Proc. Nati. Acad. Sci. USAVol. 88, pp. 741-744, February 1991Physiology/Pharmacology

Electron probe microanalysis of calcium release and magnesiumuptake 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 sacsof endoplasmic reticulum (ER) that can be located unequivo-cally in freeze-dried cryosections. The elemental composition ofthe ER was determined by electron probe x-ray microanalysisand was visualized in high-resolution x-ray maps. In the ER ofdark-adapted photoreceptors, the Ca concentration was 47.5 ±1.1 mmol/kg (dry weight) (mean ± SEM). During a 3-secnonsaturating light stimulus, -50% of the Ca content wasreleased from the ER. Light stimulation also caused a highlysignificant increase in theMg content of the ER; the ratio ofMguptake to Ca released was -0.7. Our results show unambig-uously that the ER is the source of Ca2+ release during cellstimulation and suggest that Mg2+ can nearly balance thecharge movement of Ca2+.

The mobilization of Ca2" from an intracellular store by areaction cascade comprising a cell-surface receptor, a gua-nine nucleotide binding protein, a phospholipase C, andinositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is an importantmechanism of signal transduction (1). A great deal of evi-dence indicates that the endoplasmic reticulum (ER) is thetarget of Ins(1,4,5)P3 action (2-5) and the organelle involvedin the physiological regulation of cytoplasmic Ca2" in non-muscle cells (6-8), although some authors proposed that theIns(1,4,5)P3-sensitive Ca2" pool is located in a separateorganelle ("calciosome") (9, 10). Direct measurements oftheCa2+ content and of Ca2+ changes in the ER (or the calcio-somes) have been difficult to obtain. Consequently, little isknown about the ion movements that must occur during Ca2+release, to maintain charge balance across the ER membrane.The large sacs ofER in bee photoreceptors (11) are uniquelysuitable for addressing these questions because the sizemakes them clearly identifiable in ultrathin freeze-dried cry-osections and allows unambiguous experiments in which theanalyzed area is completely within the ER cisternae. There-fore, we determined by electron probe x-ray microanalysis(EPMA) the elemental composition of the ER in situ, in thedark and during light stimulation, that, in invertebrates, isknown to be followed by Ins(1,4,5)P3-mediated (12, 13)intracellular Ca2' release (14, 15). We quantitated the amountof Ca2' released from the ER during photoresponse anddemonstrate an associated uptake of Mg2' into the ER,suggesting that Mg2+ movements make a major contributiontoward maintaining electroneutrality during Ca2' releasefrom the ER in bee photoreceptors. Preliminary reports ofthese findings have been published (16, 17).

MATERIALS AND METHODSHoney bee drones (Apis mellifera) were kept in the dark at32°C to 36°C for up to 10 days and fed a concentrated sugar

solution. Decapitation and all subsequent manipulationswere done under red light (A > 600 nm), which elicits noelectrical response of the photoreceptors (18). Thick slices(600-1000 Am) ofthe drone head (19) remained in oxygenatedphysiological saline containing 170mM NaCl, 10mM KCI, 10mM MgCl2, 1.6mM CaC12, 10mM Tris-HCl (pH 7.4), and 400mM sucrose. Sucrose was added to increase the osmolarity(815 milliosmolal compared to the 615 milliosmolal conven-tionally used) and to act as the cryoprotectant. Intracellularrecordings (data not shown) demonstrate that the incubationin hypertonic saline for =20 min had no effect on the restingpotential. The light intensity that elicits half-maximal re-sponse was not changed, but the maximum amplitude of thereceptor potential was decreased by =-30% and the kineticsof the light response was slowed down.

