Acta physiol. scand. 1975. 95. 117-125 From the Department of Medical Chemistry, University of Helsinki, Finland
Accumulation of Calcium by Retinal outer Segments BY
KARIHEMMINKI Received 18 March 1975
Abstract HEMMINKI, K. Accumulation of calcium by retinal outer segments. Acta physiol. scand. 1975. 95. 117-125. Two preparations of bovine photoreceptors, including intact and lysed outer segments, are used to study accumulation of Ca++ in order to differentiate between binding and transport. The two preparations accumulate initially roughly equal amounts of Ca++ in the presence or absence of ATP. In the absence of ATP no further accumulation occurs. With ATP accumulation continues most rapidly in intact outer segments. The addition of a Ca++-ionophore (A 23187) to assays containing ATP, results in kinetics of uptake similar to those in the absence of ATP. Thus the initial phase of Ca++ accumulation is predominantly due to binding, while that induced by ATP is due to transport. Accumulation of Caff by outer segments can be stimulated by preloading them with ATP suggesting that ATP enters the structures and acts intracellularly. Illumination decreases binding of Ca++ to outer segments but does not consistently affect transport. After illumination more Ca++ is retained by intact than lysed outer segments indicating that plasma membrane acts as a barrier to the escape of Ca++. Phospholipids and protein appear to be responsible for Ca++ binding of outer segments as suggested by a reduced binding ability after removal of these constituents. Phosphatidyl ethanolamine is the main phospholipid class of outer segments.
Calcium ions have been suggested to mediate between absorption of light and hyperpolarisation of the cell membrane in retinal photoreceptors (Tomita 1970, Hagins 1972). Evidence is now accumulating in support of the involvement of Ca++ in light-induced changes. Ca++ has been reported to be released from outer segments on illumination (Mason et ul. 1974, Hemminki 1975) and some data suggest that accumulation of Ca++by outer segments is regulated by light (Mason et al. 1974). Accumulation of Ca++ions by photoreceptors is stimulated by exogenous ATP (Bownds et al. 1971, Neufeld et al. 1972) but it has not been established whether ATP increases binding or transport of the ion. In this study accumulation of Ca++by intact and lysed bovine outer segments is investigated. A calcium ionophore, A23187 (Caswell and Pressman 1972) is applied in the separation of binding and transport. Regulation of accumulation of Ca++ by light is investigated and the Ca++ binding component is characterized. 117
118
KARI HEMMINKI
Materials and Methods Isolation of outer segments
Bovine eyes were removed immediately after slaughter and placed in a light-proof container at 2 to 5°C for transportation. After about 1 h retinas were dissected under dim red light, washed in 0.25 M sucrose and collected in 1.0 M sucrose (0.5 ml/retina at 0°C). The suspension of retinas was shaken b y h an d f o r 2 x 15 s and transferred into a beaker, which was covered with a nylon mesh (hole size 0.5 mm). The liquid was sieved through the mesh and the contents of the beaker were washed with 0.25 M sucrose (1.5 ml/retina); the wash was filtrated through the mesh and combined with the first slurry. The suspension was centrifuged at 9000 x g for 10 min and the supernatant was decanted. The pellet was resuspended in 1.05 M sucrose (1.5 ml/retina) and transferred into cellulose nitrate tubes. 5 ml of 0.85 M sucrose was overlayered on 25 ml of 1.05 M sucrose containing the sample and the gradient was centrifuged in a Spinco SW-25 rotor at 63000 X g for 30 min. The interfacial layer was collected with a Pasteur pipette, diluted with 3 vol. of 0.25 M sucrose and sedimented at 9000 g for 10 min. The pellets contained retinal outer segments. Outer segments were lysed by maintaining in distilled water for 10 min. The extent of lysis was examined by injecting samples of outer segments (0.1 ml) into 0.12 M ammonium acetate (1 ml) and rapidly monitoring the change of absorbance a t 640 nm. Procedure
