Proc. Nati. Acad. Sci. USA Vol. 88, pp. 741-744, February 1991 Physiology/Pharmacology
Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors (signal transduction/retina/invertebrates)
0. BAUMANN*, B. WALZ*, A. V. SOMLYOt, AND A. P. SOMLYOt* *Institut fur Zoologie, Universitdt Regensburg, Universitatsstrasse 31, D-8400 Regensburg, Federal Republic of Germany; and tDepartment of Physiology, Medical Center, Box 449, University of Virginia, Charlottesville, VA 22908
Communicated by Robert M. Berne, October 19, 1990
ABSTRACT Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 ± 1.1 mmol/kg (dry weight) (mean ± SEM). During a 3-sec nonsaturating light stimulus, -50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was -0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.
The mobilization of Ca2" from an intracellular store by a reaction cascade comprising a cell-surface receptor, a guanine nucleotide binding protein, a phospholipase C, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism of signal transduction (1). A great deal of evidence indicates that the endoplasmic reticulum (ER) is the target of Ins(1,4,5)P3 action (2-5) and the organelle involved in the physiological regulation of cytoplasmic Ca2" in nonmuscle cells (6-8), although some authors proposed that the Ins(1,4,5)P3-sensitive Ca2" pool is located in a separate organelle ("calciosome") (9, 10). Direct measurements of the Ca2+ content and of Ca2+ changes in the ER (or the calciosomes) have been difficult to obtain. Consequently, little is known about the ion movements that must occur during Ca2+ release, to maintain charge balance across the ER membrane. The large sacs of ER in bee photoreceptors (11) are uniquely suitable for addressing these questions because the size makes them clearly identifiable in ultrathin freeze-dried cryosections and allows unambiguous experiments in which the analyzed area is completely within the ER cisternae. Therefore, we determined by electron probe x-ray microanalysis (EPMA) the elemental composition of the ER in situ, in the dark and during light stimulation, that, in invertebrates, is known to be followed by Ins(1,4,5)P3-mediated (12, 13) intracellular Ca2' release (14, 15). We quantitated the amount of Ca2' released from the ER during photoresponse and demonstrate an associated uptake of Mg2' into the ER, suggesting that Mg2+ movements make a major contribution toward maintaining electroneutrality during Ca2' release from the ER in bee photoreceptors. Preliminary reports of these findings have been published (16, 17).
solution. Decapitation and all subsequent manipulations were done under red light (A > 600 nm), which elicits no electrical response of the photoreceptors (18). Thick slices (600-1000 Am) ofthe drone head (19) remained in oxygenated physiological saline containing 170 mM NaCl, 10 mM KCI, 10 mM MgCl2, 1.6 mM CaC12, 10 mM Tris-HCl (pH 7.4), and 400 mM sucrose. Sucrose was added to increase the osmolarity (815 milliosmolal compared to the 615 milliosmolal conventionally used) and to act as the cryoprotectant. Intracellular recordings (data not shown) demonstrate that the incubation in hypertonic saline for =20 min had no effect on the resting potential. The light intensity that elicits half-maximal response was not changed, but the maximum amplitude of the receptor potential was decreased by =-30% and the kinetics of the light response was slowed down. After 20-30 min of incubation in physiological saline, the slices were rapidly frozen in a LifeCell CF 100 slam-freezer (LifeCell, The Woodlands, TX), either while dark-adapted or 3 sec after turning on a tungsten filament light source that delivered =t0.2 mW/cm2 in the specimen plane and in the spectral region of 450-600 nm. This intensity, given in a 20-msec test flash, is 500 times dimmer than necessary to saturate the intracellularly recorded receptor potential. Thick sections (100-200 nm) were cut at - 130°C with a ReichertJung cryoultramicrotome and then freeze-dried (20). The sections were analyzed either by EPMA, in the spot mode, or by x-ray mapping. The instrumentation and methods for EPMA, including calibration, have been published (20-24). The individual spectra were collected until the measurement error was 1.2 mmol of Ca per kg (dry weight) for cytoplasm and mitochondria and until the measurement error was '5% for Ca in the ER. The probe diameter was 200 nm in the mitochondria and 200-1000 nm in the cytoplasm. In the ER the probe diameter varied between 200 and 500 nm but excluded any overlap of cytoplasm. Quantitative x-ray maps were acquired by collecting an entire energy-dispersive x-ray spectrum in 15 sec at each pixel, by using a high-brightness field emission source. The scanning system was computercontrolled and included a dark-field-image cross-correlation feedback loop to compensate for specimen drift (23). Ca2+ is used throughout the manuscript to designate the ionized element and Ca is used for total calcium, as measured by x-ray microanalysis.
