The Ciliary Muscle and Zonules of Zinn Modulate Lens Intracellular Hydrostatic Pressure Through Transient Receptor Potential Vanilloid Channels

Abstract

Purpose: Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient.

Methods: We measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure.

Results: Ciliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K.

Conclusions: These results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

Figure 1

Modulation of the ciliary muscle altered the circumference of the ciliary body. The pupil diameter observed in untreated control eyes (A) was reduced by contraction of smooth muscle by pilocarpine (B), or increased by relaxation of smooth muscle by tropicamide (C). The modulation of pupil dilation confirmed drug action. Removal of the posterior sclera, retina, and vitreous revealed the circumlental space between the ciliary processes and the lens (D, asterisk). Measurements taken on higher-power images showed that in control eyes (E), the distance between ciliary processes and the lens was 149 ± 7 μm (mean ± SD, arrow). In eyes treated with ciliary muscles contracted by pilocarpine (F), the circumlental space was reduced to 124 ± 14 μm. In eyes with ciliary muscles relaxed by tropicamide (G), the circumlental space was increased to 174 ± 7 μm. The mean differences in circumlental space (H) were statistically significant (P < 0.05, 1-way ANOVA, n = 6–10).

Figure 2

Lens hydrostatic pressure was reduced following dilation of the ciliary muscle with tropicamide (▪). (A) In eyes with intact zonules, intracellular hydrostatic pressure in mouse lenses decreased 22 to 27 mm Hg after perfusion with 0.1% tropicamide (n = 4). (B) Since initial hydrostatic pressures varied from 24 to 37 mm Hg depending on the depth of electrode penetration, pressures from individual lenses were normalized to their initial values and plotted as the net change in pressure during drug administration. (C) The mean (±SD) change recorded in lens hydrostatic pressure was 24.3 ± 2.6 mm Hg (P < 0.05, n = 4) and occurred within ∼40 minutes following tropicamide administration.

Figure 3

Intact zonules and TRPV4 activity were both required for the reduction of lens hydrostatic pressure by tropicamide. (A) Lenses dissected free of the zonules (□) had stable values of surface intracellular pressure of ∼20 mm Hg that were unaffected by perfusion with 0.1% tropicamide. (B) Application of tropicamide to free lenses reduced the hydrostatic pressure near the surface by 0.5 ± 0.6 mm Hg after 100 minutes (P > 0.05, n = 4). (C) When lenses with intact zonules were preincubated with the TRPV4 inhibitor HC-067047 (△), the addition of tropicamide to the bath solution had no effect on pressure. (D) In the presence of HC-067047, application of tropicamide to the lenses lowered the surface hydrostatic pressure by 1.5 ± 1.9 mm Hg after 100 minutes (P > 0.05, n = 4). The pressure reduction observed in lenses with intact zonules and uninhibited TRPV4 activity (▪) is shown for comparison in (B, D).

Figure 4

Lens hydrostatic pressure was increased after contraction of the ciliary muscle by pilocarpine (•). (A) When zonules were intact, surface intracellular pressure increased by 13 to 18 mm Hg within 40 minutes after the application of 0.2% pilocarpine. (B) The mean (±SD) change recorded in lens hydrostatic pressure was 16.4 ± 2.9 mm Hg (P < 0.05, n = 4).

Figure 5

Intact zonules and TRPV1 activity were required for pilocarpine to increase lens hydrostatic pressure. (A) Intracellular pressures in lenses without intact zonules (○) were unaffected by the addition of 0.2% pilocarpine to the bath. (B) Application of pilocarpine to free lenses increased the hydrostatic pressure near the surface by a mean value of 1.3 ± 2.2 mm Hg after 100 minutes (P > 0.05, n = 6). (C) When lenses with intact zonules were preincubated in the TRPV1 inhibitor A-889425 22 (▽), the subsequent addition of pilocarpine had no effect on pressure. (D) Application of pilocarpine to the TRPV1-inhibited lenses raised the mean hydrostatic pressure by 0.2 ± 1.2 mm Hg after 100 minutes (P > 0.05, n = 4). The pressure increase observed in lenses with intact zonules and uninhibited TRPV1 activity (•) is shown for comparison in (B, D).

Figure 6

The hydrostatic pressure gradient across the entire lens was modulated by tropicamide and pilocarpine. The hydrostatic pressure gradient was determined in the absence of drugs (⋄) or the presence of 0.1% tropicamide (▪) or 0.2% pilocarpine (•) and plotted against the normalized distance from the lens center. Solid lines are fits to an equation that relates intercellular water flow through gap junctions to the hydrostatic pressure gradient.

Figure 7

PI3K signaling was required for pilocarpine to increase lens hydrostatic pressure. (A) Application of 0.2% pilocarpine to lenses with intact zonules from knockout mice lacking the p110α catalytic subunit of PI3K () failed to increase intracellular hydrostatic pressure. (B) Application of pilocarpine to the PI3K knockout lenses raised the hydrostatic pressure by 0.2 ± 0.9 mm Hg after 100 minutes (P > 0.05, n = 4). The pressure increase observed in wild-type lenses with intact zonules (•) is shown for comparison in (B).

Figure 8

Lens epithelial cells showed increased Akt phosphorylation after ciliary muscle contraction with pilocarpine. (A–C) Lens capsules with adherent epithelial cells from untreated wild-type lenses had low levels of phospho-Akt. (D–F) Ten minutes after 0.2% pilocarpine treatment, epithelial phospho-Akt staining was notably increased (G–I) and remained high 20 minutes after pilocarpine was applied. (J) Lens epithelia from p110α knockout mice lacked phospho-Akt staining. (K) Lens epithelia from PTEN knockout mice had high basal levels of phospho-Akt staining.