Investigative Ophthalmology & Visual Science, Vol. 30, No. 7, July 1989 Copyright © Association for Research in Vision and Ophthalmology Oxidative Inhibition of Ca2+-ATPase in the Rabbit Lens Douglas Dorchmon, Christopher A. Parerson, and Nicholas A. Delamere Hydrogen peroxide inhibition of maximum Ca2+-ATPase and Na+,K+-ATPase activity was measured in a membrane-enriched preparation of rabbit lens cortical fibers and epithelium. At 5 X 1O~6 M hydrogen peroxide maximum Ca2+-ATPase activity was inhibited by 39%, while maximum Na+,K+- ATPase activity was stimulated. Ca2+-ATPase activity was almost completely inhibited at 5 X 10~4 M hydrogen peroxide, in comparison to Na+,K+-ATPase activity, which was only inhibited by 28% at a concentration of hydrogen peroxide an order of magnitude larger. The addition of catalase to hydrogen peroxide-pretreated samples did not reverse the inhibition of Ca2+-ATPase by hydrogen peroxide. Invest Ophthalmol Vis Sci 30:1633-1637,1989 The possible involvement of lenticular calcium Materials and Methods metabolism in the development of experimental and human cataract has been explored in several stud- Animal Tissues 1 3 ies. " Regulation of lens calcium content is accom- Rabbit eyes were obtained from healthy 2 kg New plished by restricted membrane permeability and a Zealand strain albino rabbits, about 10 weeks old, calcium pump resident in the lens cell membrane. killed painlessly by intravenous administration of 2+ The activity and distribution of Ca -ATPase in the T-61 euthanasia solution (American Hoechst Corp., 4 lens has been described by Hightower et al and Somerville, NJ). Lenses were dissected from the globe Borchman et al.5 by a posterior approach. Separation of the lens epi- Many studies provide evidence for derangement of thelium, cortex and nucleus was the same as pre- membrane function and compositional changes in viously described.5 The experiments adhered to the cataractous lenses.6 It has been suggested that oxida- ARVO Resolution on the Use of Animals in Re- tive mechanisms might account for many of these search. changes.7 Oxidative damage to the lens during cata- ractogenesis is well documented and appears to be Membrane-Enriched Preparation initiated at the cell membrane.8 Studies on lens Na+,K+-ATPase, the sodium pump Cortical and epithelial material was pooled sepa- enzyme, have demonstrated its susceptibility to inhi- rately from eight rabbit lenses. Six separate pools of bition by hydrogen peroxide.9"14 However, there is no tissue were used in this study. After Teflon douncer information available relating the sensitivity of lens homogenization, membrane-enriched microsomal Ca2+-ATPase to oxidative insult. The purpose of this preparations were made from each pool using a dif- ferential centrifugation protocol described pre- study was to evaluate the impact of hydrogen perox- 5 ide upon lens Ca2+-ATPase activity in comparison to viously. Membrane-enriched preparations, at a con- maximum Na+,K+-ATPase activity. centration of 2-7 mg/ml in buffer, were divided into 0.5 ml aliquots and stored frozen in liquid nitrogen for up to 1 week. All pools were prepared separately. After the assays for one pool of membrane material From the Department of Ophthalmology, University of Louis- were completed, another pool was prepared. We have ville School of Medicine, Kentucky Lions Eye Research Institute, 2+ Louisville, Kentucky. established that Ca -ATPase activity in our prepara- Supported by research NIH grant EY-06916, the Kentucky tion does not diminish over a 1 month period storage Lions Eye Research Foundation, and an unrestricted grant from in liquid nitrogen. Research to Prevent Blindness, Inc. Submitted for publication: June 17, 1988; accepted January 11, 1989. ATPase Activity Reprint requests: Douglas Borchman, PhD, Kentucky Lions Eye 2+ Research Institute, 301 E. Muhammad Ali Boulevard, Louisville, Ca -ATPase activity was measured at 37°C as de- KY 40202. scribed in detail previously,5 using membrane at a 1633 Downloaded from iovs.arvojournals.org on 09/25/2021 1634 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / July 1989 Vol. 30 Cortical Ca2+-ATPase Activity membrane epithelium). The reaction mixture con- tained either 5 mM EGTA (buffered with tris pH 7.4), or CaCl2-EGTA buffer to give a final free cal- cium concentration of 1 X 10"5 M, previously deter- mined to yield maximal lens Ca2+-ATPase activity.5 After a 5 min temperature equilibration the ATPase reaction was started by adding labeled (gamma- 32P)ATP (4 X 104 cpm/jimol). The reaction was ter- minated at 10, 20 and 30 min by taking aliquots of m 100 ix\ of sample and placing the aliquot into 50 jtl x ice-cold trichloroacetic acid (5% final concentration). Samples were assayed in duplicate. 