A knowledge of the mechanism by which

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1 Calcium transport in the lens K. R. Hightower, V. Leverenz, and V. N. Reddy Evidence based on the following three observations suggests the existence of a calcium transport system in the mammalian lens: calcium levels in the lens are lower than that measured, in the aqueous humor; calcium efflux is temperature-dependent and is reduced by inhibitors of Ca ++ transport; and there exists a calcium-activated, magnesium-dependent ATPase. In rat, bovine, dog, and rabbit lenses, the concentration of total calcium was found to be approximately 0.2 mm, at least an order of magnitude lower than that found in the aqueous humor. To determine the nature of the mechanism responsible for maintaining these low levels, calcium fluxes were measured. During the initial rapid phase of 45 Ca efflux, the rate at4 C was reduced, by 85% compared with that found at 37 C. Efflux was not altered in the absence of external Na +. Calcium efflux was reduced, however, by lanthanum and propranolol, inhibitors of Ca/Mg ATPase. The presence ofcalmg ATPase was also demonstrated in the rat, bovine, and rabbit lens and was likewise inhibited by both lanthum and propranolol. Key words: lens, Ca ++ -Mg ++ ATPase, 45 Ca efflux, transport, propranolol, lanthanum A knowledge of the mechanism by which internal calcium is regulated in the mammalian lens is essential to the understanding of the role of this cation in the maintenance of lens transparency. The possibility that Ca ++ may have a role in the pathogenesis of cataract is suggested by the observations that isolated a-crystallins aggregate in its presence. 1-3 The aggregation of soluble crystallins to form high-molecular-weight proteins is believed to be responsible for light scattering and the resultant lens opacity. 4 Although a number of studies have dealt with the effects of extracellular concentra- From the Institute of Biological Sciences, Oakland University, Rochester, Mich. The study was supported in part by Research grants EY and EY from the National Eye Institute of the National Institutes of Health. A preliminary report was presented at the 7th Conference on the Biochemistry of the Eye, Oakland University, October Submitted for publication Oct. 5, Reprint requests: Dr. K. R. Hightower, Institute of Biological Sciences, Oakland University, Rochester, Mich tions of calcium on lens membrane permeability, 5 little attention has been focused on intracellular calcium in the lens and the mechanisms involved in its regulation. Although it is well established that in most cells, the intracellular level of calcium is lower than in the extracellular fluids and is maintained by a calcium pump or an exchange mechanism, 6 it is not certain whether the calcium concentration in the ocular lens is deficient compared with the aqueous humor. A number of previous studies report values ranging from 0.1 to 4 mm, 7 " 10 which raises the question of whether transport of this ion occurs in the lens. The purposes of this study are (1) to reinvestigate the Ca ++ levels in the mammalian lens by atomic absorption spectroscopy, (2) to ascertain whether calcium fluxes are under metabolic control, and (3) to investigate the presence of a calcium-activated ATPase. Methods Rabbit, bovine, rat, and dog lenses were blotted on filter paper moistened with calcium-free saline or medium to remove vitreous and remain /80/ $00.80/ Assoc. for Res. in Vis. and Ophthal., Inc. 1059

