Vision
0032-6989
Res. Vol. 26. NO. 5, pp. 67’3-6‘8. 1986
Printed in Great Bnram. All rights reserved
LIGHT
CopyrIght
S
1986
86 53.00
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Pergamon Press tid
INDUCED SODIUM DEPENDENT ACCUMULATION OF CALCIUM AND POTASSIUM IN THE EXTRACELLULAR SPACE OF BEE RETINA BARUCH MIXKE*
and MMARCOS TSACOPO~LOS
Experimental ophthalmology Laboratory, University of Geneva, 22, rue Atcide-Jentzer, IZI L Geneva 4, Switzerland and Department of Physiology, Hebrew University, Hadassah Medical School. Jerusalem 9IOiO. Israel (Receiced 5 August 1985; in reriredform
2 December 1985)
Abstract-Intense illumination of long duration induced a large transient increase in extracellular calcium (A[Ca”+].) and potassium (A[K”],) during and after light in bee retina when measured with ion-selective microelectrodes. Whenever a large A[Ca2+], appeared, it was accompanied by a transient afterdepolarization (TA). Both the increase in [Ca2+Jo, [K’], and the TA were reduced or abolished when sodium was replaced by arginine, choline or lithium (Li+) ions. At O-Na conditions a Na independent decrease in [Ca”], was observed during illumination only. A pronounced transient depolarization of the photoreceptor in the dark due to transient anoxia did not result in a significant change in [Ca”L. In some retinae the elevated level of [K+g after light was absent, however a small Na-dependent TA was still observed. The above findings suggest that intense long illumination induces a large Ca’+ influx into the photoreceptors which is followed by Na-dependent CaZ+ efflux due to Na-Ca exchange. The light-induced afterdepolarization arises mainly from K+ accumulation in the extracellular space but partially from the electrogenicity of Na-Ca exchange. Na-Ca exchange
Bee photoreceptors
Ion-selective microelectrodes
membrane using the Na’ gradient as an energy source (Blaustein, 1974; Blaustein and Nelson, Intracellular free Ca’+ concentration ([Ca’+]i) 1982; R’equena and ~uilins, 1979). Since there is a key factor in controlling important cellular is evidence suggesting that 3-5 Na ions are activities. Light stimulation of the invertebrate exchanged for each Ca’+ -extruded, this mechphotoreceptor produces a large transient anism is probably electrogenic (Blaustein, 1974; increase of [Ca’+], (Brown and Blinks, 1974; Yau and Nakatani, 1985). However, recent Brown et al., 1977; Levy and Fein, 1985; Nagy investigations indicate that this transport and Stieve, 1983; Ivens and Stieve, 1984). It system is extremely complex and difficult to is still not clear how this elevated Ca?+ level characterize (Sheu and Blaustein, 1983; Lederer returns to the dark baseline level. In many and Nelson, 1983). different types of cells including photoreceptors Until now, activation of Na-Ca exchange (incertebrates: Lisman and Brown, 1972; Brown in invertebrate phosoreceptors was suggested and Lisman, 1972; Waloga et al., 1975; Bader on the basis of an increase in [Ca’+]i which was et al., 1976; ~er~ebru~e~:Yoshikami et al., 1980; induced by a decrease in the Na’ gradient Gold and Korenbrot, 1980; Schnetkamp, 1980; across the photoreceptor’s membrane (Lisman Fain and Lisman, 1981; Bastian and Fain, 1982; and Brown, 1972; Brown and Lisman, 1972; Yau et al., 1981; Hodgkin et al., 1984; Yau and Waloga et nl., 1975; Bader er al.. 1976). HOWNakatani, 1984; Yau and Nakatani, 1985) an ever, direct demonstration of light-activated important mechanism for Ca’+ extrusion is Ca?+ extrusion by Na-Ca exchange (which the Na-Ca exchange across the cytoplasmic is the expected physiological activity of the exchanger) is still lacking. A large amount of light (i.e. intense-short ‘Please address reprint requests to Professor Minke in duration or weaker-long duration) was reported Jerusalem. INTRODUCTION
to cause transienr afterdepolarization iTA)” at the end of the receptor potential In several Invertebrate species: drone (Baumann and Hadjilazaro, 1972); locust (Tsukahara and Horridge, 1978); ;Musca (Armon and IMinke, 1983): Cafliphora (Minke and Kirschfeld, 1984), though rhe origin of this intense-light-induced TA has been obscure. Recently. Minke and Armon (1984) showed that the illumination of fly photoreceptors following hypoxia or application of ruthenium red (a blocker of Ca” uptake into mitochondria; Alnaes and Rahamimoff, 1975) induced a transient afterdepolarization (TA) similar to that of the drone. The high sensitivity of this TA to Na- and Calgradients across the cell membrane suggested that it was related to an activation of Na-Ca exchange. Using intense illumination of long duration in the bee retina which has a restricted extracellular space, we measure in the present study changes in the ionic concentration of extracellular CaZT and K’ in parallel with extracellularly and intracellularly recorded voltage responses to bright lights.
