It has been suggested that elevated levels of insulin or insulinlike growth factors (IGFs) play a role in the development of diabetic vascular complications. Previously, we have shown a differential response to insulin between vascular cells from retinal capillaries and large arteries with the former being much more insulin responsive. In the present study, we have characterized the receptors and the growth-promoting effect of insulinlike growth factor I (IGF-I) and multiplication-stimulating activity (MSA, an IGF-II) on endothelial cells and pericytes from calf retinal capillaries and on endothelial and smooth muscle cells from calf aorta.We found single and separate populations of high affinity receptors for IGF-I and MSA with respective affinity constants of 1 X 10-9 M-1 and 10-8 M-l in all four cell types studied.
A B S T R A C T The pathologies of diabetic micro-and macroangiopathy are different, suggesting that diabetes affects these two types of vascular tissue in a dissimilar manner. We have compared insulin receptors and the effects of insulin on cultured endothelium from calf retinal capillaries and aorta, and the vascular supporting cells, retinal pericytes, and aortic smooth muscle cells. '251-insulin binds to high affinity insulin receptors on all four cell types. Receptor concentrations were similar except for aortic smooth muscle cells, which have 10-fold fewer receptors than the other cell types. Insulin at a concentration of 10 ng/ ml stimulated ['4C]glucose incorporation into glycogen in retinal endothelial cells and pericytes and aortic smooth muscle cells, but had no effect on aortic endothelium. Insulin over a concentration range of 10 ng/ml-10 ,ug/ml, stimulated [3H]thymidine incorporation into the DNA of retinal pericytes, and endothelial cells and aortic smooth muscle cells but had no effect on aortic endothelial cells. These data suggested that a differential response to insulin may exist between endothelium of micro-and macrovasculature, and suggest that retinal capillary endothelium and retinal pericytes are both very insulin-sensitive tissues.
Image enhancement has been shown to improve face recognition by visually impaired observers. We conducted three experiments in an effort to refine our understanding of the parameters leading to this effect. In experiment 1 we found that the band of spatial frequencies between 4 and 8 cycles/face is critical for face recognition. In experiment 2 we found that enhancement of these frequencies and the resulting image distortion actually reduced recognition performance for normal observers. Since the degradation of performance by low vision is larger than the effect of distortion, the enhancement that reduces performance for normal observers may still be beneficial for the visually impaired observer. Experiment 3 found that patients tend to prefer images enhanced at frequencies higher than the critical frequencies found in experiment 1. Such individually selected enhancement did not improve recognition in comparison with uniformly applied enhancement. The lack of an enhancement effect may be due to the small variability in enhancement frequencies selected by our subject population.
Insulin and insulin-like growth factors (IGFs) have been implicated in the pathogenesis of diabetic retinopathy and peripheral vascular complications. Previously, we have shown that retinal capillary endothelial cells responded to insulin and IGFs for metabolic and growth effects, whereas aortic endothelial cells were not responsive. In contrast, vascular supporting cells from both retinal capillaries (i.e. pericytes) and aorta (i.e. smooth muscle cells) responded equally to insulin, IGF-I, and IGF-II. The structure and ligand specificities of the receptor for these peptides were studied by covalently cross-linking 125I-labeled peptide hormones to their respective receptors using disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. The binding subunit of the insulin receptor, alpha-subunit, for all cell types was found to have a mol wt 145,000 under reduced conditions. Labeling of this band was inhibited by 10(-9) M insulin, antiinsulin receptor antibodies, and 10(-8) M IGF-I, but not by multiplication-stimulating activity (IGF-II). The beta-subunit of the insulin receptor in endothelial cells was identified by its ability to be autophosphorylated when stimulated by insulin and was found to have a mol wt of 99,000. Covalent cross-linking of IGF-I to its receptor revealed a mol wt of 145,000, similar to that of insulin receptor, except that IGF-I was 100-fold more potent than insulin in competing with [125I]IGF-I for binding. [125I]IGF-II in all cells was cross-linked to receptor with mol wt of 260,000 and 230,000 under reduced and nonreduced conditions, respectively. IGF-I competed weakly with [125I]IGF-II, whereas insulin was ineffective. [125I]IGF-II also bound to the band with alpha mol wt of 135,000, which was inhibited by insulin, IGF-I, and IGF-II. In summary, receptors for insulin, IGF-I, and IGF-II on cells from micro- and macrovessels are biochemically similar to those in other cells. Interestingly, the finding of large numbers of IGF-I and IGF-II receptors on endothelial cells suggests that these growth factors play a physiological role and are involved in vascular complications associated with diabetes.
