Regional Distribution of Free Calcium in Selenite Cataract: Relation to Calpain II

Transcription

1 Regional Distribution of Free Calcium in Selenite Cataract: Relation to Calpain II K. R. Highrower,* L. L. David,t and T. R. Shearerf The purpose of this experiment was to assess the roles of free, intracellular calcium and calcium-dependent neutral protease (calpain II, EC ) in selenite nuclear cataract. Free calcium ion concentrations within lens nuclear fibers during selenite cataractogenesis increased to 3 pm on day 2 post-injection (clear lens) and to 108 nm at day 4 (nuclear cataract). Calpain II is known to be activated in vitro by calcium levels above 50 nm. 1 Calpain II activity was present in the lens nucleus at time periods preceding formation of selenite cataract. These data suggested that after selenite injection, calpain II was activated by elevated free calcium in the nucleus, and that calpain H-induced proteolysis of nuclear proteins was an important mechanism in selenite cataract. Calpain II levels were also observed to decrease in the nucleus during selenite cataractogenesis, probably due to autolysis. This was supported by the finding that incubation of purified lens calpain II with 100 nm calcium caused partial inactivation of the protease. Invest Ophthalmol Vis Sci 28: ,1987 Calcium levels in the lens were increased in a wide variety of experimental animal models of cataract 2 " 5 and in some human cataracts. 6 Recent data from the selenite model of nuclear cataract suggested that increased lens calcium may be a cause rather than an effect of cataract. 7 Total lens calcium increased more than 10-fold (dry weight basis) prior to cataract formation, while overall sodium and potassium concentrations were not appreciably changed. Increased lenticular calcium may stimulate proteolysis observed in selenite cataract by activation of calcium-dependent neutral protease (calpain II). 7 Proteolysis of a- and /3-crystallins, insoluble proteins and major membrane proteins occurred during formation of selenite nuclear opacity. Calpain II was purified from whole rat lenses, and the enzyme was found to be activated above 50 MM calcium ion. 1 However, no data are available to show whether calpain II activity is actually present in rat lens nucleus or if the intracellular levels of free calcium ion were high enough in selenite cataract to activate calpain II enzyme. The two purposes of the present report were: (1) to determine the regional distribution of calpain II in normal rat lens and in selenite cataract; and (2) to From the ""Institute for Biological Sciences, Oakland University, Rochester, Michigan, and the fdepartments of Biochemistry and Ophthalmology, Oregon Health Sciences University, Portland, Oregon. Supported in part by grant Nos. EY-03600, EY (TRS), andey (KRH). Submitted for publication: January 15, Reprint requests: Dr. K. R. Hightower, Institute for Biological Sciences, Oakland University, Rochester, MI determine the concentrations of free, intracellular calcium in selenite cataract. The data collected were consistent with the hypothesis that calcium activation of calpain II in the nucleus contributed to development of selenite cataract. Materials and Methods Free Calcium Ion Determinations On day 10 post-partum, Sprague-Dawley rat pups (Charles River Labs, Inc., Wilmington, MA) received a single 0.05 ml subcutaneous injection of an overdose of sodium selenite at 30 mm Se/kg body weight. On days 2 and 4 post-injection, pups were killed with T-61 Euthanasia solution, and eyes were removed 1 min later. In order to measure lens voltages in situ, enucleated eyes were immediately transferred to a waterjacketed chamber maintained at 34 C. In a small incision at the optic stalk, voltage and calcium microelectrodes were separately driven at 5 /um steps into the cortex (10-50 ^m from lens surface) and nucleus ( nm). Potentials were monitored continuously on separate channels of a WPI F235 electrometer as described in detail elsewhere. 8 Both electrodes might typically show an initial 10 mv loss of potential over a 10 min period, the difference remaining constant and indicating a stable calcium level. Calcium concentrations in fiber cell cytosol were calculated from the difference in potential obtained from voltage and calcium electrodes. These electrodes did not impale the same fiber cell. However, 1702