iMETHODS
Experiments were done on slices of the retina of the compound eye of the 8 drone Appis mefflifera, superfused with oxygenated Ringer (for details, see Tsacopoulos and Poitry, 1982). Double barrelled ion-selective microelectrodes were used to record changes in either [Ca’+], (Levi and Fein, 1985) or [K’], (Coles and Tsacopoulos, 1979; Munoz et al., 1983; Coles and Orkand, 1985) resulting from intense brief (1 s) or a long (10 s) steps of light. The reference barrel in each case was used to monitor the extracellular voltage response. Single barrelled, 1 M KCl-filled microelectrodes (40 MQd.c. re sistance) were introduced intracellularly to record membrane potentials. The voltage responses were digitized @-bit resolution) and stored on floppy disks using a g-bit microcomputer (IMSAI 8080). A computer program
*The afterpotential induced by intense illumination (TA) should not be confused with the prolonged depolarizing afterpotential (PDA) which arises from rhodopsin to metarhadopsin pigment conversion, since the TA can be induced by white light when there is no net pigment conversion.
transformed rhc ~,oitagz ~len~~l~ nlc i;y using the appropriate calibr;ltlon CLITL~~.~ .j;‘ t1-1~. microelectrodes and a modified form 01 the Nzrnst equation (Coles and Tsitcapouloj. 197c); Let-i and Fein. 1985). The composition of thr control solution in mM \vils NaCI. 7SC);KCI. IU. CaCI,. 1.6: Tris buffer 10. pH 7.1. Estra~ellui~r receptor potential was recorded wirh thi: reference barrel of the double-barrelled ion-ssn%tivc electrode. The response i$‘as identified ~1sextracellular because the apparent resting porentlal was less than about 2 ml’ and the response to a light flash was a negative-going potential with an amplitude rarely greater than IO mV (Coles and Tsacopoulos, 1979; Tsacopoulos and Poitry, 1982). Extracellular porassium signal (A[K+],) was recorded Lvith a double-barrelled K’ sensitive microelectrode (tip size 1 {irn). Tht: technique of making these microelectrodzs has been extensively described (Coles and Tsacopoulos, 1979; Coles and Orkand. 1983; Munoz et al., 1983). Briefly, one barre! of theta glass micropipette was silanized with trimethyidimethylaminosilane (Fluka, Buchs, Switzrrland) and the tip filled with a K-- ion exchanger sensor solution (see also Coles and Orkand. 1983). The K- sensor ~vas made from !“,i, by weight K tetra-p-chlorophenylborate in 2.6-dimethylnitrobenzen (Fluka). The reset of the barrel was filled with Ringer solution. The reference barrel was also filled with Ringer solution. Calibration of the K L sensitive electrode was carried out in a manner similar to the procedure described in detail by Coles and Tsacopoulos (1979). Calibration solutions were modifications of the Ringer solution in which varying fraction of sodium chloride had been replaced by equimolar quantities of potassium cloride. The observed potassium potentials were plotted as a function of log potassium concentration. A modified form of the Nernst equation (which took into account the sensitivity of the electrode to sodium ions) was used to fit the data (see Coles and Tsacopoulos, 1979). Since all calibration solutions had the same ionic strength, according to the Debye--Hiickel theory the activity coefficient of K’ should remain constant in all rhe solutions including the superfusate. Accordingly, potassium concentration in the various experiments could be derived directly from the calibration curb’c. This derivation was based on the assumption that the extracellular medium has the same ionic
Light induced calcium accumulation in bee retina
