Neuron,

Vol. 4, 547-555,

April,

1990, Copyright

0 1990 by Cell Press

Feedback Inhibition by Calcium limits of Calcium by lnositol Trisphosphate in Limulus Ventral Photoreceptors Richard Payne: and Alan Fein*

Thomas M. Flores,t

* Department of Zoology +Department of Biochemistry University of Maryland College Park, Maryland 20742 *Department of Physiology University of Connecticut Health Farmington, Connecticut 06032

Center

Summary Injection of inositol 1,4,5 trisphosphate (tnsP3) into Limulus ventral photoreceptors elevates the concentration of intracellular calcium ions and as a consequence depolarizes the photoreceptor. This InsPI-induced elevation can be inhibited by a prior injection of calcium or InsP3 delivered 1 s earlier. Recovery from this inhibition has a half-time of between 1.5 and 5 s at 2O’C. Calcium released by InsP3 therefore inhibits further release of calcium from InsP3 -sensitive calcium stores. This feedback inhibition may protect the calcium stores from depletion during prolonged bright illumination. Feedback inhibition, rather than periodic depletion of calcium stores, may also underlie the oscillatory bursts of InsP,-induced calcium release that have been observed in many cell types. Introduction lnositol 1,4,5 trisphosphate (InsP$ is an intracellular messenger that releases calcium from intracellular stores within a wide variety of tissues (Berridge and Irvine, 1984, 1989). Recently, the mechanisms that control calcium release by InsP3 have been investigated (Berridge and Irvine, 1989). In this paper we provide direct evidence that calcium released by InsP3 feeds back to inhibit further calcium release (Suematsu et al., 1984; Chueh and Gill, 1986; Thierry and Klee, 1986; Payne et al., 1988; Baumann and Walz, 1989; Joseph et al., 1989; Parker and Miledi, 1989; Parker and Ivorra, 1990). The injection of InsP, into several cell types produces oscillations in the numbers of open calcium-activated ion channels (Miledi et al., 1987; Berridge and Calione, 1988; Payne et al., 1988; Wakui et al., 1989). The negative feedback loop that we describe here may play a role in producing oscillations in the intracellular concentration of calcium ions ([Caz+]J, which underlie the observed oscillations in channel openings. Light-induced production of InsP3 is thought to mediate the elevation of [Cal+], that accompanies light-induced depolarization of Limulus ventral photoreceptors (Brown et al., 1984; Fein et al., 1984). The elevation of [Caz+]r is the probable cause of light adaptation, since injection of calcium into the pho-

the Release

toreceptor desensitizes the cell to depolarization by subsequent light flashes (Lisman and Brown, 1972). Rapid pressure injection of calcium into the photoreceptor also causes a transient depolarization by somehow activating the light-sensitive conductance (Payne et al., 1986b), indicating a role for calcium in visual excitation. However, calcium-activated ion channels have yet to be identified in the photoreceptor’s plasma membrane, and other second messengers that might be produced during illumination, such as cyclic guanosine monophosphate, may also play a role in visual excitation (Johnson et al., 1986). Precise knowledge of the mechanism by which calcium depolarizes the photoreceptor is not critical for the analysis of this paper; rather, we concentrate on the control of calcium release by InsP,. Pressure injection of InsP3 into Limulus ventral photoreceptors releases calcium from intracellular stores, causing an elevation of [Caz+]i that results in a transient depolarization followed by a desensitization to subsequent stimulation by light (Brown et al., 1984; Fein et al., 1984; Brown and Rubin, 1984; Payne et al., 1986a). The regulation of the actions of InsP3 in ventral photoreceptors has been investigated previously by using the InsP3-induced depolarization as a measure of calcium release. Stimuli that elevate [Ca2+]r, such as light or the injection of InsPs, are followed by a period of insensitivity to depolarization by subsequent injections of InsP3 (Fein et al., 1984; Brown et al., 1984; Payne et al., 1986a). Injection of calcium or InsP3 under voltage-clamp conditions also causes a temporary insensitivity to subsequent injections of InsP3 (Payne et al., 1986a, 1986b). It was therefore proposed that prior elevation of [Ca2+], inhibits the ability of InsP, to release calcium (Payne et al., 1988). For this paper, we sought a direct demonstration of the inhibition of InsP3-induced calcium release by prior elevation of [Ca2+]i. We use the photoprotein aequorin (Shimomura et al., 1962) to measure InsP3induced calcium release directly. We also use doublebarreled micropipettes that allow the injection of calcium and InsP3 into the same area of the photoreceptor. We show that elevation of [Ca2+]i prior to an injection of InsP3 temporarily inhibits the ability of InsP3 to release calcium, and we describe the time course and temperature dependence of the rapid recovery from this inhibition.