2 No. 10 CA AND CALPAIN II IN SE CATARACT / Highrower er ol multiple impalements into different fibers within the same region of the lens showed variations amounting to only 3 to 5 mv (Table 1), establishing the validity of the procedure. Only after impalement into the nuclear region did electrodes occasionally show altered sensitivity or hysteresis due to loss of calcium cocktail from the electrode or plugging of the electrode tip. In these cases, electrodes were discarded, and measurements were made with fresh electrodes. Total calcium was determined by atomic absorption spectroscopy. 9 Measurement of Calpain II After cervical dislocation or CO 2 inhalation, lenses were collected from rats and separated under the dissecting microscope into three regions (capsule/epithelium, cortex, and nucleus) in homogenizing buffer containing 20 mm Tris (ph 7.5), 1 mm EDTA, 1 mm EGTA, and 1 mm dithioerythritol. Regions from two or three pairs of lenses were frozen at -70 C in 0.5 or 1.0 ml of homogenizing buffer. Thawed regions were then homogenized and centrifuged at 10,000 g for 25 or 30 min, and the supernatants were saved for assay of calpain II activity. Calpain II activity could be detected in crude supernatants of rat lens. However, attempts to precisely quantitate calpain II in crude lens homogenates were hampered because the assay was non-linear with increasing amounts of lens protein, possibly due to the presence of endogenous inhibitor. 1 To allow more accurate measurements of calpain II activity, DEAE chromatography was also used to partially purify calpain II and remove endogenous inhibitor. Chromatography of lens regions was performed by HPLC on a 7.5 X 75 mm TSK DEAE-5-PW column with elution of proteins by a linear gradient of mm NaCl in homogenizing buffer over 45 min with a 1.0 ml/min flow rate. Calpain II activity emerged at approximately 400 mm NaCl over a 3 min period. One minute fractions were assayed for calpain II, 1 and specific activity (U/mg protein) in the initial lens supernatant was calculated by assuming 100% recovery from the DEAE column. Calpain II assay was performed using FITC-labeled casein as previously described.' This assay was capable of detecting calpain in relatively small amounts of lens sample (0.3-1 mg total protein), but this may have led to somewhat increased variability. One unit of calpain activity was defined as the amount of enzyme releasing 1 ng of acid-soluble fragments from FITC-casein per min at 30 C. Protein analyses were performed with the Bio-Rad (Richmond, CA) dye-binding assay, using bovine serum albumin as standard. The amount of nucleus dissected from the lens varied from 19 to 44% of total Table 1. Voltage measurements in different fiber cells within the same region of single lenses Impalement Control lens (-mv) 51* Cataract lens (-mv) * All values are for a single voltage measurement taken after a 10 min stabilization period. lens protein, but this variability did not alter conclusions regarding calpain II activity during cataract formation. Autolysis of calpain II was tested by incubating the enzyme (after partial purification by DEAE as above and removal of chelators by ultrafiltration) with CaCl 2 for various time periods. The enzyme was then placed in an excess of EGTA to stop autolysis, and remaining calpain II activity was determined as before. 1 Rats used in this study were treated in accordance with the ARVO Resolution on the Use of Animals in Research. Results Total calcium levels in control lenses were 192 nm (Table 2, column 2). Two days after selenite injection (no nuclear cataract), total lens calcium doubled, while at 4 days (nuclear cataract present), total calcium increased to 754 nm (Table 2). These data confirmed previous reports, showing elevation of total calcium in selenite nuclear cataract. 37 ' 10 Free Intracellular Calcium Free intracellular calcium ion concentrations in cortex and nucleus of normal rat lens were 0.2 and 0.5 ^M, respectively (Table 2, columns 3, 4). These free calcium levels were an order of magnitude lower than observed in rabbit and human lenses using similar microelectrodes. 811 On day 2 following selenite injection, free calcium levels in the cortex and nucleus increased 5- to 6-fold. Moreover, a slight, but statistically significant gradient in free calcium levels existed between cortex and nucleus, viz. 1 vs 3 vm. On day 4 following selenite, free calcium levels were 43 fim in the cortex and 108 ^M in the nucleus, and the gradient between cortex and nucleus was very evident. During selenite cataract formation, an increase in the percent free calcium also occurred (Table 2, column 5).