strength as the superfusate. The detailed experiments of Coles and Orkand (1983, 1985) showed that this assumption is valid. Calcium signal was recorded extracellularly with a double-barrelled, bevelled microelectrode (tip size 2 PM). The technique of making these electrodes is identical to that for K-sensitive electrodes except that Ca-ion exchange sensor was used to fill the tip of one barrel. We used Ca-cocktail (Fluka2 1048) as Ca-ion exchange sensor. This cocktail is based on neutral carrier ligand (Fluka 21191) which was described by Oheme et al. (1976). This sensor was used in recent studies in which intracellular Ca was measured in the drone and Limufus ventral photoreceptors (Coles and Orkand, 1985; Levy and Fein, 1985). The study of Levy and Fein (1985) indicated that the Ca-sensitive electrode has a negligible sensitivity to Na+, K+, Mg’+ or H+ ions. Furthermore, calibration curves which were measured in a wide range of pCa showed a Nernstian function in the range between pCa 2-6. In the present experiments Ca calibration solutions were modification of Ringer solution in which varying fractions of sodium chloride had been replaced by calcium chloride in the range of pCa 3. The observed Ca potentials were plotted as a function of log Ca concentration and the Nernst equation could always be fitted to the data. Based on the assumption and theory used for the K+ electrode we derived also the calcium concentration directly from the calibration curves. Calibration curves were plotted for each of the K and Ca electrodes at the beginning and at the end of every experiment. Preceeding the responses there is an electrical artifact. The electrical resistance of the ionic sensor barrel is much higher (5. IO” Q for the Ca sensor and 3.10” R for the K sensor) than the resistance of the reference barrel (-6.1O’Q). Thus, due to the difference in the response time of the two barrel, when the voltage signal from the reference barrel is subtracted from the ionic sensor barrel a positive-going voltage difference remains. The [Ca’+], records were obtained from the same retina and depth as the [K’], records, after withdrawal of the intracellular micropipette and using the second micromanipulator to introduce the Ca” electrode into the retina. For light stimulation we used Xenon light beam which was delivered perpendicularly to the cut surface which stimulated all the photoreceptors simultaneously. The light intensity at the level of the photoreceptors was 6.5. IO” photons.cm-‘.s-‘.
681 RESULTS
Light -induced Na-dependent increase in [Ca ‘*j. and [K’], accompanied 6, afterdepolarkation Figure l(A) shows the intracellularly (a) and extracellularly (b) recorded receptor potentials in response to a short flash. A typical transient increase in [K’], resulting from this flash is also shown (c) (Tsacopoulos et al., 1983). Only a relatively small biphasic change in [Ca’+], (a decrease followed by an increase) was observed under these conditions (d), and the receptor potential showed fast and slow decaying phases. In the same retina, a 10 s step of light induced a pronounced TA [Fig. l(Ba)], which was also reflected as a negative deflection in the extracellular voltage recording [Fig. 1(Bb)]. During the light there was usually an initial decrease in [Ca’+], [Fig. 3(Bd)] followed by a large increase in [Ca’+], [Fig. l(Bd)] which continued to increase for more than 5 set after the light has been turned off and then decayed and returned to the baseline. The initial decrease in [Ca’+], is very clear in Fig. 3(Bd) but it is concealed by the increase in [Ca”], in Fig. 1(Bd). A[K+], usually showed two phases of decay after cessation of light. In contrast to A[Ca’+], the peak increase of [K+], in response to short [Fig. I(Ac)] and long [Fig. l(Bc)] light stimuli were roughly similar. Both the TA[I(Ba)] and AICaz+10 [l(Bd)] were dependent on the amount of light stimuli (i.e. on the product of the light intensity, I, and the stimulus duration, I). This is in contrast to the receptor potential which depends mainly on light intensity and show similar peak amplitude in response to short (in msec range) or long durations of light of the same intensity. At relatively weak or short duration stimuli both A[Ca’+], and TA were small or absent [Fig. l(A)] while increasing light intensity or duration in a relatively narrow range resulted in a pronounced TA