Chlorpromazine (CPZ) concentrations of 5 x 10(-5)M or greater were found to disrupt lens electrolyte and water balance significantly. Lens sodium and calcium levels increased while potassium decreased. These changes were accompanied by water accumulation. Such effects of CPZ were not dependent upon exposure to ultraviolet light. Electrical measurements revealed that CPZ induced depolarization of the lens potential but little change of overall lens conductance. 86Rb efflux from the lens was unaltered by a 1 hr exposure to CPZ. The active sodium pump in the lens was inhibited by CPZ, as evidenced by a reduction in Na- K-ATPase activity.
In the rabbit, cryoextraction of the lens and subsequent storage in Tyrode's solution did not alter the Na,K-ATPase activity from that determined in immediately excised rabbit lenses. Similarly, the procedures employed with the rabbit eye to simulate collection and storage of normal human eyes (eye banking) had no effect upon the Na,K-ATPase activity of the lens. These results permitted the investigation of human lenses with the knowledge that measured Na,K-ATPase activity had not been altered grossly by any manipulation procedures. Analysis of Na,K-ATPase activity in 44 eye bank lenses, 14 primary nuclear cataracts, 11 primary cortical cataracts, 18 primary posterior subcapsular cataracts, and 31 mixed cataracts revealed no significant difference in the enzyme activity between these groups. Similarly, there was no correlation between electrolyte levels and Na,K-ATPase in a further 18 mixed cataracts. It is concluded that, despite an often pronounced electrolyte imbalance, human cataract can develop without significant alteration in Na,K-ATPase activity.
We examined the ability of rabbit ciliary epithelium to metabolize arachidonic acid in vitro. The epithelium was homogenized and incubated with 14C-labeled arachidonic acid. 14C-labeled metabolites were extracted and then separated by thin layer chromatography. The range of arachidonic acid metabolites synthesized by ciliary epithelium was compared to the metabolites generated by rabbit iris-ciliary body. Ciliary epithelium produced substantial amounts of arachidonic acid metabolites that comigrated with 5-HETE and 12-HETE. Authenticity of the 12-HETE produced by ciliary epithelium was confirmed by gas chromatography/mass spectrometry. The ciliary epithelium generated only small amounts of the cyclooxygenase products, PGF2 alpha, PGE2, PGD2 and 6k-PGF1 alpha. In contrast, the iris-ciliary body produced large amounts of cyclooxygenase products such as PGF2 alpha and PGD2. The ability of the ciliary epithelium to generate 12-HETE is noteworthy since 12(R)-HETE is known to be capable of lowering intraocular pressure.
Insulin has been shown to elicit changes of Na,K-ATPase activity in various tissues. Na,K-ATPase in the nonpigmented ciliary epithelium (NPE) plays a role in aqueous humor secretion and changes of Na,K-ATPase activity impact the driving force. Because we detect a change of NPE Na,K-ATPase activity in response to insulin, studies were carried out to examine the response mechanism. Ouabain-sensitive rubidium (Rb) uptake by cultured NPE cells, measured as a functional index of Na,K-ATPase-mediated inward potassium transport, was found to increase in cells exposed for 5 min to insulin. The maximally effective concentration was 100 nM. An intrinsic increase of Na,K-ATPase activity evident as a >2-fold increase in the rate of ouabain-sensitive ATP hydrolysis in homogenates obtained from cells exposed to 100 nM insulin for 5 min was also observed. Insulin-treated cells exhibited Akt, Src family kinase (SFK), ERK1/2, and p38 activation, all of which were prevented by a pI3 kinase inhibitor LY294002. The Rb uptake and Na,K-ATPase activity response to insulin both were abolished by PP2, an SFK inhibitor which also prevented p38 and ERK1/2 but not Akt activation. The Akt inhibitor MK-2206 did not change the Na,K-ATPase response to insulin. The findings suggest insulin activates pI3K-dependent Akt and SFK signaling pathways that are separate. ERK1/2 and p38 activation is secondary to and dependent on SFK activation. The increase of Na,K-ATPase activity is dependent on activation of the SFK pathway. The findings are consistent with previous studies that indicate a link between Na,K-ATPase activity and SFK signaling. J. Cell. Physiol. 9999: 1-12, 2016. © 2016 Wiley Periodicals, Inc.