RESULTS
MATERIALS AND METHODS Honey bee drones (Apis mellifera) were kept in the dark at 32°C to 36°C for up to 10 days and fed a concentrated sugar
The morphology of the bee photoreceptor, including the submicrovillar ER cisternae (11) that range from 0.2 to 1.0 ,um in diameter, is illustrated in electron micrographs in Fig. 1; a freeze-dried cryosection is shown in Fig. 2. The large size and the significantly greater electron lucency of the ER than of
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: ER, endoplasmic reticulum; EPMA, electron probe microanalysis; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate. tTo whom reprint requests should be addressed. x-ray
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I the adjacent cytoplasm permit the unambiguous selection of a given region, ER or cytoplasm, for electron probe analysis. The results of the elemental quantitation of the ER, the cytoplasm, and the mitochondria in dark-adapted and lightstimulated photoreceptors are summarized in Table 1. The quantitation is based on the ratio of the characteristic peak to the x-ray continuum counts from freeze-dried material (21,
FIG. 2. Longitudinal unstained freeze-dried cryosection of honey bee photoreceptor. Contrast is due to the differences in dry mass between organelles. Stars indicate the electron-lucent ER next to the microvillar rhabdom (M). X-ray maps of the boxed area containing ER and cytoplasm are shown in Fig. 3. (Bar = 1 jm.)
Proc. Natl. Acad Sci. USA 88 (1991)
FIG. 1. Light (a) and electron (b and c) micrographs of chemically fixed (11) honey bee photoreceptors. The bee retina has a regular structure and is composed of glial cells (G) and receptor cells (R). The large cisternae of smooth ER at the base of the photoreceptive microvilli (M) are labeled by stars and are shown in cross (b) and longitudinal (c) sections. A detailed description of the ER in honey bee photoreceptors is given elsewhere (11). (Bars: a, 10 Am; b and c, 1 Aum.)
22) and, therefore, the data are expressed as mmol/kg (dry weight). The cellular water content of bee photoreceptors is difficult to measure. By assuming that all K [823 mmol/kg (dry weight)] is in solution, and based on very accurate ion-selective microelectrode measurements (25) of intracellular K+ [activity coefficient for potassium (aKj) = 89 mM; activity coefficient = 0.7], we can estimate a cytoplasmic water content of -85%. Given this value, the calculated cytoplasmic concentrations of Na' (8 mmol/liter ofcell H20) and Cl- (15 mmol/liter of cell H20) are in good agreement with published data obtained with ion-selective electrodes (25, 26); the K content is comparable to that obtained in a previous electron probe study (27). The mean Ca concentration in the ER in dark-adapted photoreceptors was 47.5 ± 1.1 mmol/kg (dry weight) (mean ± SEM). X-ray maps (Fig. 3) of the area outlined in the cryosection shown in Fig. 2 illustrate the high Ca concentration, evenly distributed and confined within the cisternae. The relatively high Ca concentration of the cytoplasm [3.0 ± 0.2 mmol/kg (dry weight)] may reflect the inclusion of small ER tubules not imaged in unstained cryosections and/or Ca bound to proteins such as calmodulin (28). The low Ca concentration of the mitochondria [1.0 ± 0.2 mmol/kg (dry weight)] is comparable to that found in liver mitochondria [ref. 29; 0.8 mmol/kg (dry weight)]. A 3-sec continuous illumination with light at a nonsaturating intensity caused a large and highly significant (P < 0.001) reduction in the total Ca content of the ER by 52.8 ± 6.3% (confidence interval or 2 SD). We do not know whether more Ca can be released by saturating light intensities. Given the 0.5 mass ratio [measured as the x-ray continuum ratio (20) through paired analysis, with identical probe parameters, of ER and cytoplasm] and the 0.08 volume ratio (morphometric measurements in chemically fixed specimens) of ER/ cytoplasm, the amount of Ca released should be sufficient to raise cytoplasmic Ca by -1 mmol/kg (dry weight), that is, above the detectable limits of analysis [2-3 SEM = 0.4-0.6 mmol/kg (dry weight)]. However, illumination causes a net extrusion of Ca2+ from photoreceptors (19) that is apparently
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Proc. Natl. Acad. Sci. USA 88 (1991)
Table 1. Subcellular distribution of Ca and other elements in bee photoreceptors Elemental concentration, mmol/kg (dry weight) K Cl P S Mg n Na Region Control 688 ± 9.0 1225 ± 21.0 131 ± 2.8 1353 ± 26.3 66 ± 1.3 179 106 ± 5.4 ER 732 ± 14.6 83 ± 4.3 823 ± 17.0 522 ± 13.1 65 ± 1.8 63 44 ± 4.3 Cytoplasm 61 ± 2.4 483 ± 9.4 586 ± 7.1 553 ± 5.9 53 ± 1.4 70 52 ± 3.8 Mitochondria Light-stimulated 137 ± 3.5 1116 ± 20.3 1052 ± 24.3 612 ± 10.3 83 ± 2.2 ER 138 177 ± 6.3 111 ± 3.4 772 ± 15.8 803 ± 16.4 485 ± 12.9 67 65 ± 1.6 102 ± 6.1 Cytoplasm 90 ± 3.5 427 ± 9.7 588 ± 9.2 528 ± 8.1 51 ± 1.6 67 88 ± 6.1 Mitochondria Seven control animals and six light-stimulated animals were used. Data are mean ± SEM.