and A[Ca’-I,, [Fig. l(B)]. The traces which are presented in Fig. 1 were very reproducible. Similar traces were observed in all of the 42 retinae that we examined. We found variability in the amplitudes and timecourses of the various phases described above. These variabilities are fully represented in Figs l-6 (see below). We found in all our experiments that the steady state concentration of [Ca’+], in the retina was significantly lower than its concentration in the perfusate. The reason for this observation is not clear. Figure 2 demonstrates that when successive short light stimuli were applied [see Fig. l(Ad)
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Light induced calcium accumulation in bee retina
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Fig. 2. Successive short light stimulations caused an increase in &a?+], only in response to the initial 9 light stimuli in a train of light pulses. The upper trace shows measurements of [Ca’+), and the lower trace shows simultaneous measurement of the light responses recorded intracellularly with another electrode in response to successive flashes. [Ca2+], reached a peak after the ninth stimulus and then declined during the repetitive stimulations. This result suggests that the source for [Ca’+], accumulation is limited and that this source is replenished in a rate which is slower than the inter Rash interval (6sec).
for the response to a single flash] the concentration of Ca2+ in the extracehular space increased. The increase in [Ca2+], due to the light flashes was slower (time to peak 60 set) and smaller (A[Ca’+], N 1 mM) as compared to the changes in fCa*+], after continuous illumination [see Fig. l(Bd)]. After the ninth light pulse, additional light pulses did not cause further increase in [Cal+], and the level of [Ca2+],
decreased in spite of additional stimulations. This result suggests that the source of Ca2+ accumulation in the extraceilular space is rather limited and the replenishing process is relatively slow (see Discussion). To investigate the ionic dependence of A[Ca*+], and of the TA Na+ was replaced in the perfusate by arginine (8 retinae), choline (3 retinae) or Li+ (2 retinae). Figure 3(A) presents
Fig. I. Intense illumination of long duration induced a large increase in extracellular [Ca”] and fK*] accompanied by depolarization. The figure compares voltage responses and extracellular ionic changes to a single I set flash (A), or to a IO set step of intense white light (B). In this and Figs 3, 6, the records are displayed by the computer on the same time-scale. (a) Intracellular receptor potential recorded with fine micropipette (40 MR d.c. resistance). Voltage scale represents the voltage with reference to the extracellular fluid. (b) Extracellular receptor potential made with the reference barrel of the doubiebarreled K+-sensitive electrode. The response was identified as extracellular because the apparent resting potential was less than about 2 mV and the response to a light Rash was a negative-going potential with an amplitude rarely greater then IOmV. (c) Extracellular potassium signal (A[K+],) was recorded with a double-barreled K+-sensitive microelectrode (tip size 5 I pm). (d) Calcium signal was recorded extracellularly with a double-barrelled, bevelled microelectrode (tip size 2 pm). Preceeding the response, there is an electrical artifact. This record was obtained from the same retina and depth as the K+ records. after withdrawal of the intracellular micropipette and using the second micromanipulator to introduce the Ca’+ electrode into the retina. The extracelluiarly recorded receptor potential is shown in (bf. Comparing the records in A and B, it is clear that the amplitude of A[Ca”f, increased when the duration of the light stimulus was increased from I to IO sec. The light monitor is indicated at the bottom of traces (d). A vertical dotted line is traced in order to facilitate examination of the event following cessation of the light.
