. Metabolism would only be coupled to the Na pump, and the effect of metabolic inhibitors on the movement of K would be mediated by a primary effect on the Na P-P.
MICROELECTROD
E AND ION TRANSPORT
IN LENS
223
ACKNOWLEDGMENTS
This work was mainly supported by a National Institutes of Health Grant EY 00160, and in part by a Grant from Fight for Sight, Inc. New York City. Most of the experimental results describedin this paper were carried out partially in collaboration with P. J. Bentley, Carl D. Mills, Hidenao Toyofuku, and Laura Fox. REFERENCES Andree, G. (1958. Arch. Ges. Physkl. 267, 109. Bonting, S. L. (1970). In Yembranes and Ion Transport (Ed. Bittar, E. E.) John Wiley & Sons, New York. Brindley, G. S. (1956). Brit. J. Ophthdmol. 40, 385. Candia, 0. A., Bentley, P. J., Milis, C. D. and Toyofuku, H. (1970). Nature (London) 227, 852. Duncan, G. (1969). Eq. Eye Res. 8,406. Harris, E. J. (1960). In Tranqort and Accumulation in Biologid fly&ems. Butterworths, London. Harris, J. E. and Becker, B. (1965). In Symposium 012the Lens (Ed. Harris, J. E.) Mosby, St. Louis. Toyofuku, H. and Bentley, P. J. (1970). Invest. Ophthdmol. 9,959.
Microelectrode and Short-Circuiting Ttxlm@es for the Study of Ion Tramp-t in the Lens OSCAR
A.
CANDIA
Department of Ophthalmology, Mount Sinai School of Medicine City University of New York, New York 10029, U.S.A.
of
the
Ion transport was studied by considering the lens as a composite asymmetrical system rather than a closed system. When the toad lens is isolated in a chamber, the anterior surface of the lens is 30 mV positive with respect to the posterior surface. ‘Iranslentioular unidirectional fluxes of Na, c1, and K were measured under short-circuit conditions. It was found that only Na is actively transported across the lens; the transport being in a posterior-anterior direction. A discrepancy between net Na flux and short-circuit-current is interpreted on the basis of Na being gained by the lens across its posterior surface and K being lost across its anterior surface. With the aid of a glass microelectrode the potential difference across the epithelial layer under the anterior capsule was determined and a larger short-circuit-current obtained. It is concluded that in the amphibian lens the movement of K appears to be secondary to electrical gradients created by an electrogenic Na pump.
1. Introduction Active ionic transport is of great relevance to most biological membranes including ocular membranes such as the cornea and the ciliary body. In the crystalline lens, although the maintenance of a proper ionic composition seems to be necessary for the control of a normal degree of hydration and transparency, no direct relationship has been established. One of the reasons is, perhaps, that techniques widely used on other transport systems to characterize ionic transport have not been applied in the lens, until very recently. I am referring in particular to the short-circuiting technique and the measurement of transepithelial unidirectional fluxes. The movement of ions across membranes depends basically on two factors: the driving forces and the permeability of the membrane. Depending upon the nature of the driving forces, movement of ions can be separated into two categories: passive and active. Passive driving forces are, for example, the electrical potential difference (PD) and the chemical gradient. Active forces usually involve metabolic energy coupled to the transport system by resistance cross-coefficients. Depending upon their geometrical characteristics, transport systems can be classified into two main categories : (1) closed or symmetrical systems ; and (2) open or asymmetrical systems. An example of a closed system is the red cell where the membrane symmetrically surrounds the red cell compartment. Examples of open asymmetrical systems are the classical models: isolated frog skin and toad urinary bladder. In closed systems, in a steady state, opposite fluxes are equal and there is no net flux across the membrane. Nevertheless, concentration gradients and PD are found across the cell membrane. Metabolic energy is expended to maintain this electrochemical gradient even though no net %ux normally occurs across the membrane. In asymmetrical open systems a net transfer of ions between compartments usually occurs. Because of the accessibility to the compartments, transport properties of open systems are generally easier to characterize. F
219
220
0.
A.