sufficient to reduce the remaining increase in total cytoplasmic Ca below the level of detectability by EPMA. The concentrations [expressed in mmol/kg (dry weight)] of Na, Cl, and K, in both control and light-stimulated photoreceptors, were higher in the ER than in the cytoplasm. This finding is not necessarily indicative of a Na, Cl, or K concentration gradient across the ER membrane, because an "excess" of ions in solution is associated with the greater
SEM
S
FIG. 3. X-ray maps (103 x 30 pixels at 15 sec per pixel 13 hr) of the cryosection shown in Fig. 2 of a dark-adapted bee photoreceptor. (a) The dark-field scanning transmission image was inverted (STEM) to make it comparable to the bright-field transmission electron microscope image in Fig. 2. In the STEM image the regions having lower density (the ER) are bright whereas in the continuum x-ray image (Cont) these areas appear dark due to the smaller number of continuum counts (higher hydration in vivo). The calcium (red) and phosphorus (green) maps illustrate the sharp gradients across the ER membrane. Note the more diffuse distribution of sulfur (yellow). The intensities of the colors were contrast enhanced to indicate the relative elemental distribution, with the weakest intensity representing the lowest and the brightest intensity representing the highest elemental concentration in the scanned area.
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47.5 ± 1.1 3.0 ± 0.2 1.0 ± 0.2 22.4 ± 1.0 3.3 ± 0.2 1.4 ± 0.2
hydration of the ER and because EPMA measures total (free and bound) elemental concentrations, rather than activities. During photostimulation the concentration of Na increased and that of K decreased in all probed compartments (ER, cytoplasm, and mitochondria). Except for the cytoplasmic K decrease, which is just statistically significant (P < 0.05), all these changes are highly significant (P < 0.001). They reflect the light-induced Na' influx and K+ efflux in invertebrate photoreceptors (25) and suggest that the permeability of the ER membrane to these ions permits their rapid redistribution among the compartments. In addition, there was an increase in cytoplasmic Cl during illumination (P < 0.05), in agreement with ion-selective microelectrode measurements (26). Light stimulation also caused Mg uptake into the ER (P < 0.001), suggesting that Mg2+ moves as a counterion during Ca2' release to maintain the charge balance across the ER membrane. The amount of Mg uptake (16.7 ± 5.1 mmol/kg; confidence interval or 2 SD) was slightly lower than the amount of Ca release (25.1 ± 3.0 mmol/kg). Given the aforementioned ER/cytoplasm volume ratio, the amount of Mg moving out of the cytoplasm, about 1 mmol/kg (dry weight), would have been below the level of detectability (Table 1), even in the absence of an extracellular Mg influx that could occur.
DISCUSSION The present study demonstrates directly that the wellcharacterized submicrovillar cisternae of ER in invertebrate photoreceptors (11, 29) are the source of Ins(1,4,5)P3mediated (12, 13) Ca2" release during photostimulation. Evidence, although indirect, for this conclusion was also obtained in Limulus ventral photoreceptors (13, 30): In aequorin-injected Limulus photoreceptors, light flashes or brief pressure injections of Ins(1,4,5)P3 elicit aequorin luminescence that originates in the cell area where the submicrovillar ER cisternae are located. In addition, the studies on Limulus ventral photoreceptors suggest that, although all ER accumulates Ca2' actively, not all ER is competent to release Ca2+ in response to Ins(1,4,5)P3. The present results support the conclusion derived from EPMA studies on hepatocytes (31), retinal rods (32), and a variety of other cells (33) that the ER is the major intracellular Ca store in nonmuscle cells. However, the Ca concentrations obtained in those studies [5 and 13 mmol/kg (dry weight), respectively] were underestimates of the true Ca content of the ER, because the probe diameter was larger than the ER tubules. The Ca concentration in bee photoreceptor ER [47.5 mmol/kg (dry weight)] is lower than in the sarcoplasmic reticulum of toadfish muscle [ref. 34; 77 mmol/kg (dry weight)] or the terminal cisternae of frog muscle (ref. 20; 117 mmol/kg (dry weight)] but comparable to the steady-state Ca content of 32 nmol/mg of protein in liver microsomes under optimal conditions (35).