2 1060 Hightower, Leverenz, and Reddy Invest. Ophthalmol. Vis. Sci. September 1980 Table I. Incubation media Substance KC1 NaCl MgCl ATP (Tris) Quabain EGTA Histidine KCN K + from buffer Cl" from buffer Concentration 52mM 0 or 20 mm 2 mm 2 mm 10" 5 or 10-4 M 0.36 mm 56 mm 9 mm 54 mm 58 mm Table II. Ca ++ levels in mammalian lenses Species Rabbit (7) Bovine (5) Rat (4) Dog (2) Concentration (fimol/kg lens H 2 O) 235 ± ± ± Number of experiments in parentheses. Content (ng/mgdrywt.) 17.5 ± ± ± 2 ing zonules. They were either homogenized immediately in 2 ml of chilled histidine-ethylene glycol (EGTA) solution, (300 milliosm) or separated first into capsule-epithelium, cortex, and nucleus. The capsule-epithelium preparation, composed of posterior and anterior capsule with adhering epithelial cells and small amounts of cortical fibers, was obtained by peeling off the capsule with a fine forceps from a lens positioned on a glass surface. Initial experiments involving ATPase measurements were designed to optimize the dissection procedure and produce uniform capsuleepithelium weights of 6 to 8 mg. The outermost cortical fibers were obtained by pinching anterior and posterior fiber fragments with a fine forceps. Nuclear fibers were collected by peeling and tearing away cortical fibers in concentric layers until a 70 to 90 mg mass remained. The consistency and rigidity of the nucleus was markedly different from those of the surrounding cortical tissue, which proved useful in obtaining a reproducible, well-defined nucleus. Tissue fractions were homogenized at 4 C in different volumes of buffer. Homogenates of localized lens tissue were generally obtained by grinding epithelial preparations (6 to 8 mg) in 450 /JL\ of buffer, nucleus (70 to 90 mg) in 500 /xl, and cortical fibers (160 to 200 mg) in 1 ml. Since the resulting homogenates contained both soluble and insoluble protein in a suspension, constant stirring of the viscous homogenate was necessary while aliquots were obtained for incubation. A typical incubation medium consisted of the homogenate, histidine-egta solution, and various electrolytes shown in Table I. Stock solutions of labile substances such as ouabain, KCN, or ATP (Tris) were made up fresh just prior to use. Incubation media were assayed for calcium levels with atomic absorption spectrophotometer (described below), and total calcium concentrations were varied by the addition of EGTA and calcium. Free calcium was calculated according to Shatzmann." Since low levels of Ca ++ were involved in assay procedures, all precautions were taken to ensure that contamination was minimized. The use of glass was limited to grinding rods, homogenizing tubes, and pipettes, which were cleaned and soaked in EDTA and rinsed in deionized H 2 O having a measured calcium concentration of less than 10~ 8 M. Polystyrene incubation tubes were used routinely and examined for calcium levels. Inorganic phosphate, measured by the method of Fiske and SubbaRow, 12 was measured in incubation media at 37 and 4 C, the difference being that liberated by the enzymatic hydrolysis of ATP. The reaction was terminated by the addition of cold 50% trichloraacetic acid. Measurement of calcium in lens and all standards was performed with the Perkin Elmer atomic absorption spectrophotometer (Model 272) operated with a slit width of 0.7 nm, wavelength set at nm, and an aspiration rate of 15 (x\l sec. It was necessary to warm up the calcium bulb for at least 15 min to achieve sufficient stability for reproducibility. Stock standard solutions were prepared from CaCO 3 and deionized water. For efflux measurements, lenses were preincubated for 3 hr at 37 C in KEI-4 medium 13 containing 45 Ca. The stock solution of 45 Ca in H 2 O has a specific activity at 20 mci/mg at.a concentration of 20 mci/ml. Lenses were then transferred to tracer-free KEI-4 at 37 C. Aliquots of 100 /xl were sampled periodically over a 1 hr period and assayed for radioactivity with a Tricarb scintillation counter. In temperature reduction experiments, lenses were preincubated for 3 hr at 37 C and Vi hr at 4 C in media containing 45 Ca. They were then transferred to tracer-free KEI-4 at 4 C for efflux measurement. Results In an effort to establish whether or not there exists a calcium concentration gradient in the lens and thus a need for a calcium