CAKDIA
A superficial look at the lens geometry and ionic gradients may suggest its classification as a closed symmetrical system. Indeed, the lens has been compared by many to a giant cell (Harris, 1960; Bonting, 1970). However, both anatomical and functional asymmetrical characteristics are immediately apparent, due to the presence of a layer of epithelial cells only under the anterior capsule. The lens should really be classified as a composite asymmetrical system. Because of this we decided to adapt to the lens two of the most profitable techniques for studying ion transport, namely short-circuiting and measurements of unidirectional fluxes. The basic consideration in applying the short-circuiting technique is that regardless of the complexity of the membrane (that can be considered as a black box), passive unidirectional fluxes should be the samein opposite directions if the electrochemical gradient between the two compartments is zero. This is commonly accomplished by using identical solutions on both sides of the membrane and cancelling any PD created by the membrane by means of an external battery. On the other hand, a net ionic flux occurring in this condition is compelling evidence of an active transport of that ion. Equality between the short-circuit current (SCC) and the net flux of the ion is further confirmation of the transport. In studying ion transport in the lens, two general approaches were commonly used: one was to measure ionic uptake and washouts in various experimental conditions, and the other was to introduce a microelectrode into the lens and measure the PD in reference to the outside bathing medium in which ionic composition was modified. 2. Results and Discussion About three years ago we decided to investigate the possibility that a PD existed acrossthe entire lens. We isolated the lens of the toad in a chamber (seeFig. 1) and found a translenticular PD of 30 mV in the anterior-posterior direction, the anterior side being positive. We examined this PD in a variety of bathing solutions in which the ionic composition was altered. Complete removal of Cl from either/or both bathing solutions had little effect on the PD. Removal of K from both bathing solutions increased the translenticular PD by 5 mV, while removal of Na reduced it by 12 mV. These results pointed to a possibletransport of Na, but were inconclusive
tiole
fia. 1. Glass chamber solution.
for
microelectrode
iu which the lens is mounted separating
the anterior
from
the posterior
bathing
MICROELECTRODE
AND
ION
TRANSPORT
IN
LENS
221
about the participation of K. The next logical step was to short-circuit the translenticular PD and measure unidirectional fluxes across the entire lens. Chloride fluxes were equal in both directions and amounted to 0.33 pequiv/cm2-hr. K fluxes were also equal in both directions and amounted to only O~03~equiv/cm2-hr. Na fluxes were different, 0.38 pequiv/cm2-hr in the posterior-anterior direction and 0.17 pequiv/cms-hr in the anterior-posterior direction. The difference between Na fluxes expressed as an electrical current is equal to 5.6 pA/cm2. However, the average SCC measured during the Na flux determination was much larger, 25.4 pA/cm2. The equality of the two unidirectional K fluxes and the higher PD measured in Kfree solutions are two strong arguments against an active transport of K. If there was a K transport into the lens across the anterior surface, part of it would leak out across the posterior surface and a net flux would be observed. The fact that removal of Na reduces the PD only partially is in good agreement with the finding that the net Na transport is only about 25% of the SCC. We have been unsuccessful in determining which ion is responsible for the remaining PD and SCC. Because of that, we decided to consider the possibility that the lens compartment is not in a steady state situation when isolated in the chamber. It is well known that during prolonged incubation the lens gains Na and loses K. It is also known that the anterior face is more permeable to K than the posterior, while the opposite occurs for Na (Harris and Becker, 1965). A net transfer of positive charges would occur from the posterior to the anterior solution if the net loss of K across the anterior surface exceeded the loss across the posterior surface and if Na gain across the posterior surface was larger than across the anterior face. The SCC would be carried by sodium moving in across the posterior face and by K moving out across the anterior face. We have done some experiments with the frog lens isolated in a chamber in which the uptake of Na22 by either surface was measured simultaneously with the total K loss towards both bathing solutions. We found that in a period of 10 hr a lens of about 200 mg (7 mm in diameter) loses 6 pequiv of K when short-circuited in a chamber. Four pequiv are lost to the anterior bathing solution and 2 pequiv to the posterior bathing solution. We did not measure the total gain of Na, but the relative uptake at each surface. Toyofuku and Bentley (1970) found that toad lenses gain 1.5 times as much Na as they lose K. The difference is made up by a gain of Cl and other anions (Bentley, pers. comm.). From our data and those of Toyofuku and Bentley we estimate a net gain of Na of 6.5 pequiv across the posterior surface, and 2.5 pequiv across the anterior surface. A complete balance of the movement of K and Na is shown in Fig. 2. As a result, there is a net movement of positive charges in the post,erior-anterior direction equivalent to 3.5 pequiv/lens during a period of 10 hr. This represents 0.35 pequiv/hr per lens, or 0.83 pequivlhr-cma, which is surprisingly similar to the difference between the net translenticular Na flux and the SCC (0.74 pequiv/hr-cm2). Ouabain and iodoaeetate depress the SCC in the isolated lens, but it takes 4-10 hr before complete inhibition is achieved. This is also in agreement with our interpretation that part of the SCC is produced by ionic diffusion. The measurement of SCC and ionic fluxes across the entire lens can determine the amount and direction of ionic fluxes, but it does not provide information on the anatomic location of the pump. Previous studies have indicated that Na-K ATPase activity and ionic pumps are found in the epithelial cells of the anterior face. It was expected then, that the PD across the layer of epithelial cells in the anterior face would he larger than across the posterior capsule. To confirm this assumption we
0.