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The finding that, in bee photoreceptors, Ca release is associated with Mg uptake suggests that Mg2" moves as a counterion during Ca2" release to maintain the charge balance across the ER membrane. In striated muscle, K+ is the major counterion moving into the sarcoplasmic reticulum during Ca2` release (20), and monovalent cation movement may play a similar role during Ca2+ release from the ER in permeabilized hepatocytes (36). However, in vivo measurements, comparable to those in the present study, are yet to be made on vertebrate cells. In the sarcoplasmic reticulum of tetanized skeletal muscle, Mg2+ makes a relatively small contribution to counterion movement (20). In bee photoreceptors, Mg2+ seems to be the major counterion providing charge neutralization. Further tests of this possibility will require measurements during the early phase of photostimulation, before the onset of the recovery process. However, it is already clear from this and previous studies (37) that not only the Ca2+ but also the Mg2+ content of cell organelles (mitochondria and ER) is subject to dynamic changes that may play a role in physiological regulation, and the possibility should be explored that, under physiological conditions in the ER of nonpermeabilized liver and other vertebrate cells, Mg2+ is also the source of counterion movement during Ca2+ release. Thus, our results demonstrate unambiguously that the ER is the organelle involved in the regulation of cytoplasmic Ca2 . Our data are consistent with the view that the ER contains a Ca2+-ATPase (5-8) that maintains a high Ca2' gradient across the ER membrane, and a Ca2+ release channel sensitive to Ins(1,4,5)P3 (2-5). These findings do not support the view that an ER-independent cell organelle contains the Ins(1,4,5)P3-sensitive Ca2+ pool.
Proc. NatL Acad. Sci. USA 88 (1991) 9. Volpe, P., Krause, K.-H., Hashimoto, S., Zorzato, F., Pozzan, T., Meldolesi, J. & Lew, D. P. (1988) Proc. Natl. Acad. Sci. USA 85, 1091-1095. 10. Krause, K.-H., Pittet, D., Volpe, P., Pozzan, T., Meldolesi, J. & Lew, D. P. (1989) Cell Calcium 10, 351-361. 11. Baumann, 0. & Walz, B. (1989) Cell Tissue Res. 255, 511-522. 12. Payne, R. (1986) Photobiochem. Photobiophys. 13, 373-397. 13. Payne, R., Walz, B., Levy, S. & Fein, A. (1988) Philos. Trans. R. Soc. London Ser. B 320, 359-379. 14. Brown, J. E. & Blinks, J. R. (1974) J. Gen. Physiol. 64, 643-665. 15. Ziegler, A. & Walz, B. (1990) Neurosci. Lett. 111, 87-91. 16. Baumann, O., Walz, B. & Somlyo, A. P. (1990) in Electron Microscopy 1990, eds. Peachey, L. D. & Williams, D. B. (San Francisco Press, San Francisco), Vol. 2, pp. 152-153. 17. Baumann, O., Somlyo, A. V., Walz, B. & Somlyo, A. P. (1990) Cell Biol. Int. Rep. 14, 44 (abstr.). 18. Bertrand, D., Fuortes, G. & Muri, R. (1979) J. Physiol. (London) 296, 431-441. 19. Ziegler, A. & Walz, B. (1989) J. Comp. Physiol. A 165,697-709. 20. Somlyo, A. V., Gonzalez-Serratos, H., Shuman, H., McClellan, G. & Somlyo, A. P. (1981) J. Cell Biol. 90, 577-594. 21. Hall, T. A. & Gupta, B. L. (1983) Q. Rev. Biophys. 16, 279330. 22. Shuman, H., Somlyo, A. V. & Somlyo, A. P. (1976) Ultramicroscopy 1, 317-339. 23. Somlyo, A. V., Broderick, R., Shuman, H., Buhle, E. L. & Somlyo, A. P. (1988) Proc. Natl. Acad. Sci. USA 85, 62226226. 24. Kitazawa, T., Shuman, H. & Somlyo, A. P. (1983) Ultramicroscopy 11, 251-262. 25. Coles, J. A. & Orkand, R. K. (1985) J. Physiol. (London) 362, 415-435. 26. Coles, J. A., Orkand, R. K. & Yamate, C. L. (1989) Glia 2, 287-297. 27. Coles, J. A. & Rick, R. (1985) J. Comp. Physiol. A 156,
We thank Dr. Z. Shao for consultations in x-ray mapping and Mr. S. Majewski for writing the slow-scan mapping program and for help in data processing. We thank Mr. J. Silcox for technical assistance, Dr. R. Kretsinger, Dr. J. Herr, and Mrs. S. Cobey for supplying honey bees, and LifeCell for kindly providing the slam-freezer. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 4, I1), NATO, and National Institutes of Health Grant HL-15835 to the Pennsylvania Muscle Institute.
