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Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling.

Gao J, Sun X, Moore LC, White TW, Brink PR, Mathias RT - J. Gen. Physiol. (2011)

Bottom Line: Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels.When the lens' circulation of Na(+) was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally.These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794, USA.

ABSTRACT
We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na(+) that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens' circulation of Na(+) was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.

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The intracellular pressure measuring system. (A) A sketch of the effect of intracellular pressure on the electrode–intracellular solution interface. An elevated intracellular pressure (e.g., 100 mmHg) over that in the bathing solution causes the interface to move up the shank of the electrode, thus filling the narrow tip with relatively high resistance cytoplasm. (B) When the same 100-mmHg pressure is applied to the port of the microelectrode, the interface moves back to the tip as shown, and the electrode resistance is restored to its relatively lower value recorded in the bathing solution. (C) The manometer used to adjust the hydrostatic pressure at the electrode port. The crank drives a piston to create the pressure, which is connected to the electrode port through the plastic tubing. When the electrode resistance is restored to its value in the bathing solution, such that an increase in applied pressure has no effect on resistance, whereas a small reduction in pressure causes a small increase in resistance, we assume the applied pressure equals the intracellular pressure. The value of applied pressure is then read from the column of mercury.
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fig2: The intracellular pressure measuring system. (A) A sketch of the effect of intracellular pressure on the electrode–intracellular solution interface. An elevated intracellular pressure (e.g., 100 mmHg) over that in the bathing solution causes the interface to move up the shank of the electrode, thus filling the narrow tip with relatively high resistance cytoplasm. (B) When the same 100-mmHg pressure is applied to the port of the microelectrode, the interface moves back to the tip as shown, and the electrode resistance is restored to its relatively lower value recorded in the bathing solution. (C) The manometer used to adjust the hydrostatic pressure at the electrode port. The crank drives a piston to create the pressure, which is connected to the electrode port through the plastic tubing. When the electrode resistance is restored to its value in the bathing solution, such that an increase in applied pressure has no effect on resistance, whereas a small reduction in pressure causes a small increase in resistance, we assume the applied pressure equals the intracellular pressure. The value of applied pressure is then read from the column of mercury.

Mentions: A pulse generator (model 182A; Wavetec) and a digital real-time oscilloscope (TDS 210; Tektronix) interfaced with a self-built amplifier were used to measure microelectrode resistances. The resistance of the voltage electrode was 1.5–2.0 MΩ when filled with 3 M KCl, and that of the reference electrode was 0.2 MΩ when filled with agar (dissolved in 3 M KCl) to prevent the Cl−-induced liquid junction potential. About 30% of the experiments were terminated because the electrode clogged or occasionally the tip broke. We use low resistance microelectrodes for intracellular recordings in the lens because sharper, higher resistance microelectrodes usually break before they penetrate the lens capsule, and the higher the resistance the more prone the electrode is to irreversible clogging. Serial current pulses with amplitudes of 100 nA and durations of 15 ms were applied during the measurements of intracellular pressure within lenses. The amplitude of voltage steps responding to current pulses indicated the resistance of the tip of the electrode. First, the resistance of the voltage electrode was recorded when the voltage electrode was immersed in the bath (normal Tyrode’s solution). Then, the electrode was inserted into the lens. Pressure within the lens was higher than that in the tip of the electrode, so the higher pressure pushed intracellular cytoplasm into the tip of the electrode (Fig. 2 A). Because cytoplasm has relatively high resistance, the amplitude of the electrode resistance increased. The port of the tightly sealed electrode holder was connected to a mercury manometer by plastic tubing, which allowed us to adjust the pressure within the electrode. The manometer can record a pressure of about ±400 mmHg. The pressure is generated by turning the crank shown in Fig. 2 C. We increased pressure within the electrode to push cytoplasm back into the intracellular space. When pressure within the electrode is equal to intracellular pressure, cytoplasm should be completely pushed out of the electrode, and the amplitude of the electrode resistance should return to its original level measured in the bathing solution (Fig. 2 B). Thus, the reading from the manometer when the applied pressure caused the resistance to return to its original value represented the intracellular pressure within the lens. However, if the applied pressure exceeded that within the lens cell, the electrode resistance should remain the same as recorded in the bath. The pressure generated by the manometer was therefore backed off until the resistance just began to increase. This was the value recorded for the intracellular pressure.


Lens intracellular hydrostatic pressure is generated by the circulation of sodium and modulated by gap junction coupling.