Ca++ accumulation was assayed as described recently (Hemminki, 1974) by incubating samples of outer segments (50 PI) in ‘physiological medium’ containing 0.12 M NaCl, 10 mM KCI, 1 mM MgSO,, 1 mM CaCI, and 10 m M sodium phosphate buffer, pH 7.4; 46CaC1, (s.a. 1040 mCi/mg Ca+f, Amersham) was used at 0.5pCi. Incubations were performed in room light at 37°C. Incubations were terminated by adding 3 ml of 0.1 M Tris-HCI, pH 7.5 and transferring the mixtures onto Millipore filters (0.45 ,urn), previously soaked in 0.25 M KCI. The membranes were washed with 2 x 4 ml batches of Tris with vacuum suction and counted in 5 ml of Bray’s scintillation fluid. Blanks contained no outer segments. A calcium ionophore, A 23187 (Cashwell and Pressman 1972; Foreman er al. 1973), was a gift from Eli Lilly. Purity of outer segments was investigated by assaying for marker enzymes including c holinesterase (E.C. 3.1.1.81, a marker of neuronal membranes, succinate dehydrogenase (E.C. 1.3.99.1), a mitochondria1 marker and rotenone-insensitive NADH-cytochrome C oxidoreductase (E.C. 1.6.99.3) a microsomal marker. The enzyme assays are described elsewhere (Hemminki and Suovaniemi 1973). Phospholipids were extracted by the system of Folch and analysed as described previously (Hemminki 1973). Cholesterol was determined according to Pearson et al. (1953). Electron microscopy was performed according to Sabatini et al. (1963).
Results Characterization of outer segments
Intact bovine retinal outer segments were prepared by gradient centrifugation and for the preservation of structure they were kept in isotonic or, for short times, in hypertonic solutions. Outer segments of this preparation appeared intact and were surrounded by undisrupted plasma membranes (Fig. 1). Lysed outer segments (Fig. 2) were prepared by suspending outer segments in water. The fraction composed of vesicularized membranes. The degree of lysis of outer segments was checked by an osmotic test. Outer segments were suspended in ammonium acetate, which passes freely into these structures causing osmotic swelling (Heller et al. 1971). These results are shown in Table 1. Absorbances of the fractions (A/mg protein) were 1.26 and 0.03 in intact, and lysed outer segments, respectively, in good agreement with the electron microscope observations. Purity of outer segments was examined with marker enzymes and spectral ratios (Table 11). Markers of neuronal and microsomal membranes showed high purity of outer segments
CA"
ACCUMULATION BY OUTER SEGMENTS
119
Fig. 2
Fig. 1
Fig. 1. An electron micrograph of intact bovine retinal outer segments, x 6 003. Fig. 2. Lysed outer segments, prepared by maintaining outer segments in water for over 10 min, x 3 900.
as the specific activities of these enzymes in the samples were only 7 % of the homogenate activities. No mitochondria1 contamination was detected in outer segments. The spectral ratios indicated concentration of rhodopsin by 6.5 to 10-fold which is among the best purifications cited in the literature (Zorn and Futterman 1973, Daemen 1973). Accumulation of calcium by outer segments The effect of added ATP on the accumulation of calcium was studied with intact and lysed outer segments (Fig. 3). At 1 mM concentration ATP increased the initial accumulation of 'Ca++ only slightly; within 30 min a 4-fold increase was observed with intact outer segments as compared to a less than 2-fold increase with lysed outer segments. As lysed outer segments are osmotically inactive, they are likely to lack transport functions. Thus ATP may influence a transport mechanism for Ca++rather than facilitate binding of the ion. In order to differentiate between binding and transport a calcium ionophore A 231 87 (Cashwell and Pressman 1972) was used (Fig. 4). Kinetics of Ca++ uptake in the presence TABLE I. Osmotic properties of intact and lysed bovine outer segments. Samples were mixed with 0.12 M ammonium acetate and absorbance was monitored at 640 nm.
A absorbance/mg protein Intact outer segments Lysed outer segments
1.26 0.03
120
KARI HEMMINKI
TABLE 11. Enzymatic and spectral characterization of lysed retinal outer segments. Enzyme activities are expressed in nmol/min/mg protein. Means +S.E. of 4 to 5 determinations.