213-222. 28. DeCouet, H. G., Jablonski, P. P. & Perkin, J. L. (1986) Cell Tissue Res. 244, 315-319. 29. Whittle, A. C. (1976) Zool. Scr. 5, 191-206. 30. Payne, R. & Fein, A. (1987) J. Cell Biol. 104, 933-937. 31. Somlyo, A. P., Bond, M. & Somlyo, A. V. (1985) Nature (London) 314, 622-625. 32. Somlyo, A. P. & Walz, B. (1985) J. Physiol. (London) 358,
1. Berridge, M. J. (1987) Annu. Rev. Biochem. 56, 159-193. 2. Streb, H., Irvine, R. F., Berridge, M. J. & Schulz, I. (1983) Nature (London) 306, 67-69. 3. Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N. & Mikoshiba, K. (1989) Nature (London) 342, 32-38. 4. Mignery, G. A., Sudhof, T. C., Takei, K. & De Camilli, P. (1989) Nature (London) 342, 192-195. 5. Baumann, 0. & Walz, B. (1989) J. Comp. Physiol. A 165, 627-636. 6. Somlyo, A. P. (1984) Nature (London) 308, 516-517. 7. Carafoli, E. (1987) Annu. Rev. Biochem. 56, 395-433. 8. Walz, B. & Baumann, 0. (1989) Prog. Histochem. Cytochem. 20, No. 2.
33. Somlyo, A. P., Somlyo, A. V., Bond, M., Broderick, R., Goldman, Y. E., Shuman, H., Walker, J. W. & Trentham, D. R. (1987) in Cell Calcium and the Control of Membrane Transport, eds. Eaton, D. C. & Mandel, L. J. (Rockefeller University Press, New York), Vol. 42, pp. 77-92. 34. Somlyo, A. V., Shuman, H. & Somlyo, A. P. (1977) Nature
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35. Brattin, W. J., Waller, R. L. & Recknagel, R. 0. (1982) J. Biol. Chem. 257, 10044-10051. 36. Joseph, S. K. & Williamson, J. R. (1986) J. Biol. Chem. 261, 14658-14664. 37. Bond, M., Vadasz, G., Somlyo, A. V. & Somlyo, A. P. (1987) J. Biol. Chem. 262, 15630-15636.
Electron probe microanalysis of calcium release and magnesium uptake by endoplasmic reticulum in bee photoreceptors (signal transduction/retina/invertebrates)
0. BAUMANN*, B. WALZ*, A. V. SOMLYOt, AND A. P. SOMLYOt* *Institut fur Zoologie, Universitdt Regensburg, Universitatsstrasse 31, D-8400 Regensburg, Federal Republic of Germany; and tDepartment of Physiology, Medical Center, Box 449, University of Virginia, Charlottesville, VA 22908
Communicated by Robert M. Berne, October 19, 1990
ABSTRACT Honey bee photoreceptors contain large sacs of endoplasmic reticulum (ER) that can be located unequivocally in freeze-dried cryosections. The elemental composition of the ER was determined by electron probe x-ray microanalysis and was visualized in high-resolution x-ray maps. In the ER of dark-adapted photoreceptors, the Ca concentration was 47.5 ± 1.1 mmol/kg (dry weight) (mean ± SEM). During a 3-sec nonsaturating light stimulus, -50% of the Ca content was released from the ER. Light stimulation also caused a highly significant increase in the Mg content of the ER; the ratio of Mg uptake to Ca released was -0.7. Our results show unambiguously that the ER is the source of Ca2+ release during cell stimulation and suggest that Mg2+ can nearly balance the charge movement of Ca2+.
The mobilization of Ca2" from an intracellular store by a reaction cascade comprising a cell-surface receptor, a guanine nucleotide binding protein, a phospholipase C, and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an important mechanism of signal transduction (1). A great deal of evidence indicates that the endoplasmic reticulum (ER) is the target of Ins(1,4,5)P3 action (2-5) and the organelle involved in the physiological regulation of cytoplasmic Ca2" in nonmuscle cells (6-8), although some authors proposed that the Ins(1,4,5)P3-sensitive Ca2" pool is located in a separate organelle ("calciosome") (9, 10). Direct measurements of the Ca2+ content and of Ca2+ changes in the ER (or the calciosomes) have been difficult to obtain. Consequently, little is known about the ion movements that must occur during Ca2+ release, to maintain charge balance across the ER membrane. The large sacs of ER in bee photoreceptors (11) are uniquely suitable for addressing these questions because the size makes them clearly identifiable in ultrathin freeze-dried cryosections and allows unambiguous experiments in which the analyzed area is completely within the ER cisternae. Therefore, we determined by electron probe x-ray microanalysis (EPMA) the elemental composition of the ER in situ, in the dark and during light stimulation, that, in invertebrates, is known to be followed by Ins(1,4,5)P3-mediated (12, 13) intracellular Ca2' release (14, 15). We quantitated the amount of Ca2' released from the ER during photoresponse and demonstrate an associated uptake of Mg2' into the ER, suggesting that Mg2+ movements make a major contribution toward maintaining electroneutrality during Ca2' release from the ER in bee photoreceptors. Preliminary reports of these findings have been published (16, 17).