After 20-30 min of incubation in physiological saline, theslices were rapidly frozen in a LifeCell CF 100 slam-freezer(LifeCell, The Woodlands, TX), either while dark-adapted or3 sec after turning on a tungsten filament light source thatdelivered =t0.2 mW/cm2 in the specimen plane and in thespectral region of 450-600 nm. This intensity, given in a20-msec test flash, is 500 times dimmer than necessary tosaturate the intracellularly recorded receptor potential. Thicksections (100-200 nm) were cut at - 130°C with a Reichert-Jung cryoultramicrotome and then freeze-dried (20). Thesections were analyzed either by EPMA, in the spot mode, orby x-ray mapping. The instrumentation and methods forEPMA, including calibration, have been published (20-24).The individual spectra were collected until the measurementerror was 1.2 mmol of Ca per kg (dry weight) for cytoplasmand mitochondria and until the measurement error was '5%for Ca in the ER. The probe diameter was 200 nm in themitochondria and 200-1000 nm in the cytoplasm. In the ERthe probe diameter varied between 200 and 500 nm butexcluded any overlap of cytoplasm. Quantitative x-ray mapswere acquired by collecting an entire energy-dispersive x-rayspectrum in 15 sec at each pixel, by using a high-brightnessfield emission source. The scanning system was computer-controlled and included a dark-field-image cross-correlationfeedback 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 measuredby x-ray microanalysis.

RESULTSThe morphology of the bee photoreceptor, including thesubmicrovillar ER cisternae (11) that range from 0.2 to 1.0 ,umin diameter, is illustrated in electron micrographs in Fig. 1; afreeze-dried cryosection is shown in Fig. 2. The large size andthe significantly greater electron lucency of the ER than of

Abbreviations: ER, endoplasmic reticulum; EPMA, electron probex-ray microanalysis; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate.tTo whom reprint requests should be addressed.

741

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

0

742 Physiology/Pharmacology: Baumann et al.

FIG. 1. Light (a) and electron(b and c) micrographs of chemi-cally fixed (11) honey bee photo-receptors. The bee retina has aregular structure and is composedof glial cells (G) and receptor cells(R). The large cisternae of smoothER at the base of the photorecep-tive microvilli (M) are labeled bystars and are shown in cross (b)and longitudinal (c) sections. Adetailed description of the ER inhoney bee photoreceptors is givenelsewhere (11). (Bars: a, 10 Am; band c, 1 Aum.)I

the adjacent cytoplasm permit the unambiguous selection ofa 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 light-stimulated photoreceptors are summarized in Table 1. Thequantitation is based on the ratio of the characteristic peak tothe x-ray continuum counts from freeze-dried material (21,

FIG. 2. Longitudinal unstained freeze-dried cryosection ofhoneybee photoreceptor. Contrast is due to the differences in dry massbetween organelles. Stars indicate the electron-lucent ER next to themicrovillar rhabdom (M). X-ray maps of the boxed area containingER and cytoplasm are shown in Fig. 3. (Bar = 1 jm.)

22) and, therefore, the data are expressed as mmol/kg (dryweight). The cellular water content of bee photoreceptors isdifficult to measure. By assuming that all K [823 mmol/kg(dry weight)] is in solution, and based on very accurateion-selective microelectrode measurements (25) of intracel-lular K+ [activity coefficient for potassium (aKj) = 89 mM;activity coefficient = 0.7], we can estimate a cytoplasmicwater content of -85%. Given this value, the calculatedcytoplasmic concentrations ofNa' (8 mmol/liter ofcell H20)and Cl- (15 mmol/liter of cell H20) are in good agreementwith published data obtained with ion-selective electrodes(25, 26); the K content is comparable to that obtained in aprevious 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 thecryosection shown in Fig. 2 illustrate the high Ca concentra-tion, 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 smallER tubules not imaged in unstained cryosections and/or Cabound to proteins such as calmodulin (28). The low Caconcentration of the mitochondria [1.0 ± 0.2 mmol/kg (dryweight)] 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 nonsaturat-

ing 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 moreCa can be released by saturating light intensities. Given the0.5 mass ratio [measured as the x-ray continuum ratio (20)through paired analysis, with identical probe parameters, ofER and cytoplasm] and the 0.08 volume ratio (morphometricmeasurements in chemically fixed specimens) of ER/cytoplasm, the amount of Ca released should be sufficient toraise cytoplasmic Ca by -1 mmol/kg (dry weight), that is,above the detectable limits of analysis [2-3 SEM = 0.4-0.6mmol/kg (dry weight)]. However, illumination causes a netextrusion of Ca2+ from photoreceptors (19) that is apparently

Proc. Natl. Acad Sci. USA 88 (1991)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