222
A.
CANDIA
introduced a fine glass pipette with a tip diameter of about 80 p into the lens fibers while the lens was isolated in the chamber. This pipette served as a third electrode to record the PD across either the anterior or posterior surface. The inside of the lens was found, as expected from previous work (Brindley, 1956; Andree, 1958; Duncan, 1969), to be negative with respect to either side. However, the PD across the anterior side was 61 mV and the PD across the posterior side was 32 mV. The electrical resistance of the anterior side was about half of the resistance across the posterior side. Because of this, when the PD across the entire lens was short-circuited, the inside of the lens remained about 48 mV negative. Consequently, the layer of epithelial cells where the pump is located is not really completely short-circuited when the translenticular PD is cancelled. When the PD across the epithelial cells is short-circuited the posterior solution becomes 107 mV positive with respect to the inside of the lens or the anterior solution. The SCC when the epithelial cells are short-circuited, is about 117 pA/cm2; an extraordinary amount compared to SCC found in other membranes. Anterior
Posterior
2~57-T‘\“~5 Na + 4-f
2---w-l
Fro.
2. Balance
of
ionic movement between
-72
K
t
I
anions
-
3.5
net
+
8 lens mounted in
a chamber
and bathing
solutions.
The anatomical complexity of the lens poses the following question: which is the real SCC, the one measured with the whole lens short-circuited or when the epithelial cells are short-circuited? The SCC measured with the entire lens short-circuited is affected by the extra load imposed by the resistance of the posterior capsule and lens fibers, and does not represent the full capability of the epithelial pump. However, when cells themselves are short-circuited they are not in the ideal condition of electrochemical neutrality. The PD is zero, but a chemical gradient exists across the cells because of the difference between the ionic composition of the lens and the anterior bathing solution. To solve this problem, the anterior face of the lens was bathed with a solution in which the concentration of Na, K, and Cl were 31, 75, 33 mV, respectively. These ionic concentrations were similar to those found in the toad lens (Candia et al., 1970). With this solution bathing the anterior face of the lens, the PD across the anterior cell layer was reduced from 61 mV to about 30 mV and the average SCC was 60 pA/cm2. We believe this value to represent the real short-circuit current capability of the pump in the epithelial cells. Under this condition, if the measurements of unidirectional Na fluxes across the anterior face were possible, we would expect the net Na flux to account for the total SCC. Our results and analysis suggest that the movement of K is secondary to electrochemical gradients created by an electrogenic Na pump located at the epithelial cells. Metabolism would only be coupled to the Na pump, and the effect of metabolic inhibitors on the movement of K would be mediated by a primary effect on the Na P-P.
MICROELECTROD
E AND ION TRANSPORT
IN LENS
223
ACKNOWLEDGMENTS
This work was mainly supported by a National Institutes of Health Grant EY 00160, and in part by a Grant from Fight for Sight, Inc. New York City. Most of the experimental results describedin this paper were carried out partially in collaboration with P. J. Bentley, Carl D. Mills, Hidenao Toyofuku, and Laura Fox. REFERENCES Andree, G. (1958. Arch. Ges. Physkl. 267, 109. Bonting, S. L. (1970). In Yembranes and Ion Transport (Ed. Bittar, E. E.) John Wiley & Sons, New York. Brindley, G. S. (1956). Brit. J. Ophthdmol. 40, 385. Candia, 0. A., Bentley, P. J., Milis, C. D. and Toyofuku, H. (1970). Nature (London) 227, 852. Duncan, G. (1969). Eq. Eye Res. 8,406. Harris, E. J. (1960). In Tranqort and Accumulation in Biologid fly&ems. Butterworths, London. Harris, J. E. and Becker, B. (1965). In Symposium 012the Lens (Ed. Harris, J. E.) Mosby, St. Louis. Toyofuku, H. and Bentley, P. J. (1970). Invest. Ophthdmol. 9,959.