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

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.

Figure 1.

A sketch of the hypotheses being tested in this study. (A) The net flux of Na⁺, followed by fluid, enters the lens at both poles and exits at the equator. The interesting pattern of circulation ensures maximum stirring of the fluid, which is hypothesized to act as a micro circulatory system for the avascular lens (Mathias et al., 2007). (B) A more detailed view of Na⁺ and K⁺ fluxes. Na⁺ flows into the lens along the extracellular spaces between cells, moves down its electrochemical gradient to enter fiber cells, reverses its direction, and flows back to the lens surface, where the Na/K ATPase transports it out of the lens to complete the circulation. The circulating pattern of flow shown in A occurs because gap junctions coupling DF cells direct the intracellular leg of the circulation to the equator. (C) Our first hypothesis is that water circulates through the lens as shown in this panel. Our second hypothesis is that the water circulation follows and is driven by the Na⁺ flux. Water enters each fiber cell through AQP0 resulting from local osmotic gradients created by the transmembrane Na⁺ flux, and leaves the lens through AQP1 resulting from local osmotic gradients generated by the Na/K ATPase. For the intracellular water to flow back to the surface of the lens, we hypothesize that a hydrostatic pressure (p_i mmHg) develops that drives the water from cell to cell through gap junctions. (D) The predicted hydrostatic pressure gradient. The intracellular hydrostatic pressure is graphed as a function of normalized distance (r/a) from the lens center, where a (cm) is the lens radius, and r (cm) is the distance from the lens center. The equation relates intracellular water flow (u_i cm/s) to the hydrostatic pressure gradient (dp_i/dr mmHg/cm), the hydraulic conductivity of a single gap junction channel (L_j (cm³/s)/(mmHg)), the number of gap junction channels per area of cell-to-cell contact (N_j cm⁻²), and the fiber cell width (w cm).

Figure 2.

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.

Figure 3.

The standing hydrostatic pressure gradient in lenses from WT mice, which were ∼2 mo old. The hydrostatic pressure (p_i mmHg) is graphed as a function of normalized distance (r/a) from the lens center, where a (cm) is the lens radius, and r (cm) is the distance from the lens center. The data are from 14 lenses from seven mice. The pressures at two to six radial locations were recorded from each lens. The smooth curve is the best fit of Eq. 7 to the data.

Figure 4.

The effect on intracellular hydrostatic pressure of increasing the number of gap junction channels coupling the MFs. (A) The standing hydrostatic pressure gradient in lenses from Cx46 KI mice, which were ∼2 mo old. The hydrostatic pressure (p_i mmHg) is graphed as a function of normalized distance (r/a) from the lens center, where a (cm) is the lens radius, and r (cm) is the distance from the lens center. The data are from 12 lenses from six mice. The pressures at two to six radial locations were recorded from each lens. The smooth curve is the best fit of Eq. 7 to the data. Based on previous studies, the MF coupling conductance in the Cx46 KI lenses is approximately double that in WT lenses (Mathias et al., 2010). Based on the derivation of Eq. 7, if fluid flow in WT and KI lenses is the same, the pressure gradient in the KI lenses should be approximately half that in WT lenses. The best fits of the model to the data give the ratio of p_i(0) in Cx46 KI/WT lenses as 0.57. (B) An over-plot of the Cx46 KI and WT data.

Figure 5.

The effect on intracellular hydrostatic pressure of reducing the number of gap junction channels coupling the differentiating and MFs. (A) The standing hydrostatic pressure gradient in lenses from GPX-1 KO mice, which were ∼2 mo old. The hydrostatic pressure (p_i mmHg) is graphed as a function of normalized distance (r/a) from the lens center, where a (cm) is the lens radius, and r (cm) is the distance from the lens center. The data are from 10 lenses from five mice. The pressures at two to six radial locations were recorded from each lens. The smooth curve is the best fit of Eq. 7 to the data. Because the manometer can only measure ∼400 mmHg, the pressures at locations closer to the lens center than ∼0.4a could not be determined, other than that they exceeded 400 mmHg. The MF coupling conductance in the GPX-1 KO lenses was ∼60% of that in WT lenses (Wang et al., 2009). Based on the derivation of Eq. 7, the pressure gradient should be approximately inversely proportional to the MF coupling conductance, or ∼1.67 times greater than in WT. The best fits of the model to the data give the ratio of p_i(0) in GPX-1 KO/WT lenses as 1.52. (B) An over-plot of the GPX-1 KO and WT data.

Figure 6.

The effect on intracellular hydrostatic pressure of approximately halving the number of gap junction channels coupling the MFs. (A) The standing hydrostatic pressure gradient in lenses from Cx46+/− KO mice, which were ∼2 mo old. The hydrostatic pressure (p_i mmHg) is graphed as a function of normalized distance (r/a) from the lens center, where a (cm) is the lens radius, and r (cm) is the distance from the lens center. The data are from 12 lenses from six mice. The pressures at two to six radial locations were recorded from each lens. The smooth curve is the best fit of Eq. 7 to the data. Because the manometer can only measure ∼400 mmHg, the pressures at locations closer to the lens center than ∼0.55a could not be determined, other than that they exceeded 400 mmHg. The MF coupling conductance in Cx46+/− KO lenses was ∼50% of that in WT lenses (Mathias et al., 2010). Based on the derivation of Eq. 7, the pressure gradient should be approximately inversely proportional to the MF coupling conductance, or approximately two times greater in the KO than WT lenses. The best fits of the model to the data give the ratio of p_i(0) in Cx46+/− KO/WT lenses as 1.92. (B) An over-plot of the Cx46+/− and WT data.

Figure 7.

The effect of approximately eliminating the transmembrane electrochemical gradient for Na⁺ on central hydrostatic pressure in WT mouse lenses. The high K⁺/low Na⁺ external solution contained 140 mM K⁺ and 5 mM Na⁺. (A) Typical data from one lens showing the time course of the reduction in central hydrostatic pressure associated with elimination of the electrochemical gradient for Na⁺. After ∼120 min in 5 mM of extracellular Na⁺, the pressure near the center of the lens dropped to near zero. Upon restoration of normal extracellular Na⁺, the pressure began to recover, but we did not wait for full recovery, which was unlikely because the long exposure to low extracellular Na⁺ affected many transport systems and was probably not completely reversible. (B) The average hydrostatic pressure in lenses immersed in high K⁺/low Na⁺ solution (seven lenses from seven mice) compared with the pressure in normal Tyrode’s solution (five lenses from five mice). The central pressure in lenses immersed in high K⁺/low Na⁺ solution consistently fell to near zero in a time period of ∼2 h. In control lenses, the hydrostatic pressure remained relatively constant over a period of more than 2 h. After a period of 140 min, the average pressure was 85% of its initial value.

Figure 8.

The effect of Na/K ATPase inhibition with a saturating concentration of ouabain on central intracellular pressure in WT lenses. (A) Typical data from one lens showing the time course of the reduction in central hydrostatic pressure after blockade of the Na/K ATPase. After ∼30 min in ouabain, pressure near the center of the lens dropped to about half its original value. (B) The average time course of reduction in hydrostatic pressure in six lenses from six mice. The time course represents several events with different time scales, as described in Results.