0

Proc. Natl. Acad. Sci. USA 88 (1991) 743

Table 1. Subcellular distribution of Ca and other elements in bee photoreceptorsElemental concentration, mmol/kg (dry weight)

Region n Na Mg P S Cl K Ca

ControlER 179 106 ± 5.4 66 ± 1.3 688 ± 9.0 1225 ± 21.0 131 ± 2.8 1353 ± 26.3 47.5 ± 1.1Cytoplasm 63 44 ± 4.3 65 ± 1.8 522 ± 13.1 732 ± 14.6 83 ± 4.3 823 ± 17.0 3.0 ± 0.2Mitochondria 70 52 ± 3.8 53 ± 1.4 553 ± 5.9 586 ± 7.1 61 ± 2.4 483 ± 9.4 1.0 ± 0.2

Light-stimulatedER 138 177 ± 6.3 83 ± 2.2 612 ± 10.3 1052 ± 24.3 137 ± 3.5 1116 ± 20.3 22.4 ± 1.0Cytoplasm 67 102 ± 6.1 65 ± 1.6 485 ± 12.9 803 ± 16.4 111 ± 3.4 772 ± 15.8 3.3 ± 0.2Mitochondria 67 88 ± 6.1 51 ± 1.6 528 ± 8.1 588 ± 9.2 90 ± 3.5 427 ± 9.7 1.4 ± 0.2Seven control animals and six light-stimulated animals were used. Data are mean ± SEM.

sufficient to reduce the remaining increase in total cytoplas-mic 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 photore-ceptors, were higher in the ER than in the cytoplasm. Thisfinding is not necessarily indicative of a Na, Cl, or Kconcentration gradient across the ER membrane, because an"excess" of ions in solution is associated with the greater

SEM~~~~~~~~~A:4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...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 photorecep-tor. (a) The dark-field scanning transmission image was inverted(STEM) to make it comparable to the bright-field transmissionelectron microscope image in Fig. 2. In the STEM image the regionshaving lower density (the ER) are bright whereas in the continuumx-ray image (Cont) these areas appear dark due to the smaller numberof continuum counts (higher hydration in vivo). The calcium (red) andphosphorus (green) maps illustrate the sharp gradients across the ERmembrane. Note the more diffuse distribution of sulfur (yellow). Theintensities of the colors were contrast enhanced to indicate therelative elemental distribution, with the weakest intensity represent-ing the lowest and the brightest intensity representing the highestelemental concentration in the scanned area.

hydration of the ER and because EPMA measures total (freeand bound) elemental concentrations, rather than activities.During photostimulation the concentration ofNa increased

and that of K decreased in all probed compartments (ER,cytoplasm, and mitochondria). Except for the cytoplasmic Kdecrease, which is just statistically significant (P < 0.05), allthese changes are highly significant (P < 0.001). They reflectthe light-induced Na' influx and K+ efflux in invertebratephotoreceptors (25) and suggest that the permeability of theER membrane to these ions permits their rapid redistributionamong the compartments. In addition, there was an increasein cytoplasmic Cl during illumination (P < 0.05), in agree-ment 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 duringCa2' release to maintain the charge balance across the ERmembrane. The amount of Mg uptake (16.7 ± 5.1 mmol/kg;confidence interval or 2 SD) was slightly lower than theamount of Ca release (25.1 ± 3.0 mmol/kg). Given theaforementioned ER/cytoplasm volume ratio, the amount ofMg moving out of the cytoplasm, about 1 mmol/kg (dryweight), would have been below the level of detectability(Table 1), even in the absence of an extracellular Mg influxthat could occur.