A Outer segments Choiinesterase Succinate dehydrogenase Rotenone-insensitive NADHcytochrome C reductase A278/A498 A400/A498
2.1k0.8 0 8.0k2.4 2.24f0.07 0.294k0.04
B Homogenate
B/A
29.3k2.2 49.2k2.9
14.0
124+ 10.1 14.5k0.7 3.01 k0.07
15.5 6.5 10.3
03
of A 23187 were largely similar to those observed in the absence of ATP (Fig. 3 A). No net accumulation of Ca++ occurred after 1 min. It therefore appeared likely that the initial uptake was largely due to binding of Cat+ while the delayed uptake, induced by ATP, would be due to transport. If it is assumed that A 23187 makes membranes of outer segments freely permeable to Ca++, Fig. 4 B would then describe the maximal Ca++ binding capacity of the membranes: about 5 nmol Ca++/mgprotein. It is of interest to refer to Fig. 3 A showing that even intact outer segments acquire about 50% of the maximal binding capacity within 1 min. This indicates ready accessibility of a number of Ca++binding sites. Accumulation of Ca++by outer segments was studied at 2 different Ca++-concentrations, 1 mM and 4 x M as shown in Fig. 5. At low Ca++ concentrations the kinetics were largely similar to those at the high concentration of Cat+. Stimulation of Ca++uptake by outer segments, described in Fig. 3, was suggested to reflect stimulation of transport. It was therefore of interest to show, whether ATPentered outer segments. Outer segments were incubated in the presence of ATP at 0°C for 30 min and an assay for Ca++uptake was performed. If the uptake was stimulated as compared to the control samples, where ATP was added immediately before the assay, this would then suggest that ATP slowly diffused into outer segment. This appeared to be the case (Table 111) as uptake was stimulated by a factor of 1.3 in intact outer segments. The stimulation of transport was possibly even higher since binding, as measured in the presence of the calcium ionophore was somewhat lower in the experimental samples. These data suggest that ATP stimulates transport of Ca++into outer segments intracellularly.
Fig. 3. Effect of ATP on the accumulation of radioactive calcium into retinal outer segments. Outer segments containing about 100 p g of protein were incubated in physiological medium containing 0.5 pCi 46CaC1, in a total volume of 450 p1. In (B) 1 mM ATP was included. ( - 0 -) intact outer segments, ( - o -) lysed outer segments. Means +S.E. of 4 expts.
121
CA++ ACCUMULATION BY OUTER SEGMENTS
Fig. 4. Effect of A23187, a calcium ionophore. on the accumulation of radioactive calcium into retinal outer segments. Incubation conditions as described in Fig. 3. All assays were performed in the presence of 1 mM ATP (B) contained 0.9 p M A23187. (-.-) intact outer segments, (- 0-) lysed outer segments. Means S.E. of 4 expts.
B Fig. 5. Effect of calcium concentration on the accumulation of radioactive calcium in retinal outer segments. (A) was incubated in physiological medium containing 1 mM %aC12 and 0.5 pCi 45CaCI, while in (B) non-radioactive calcium was omitted. The incubations were performed in the presence of 1 mM ATP. (- -) intact outer segments. ( - 0-) lysed outer segments. Means f.S.E.of 4 expts.