3 Volume 19 Number 9 Calcium transport in lens TIME (min.) Fig. 1. Efflux of 45 Ca from rabbit lenses at 37 C (circles) and 4 C (squares). Each point is the mean ± S.E., n = 6 to 10. pump, the Ca ++ content of mammalian lenses was determined. The results obtained from rat, bovine, dog, and rabbit lenses shown in Table II indicate that the value of total calcium was approximately 200 /xm, considerably less than the range of 1.2 to 1.7 mm in the aqueous humor of these species. H To investigate the mechanism responsible for maintaining low calcium in the lens, flux measurements of 45 Ca were made in the rabbit lens. Fig. 1 shows that the initial rate of calcium extrusion at 37 C was reduced from a value of 8.7 to 1.3 hr" 1 at 4 C (Q 10 = 1.9), suggesting that calcium efflux may be active. Active efflux of calcium is often mediated by sodium-calcium exchange in many tissues, the source of energy being supplied by the sodium gradient. Therefore countertransport in the rabbit lens was studied by measuring the efflux of calcium under conditions in which the sodium gradient was changed. Fig. 2 shows that calcium efflux was unaffected when sodium was removed from the medium. In experiments in which Na ++ content of the medium was altered, the initial ratio of the internal to external sodium was 17:10, Table III. Ca-ATPase in rabbit, bovine, and rat lenses* Species Whole lens Per mg wet wt. Rabbit (25) 200 ± 20 Bovinet ( 5) 518 ± 22 Rat (6) 95 ± Number of experiments in parentheses. * Expressed as nanomoles inorganic phosphate liberated per hour. t Stored slaughter-house lenses. which was significantly different from the corresponding ratio of 17:145 in the normal gradient. In contrast to the lack of effect of sodium, the efflux of 45 Ca in the lens was inhibited by lanthanum and propranolol (Fig. 3), which have been shown to inhibit Ca-ATPase - mediated transport in other cells. 15 ' 16 La 3+ appeared to be a more effective inhibitor than propranolol, decreasing the turnover rate of calcium by a factor of 10. These results suggest that Ca ++ transport in the lens may be mediated by a Ca-ATPase rather than by an exchange process involving sodium. In view of these findings, it was decided to determine whether the enzyme Ca-ATPase

4 1062 Hightower, Leverenz, and Reddy Invest. Ophthalmol. Vis. Sci. September u mm No + (CONTROL) No-FREE TIME (min.) Fig. 2. Efflux of 45 Ca from rabbit lenses in the presence of 145 mm Na + and in the absence of Na +. Mean ± S.E., n = o 4O i 30 ui o n 20 LANTHANUM PROPRANOLOL 10 CONTROL TIME (min.) I Fig. 3. Effect of lanthanum (1 x 10" 4 M) and propranolol (3 x 10" 4 M) on efflux of 45 Ca from rabbit lens epithelium. P h Inorganic phosphate. Mean ± S.E., n = 6 to 8.

5 Volume 19 Number 9 Calcium transport in lens } J I o -100 Ii -200 I 10 10' i CONC OF Co +t (M) IN INCUBATION MEDIUM Fig. 4. Calcium-activated Mg ++ -dependent ATPase in whole lens plotted as inorganic phosphate (P,) liberated when various amounts of Ca ++ are added to the assay medium. Mean ± S.E., n = 6 to 12. Table IV. Effect of calcium concentration on the distribution of Ca-ATPase activity* in rabbit lenses Concentration calcium (M) Lens fraction Ca-ATPase (nmoles/hr) Whole tissue Per mg wet wt. Per nig protein 10' 10' Epithelium (25) Cortex (8) Nucleus (10) Epithelium (25) Cortex (8) Nucleus (10) 77 ± ± ± ± ± ± 20 ± 20 ± 5 ± 2 Number of experiments in parentheses. * Expressed as nanomoles inorganic phosphate liberated per hour. is, in fact, present in the lens. Ca-ATPase activity determined in the assay is the difference in the activities of Mg-ATPase in the presence and absence of added calcium. Fig. 4 shows that the maximum enzyme activity was observed at a calcium concentration of approximately 10~ 5 M, whereas a further increase in calcium appeared to have an inhibitory effect, with the activity falling markedly below the baseline level at a calcium concentration of 10~ 3 M. The relative activity of the enzyme at a calcium concentration of 10~ 5 M in rat, rabbit, and bovine lenses is shown in Table III. In an effort to localize Ca-ATPase in the lens, enzyme activities were measured in rabbit lens homogenates of capsule-epithelium, cortex, and nucleus. Table IV shows that at a calcium concentration of 10~ 5 M, measurable amounts of Ca-ATPase were present in both the epithelium and cortex but absent in the nucleus. However, at a Ca ++ concentration of 10~ 7 M, the nucleus was found to have significant enzyme activity (Table IV). At this calcium concentration, total enzyme activity in the nucleus represented 26% of the total activity, the cortex nearly 55%, and the epithelium 18%. In terms