Partial separation of protein kinase activity from rhodopsin in isolated bovine retinal photoreceptor outer segments was accomplished by mild ultrasonic treatment followed by ultracentrifugation. Residual kinase activity in the rhodopsin-rich sediment was destroyed by chemical denaturation which did not affect the spectral properties of the rhodopsin. The retinal outer segment kinase was found to be specific for rhodopsin, since in these preparations it alone of several bovine protein kinases was capable of phosphorylating rhodopsin in the light. The phosphorylation reaction apparently requires a specific conformation of the rhodopsin molecule since it is abolished by heat denaturation of rhodopsin, and it is greatly reduced or abolished by treatment of the visual pigment protein with potassium alum after the rhodopsin has been "bleached" by light. When kinase and rhodopsin or opsin fractions were prepared from dark-adapted and bleached outer segments and the resultant fractions were mixed in various combinations of bleached and unbleached preparations, the observed pattern of light-activated phosphorylation was consistent only with the interpretation that a conformational change in the rhodopsin molecule in the light exposes a site on the visual pigment protein to the kinase and ATP. These results rule out the possibility of a direct or indirect (rhodopsin-mediated) light activation of the kinase. Finally, phosphorylation of retinal outer segment protein in monochromatic lights of various wavelengths followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that both rhodopsin and the higher molecular weight visual pigment protein reported by several laboratories have the same action spectrum for phosphorylation. This result is consistent with the suggestion that the higher molecular weight species is a rhodopsin dimer.
Glaucoma and vitreoretinal surgeries have long been associated with cataract progression. 1-10 There have been many theories to explain this phenomenon. These theories include direct or indirect mechanical damage from instrumentation, light toxicity from the operating scope, intraocular irrigating solution, use of intraocular gas, intraocular inflammation, and advanced patient age. 11,12 These theories, however, do not distinguish between the different types of cataracts that form postoperatively, nor do they provide a unified coherent explanation for the observed cataract progression. In this chapter, we propose a mechanism of cataractogenesis, specifically nuclear sclerosis formation, which we believe occurs as a function of altered aqueous fluid dynamics after intraocular surgery. We present this as the nutrient theory of cataractogenesis.Before detailed discussion of the nutrient theory of cataractogenesis as a function of altered aqueous fluid dynamics, a background review of the following concepts will serve as a vital conceptual framework for its understanding: 1) anatomy and metabolism of the lens, 2) aqueous humor and vitreous biochemistry, 3) fluidics of the aqueous humor and vitreous, 4) types of cataracts, 5) recognized causes of cataracts, and 6) current theories of nuclear sclerosis formation after pars plana vitrectomy. 173
The effect of several naturally-occurring retinoids and 13-cis-retinoic acid on the proliferation of cultured bovine retinal pigment epithelial (RPE) cells was investigated. None of the retinoids tested were toxic to the cultures and all, except retinylpalmitate, inhibited cell proliferation when given for more than 3 days. The relative potencies of the retinoids were; all-trans-retinoic acid greater than 13-cis-retinoic acid greater than all-trans-retinol approximately equal to all-trans-retinaldehyde. Uptake of retinoic acid by cultured RPE cells was 10-fold less than the uptake of retinol. Although retinoic acid-treated cultures showed strong density-dependent growth inhibition, cellular proliferation was inhibited more in sparse cultures than in dense ones. Retinoic acid did not significantly inhibit the proliferation of first passage bovine or rabbit RPE cells, but partially inhibited the proliferation of first passage human RPE cells. The sensitivity of all these cultures to growth inhibition by retinoic acid increased in subsequent subcultures, yet there was no effect of passage number on retinoic acid uptake. This study demonstrates that RPE cell proliferation can be inhibited by retinoic acid but the sensitivity of these cells to the retinoid's effects are modulated by incubation time, in vitro aging, and cell density.
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