0.3
Effect of illumination on the accumulation of calcium
Light regulation of Ca++ accumulation by outer segments was investigated by incubating illuminated and unilluminated preparations in the presence of radioactive Ca++(Table IV). Incubation conditions were such that mainly binding of Ca++was probed, e.g. absence of ATP and presence of A 23187. Illuminated preparations bound consistently less calcium. When the assays were carried out in conditions suitable to monitor transport, no light reguTABLE 111. Accumulation of calcium by outer segments preloaded with ATP. Outer segments were preincubated in physiological medium with or without 2 mM ATP at 0°C in the dark. After 30 min 2 mM ATP was added to the control samples; 0.5 pCi of *TaCl, and in indicated cases 1 p M A 23187 were added. Samples were incubated at 37°C for 10 min and collected on filters. Means 5S.E. of 8 determinations. * Significant at p
Accumulation of Calcium by Retinal outer Segments BY
KARIHEMMINKI Received 18 March 1975
Abstract HEMMINKI, K. Accumulation of calcium by retinal outer segments. Acta physiol. scand. 1975. 95. 117-125. Two preparations of bovine photoreceptors, including intact and lysed outer segments, are used to study accumulation of Ca++ in order to differentiate between binding and transport. The two preparations accumulate initially roughly equal amounts of Ca++ in the presence or absence of ATP. In the absence of ATP no further accumulation occurs. With ATP accumulation continues most rapidly in intact outer segments. The addition of a Ca++-ionophore (A 23187) to assays containing ATP, results in kinetics of uptake similar to those in the absence of ATP. Thus the initial phase of Ca++ accumulation is predominantly due to binding, while that induced by ATP is due to transport. Accumulation of Caff by outer segments can be stimulated by preloading them with ATP suggesting that ATP enters the structures and acts intracellularly. Illumination decreases binding of Ca++ to outer segments but does not consistently affect transport. After illumination more Ca++ is retained by intact than lysed outer segments indicating that plasma membrane acts as a barrier to the escape of Ca++. Phospholipids and protein appear to be responsible for Ca++ binding of outer segments as suggested by a reduced binding ability after removal of these constituents. Phosphatidyl ethanolamine is the main phospholipid class of outer segments.
Calcium ions have been suggested to mediate between absorption of light and hyperpolarisation of the cell membrane in retinal photoreceptors (Tomita 1970, Hagins 1972). Evidence is now accumulating in support of the involvement of Ca++ in light-induced changes. Ca++ has been reported to be released from outer segments on illumination (Mason et ul. 1974, Hemminki 1975) and some data suggest that accumulation of Ca++by outer segments is regulated by light (Mason et al. 1974). Accumulation of Ca++ions by photoreceptors is stimulated by exogenous ATP (Bownds et al. 1971, Neufeld et al. 1972) but it has not been established whether ATP increases binding or transport of the ion. In this study accumulation of Ca++by intact and lysed bovine outer segments is investigated. A calcium ionophore, A23187 (Caswell and Pressman 1972) is applied in the separation of binding and transport. Regulation of accumulation of Ca++ by light is investigated and the Ca++ binding component is characterized. 117
118
KARI HEMMINKI
Materials and Methods Isolation of outer segments
Bovine eyes were removed immediately after slaughter and placed in a light-proof container at 2 to 5°C for transportation. After about 1 h retinas were dissected under dim red light, washed in 0.25 M sucrose and collected in 1.0 M sucrose (0.5 ml/retina at 0°C). The suspension of retinas was shaken b y h an d f o r 2 x 15 s and transferred into a beaker, which was covered with a nylon mesh (hole size 0.5 mm). The liquid was sieved through the mesh and the contents of the beaker were washed with 0.25 M sucrose (1.5 ml/retina); the wash was filtrated through the mesh and combined with the first slurry. The suspension was centrifuged at 9000 x g for 10 min and the supernatant was decanted. The pellet was resuspended in 1.05 M sucrose (1.5 ml/retina) and transferred into cellulose nitrate tubes. 5 ml of 0.85 M sucrose was overlayered on 25 ml of 1.05 M sucrose containing the sample and the gradient was centrifuged in a Spinco SW-25 rotor at 63000 X g for 30 min. The interfacial layer was collected with a Pasteur pipette, diluted with 3 vol. of 0.25 M sucrose and sedimented at 9000 g for 10 min. The pellets contained retinal outer segments. Outer segments were lysed by maintaining in distilled water for 10 min. The extent of lysis was examined by injecting samples of outer segments (0.1 ml) into 0.12 M ammonium acetate (1 ml) and rapidly monitoring the change of absorbance a t 640 nm. Procedure