6 1064 Hightower, Leverenz, and Reddy Invest. Ophthalmol. Vis. Sci. September 1980 CONTROL 20 0 PROPRANOLOL S LANTHANUM Mg* -ATPose Co**-ATPase Fig. 5. Effect of lanthanum and propranolol on Mg ++ -ATPase and Ca ++ -ATPase activity in rabbit lens epithelium. P h Inorganic phosphate. Mean ± S.E., n = 6 to 8. Table V. Effect of lanthanum Experiment Control Lanthanum (1 X 10~ 4 M) Proiiranolol (3 X 10-"M) Number of experiments in parentheses. and propranolol on lens calcium Concentration (funol/kg lens H 2 O) 174 ± 11 (16) 328 ± 37 ( 5) 1483 ± 260 ( 6) Lens calcium Content (ng/mg dry wt.) 12.9 ± 1 (6) 30.1 ± 4 (5) ± 20 (6) of specific activity, however, Ca-ATPase was some 30-fold higher in the epithelium than in the cortical or nuclear fibers. The results shown in Fig. 5 demonstrate that CA-ATPase activity in the epithelium was depressed by lanthanum and propranolol, whereas Mg-ATPase was unaffected. Consistent with the observations on Ca ++ efflux (Fig. 3), La 3+ appeared to be a more effective inhibitor of Ca-ATPase than propranolol. In cultured rabbit lenses, lanthanum and propranolol were also effective in elevating internal calcium concentrations (Table V). However, in this instance, lens calcium was more elevated by propranolol than La 3+. On further examination it was found that lenses exposed to propranolol became hydrated, which could explain, in part, the high calcium level. In any case the changes in lens calcium would appear to be the result of inhibiting the transport enzyme Ca-ATPase, particularly in the case of La 3+. Discussion The results of the present investigation clearly demonstrate that in the mammalian lens, the level of calcium is lower than in the aqueous humor, probably as a result of a calcium pump, the existence of which is supported by the observation that 45 Ca efflux is significantly reduced by temperature reduction. The mechanism for active transport of calcium out of the lens may involve either Na/Ca exchange or the enzyme Ca/ATPase, since these are the known mechanisms by which Ca ++ is transported out of other cells. 16 In the lens, it appears that Na/Ca exchange may be absent, since Ca ++ efflux is unaffected by a change in the Na + gradient. It is known that a small change in the external sodium concentration results in a sizable change in Ca ++ efflux when countertransport is involved. 18 The alternate mechanism involving Ca-ATPase is more likely because the enzyme is, in fact, present in the lens and is