3 1704 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1987 Vol. 28 Table 2. Concentrations of total and free calcium in rat lens during development of selenite cataract Group Free Co 1 Total calcium Cortex Nucleus Free Ca + + in lens} 0.30 Control 192 ± 25* Se-2days 380 ± 50 Se-4days 754 ± ± ± ± ± ± ± Mean ± SE (n = 4-6). weighted avg. of free Ca ++ in cortex or nucleus t % Free = X 100 total calcium (assuming nucleus = 20% by weight). Calpain II Enzyme Activity In the present study, calpain II enzyme activity was found in both cortex and nucleus of normal rat lens (Figs. 1, 2), while no activity could be detected in the epithelium (data not shown). In 10-day-old control rats receiving no selenite injection, the specific activity of calpain II after DEAE chromatography was U/mg in cortex (Fig. 1) and U/mg in nucleus (Fig. 2). Therefore, cortex contained over twice the specific activity of calpain II found in the nucleus of control animals. Calpain II activity was not significantly altered in DAYS POST-INJECTION Fig. 1. Calpain II activity in cortex of young rat lens after DEAE chromatography. (O O) = control and (X X) = seleniteinjected rats. Error bars show 95% confidence intervals (1.96 X standard error) for location of true means. 3-8 pools of lenses were analyzed: 6-8 lens regions/pool DAYS POST-INJECTION Fig. 2. Calpain II activity in nucleus of young rat lens after DEAE chromatography. See Figure 1 for legend. The means at day 3 were statistically different (P < 0.05). the cortex during cataract formation (Fig. 1), but selenite injection caused major changes in calpain II activity in the lens nucleus (Fig. 2). A precipitous drop occurred in nuclear calpain II activity by day 3 after selenite injection. Nuclear calpain II activity was still appreciable at 2 days post-selenite injection. Results roughly similar to those above were obtained from calpain assays on crude lens homogenates (data not shown). Calpain II activity in nucleus 2 days after selenite decreased to 49% of control, while calpain activity was only 19% of control at 4 days after selenite. To investigate whether the observed decrease in calpain II activity could be due to calcium-induced autolysis of calpain, purified lens calpain II was incubated in the presence of 100 nm calcium. One hundred ^M calcium caused over 40% loss of calpain II activity in 2.5 hr (Fig. 3, lower curve). Incubation of calpain II without calcium led to no significant inactivation (Fig. 3, upper curve). Discussion The present study provided three pieces of information consistent with the idea that activation of calpain II enzyme occurred during the formation of selenite nuclear cataract. First, calpain II activity was found in the nucleus of rat lens before onset of selenite nuclear opacity. This was true when calpain II

4 No. 10 CA AND CALPAIN II IN SE CATARACT / Highrower er ol was assayed in either crude lens homogenates or in samples of lenses where the endogenous inhibitor of calpain II was removed prior to enzyme assay. Such data are important because the only previous information on the regional distribution of calpain II was in bovine lens, where no calpain II activity was found in the nucleus. 12 The second important piece of information provided by these experiments was that free calcium levels started to rise at least 2 days before onset of cataract. Free calcium levels eventually rose much higher in the nucleus than in the cortex in selenite cataract. These free calcium levels reflected the distribution of total calcium previously assessed in selenite cataract by the electron probe 3 and by atomic absorption spectroscopy. 7 At day 2 post-injection, free calcium levels in the nucleus increased 6-fold to 3 nm, and by day 4, nuclear free calcium was 108 jum. Previous studies showed that purified calpain II from rat lens was activated in vitro by calcium ion concentrations greater than 50 ixm. 1 We surmise that after selenite injection, free calcium levels in the nucleus rose high enough before onset of visible opacity to activate calpain II and initiate the extensive proteolysis observed in selenite cataract. Another major difference between the clear lens (day 2) and the opaque lens (day 4) was the dramatic increase in the fraction of free calcium occurring on day 4. Excess calcium was apparently initially sequestered by calcium-binding proteins and other con-, stituents so that free calcium was less than 0.4%. On day 4, however, the fraction of free calcium increased markedly to 7%, as if calcium-binding sites were saturated. This is important because free calcium is the physiologically active form of calcium capable of activating calpain II. The third important finding in this study was the decrease in calpain II activity occurring in the lens nucleus following selenite injection. Presumably this was due to the well known autolysis of calpain II noted after exposure of the enzyme to elevated calcium. 13 Loss of calpain activity after formation of cataract in the Nakano mouse was also postulated to be due to autolysis. 14 Autolysis was further supported in the present experiment by observing loss of calpain II activity after incubation of calpain II in the presence of calcium at concentrations found in the lens nucleus with selenite cataract. Autoproteolytic inactivation of rabbit muscle calpain II also occurred after incubation with calcium. 