Gao J, Sun X, Moore LC, White TW, Brink PR, Mathias RT - J. Gen. Physiol. (2011)

The intracellular pressure measuring system. (A) A sketch of the effect of intracellular pressure on the electrode–intracellular solution interface. An elevated intracellular pressure (e.g., 100 mmHg) over that in the bathing solution causes the interface to move up the shank of the electrode, thus filling the narrow tip with relatively high resistance cytoplasm. (B) When the same 100-mmHg pressure is applied to the port of the microelectrode, the interface moves back to the tip as shown, and the electrode resistance is restored to its relatively lower value recorded in the bathing solution. (C) The manometer used to adjust the hydrostatic pressure at the electrode port. The crank drives a piston to create the pressure, which is connected to the electrode port through the plastic tubing. When the electrode resistance is restored to its value in the bathing solution, such that an increase in applied pressure has no effect on resistance, whereas a small reduction in pressure causes a small increase in resistance, we assume the applied pressure equals the intracellular pressure. The value of applied pressure is then read from the column of mercury.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig2: The intracellular pressure measuring system. (A) A sketch of the effect of intracellular pressure on the electrode–intracellular solution interface. An elevated intracellular pressure (e.g., 100 mmHg) over that in the bathing solution causes the interface to move up the shank of the electrode, thus filling the narrow tip with relatively high resistance cytoplasm. (B) When the same 100-mmHg pressure is applied to the port of the microelectrode, the interface moves back to the tip as shown, and the electrode resistance is restored to its relatively lower value recorded in the bathing solution. (C) The manometer used to adjust the hydrostatic pressure at the electrode port. The crank drives a piston to create the pressure, which is connected to the electrode port through the plastic tubing. When the electrode resistance is restored to its value in the bathing solution, such that an increase in applied pressure has no effect on resistance, whereas a small reduction in pressure causes a small increase in resistance, we assume the applied pressure equals the intracellular pressure. The value of applied pressure is then read from the column of mercury.
Mentions: A pulse generator (model 182A; Wavetec) and a digital real-time oscilloscope (TDS 210; Tektronix) interfaced with a self-built amplifier were used to measure microelectrode resistances. The resistance of the voltage electrode was 1.5–2.0 MΩ when filled with 3 M KCl, and that of the reference electrode was 0.2 MΩ when filled with agar (dissolved in 3 M KCl) to prevent the Cl−-induced liquid junction potential. About 30% of the experiments were terminated because the electrode clogged or occasionally the tip broke. We use low resistance microelectrodes for intracellular recordings in the lens because sharper, higher resistance microelectrodes usually break before they penetrate the lens capsule, and the higher the resistance the more prone the electrode is to irreversible clogging. Serial current pulses with amplitudes of 100 nA and durations of 15 ms were applied during the measurements of intracellular pressure within lenses. The amplitude of voltage steps responding to current pulses indicated the resistance of the tip of the electrode. First, the resistance of the voltage electrode was recorded when the voltage electrode was immersed in the bath (normal Tyrode’s solution). Then, the electrode was inserted into the lens. Pressure within the lens was higher than that in the tip of the electrode, so the higher pressure pushed intracellular cytoplasm into the tip of the electrode (Fig. 2 A). Because cytoplasm has relatively high resistance, the amplitude of the electrode resistance increased. The port of the tightly sealed electrode holder was connected to a mercury manometer by plastic tubing, which allowed us to adjust the pressure within the electrode. The manometer can record a pressure of about ±400 mmHg. The pressure is generated by turning the crank shown in Fig. 2 C. We increased pressure within the electrode to push cytoplasm back into the intracellular space. When pressure within the electrode is equal to intracellular pressure, cytoplasm should be completely pushed out of the electrode, and the amplitude of the electrode resistance should return to its original level measured in the bathing solution (Fig. 2 B). Thus, the reading from the manometer when the applied pressure caused the resistance to return to its original value represented the intracellular pressure within the lens. However, if the applied pressure exceeded that within the lens cell, the electrode resistance should remain the same as recorded in the bath. The pressure generated by the manometer was therefore backed off until the resistance just began to increase. This was the value recorded for the intracellular pressure.

Bottom Line: Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels.When the lens' circulation of Na(+) was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally.These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, SUNY at Stony Brook, NY 11794, USA.

ABSTRACT
We recently modeled fluid flow through gap junction channels coupling the pigmented and nonpigmented layers of the ciliary body. The model suggested the channels could transport the secretion of aqueous humor, but flow would be driven by hydrostatic pressure rather than osmosis. The pressure required to drive fluid through a single layer of gap junctions might be just a few mmHg and difficult to measure. In the lens, however, there is a circulation of Na(+) that may be coupled to intracellular fluid flow. Based on this hypothesis, the fluid would cross hundreds of layers of gap junctions, and this might require a large hydrostatic gradient. Therefore, we measured hydrostatic pressure as a function of distance from the center of the lens using an intracellular microelectrode-based pressure-sensing system. In wild-type mouse lenses, intracellular pressure varied from ∼330 mmHg at the center to zero at the surface. We have several knockout/knock-in mouse models with differing levels of expression of gap junction channels coupling lens fiber cells. Intracellular hydrostatic pressure in lenses from these mouse models varied inversely with the number of channels. When the lens' circulation of Na(+) was either blocked or reduced, intracellular hydrostatic pressure in central fiber cells was either eliminated or reduced proportionally. These data are consistent with our hypotheses: fluid circulates through the lens; the intracellular leg of fluid circulation is through gap junction channels and is driven by hydrostatic pressure; and the fluid flow is generated by membrane transport of sodium.

Show MeSH
Related in: MedlinePlus