Ca++ accumulation was assayed as described recently (Hemminki, 1974) by incubating samples of outer segments (50 PI) in ‘physiological medium’ containing 0.12 M NaCl, 10 mM KCI, 1 mM MgSO,, 1 mM CaCI, and 10 m M sodium phosphate buffer, pH 7.4; 46CaC1, (s.a. 1040 mCi/mg Ca+f, Amersham) was used at 0.5pCi. Incubations were performed in room light at 37°C. Incubations were terminated by adding 3 ml of 0.1 M Tris-HCI, pH 7.5 and transferring the mixtures onto Millipore filters (0.45 ,urn), previously soaked in 0.25 M KCI. The membranes were washed with 2 x 4 ml batches of Tris with vacuum suction and counted in 5 ml of Bray’s scintillation fluid. Blanks contained no outer segments. A calcium ionophore, A 23187 (Cashwell and Pressman 1972; Foreman er al. 1973), was a gift from Eli Lilly. Purity of outer segments was investigated by assaying for marker enzymes including c holinesterase (E.C. 3.1.1.81, a marker of neuronal membranes, succinate dehydrogenase (E.C. 1.3.99.1), a mitochondria1 marker and rotenone-insensitive NADH-cytochrome C oxidoreductase (E.C. 1.6.99.3) a microsomal marker. The enzyme assays are described elsewhere (Hemminki and Suovaniemi 1973). Phospholipids were extracted by the system of Folch and analysed as described previously (Hemminki 1973). Cholesterol was determined according to Pearson et al. (1953). Electron microscopy was performed according to Sabatini et al. (1963).
Results Characterization of outer segments
Intact bovine retinal outer segments were prepared by gradient centrifugation and for the preservation of structure they were kept in isotonic or, for short times, in hypertonic solutions. Outer segments of this preparation appeared intact and were surrounded by undisrupted plasma membranes (Fig. 1). Lysed outer segments (Fig. 2) were prepared by suspending outer segments in water. The fraction composed of vesicularized membranes. The degree of lysis of outer segments was checked by an osmotic test. Outer segments were suspended in ammonium acetate, which passes freely into these structures causing osmotic swelling (Heller et al. 1971). These results are shown in Table 1. Absorbances of the fractions (A/mg protein) were 1.26 and 0.03 in intact, and lysed outer segments, respectively, in good agreement with the electron microscope observations. Purity of outer segments was examined with marker enzymes and spectral ratios (Table 11). Markers of neuronal and microsomal membranes showed high purity of outer segments
CA"
ACCUMULATION BY OUTER SEGMENTS
119
Fig. 2
Fig. 1
Fig. 1. An electron micrograph of intact bovine retinal outer segments, x 6 003. Fig. 2. Lysed outer segments, prepared by maintaining outer segments in water for over 10 min, x 3 900.
as the specific activities of these enzymes in the samples were only 7 % of the homogenate activities. No mitochondria1 contamination was detected in outer segments. The spectral ratios indicated concentration of rhodopsin by 6.5 to 10-fold which is among the best purifications cited in the literature (Zorn and Futterman 1973, Daemen 1973). Accumulation of calcium by outer segments The effect of added ATP on the accumulation of calcium was studied with intact and lysed outer segments (Fig. 3). At 1 mM concentration ATP increased the initial accumulation of 'Ca++ only slightly; within 30 min a 4-fold increase was observed with intact outer segments as compared to a less than 2-fold increase with lysed outer segments. As lysed outer segments are osmotically inactive, they are likely to lack transport functions. Thus ATP may influence a transport mechanism for Ca++rather than facilitate binding of the ion. In order to differentiate between binding and transport a calcium ionophore A 231 87 (Cashwell and Pressman 1972) was used (Fig. 4). Kinetics of Ca++ uptake in the presence TABLE I. Osmotic properties of intact and lysed bovine outer segments. Samples were mixed with 0.12 M ammonium acetate and absorbance was monitored at 640 nm.
A absorbance/mg protein Intact outer segments Lysed outer segments
1.26 0.03
120
KARI HEMMINKI
TABLE 11. Enzymatic and spectral characterization of lysed retinal outer segments. Enzyme activities are expressed in nmol/min/mg protein. Means +S.E. of 4 to 5 determinations.