2 i\
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_ 0-Na
Fig. 3. Both the increase in extracellular Cal+ and the TA are Na+ dependent. The effect of superfusion with O-Na Ringer solution on receptor potential [intracellular (a), and extracellular (b)], A[K’], and A[Ca’“l, induced by a IO set step of light are shown. For details, see legend of Fig. 1. (A) Illustrates the effect and (B) the recovery. In (A) two traces are superposed by the computer: one [the upper in (a, C) and (d); the lower in (b)] is the controi and the other is a selected response recorded after IOmin of replacement of all the Na+ by arginine. Arginine was used because the K + electrode is sensitive to choline (the cation usually used to replace Na+). Previous experiments, however, have shown that the effects of arginine and choline are the same (Tsacopoulos and Poitry, 1982). Traces (b). (c) and (d) were recorded simultaneously. Trace (a) was recorded independently but under identical condition simultaneously with Ca?+ sensitive eiectrode which showed responses similar to those of traces (d). In addition, it was first established that the effect of 0-Na+ on intracellular and extracellular receptor potential was parallel.
the receptor potential recorded intracellularly (a) and extraceilularly (b) in control Ringer (larger responses) and in Ringer where Na+ was replaced for 10 min by arginine or choline (smaller responses). In the upper traces (3a) Na ions were replaced by choline ions in one retina. Traces (c) and (d) are the corresponding measurements of [Kilo and [Ca*+&,which were taken simultaneously with traces (b). In these traces Na ions were replaced by arginin ions in another retina. Traces very similar to (d) were recorded simultaneously with traces (a) (see Fig. 4). Removing Na* for 10 min from the perfusate resulted in a reduction of both the receptor potential (especially of the plateau phase) and in a relatively much larger reduction
of the TA [Fig. 3(Aa)]. In parallel with these voltage changes the amplitude of both A[K+]* and A[Ca’+], were decreased by about 70% and the slower decaying phase in A[K+], disappeared. Figure 3{B) shows partial recovery (in the same experiments) from the 0-Na solution -20 min after control Ringer was applied. When the O-Na solution was used for 30 min or more, a receptor potential with -30% amptitude (see also Coles and Orkand, 1982) and A[K], with - 35% amplitude were still observed but the TA was abolished completely together with the increase in [Ca2+&,. We also tried to replace Na’ with Li+. Replacement of Na’ with Li+ for - 13 min resulted in -..40% reduction in the amplitudes of both the receptor
Light induced calcium accumulation in bee retina
potential and the TA and -50% reduction in A[Ca’*],. Unlike the experiments with choline or arginine, in the presence of Li’ the photoreceptors slowly depolarized in the dark (in agreement with the data of Fulpius and Baumann, 1969) by 15 mV in 9 min and by additional 7 mV after giving the standard IO set light pulse. These effects were not reversed after the return to control conditions for additional 30min. For these reasons arginine and choline were mainly used to study AICaz+]Oand the TA. In the drone retina persistence of an appreciable fraction of the receptor potential was observed by Fulpius and Baumann (1969) even after 12 hr in solution in which all the Na’ has been replaced by choline. Tris or sucrose. The increase in [Ca’+], Ca ’ + e&%.x
arisesfrom
Na-dependent
The accumulation of Cal+ in the extracellular
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space can arise from the release of intracellular Ca’+ or the cessation of an inward Ca” flux. We found a systematic way to distinguish between these two mechanisms. The initial decrease and the following increase in [Ca’+], in response to IO set bright light (Fig. 3) seem to arise from two different mechanisms. While the increase in [Ca’+],, was Na-dependent and continued to increase after the light, the decrease in [Ca’+10was not affected by removing [Na+], and persisted during illumination only. This fact is illustrated in Fig. 4 which shows the effect of prolonged 0-Na conditions on A[Ca’+],,. The figure shows that the increase in [Ca’+], has two phases: one phase during light and the other phase after the cessation of the light. The phase during light shows a strong Na-dependence. It became much smaller and slower during prolonged 0-Na conditions until it almost disappeared after 22 min in 0-Na [Fig. 4(d)].
0-Na
15mcn -
Fig. 4. The light induced Ca?+ influx can be separated from the light induced Ca efflux by removing [NaclO. The experimental paradigm was similar to that of Fig. 3(d) except that extracellular Na+ was replaced by choline instead of arginine. Traces (a) and (e) were measured before application of (a) and 24 min after choline Ringer was replaced by control solution (e). Traced (b-d) show the effects of prolonged 0-Na conditions on A[Ca*‘+],at various times as indicated. The vertical bars on the left indicate [Ca’], level between I and 3 mM.