15 SDS-PAGE demonstrated autolysis of both subunits in calpain II from chicken gizzard smooth muscle, 16 and approximately 20 and 90 amino acids from the N-termini of the 30 and 80 kd subunits of calpain II from rabbit skeletal muscle were cleaved off by autolysis. 17 In the present experi OO TIME. MIN Fig. 3. Inactivation of calpain II by incubation with 100 nm calcium. Error bars show 95% confidence intervals for location of true mean (n = 4). ment, the autolysis data and decreases in calpain II activity during selenite cataractogenesis were taken as further evidence that calpain II was indeed activated in the lens nucleus during selenite cataractogenesis. Activation of calpain II may eventually led to cleavage of calpain II itself along with hydrolysis of structural proteins observed in selenite lenses. 7 Sufficient time for calpain II to induce enough proteolysis to influence cataract formation may have occurred between days 2 and 3 after selenite injection. Most of the proteolysis observed during the formation of the nuclear cataract had already occurred by 3 days following selenite injection. 7 In rat lenses, we propose that the hydrolysis of nuclear proteins by calpain II interrupts normal interaction between proteins, leading to aggregation and light scatter. 7 A similar proteolytic mechanism may operate in the Nakano mouse cataract, where calpain II activity was normal before and during onset of cataract formation, and decreased rapidly along with other enzymes as cataract progressed. 14 Several questions remain concerning selenite nuclear cataract. The initial lesion caused by selenite leading to accumulation of calcium is unknown. Histochemical and enzymatic measurements of Ca- ATPase activity in specific regions of lens may reveal selenite inhibition of cation pumps. The possible role of selenite in opening calcium ion channels should be investigated. The reason the cortex does not accumulate as much calcium as the nucleus is unknown. Perhaps cortical and nuclearfibercell membranes are characterized by different pump-leak parameters; cal-

5 1706 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1987 Vol. 28 cium may be less actively transported from nuclear fiber cells than cortical cells. Significant amounts of a novel, low molecular weight (9 kd) inhibitor of calpain II were found in rat lens. 18 The present communication showed that calpain II activity could be detected after selenite injection even in crude lens homogenates. This suggested that the calpain II/inhibitor ratio in vivo was high enough to allow expression of calpain II activity during the initial stages of selenite cataractogenesis. Changes in the concentration of inhibitor also need to be specifically assayed, but the present data were consistent with the observation that proteolysis could be initiated in crude rat lens homogenates by addition of calcium. 7 Finally, the role of calpain II and proteolysis, as exemplified by the selenite model, needs to be evaluated in human cataractogenesis. Key words: cataract, calcium, calpain II, selenite, lens References 1. David LL and Shearer TR: Purification of calpain II from rat lens and determination of endogenous substrates. Exp Eye Res 42:227, Hightower KR: Cytotoxic effects of internal calcium on lens physiology: A review. Curr Eye Res 4:453, Shearer TR and David LL: Role of calcium in selenium cataract. Curr Eye Res 2:777, Ruck JFR and Kuck K.O: The emory mouse cataract: Loss of soluble protein glutathione, protein sulfhydryl and other changes. Exp Eye Res 36:351, Kador PF, Fukui HN, Fukushi S, Jernigan HM Jr, and Kinoshita JH: Philly mouse: A new model of hereditary cataract. Exp Eye Res 30:59, Hightower KR and Reddy UN: Calcium content and distribution in human cataract. Exp Eye Res 34:413, David LL and Shearer TR: Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis. Invest OphthalmolVisSci25:1275, Hightower KR, Duncan G, and Harrison SE: Intracellular calcium concentration and calcium transport in the rabbit lens. Invest Ophthalmsl Vis Sci 26:1032, Hightower KR, Leverenz V, and Reddy UN: Calcium Transport in the lens. Invest Ophthalmol Vis Sci 19:1059, Bunce GE, Hess JL, and Barta R: Lens calcium and seleniteinduced cataract. Curr Eye Res 3:315, Duncan G and Jacob T: Calcium and the physiology of cataract. In Human Cataract Formation, Ciba Symposium 106. London, Pitman, 1984, pp Yoshida H, Murachi T, and Tsukahara I: Distribution of calpain-i, calpain II and calpastatin in bovine lens. Invest Ophthalmol Vis Sci 26:953, Suzuki K: Calcium activated neutral protease: Domain, structure and activity regulation. Trends Biol Sci 12, 103, Yoshida H, Murachi T, and Tsukahara I: Age-related changes in calpain II and a-crystallin in the lens of hereditary cataract (Nakano) mouse. Curr Eye Res 4:983, Mellgren RL, Repetti A, Muck TC, and Easly J: Rabbit skeletal muscle calcium dependent protease requiring millimolar Ca: Purification, subunit structure, and Ca dependent autoproteolysis. J Biol Chem 257, 7203, Hathaway DR, Werth DK, and Haeberle JR: Limited autolysis reduces the Ca 2+ requirement of a smooth muscle Ca 2+ -activated protease. J Biol Chem 257, 9072, Imajoh S, Kawasaki H, and Zuzuki K: Limited autolysis of calcium-activated neutral protease (CANP): Reduction of Ca 2+ -requirement is due to the NH 2 -terminal processing of the large subunit. J Biochem 99, 1281, David LL, Shearer TR, and Palmer EA: Characterization of a novel inhibitor of calcium-dependent neutral protease from rat lens. ARVO Abstracts. Invest Ophthalmol Vis Sci 27(Suppl):274, 1986.