7 Volume 19 Number 9 Calcium transport in lens 1065 inhibited by La 3+ and propranolol, known inhibitors of Ca ++ transport. Moreoever, in the presence of these inhibitors, the calcium concentration was elevated in cultured lenses. These findings are consistent with the idea that Ca ++ in the lens may be regulated by an active transport system involving Ca-ATPase. It should be emphasized that the enzyme Ca-ATPase measured in lens homogenates refers to a calcium-activated, magnesiumdependent ATPase similar to that of the red blood cell. 19 In each homogenate preparation of epithelium, cortex, and nucleus, Ca ++ was able to stimulate Mg-ATPase only when Mg ++ was present. This is in contrast to the enzyme in heart tissue 20 in which Mg + is not obligatory, since calcium substitutes for this ion. It is interesting to note that Ca ++ stimulation of the enzyme in the lens nucleus could not be detected at a calcium concentration of 10~ 5 M, yet was measurable at 10~ 7 M. In contrast, the maximum stimulation occurs at a Ca ++ concentration of 10~ 5 M in whole lens, cortex, and epithelium. Although the reason for these differences in activation levels among the various tissue preparations is not apparent, it is possible that the added calcium may not represent the true concentration in the assay medium, since Ca ++ may be bound differentially to homogenates from different regions of the lens. The activation curve for the whole lens (Fig. 4) demonstrates a saturation phenomenon characteristic of a carrier-mediated transport system. Assuming that calcium participates stoichiometrically in the splitting of ATP, the rate of hydrolysis of ATP does not increase with calcium concentration in a hyperbolic manner consistent with a simple Michaelis-Menten model. Thus we believe that the enzyme kinetics is complex and probably involves more than one binding site. Although typical kinetic parameters cannot be obtained from the present data, the apparent half-maximal activation concentration for Ca ++ can be estimated to be 10~ 7 M, which may well be the level of free calcium in an intact lens. At nearly all concentrations of calcium used in the assay, the specific enzyme activity is some 30 times higher in the epithelium than in the fibers. This is not surprising, since the epithelium is the primary site of transport for other substances as well as the enzyme Na-K-ATPase. Although the total activity of Na-K-ATPase is nearly equal in the epithelium and fibers, the fraction of total Ca-ATPase activity is higher in the fibers than in the epithelium, suggesting that fibers may also contribute to active transport of calcium. REFERENCES 1. Jedziniak JA, Kinoshita JH, Yates EM, Hocker LO, and Benedek GB: Calcium-induced aggregation of bovine lens alpha crystallins. INVEST OPHTHALMOL 11:905, Spector A and Rothschild C: The effect of calcium upon the reaggregation of bovine alpha crystallin. INVEST OPHTHALMOL 12:225, Jedziniak JA, Nicoli DF, Yates EM, and Benedek GB: On the calcium concentration of cataractous and normal human lenses and protein fractions of cataractous lenses. Exp Eye Res 23:325, Benedek GB: Theory of transparency of the eye. Appl Optics 10:459, Delamere NA and Paterson CA: The influence of calcium-free solutions upon permeability characteristics of rabbit lens. Exp Eye Res 28:45, Mela L: Mechanism and physiological significance of calcium transport across mammalian mitochondrial membranes. In Current Topics in Membranes and Transport, Bronner F and Kleinzeller A, editors. New York, 1977, Academic Press, Inc., vol. 9, pp Bushell A and Duncan G: The distribution of soluble, insoluble and high molecular weight fractions of senile normal and cataractous human lenses as a function of internal calcium. Exp Eye Res 26:223, Spector A, Adams D, and Krul K: Calcium and high-molecular weight protein aggregates in bovine and human lens. INVEST OPHTHALMOL 13:982, Rink H, Munnighoff J, and Hockwin O: Sodium, potassium and calcium contents of bovine lenses in dependence on age. Ophthalmic Res 9:129, Hart W, Peckman R, and Kimel H: Sodium, potassium, and calcium in the normal, maturing crystalline lens. Arch Ophthalalmol 69:76, Schatzmann HS: Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells. J Physiol (Lond) 235:551, Fiske CH and SubbaRow YJ: The colorimetric determination of phosphorus. J Biol Chem 66:375, Wachtl C and Kinsey VE: Studies on the crystalline

8 1066 Hightower, Leverenz, and Reddy Invest. Ophthalmol. Vis. Sci. September 1980 lens. VIII. A synthetic medium for lens culture and the effects of various constituents on cell division in the epithelium. Am J Ophthalmol 46 (II):288, Bito LZ: Intraocular fluid dynamics. I. Steady state concentration gradients of magnesium, potassium, and calcium in relation to the sites and mechanisms of ocular cation transport processes. Exp Eye Res 10:102, Parzig H: Comparative study of the effects of propranolol and tetracaine on cation movements in resealed human red cell ghosts. J Physiol (Lond) 249:27, Krasnow N: Effects of lanthanum and gadolinium ions on cardiac sarcoplasmic reticulum. Biochem Biophys Acta 282:187, Williams RJP: Calcium chemistry and its relation to biological function. Symp Soc Exp Biol 1:1, Reuter H and Seitz N: The dependence of calcium efflux from cardiac muscle on temperature and external ion composition. J Physiol (Lond) 195:451, Schatzman HJ and Rossi GL: (Ca ++ + Mg ++ )-Activated membrane ATPase in human red cells and their possible relations to cation transport. Biochim Biophys Acta 241:379, Anand MB, Chauhan MS, and Dhalla NS: Ca +2 / Mg 2+ ATPase activities of heart sarcolemma, microsomes, and mitochondria. J Biochem 82:1731, Copyright information The appearance of a code at the bottom of the first page of an original article in this journal indicates the copyright owner's consent that copies of the article may be made for personal or internal use, or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., 21 Congress Street, Salem, Mass , , for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. For reprint quantities of 50 or more, please contact Publisher.

DIDS INHIBITION OF SARCOPLASMIC RETICULUM ANION EFFLUX AND CALCIUM TRANSPORT

DIDS INHIBITION OF SARCOPLASMIC RETICULUM ANION EFFLUX AND CALCIUM TRANSPORT Kevin P. Campbell and David H. MacLennan Reprinted from ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Volume 358 Pages 328-331