A Outer segments Choiinesterase Succinate dehydrogenase Rotenone-insensitive NADHcytochrome C reductase A278/A498 A400/A498
2.1k0.8 0 8.0k2.4 2.24f0.07 0.294k0.04
B Homogenate
B/A
29.3k2.2 49.2k2.9
14.0
124+ 10.1 14.5k0.7 3.01 k0.07
15.5 6.5 10.3
03
of A 23187 were largely similar to those observed in the absence of ATP (Fig. 3 A). No net accumulation of Ca++ occurred after 1 min. It therefore appeared likely that the initial uptake was largely due to binding of Cat+ while the delayed uptake, induced by ATP, would be due to transport. If it is assumed that A 23187 makes membranes of outer segments freely permeable to Ca++, Fig. 4 B would then describe the maximal Ca++ binding capacity of the membranes: about 5 nmol Ca++/mgprotein. It is of interest to refer to Fig. 3 A showing that even intact outer segments acquire about 50% of the maximal binding capacity within 1 min. This indicates ready accessibility of a number of Ca++binding sites. Accumulation of Ca++by outer segments was studied at 2 different Ca++-concentrations, 1 mM and 4 x M as shown in Fig. 5. At low Ca++ concentrations the kinetics were largely similar to those at the high concentration of Cat+. Stimulation of Ca++uptake by outer segments, described in Fig. 3, was suggested to reflect stimulation of transport. It was therefore of interest to show, whether ATPentered outer segments. Outer segments were incubated in the presence of ATP at 0°C for 30 min and an assay for Ca++uptake was performed. If the uptake was stimulated as compared to the control samples, where ATP was added immediately before the assay, this would then suggest that ATP slowly diffused into outer segment. This appeared to be the case (Table 111) as uptake was stimulated by a factor of 1.3 in intact outer segments. The stimulation of transport was possibly even higher since binding, as measured in the presence of the calcium ionophore was somewhat lower in the experimental samples. These data suggest that ATP stimulates transport of Ca++into outer segments intracellularly.
Fig. 3. Effect of ATP on the accumulation of radioactive calcium into retinal outer segments. Outer segments containing about 100 p g of protein were incubated in physiological medium containing 0.5 pCi 46CaC1, in a total volume of 450 p1. In (B) 1 mM ATP was included. ( - 0 -) intact outer segments, ( - o -) lysed outer segments. Means +S.E. of 4 expts.
121
CA++ ACCUMULATION BY OUTER SEGMENTS
Fig. 4. Effect of A23187, a calcium ionophore. on the accumulation of radioactive calcium into retinal outer segments. Incubation conditions as described in Fig. 3. All assays were performed in the presence of 1 mM ATP (B) contained 0.9 p M A23187. (-.-) intact outer segments, (- 0-) lysed outer segments. Means S.E. of 4 expts.
B Fig. 5. Effect of calcium concentration on the accumulation of radioactive calcium in retinal outer segments. (A) was incubated in physiological medium containing 1 mM %aC12 and 0.5 pCi 45CaCI, while in (B) non-radioactive calcium was omitted. The incubations were performed in the presence of 1 mM ATP. (- -) intact outer segments. ( - 0-) lysed outer segments. Means f.S.E.of 4 expts.
0.3
Effect of illumination on the accumulation of calcium
Light regulation of Ca++ accumulation by outer segments was investigated by incubating illuminated and unilluminated preparations in the presence of radioactive Ca++(Table IV). Incubation conditions were such that mainly binding of Ca++was probed, e.g. absence of ATP and presence of A 23187. Illuminated preparations bound consistently less calcium. When the assays were carried out in conditions suitable to monitor transport, no light reguTABLE 111. Accumulation of calcium by outer segments preloaded with ATP. Outer segments were preincubated in physiological medium with or without 2 mM ATP at 0°C in the dark. After 30 min 2 mM ATP was added to the control samples; 0.5 pCi of *TaCl, and in indicated cases 1 p M A 23187 were added. Samples were incubated at 37°C for 10 min and collected on filters. Means 5S.E. of 8 determinations. * Significant at p