solution. Decapitation and all subsequent manipulations were done under red light (A > 600 nm), which elicits no electrical response of the photoreceptors (18). Thick slices (600-1000 Am) ofthe drone head (19) remained in oxygenated physiological saline containing 170 mM NaCl, 10 mM KCI, 10 mM MgCl2, 1.6 mM CaC12, 10 mM Tris-HCl (pH 7.4), and 400 mM sucrose. Sucrose was added to increase the osmolarity (815 milliosmolal compared to the 615 milliosmolal conventionally used) and to act as the cryoprotectant. Intracellular recordings (data not shown) demonstrate that the incubation in hypertonic saline for =20 min had no effect on the resting potential. The light intensity that elicits half-maximal response was not changed, but the maximum amplitude of the receptor potential was decreased by =-30% and the kinetics of the light response was slowed down. After 20-30 min of incubation in physiological saline, the slices were rapidly frozen in a LifeCell CF 100 slam-freezer (LifeCell, The Woodlands, TX), either while dark-adapted or 3 sec after turning on a tungsten filament light source that delivered =t0.2 mW/cm2 in the specimen plane and in the spectral region of 450-600 nm. This intensity, given in a 20-msec test flash, is 500 times dimmer than necessary to saturate the intracellularly recorded receptor potential. Thick sections (100-200 nm) were cut at - 130°C with a ReichertJung cryoultramicrotome and then freeze-dried (20). The sections were analyzed either by EPMA, in the spot mode, or by x-ray mapping. The instrumentation and methods for EPMA, including calibration, have been published (20-24). The individual spectra were collected until the measurement error was 1.2 mmol of Ca per kg (dry weight) for cytoplasm and mitochondria and until the measurement error was '5% for Ca in the ER. The probe diameter was 200 nm in the mitochondria and 200-1000 nm in the cytoplasm. In the ER the probe diameter varied between 200 and 500 nm but excluded any overlap of cytoplasm. Quantitative x-ray maps were acquired by collecting an entire energy-dispersive x-ray spectrum in 15 sec at each pixel, by using a high-brightness field emission source. The scanning system was computercontrolled and included a dark-field-image cross-correlation feedback loop to compensate for specimen drift (23). Ca2+ is used throughout the manuscript to designate the ionized element and Ca is used for total calcium, as measured by x-ray microanalysis.
RESULTS
MATERIALS AND METHODS Honey bee drones (Apis mellifera) were kept in the dark at 32°C to 36°C for up to 10 days and fed a concentrated sugar
The morphology of the bee photoreceptor, including the submicrovillar ER cisternae (11) that range from 0.2 to 1.0 ,um in diameter, is illustrated in electron micrographs in Fig. 1; a freeze-dried cryosection is shown in Fig. 2. The large size and the significantly greater electron lucency of the ER than of
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviations: ER, endoplasmic reticulum; EPMA, electron probe microanalysis; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate. tTo whom reprint requests should be addressed. x-ray
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I the adjacent cytoplasm permit the unambiguous selection of a given region, ER or cytoplasm, for electron probe analysis. The results of the elemental quantitation of the ER, the cytoplasm, and the mitochondria in dark-adapted and lightstimulated photoreceptors are summarized in Table 1. The quantitation is based on the ratio of the characteristic peak to the x-ray continuum counts from freeze-dried material (21,
FIG. 2. Longitudinal unstained freeze-dried cryosection of honey bee photoreceptor. Contrast is due to the differences in dry mass between organelles. Stars indicate the electron-lucent ER next to the microvillar rhabdom (M). X-ray maps of the boxed area containing ER and cytoplasm are shown in Fig. 3. (Bar = 1 jm.)
Proc. Natl. Acad Sci. USA 88 (1991)
FIG. 1. Light (a) and electron (b and c) micrographs of chemically fixed (11) honey bee photoreceptors. The bee retina has a regular structure and is composed of glial cells (G) and receptor cells (R). The large cisternae of smooth ER at the base of the photoreceptive microvilli (M) are labeled by stars and are shown in cross (b) and longitudinal (c) sections. A detailed description of the ER in honey bee photoreceptors is given elsewhere (11). (Bars: a, 10 Am; b and c, 1 Aum.)