DISCUSSIONThe present study demonstrates directly that the well-characterized submicrovillar cisternae of ER in invertebratephotoreceptors (11, 29) are the source of Ins(1,4,5)P3-mediated (12, 13) Ca2" release during photostimulation.Evidence, although indirect, for this conclusion was alsoobtained in Limulus ventral photoreceptors (13, 30): Inaequorin-injected Limulus photoreceptors, light flashes orbrief pressure injections of Ins(1,4,5)P3 elicit aequorin lumi-nescence that originates in the cell area where the submi-crovillar ER cisternae are located. In addition, the studies onLimulus ventral photoreceptors suggest that, although all ERaccumulates Ca2' actively, not all ER is competent to releaseCa2+ 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 avariety ofother cells (33) that the ER is the major intracellularCa store in nonmuscle cells. However, the Ca concentrationsobtained in those studies [5 and 13 mmol/kg (dry weight),respectively] were underestimates of the true Ca content ofthe ER, because the probe diameter was larger than the ERtubules. The Ca concentration in bee photoreceptor ER [47.5mmol/kg (dry weight)] is lower than in the sarcoplasmicreticulum of toadfish muscle [ref. 34; 77 mmol/kg (dryweight)] or the terminal cisternae of frog muscle (ref. 20; 117mmol/kg (dry weight)] but comparable to the steady-state Cacontent of 32 nmol/mg of protein in liver microsomes underoptimal conditions (35).

Physiology/Pharmacology: Baumann et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

0

744 Physiology/Pharmacology: Baumann et al.

The finding that, in bee photoreceptors, Ca release isassociated with Mg uptake suggests that Mg2" moves as acounterion during Ca2" release to maintain the charge bal-ance across the ER membrane. In striated muscle, K+ is themajor counterion moving into the sarcoplasmic reticulumduring Ca2` release (20), and monovalent cation movementmay play a similar role during Ca2+ release from the ER inpermeabilized hepatocytes (36). However, in vivo measure-ments, comparable to those in the present study, are yet tobe made on vertebrate cells. In the sarcoplasmic reticulum oftetanized skeletal muscle, Mg2+ makes a relatively smallcontribution to counterion movement (20). In bee photore-ceptors, Mg2+ seems to be the major counterion providingcharge neutralization. Further tests of this possibility willrequire measurements during the early phase of photostim-ulation, before the onset of the recovery process. However,it is already clear from this and previous studies (37) that notonly the Ca2+ but also the Mg2+ content of cell organelles(mitochondria and ER) is subject to dynamic changes thatmay play a role in physiological regulation, and the possibilityshould be explored that, under physiological conditions in theER 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 cytoplasmicCa2 . Our data are consistent with the view that the ERcontains a Ca2+-ATPase (5-8) that maintains a high Ca2'gradient across the ER membrane, and a Ca2+ release chan-nel sensitive to Ins(1,4,5)P3 (2-5). These findings do notsupport the view that an ER-independent cell organellecontains the Ins(1,4,5)P3-sensitive Ca2+ pool.

We thank Dr. Z. Shao for consultations in x-ray mapping and Mr.S. Majewski for writing the slow-scan mapping program and for helpin data processing. We thank Mr. J. Silcox for technical assistance,Dr. R. Kretsinger, Dr. J. Herr, and Mrs. S. Cobey for supplyinghoney bees, and LifeCell for kindly providing the slam-freezer. Thiswork was supported by the Deutsche Forschungsgemeinschaft (SFB4, I1), NATO, and National Institutes of Health Grant HL-15835 tothe Pennsylvania Muscle Institute.

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.

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. (SanFrancisco 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., McClel-

lan, G. & Somlyo, A. P. (1981) J. Cell Biol. 90, 577-594.21. Hall, T. A. & Gupta, B. L. (1983) Q. Rev. Biophys. 16, 279-

330.22. Shuman, H., Somlyo, A. V. & Somlyo, A. P. (1976) Ultrami-

croscopy 1, 317-339.23. Somlyo, A. V., Broderick, R., Shuman, H., Buhle, E. L. &

Somlyo, A. P. (1988) Proc. Natl. Acad. Sci. USA 85, 6222-6226.

24. Kitazawa, T., Shuman, H. & Somlyo, A. P. (1983) Ultrami-croscopy 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 156,213-222.

28. DeCouet, H. G., Jablonski, P. P. & Perkin, J. L. (1986) CellTissue 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,

183-195.33. 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 MembraneTransport, eds. Eaton, D. C. & Mandel, L. J. (RockefellerUniversity Press, New York), Vol. 42, pp. 77-92.

34. Somlyo, A. V., Shuman, H. & Somlyo, A. P. (1977) Nature(London) 268, 556-558.

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.

Proc. NatL Acad. Sci. USA 88 (1991)

Dow

nloa

ded

by g

uest

on

Dec

embe

r 19

, 202

0