Nicholas A Delamere
Publications
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.
The regional distribution of cholesterol, phospholipid and protein content was determined on pools of human lenses ranging from 13 to 68 years old. The study was undertaken to establish age matched controls for comparison with cataractous lenses. Future spectroscopic structure analysis of human lenses will be performed and the results related to chemical composition. The molar cholesterol to phospholipid ratio was 3.5 +/- 0.3 for human lens. This ratio is high for human tissue. The lens ratio increased from 2.2 +/- 0.3 in the equatorial region to 9.2 +/- 1.6 in the nuclear region. This trend was also observed in the bovine lens. The relative amount of protein increased concomitantly from 0.13 +/- 0.02 Kg protein per gram lipid in the equatorial region to 0.33 +/- 0.06 in the nucleus. The cholesterol to protein ratio remained constant throughout the lens at 0.073 +/- 0.003 Kg suggesting cholesterol could be associated with the crystallin proteins. In partially purified membrane preparations the cholesterol to phospholipid molar ratio was 2.6 +/- 0.2 and 3.2 +/- 0.2 for the cortex and nucleus respectively, three times lower than for the whole tissue. The high cholesterol content could account for the observed rigidity of membranes measured by infrared spectroscopic examination of the CH stretching band.