22) and, therefore, the data are expressed as mmol/kg (dry weight). The cellular water content of bee photoreceptors is difficult to measure. By assuming that all K [823 mmol/kg (dry weight)] is in solution, and based on very accurate ion-selective microelectrode measurements (25) of intracellular K+ [activity coefficient for potassium (aKj) = 89 mM; activity coefficient = 0.7], we can estimate a cytoplasmic water content of -85%. Given this value, the calculated cytoplasmic concentrations of Na' (8 mmol/liter ofcell H20) and Cl- (15 mmol/liter of cell H20) are in good agreement with published data obtained with ion-selective electrodes (25, 26); the K content is comparable to that obtained in a previous electron probe study (27). The mean Ca concentration in the ER in dark-adapted photoreceptors was 47.5 ± 1.1 mmol/kg (dry weight) (mean ± SEM). X-ray maps (Fig. 3) of the area outlined in the cryosection shown in Fig. 2 illustrate the high Ca concentration, evenly distributed and confined within the cisternae. The relatively high Ca concentration of the cytoplasm [3.0 ± 0.2 mmol/kg (dry weight)] may reflect the inclusion of small ER tubules not imaged in unstained cryosections and/or Ca bound to proteins such as calmodulin (28). The low Ca concentration of the mitochondria [1.0 ± 0.2 mmol/kg (dry weight)] is comparable to that found in liver mitochondria [ref. 29; 0.8 mmol/kg (dry weight)]. A 3-sec continuous illumination with light at a nonsaturating intensity caused a large and highly significant (P < 0.001) reduction in the total Ca content of the ER by 52.8 ± 6.3% (confidence interval or 2 SD). We do not know whether more Ca can be released by saturating light intensities. Given the 0.5 mass ratio [measured as the x-ray continuum ratio (20) through paired analysis, with identical probe parameters, of ER and cytoplasm] and the 0.08 volume ratio (morphometric measurements in chemically fixed specimens) of ER/ cytoplasm, the amount of Ca released should be sufficient to raise cytoplasmic Ca by -1 mmol/kg (dry weight), that is, above the detectable limits of analysis [2-3 SEM = 0.4-0.6 mmol/kg (dry weight)]. However, illumination causes a net extrusion of Ca2+ from photoreceptors (19) that is apparently
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Table 1. Subcellular distribution of Ca and other elements in bee photoreceptors Elemental concentration, mmol/kg (dry weight) K Cl P S Mg n Na Region Control 688 ± 9.0 1225 ± 21.0 131 ± 2.8 1353 ± 26.3 66 ± 1.3 179 106 ± 5.4 ER 732 ± 14.6 83 ± 4.3 823 ± 17.0 522 ± 13.1 65 ± 1.8 63 44 ± 4.3 Cytoplasm 61 ± 2.4 483 ± 9.4 586 ± 7.1 553 ± 5.9 53 ± 1.4 70 52 ± 3.8 Mitochondria Light-stimulated 137 ± 3.5 1116 ± 20.3 1052 ± 24.3 612 ± 10.3 83 ± 2.2 ER 138 177 ± 6.3 111 ± 3.4 772 ± 15.8 803 ± 16.4 485 ± 12.9 67 65 ± 1.6 102 ± 6.1 Cytoplasm 90 ± 3.5 427 ± 9.7 588 ± 9.2 528 ± 8.1 51 ± 1.6 67 88 ± 6.1 Mitochondria Seven control animals and six light-stimulated animals were used. Data are mean ± SEM.
sufficient to reduce the remaining increase in total cytoplasmic Ca below the level of detectability by EPMA. The concentrations [expressed in mmol/kg (dry weight)] of Na, Cl, and K, in both control and light-stimulated photoreceptors, were higher in the ER than in the cytoplasm. This finding is not necessarily indicative of a Na, Cl, or K concentration gradient across the ER membrane, because an "excess" of ions in solution is associated with the greater
SEM
S
FIG. 3. X-ray maps (103 x 30 pixels at 15 sec per pixel 13 hr) of the cryosection shown in Fig. 2 of a dark-adapted bee photoreceptor. (a) The dark-field scanning transmission image was inverted (STEM) to make it comparable to the bright-field transmission electron microscope image in Fig. 2. In the STEM image the regions having lower density (the ER) are bright whereas in the continuum x-ray image (Cont) these areas appear dark due to the smaller number of continuum counts (higher hydration in vivo). The calcium (red) and phosphorus (green) maps illustrate the sharp gradients across the ER membrane. Note the more diffuse distribution of sulfur (yellow). The intensities of the colors were contrast enhanced to indicate the relative elemental distribution, with the weakest intensity representing the lowest and the brightest intensity representing the highest elemental concentration in the scanned area.