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Fig. 5. The Sa-dependence of the increase of [Ca’+], is not secondary to membrane voltage changes. The upper trace shows measurements of A[Ca?+], and the lower trace shows intracellular recording of membrane voltage as in Figs I (a), 3(a). At the time indicated by two arrows a pulse of anoxia was applied by replacing oxygen for nitrogen in the solution. The effect of anoxia was to depolarize the photoreceptor in the dark by - 30 mV. No significant change in A[Ca’+], was observed at that time (upper trace). Illumination resulted in the typical changes in [Ca”], as in Figs l-3. The spikes seen on the anoxia-induced depolarization are regenerative action potentials.
When this phase became smaller the initial liecrease in [Ca’-1, became very pronounced [Fig. 4(&i)]. Accordingly A[Ca’+], during light seems to reflect the sum of Ca” influx and efflux while the peak amplitude of the increase of [CaZ+Jo, which occurred a few second after the cessation of the light [Fig. l(B)], reflects only the efflux of Ca’-. Two mechanisms might account for lightactivated Ca’+ efflux against a large eiectrochemical gradient: ATP-dependent Ca’+ pump or Na-Ca exchange. The dependence of A[Ca”], on [Na-1, can distinguish between these two mechanisms. However, the Nadependence of Ca” accumulation (Figs 3 and 4) might be only secondary to membrane voltage changes [Fig. 3(A)]. Thus reduction in A[Ca”], after Naremoval might result from the abolishment of the TA. In Fig. 5 we examined the effect of depolarization without illumination. Figure 5 shows that 130 mV of transient depolarization, which was induced by transient anoxia, did not result in a significant change in [CaZ-]O while the light-induced increase in [Ca’-10 was normal. Figure 5 thus supports the hypothesis that the low [Na-1, and not the lack of after depolarization is responsible for the large reduction in [Ca”],, at 0-Na condition, in consistence with the Na-Ca exchange mechanism. The origin of’ the transient
after depolarization
The specific Na’-dependent-Ca’accumulation observed during the TA is an evidence for a Na-Ca exchange mechanism. The observations that this Ca’- accumulation vvas accom-
panied by depolarization (TA) and that both the change of [Ca’+], and the TA were abolished at zero [Na+10 suggest that part (see below) of the TA arises, from an electrogenic exchange. Unfortunately it was not possible to test the electrogenic exchange hypothesis by measuring a conductance or current changes during the TA since in the membrane of drone photoreceptors the current-voltage relationship is highly nonlinear and a voltage clamp technique is not feasible in drone photoreceptors due to strong electrical coupling among adjacent photoreceptors (Shaw, 1969). We hypothesized that both the electrogenicity of the Na-Ca exchange (as manifested in the increase of [Ca”],) and the elevated level of [K”], (Fig. 3) could depolarize the photoreceptors and hence, contribute to the TA. By using the Nernst equation we calculated the contribution of [K+], to membrane depolarization during the TA by assuming values of [K ‘1, in the range of 87-l 15 mM (Tsacopoulos et nl.. 1983; Coles and Tsacopoulos, 1979) and the [K+], value from Figs 1(Bc), 3(c). Using these values we obtained membrane depolarizations in the range of IO-20 mV. Accordingly, we expect that the main part of the TA is due to increase in [K+], level. The increase in [K-l, however, cannot account for the entire TA. In Fig. 6, we present an experiment in which the contribution of [K+], to the generation of the TA was missing. Figure 6 shows simultaneous measurements of [K’], (b), [Ca’-1, (c) and extracellular voltage recorded from the reference of the Ca’+-sensitive electrode (a). In this particular retina, the slower decaying phase of
Light induced calcium accumulation in bee retina
687
b
O-No
/. O-No
Fig. 6. A small TA is still observed when [K ‘1, already returned to baseline: the effect of intense activity of the Na/K pump. The experimental paradigm was similar to that of Fig. I(Bb-d). (a) Extracellular receptor potentiat (with the reference barrel of the Ca-sensitive microelectrode)~ (b) [K*], and (c) [Car*k. Additional responses were recorded in the same reiina IOmin after the onset of superfusion with 0-Na (smaller response) and showed results similar to those of Fig. 3. In this retina A[K+], showed only the fast decaying phase (see Figs I and 3 for comparison) presumably due to intense activity of the Na/K pump which was reflected in a larger undershoot in A[K+],. Note the slow decay of A[Caz+], (SW text). Also note the similarity between A[Ca’+], at normal and at 0-Na, from the time of light off (dotted line) to the time of peak amplitude. This similarity indicates that the iight-induced Cal” influx has a similar magnitude during normal and O-Na conditions.