32P liberation was determined by a method based on the extraction of a phosphomolybdate complex in isobutyl-alcohol. Ca2+-activated ATPase activity was defined as the difference between the rate of P; liberation measured in the presence and absence of calcium. 100 oo -6 -5 -4 -3 -2 To determine the effect of hydrogen peroxide on Ca2+-ATPase activity, hydrogen peroxide at specified Log [Peroxide] (M) concentration, or water for the controls, was added Fig. 1. Inhibition of lens Ca2+-ATPase activity by hydrogen per- prior to temperature equilibration at 37°C. The hy- oxide in a membrane-enriched sample from rabbit lens cortical drogen peroxide concentration in the reaction mix- fibers. Ca2+-ATPase activity was measured at 37°C, pH 7.4 at a free ture was determined15 before and after the calcium calcium concentration of 1 X 10~5 M. Calcium was buffered with 6 ATPase determinations and found to be unchanged. EGTA, Kb 1 X 10 . Error bars are ± standard error, n = 5 pools. To determine whether the effect of hydrogen per- oxide on Ca2+-ATPase activity was reversible, mem- sample protein concentration of 0.1 mg/ml protein brane samples were treated for 5 min with 5 X 10"4 M for cortical samples, and 0.03 to 0.07 mg/ml for epi- hydrogen peroxide and then catalase was added 5 thelial samples (because of a limited amount of min prior to starting the Ca2+-ATPase reaction. A catalase concentration of 0.9 Mg/ml was determined 2 to completely remove hydrogen peroxide from our Epithelial Ca +-ATPase Activity sample in 1 min; we used a concentration of catalase -25 ten times that level and allowed 5-fold additional time, to ensure complete removal of hydrogen perox- ide from our samples. Catalase did not interfere with Ca2+-ATPase activity. Na+,K+-ATPase activity was measured in the same samples, as detailed pre- viously.5 The methodology is similar to that de- scribed for Ca2+-ATPase except that Na+,K+-ATPase activity was defined as the difference in the rate P; liberation measured in the presence or absence of ouabain. Protein was determined by the Peterson16 modifi- cation of the Lowry assay.17 Results 100 The basal cortical ATPase activity, in the absence oo -6 -5 -4 -3 -2 of stimulation by calcium or sodium, was 340 ± 15 Log [Peroxide] (M) nmol/mg/hr. Maximal cortical calcium ATPase ac- tivity, in the presence of 10~5 M calcium, was 88 ± 30 2+ Fig. 2. Inhibition of lens Ca -ATPase activity by hydrogen per- nmol/mg/hr, matching the value of 78 ± 12 nmol/ oxide in a membrane-enriched sample from rabbit lens epithelial mg/hr previously measured for pooled rabbit cortex.5 fibers. Ca2+-ATPase activity was measured at 37°C, pH 7.4 at a free calcium concentration of 1 X 10~5 M. Calcium was buffered with The effect of hydrogen peroxide on cortical lens EGTA, K* 1 X 106. Error bars are ± standard error, n = 5 pools. membrane calcium ATPase activity is shown in Fig- Downloaded from iovs.arvojournals.org on 09/25/2021 No. 7 H2O2 INHIBITION OF LENS ATPASE ACTIVITY / Dorchmon er ol 1635 ure 1. At low concentrations of hydrogen peroxide (5 Cortical Ca2+-ATPase Activity X 10~6 M), calcium ATPase activity was inhibited by 43%. Calcium ATPase activity was almost com- pletely inhibited at 5 X 10~4 M hydrogen peroxide. 100 Q) The decrease in calcium ATPase activity in the pres- ence of hydrogen peroxide concentrations ranging x from 5 X 10~6 to 10~4 M was found to be statistically 80 significant with a P value of 0.018 (paired variants Pero t-test). The degree of variability in the sensitivity of n different pools of membrane material to hydrogen 60 •oge peroxide is reflected in the standard error. T) Calcium ATPase activity in preparations of lens X epithelium was 250 ± 150 nmol/mg/hr. The effect of 40 hydrogen peroxide on lens epithelial membrane cal- c cium ATPase activity is shown in Figure 2. The linear o concentration-dependent inhibition of calcium ATP- 20 ase by hydrogen peroxide was found to be significant i with a P value equal to 0.005. Inh The inhibition of calcium ATPase by hydrogen 0 peroxide was not reversed when hydrogen peroxide Fig. 3. Inhibition of lens Ca2+-ATPase activity by 5 X 10"" M was removed by adding catalase to the reaction mix- 4 hydrogen peroxide with and without catalase added to remove ture (Fig. 3). In the presence of 5 X 10~ M hydrogen hydrogen peroxide prior to measuring Ca2+-ATPase activity. Ca2+- peroxide, the inhibition of cortical membrane cal- ATPase activity was measured at 37°C, pH 7.4 at a free calcium concentration of 1 X 10~5 M. Calcium was buffered with EGTA, cium ATPase activity was 47 ± 10% in the sample 6 pool tested. When the concentration of hydrogen per- Kb 1 X 10 .