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Ca
47.5 ± 1.1 3.0 ± 0.2 1.0 ± 0.2 22.4 ± 1.0 3.3 ± 0.2 1.4 ± 0.2
hydration of the ER and because EPMA measures total (free and bound) elemental concentrations, rather than activities. During photostimulation the concentration of Na increased and that of K decreased in all probed compartments (ER, cytoplasm, and mitochondria). Except for the cytoplasmic K decrease, which is just statistically significant (P < 0.05), all these changes are highly significant (P < 0.001). They reflect the light-induced Na' influx and K+ efflux in invertebrate photoreceptors (25) and suggest that the permeability of the ER membrane to these ions permits their rapid redistribution among the compartments. In addition, there was an increase in cytoplasmic Cl during illumination (P < 0.05), in agreement with ion-selective microelectrode measurements (26). Light stimulation also caused Mg uptake into the ER (P < 0.001), suggesting that Mg2+ moves as a counterion during Ca2' release to maintain the charge balance across the ER membrane. The amount of Mg uptake (16.7 ± 5.1 mmol/kg; confidence interval or 2 SD) was slightly lower than the amount of Ca release (25.1 ± 3.0 mmol/kg). Given the aforementioned ER/cytoplasm volume ratio, the amount of Mg moving out of the cytoplasm, about 1 mmol/kg (dry weight), would have been below the level of detectability (Table 1), even in the absence of an extracellular Mg influx that could occur.
DISCUSSION The present study demonstrates directly that the wellcharacterized submicrovillar cisternae of ER in invertebrate photoreceptors (11, 29) are the source of Ins(1,4,5)P3mediated (12, 13) Ca2" release during photostimulation. Evidence, although indirect, for this conclusion was also obtained in Limulus ventral photoreceptors (13, 30): In aequorin-injected Limulus photoreceptors, light flashes or brief pressure injections of Ins(1,4,5)P3 elicit aequorin luminescence that originates in the cell area where the submicrovillar ER cisternae are located. In addition, the studies on Limulus ventral photoreceptors suggest that, although all ER accumulates Ca2' actively, not all ER is competent to release Ca2+ in response to Ins(1,4,5)P3. The present results support the conclusion derived from EPMA studies on hepatocytes (31), retinal rods (32), and a variety of other cells (33) that the ER is the major intracellular Ca store in nonmuscle cells. However, the Ca concentrations obtained in those studies [5 and 13 mmol/kg (dry weight), respectively] were underestimates of the true Ca content of the ER, because the probe diameter was larger than the ER tubules. The Ca concentration in bee photoreceptor ER [47.5 mmol/kg (dry weight)] is lower than in the sarcoplasmic reticulum of toadfish muscle [ref. 34; 77 mmol/kg (dry weight)] or the terminal cisternae of frog muscle (ref. 20; 117 mmol/kg (dry weight)] but comparable to the steady-state Ca content of 32 nmol/mg of protein in liver microsomes under optimal conditions (35).
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The finding that, in bee photoreceptors, Ca release is associated with Mg uptake suggests that Mg2" moves as a counterion during Ca2" release to maintain the charge balance across the ER membrane. In striated muscle, K+ is the major counterion moving into the sarcoplasmic reticulum during Ca2` release (20), and monovalent cation movement may play a similar role during Ca2+ release from the ER in permeabilized hepatocytes (36). However, in vivo measurements, comparable to those in the present study, are yet to be made on vertebrate cells. In the sarcoplasmic reticulum of tetanized skeletal muscle, Mg2+ makes a relatively small contribution to counterion movement (20). In bee photoreceptors, Mg2+ seems to be the major counterion providing charge neutralization. Further tests of this possibility will require measurements during the early phase of photostimulation, before the onset of the recovery process. However, it is already clear from this and previous studies (37) that not only the Ca2+ but also the Mg2+ content of cell organelles (mitochondria and ER) is subject to dynamic changes that may play a role in physiological regulation, and the possibility should be explored that, under physiological conditions in the ER of nonpermeabilized liver and other vertebrate cells, Mg2+ is also the source of counterion movement during Ca2+ release. Thus, our results demonstrate unambiguously that the ER is the organelle involved in the regulation of cytoplasmic Ca2 . Our data are consistent with the view that the ER contains a Ca2+-ATPase (5-8) that maintains a high Ca2' gradient across the ER membrane, and a Ca2+ release channel sensitive to Ins(1,4,5)P3 (2-5). These findings do not support the view that an ER-independent cell organelle contains the Ins(1,4,5)P3-sensitive Ca2+ pool.
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We thank Dr. Z. Shao for consultations in x-ray mapping and Mr. S. Majewski for writing the slow-scan mapping program and for help in data processing. We thank Mr. J. Silcox for technical assistance, Dr. R. Kretsinger, Dr. J. Herr, and Mrs. S. Cobey for supplying honey bees, and LifeCell for kindly providing the slam-freezer. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 4, I1), NATO, and National Institutes of Health Grant HL-15835 to the Pennsylvania Muscle Institute.
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