A[K+],, was missing possibly due to intense activity of the Na/K pump as reflected in the large undershoot in [K+],. Similar observations were found in 4 other retinae. The small extracellular TA observed in Fig. 6 could not arise from accumulation of K+ ions in the extracellular space. However part of this small TA could arise from a decrease in [K+],. Thus a component due to the electrogenic Na-Ca exchange could be even smaller than that observed in the TA of Fig. 6. Removing Na’ from the perfusate reversibly abolished this TA [Fig. 6(a), arrow] and the undershoot in [K’10 [Fig. 6(b) smaller response] as well as reducing A[Ca2-I,. Figure 6 suggests that a small fraction of the TA may arise directly from an inward current possibly due to the eiectrogenicity of the Na-Ca exchange. An electrogenic Na-Ca exchange should
depend on membrane voltage (Requena and Mullins, 1979). Therefore we expect that the anoxia-induced depolarization (Fig. 5) would reduce the [Ca2+], level. Our failure to observe such reduction (Fig. 5) suggests that this effect is small or that it is cancelled by the small shrinkage of the extracellular space (see Discussion). DISCUSSION
The light induced Na-dependent accumulation of Ca2+ in the extracellular space is a strong indication for Na-Ca exchange. However, it is possible that the observed increase in [Ca’+]O (Figs l-3) may arise partialty from changes in the volume of the extracellular space and not entirely from Ca’+ efflux. Thus during light stimulation, drone photoreceptors may swell
a result of an increase in rrirracrlluiar osrnoticaily active particles as found in other preparations (Connors t't d.. !%?I!; Dicrzrl PI tri.. 1980). This possible swelling could lead to a shrinkage of the extracellular space and. therefore. a secondary increase in [Cal-1,. Direct measurements of the extracellular space made by measuring changes in the concentration of the cellular impermeable ion, tetraethylammonium showed maximal shrinkage of 3290 in the extracellular space of retinal slices in the drone during repetitive intense flashes (Coles er nl., 198 1; Orkand rz cri., 1984). Thus shrinkage of the extracellular space cannot account for the large (300’~) increase in [Ca’+lO. Exposure of the retina in the dark to transient anoxia (Fig. 5) a condition known to produce swelling of other types of ceils, resulted in a -30 mV transient depolarization of the photoreceptors, but without a significant changes in [Caz’]“. Thus, we conclude that the major part of the increase in [Ca”], reflects an efhux of Ca’- ions from the photoreceptors. The amount of Ca’* ions released into the extracellular space following prolonged intense light was calculated by using the peak of AICa’+10 [Fig. l(Bd)] and using the volume of the extracellular space around a single photoreceptor which is about 5.7. lO’~lrn’ (Coles and Tsacopoulos, 1979) (about 4.8% the volume of a photoreceptor which is 1.2.tO’/lm’; Coles and Tsacopoulos, 1979). This calculation shows (taking into account 32% shrinkage of the extracellular space) that at least 3.6. IO’Ca’ions are extruded from the time of light onset until the time of the peak increase of A[Ca’“],,. Due to the special geometry of the extraceltular space (see Coles and Tsacopoulos, 1979) this amount of Ca” is close to the amount extruded from a single cell. This figure is by more than an order of magnitude larger than the amount of Ca’” extruded from vertebrate rods following saturating light (Gold and Korenbrot, 1982). Using the same preparation (the drone retina) and similar experimental setup revealed that A[Ca!-1, rose from a dark level of 3 PM to light level of 15 !iM during strong short flashes of light (Levy and Coles personal communication). Calculations of the maximal amount of increase in [Ca’?], gave a value of 0.9.10’ Ca?- ions. ___________.__I_~~ -. as
“When ;! very prolonged intense illumination (60 set) was used. a condition that increased [Sa+), to a relatively high level, we observed a very large undershoot in (Ca! ‘I, - 30 set after light onset. during perfusion with normal Ringer.
By using the decrease m [Ca’-1,. durmg ii-U,i conditions. (-0.5 m&f; Figs 3 and 2; :: I; possible to estimate that 9. IO’Ca“ ion\ enter the cell at the peak decrease in [Ca’- j,, The factor 4 difference between Cal- intfuv and efflux may be explained by assuming that the Ca“ efflux includes Ca’- released from intracellular stores (Brown and Blinks. 1974; Walz, 1982). Tf A(Ca’-]O includes Ca’- released from intracellular stores and the replenishing process of these stores is slow it is expected that A[Ca’*], in response to single flashes will be reduced after stimulations by several successive flashes. Figure 2 shows that this expectation is fulfiiled, thus supporting the above suggestion. The slow decline of A[Ca’+], in Fig. 6(c) relative to the decline in Figs 1 and 3 needs some explanation. We found consistently that in retinae with presumably strong activity of the NajK pump the decline of A[Ca”], was significantly slower than in retinae with weaker pump activity [compare Figs 3(d) and 6(c)]. We assume that slower reduction in A[K-I<, after light reflects weaker activity of the Na/K pump. The Na-Ca exchange is known to operate in two opposite directions (Requena and Mullins, 1979) i.e. extruding Ca’+ (forward mode) or introducing Ca” into the ceil (reverse mode) depending on the ratio ([Na+],/[Na+],)” (n = 3-5). Part of the decline in A[Ca’+], in our experiments may arise from the exchanger operating at the “reverse mode” when the Ca’+ gradient is high and the Na+ gradient is relatively low. When [Na+], is doubled by intense light of long duration (Coles and Orkand, 19S5). Ca’. efflux due to Na-Ca exchange is expected to change into a influx as observed in the undershoot in A[Ca’&], [Fig. 3(d)]*. Consistent with this view we found that when the ratio [Na”],/[Na’], is expected to return quickly to the resting value after light, in retinae which showed intense activity of the NajK pump, a reiatively siow decay rate of A[Ca’+], was observed [Fig. 6(,c)]. In this case the Na--Ca exchanger is not expected to operate in the “reverse mode”. Thus the slow decline in A[Ca’-1, in Fig. 6(c) is fully in accord with the known properties of Na-Ca exchange. The influx of Ca’+ after light due to the exchanger, as manifested in the decline and the undershoot of A[Ca’*], can fill the intracellular and can account for the stores of Ca?’ difference between the calculated amount of Ca”entery during iIfumination and Ca” estrustion after illumination.
Light induced
calcium
act :umulation
The present study suggests that a large fracof the light induced increase in [Cal*], is due to Ca?- influx as demonstrated before in the Limufus ventral photoreceptors by Ivens and Stieve (1984). The increase in [Ca?+], seen after exposure to intense light is an essential step in light adaptation and possibly in excitation (Brown et al., 1984; Fein et al., 1984) of invertebrate photoreceptors. Na-Ca exchange then provides an efficient mechanism for controlling [Ca?+], and thus regulating sensitivity to light in these photoreceptors. tion
Acknowledgemenrs-We thank Drs R. Werman. J. E. Lisman. M. P. Blaustein, H. Stieve, P. Hillman and J. Korenbrot for very helpful comments on various versions of the manuscript and Messrs Ph. Perrottet, J.-L. Munoz and D. Pallet for technical assistance. Supported by Swiss NSF Grant 3.119-g I and George Kernen Foundation; the Swiss Foundation for Fight against Blindness, the “Societe Acedemique” of Geneva to M.T. and by NIH grant EY 03529 to B.M.; Mr N. Gaon also provided financial support for this project.
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