Results Inhibition of Responses to InsP3 Caused by the Prior Injection of Calcium or InsP3: Temperature Sensitivity Ventral photoreceptors are clearly segmented into two lobes (Calman and Chamberlain, 1982; Stern et al., 1982), a light-sensitive rhabdomeral (R) lobe and an arhabdomeral lobe insensitive to light. The R lobe

a

injections IP3 after

of IP3

b

lnjectlons IP3 after

a

of Ca

Injections peak

Figurel. sponse hibition

Prior Injection of Calcium or InsPi Inhibits to Subsequent InsP, Injections, and Recovery is Slowed by Lowering the Temperature

the from

ReIn-

The panels show recordings of the membrane potential during paired injections of calcium and InsP1 at 20°C and 8%. The bottom trace of membrane potential in each panel is a complete record showing the response to a pair of injections. For clarity, the other traces show only the response to the second of the pair of injections. The response to the first injection remained essentially unchanged. The lowest traces in each panel mark the time of the injections. (a) An injection of 100 PM InsP, was followed by another injection of InsP3 after an interval of 1, 3, 5, 10, and 20 s at 20°C. (b) An injection of 1 mM calcium was followed by an injection of 100 uM InsP3 after an interval of 1, 3, 5, 10, and 20 s at 20% (c)An injection of 100 PM InsP, was followed by another injection of InsPs after an interval of 5, 10, 20, and 40 s at 8°C. (d) An injection of 1 mM calcium was followed by an injection of 100 uM InsP3 after an interval of 5, 10, 20, and 40 s at 8°C. The duration of the injections of InsP, was 75 ms, pressure 30 psi, and that of the injections of calcium was 220 ms, pressure 40 psi. The duration and pressure of the injections of calcium were chosen so as to produce an inhibition of the response to InsPx 1 s after the injection of calcium that was similar to the inhibition observed 1 s after an injection of InsP+

contains the InsPrsensitive calcium stores (Payne and Fein, 1987). To ensure that calcium and InsP, were delivered directly to these calcium stores, the photoreceptors were impaled by micropipettes placed in the R lobe, where injections of InsP, produced a depolarization after a delay of less than 250 ms. The use of double-barreled micropipettes allowed us to inject calcium and InsP3 into the same region of the R lobe. We could then directly compare the ability of prior injections of InsP, with that of prior injections of calcium to inhibit the response to subsequent injections of InsPa. A previous qualitative study in which two single micropipettes were used (Payne et al., 1986b) did not allow such a comparison, owing to the localized nature of the rise in [Ca2+]r caused by the injections (Payne and Fein, 1987). Figures 1 and 2 illustrate recordings obtained from a cell that was impaled with a double-barreled micropipette containing 100 uM InsP, in one barrel and 1 mM calcium aspartate in the other. The cell was held in the dark throughout the experiment. At 20°C, the first of a pair of injections of InsP, depolarized the photoreceptor by between 20 and 30 mV. However, a second injection of InsP,, if delivered 1 s after the first, was ineffective in depolarizing the photorecep-

o”0

response

10

b

of IP3 after

(mV)

P

20 time after

Injections peak

0

response

10

IP3

30 first

40 injection

of

IP3 after

30 first

40 injection

50

60

(9)

Ca

(mV)

20 time after

Figure 2. The Temperature Inhibition of the Response

Dependence to lnsPj

of the

60

60

(5) Recovery

from

(a) The peak depolarization resulting frcm the second of a pair of 100 PM InsP, injections is plotted against the time interval between the paired injections. (b) The peak depolarization resulting from an injection of 100 PM InsP1 that followed an injection of 1 mM calcium is plotted against the time interval between the calcium and InsPi injections. All data were collected from the cell of Figure 1. Data at 8OC were collected first (triangles), followed by those at 20°C (circles), and finally 14’C (diamonds). The isolated points at the extreme right-hand side of each plot represent the peak depolarization caused by control injections of InsP,, which were no+ preceded by injections of calcium or insP,.

tor (Figures ?a and 2a). The time for the response to InsP3 to recover 50% of its initial amplitude varied from cell to cell within the range 1.5-5 s at 20°C. The response to the second injection usually recovered completely if the interval between the injections of InsP, was extended beyond 20 s. Lowering the temperature to 8OC greatly prolonged the recovery time of the response to InsP, (Figures ?c and 2a). The response at 8°C to the first injection of InsP,, however, was only slightly diminished in amplitude compared with that at 20°C (Figures la and Ic). Thus temperature had a greater effect on the recovery time of the response to lnsP3 than it did on

Feedback 549

Inhibition

of Calcium

Release

the first

amplitude of the depolarization caused by the injection of InsP,. Injections of 1 mM calcium into the same region of the R lobe through the other barrel of the micropipette also depolarized the photoreceptor by 20-30 mV and inhibited the response to subsequent injections of InsP, (Figures lb, Id, and 2b). The duration and pressure of the InsP, injection that followed the injection of calcium were identical to those used in the experiment of Figures la and 2a. At 20°C, the calcium injection completely inhibited the response to an injection of InsP, delivered 1 s later. At all three temperatures, 20°C, 14OC, and 8”C, the response to InsP3 recovered after the injection of calcium with the same time course as was observed after an injection of InsP3 (compare Figures 2a and 2b). In six other cells, the first of a pair of injections of 100 uM InsP3 at 20°C caused depolarizations of 26 f 5 mV (mean f SEM). The second InsP3 injection of the pair, delivered 1 s later, caused depolarizations of only 1.4 f 0.4 mV Similarly, injections of InsP, caused depolarizations of only 2.5 k 1 mV when they were delivered 1 s after injections of calcium that caused depolarizations of 21 +_ 5 mV. For five of these cells, it was possible to measure the time course of recovery at two or more temperatures between 20°C and 75°C. In all cases, the time course of recovery of the sensitivity to InsP, proved to be temperature dependent. The Q,,, of the time taken for the responses to second injections of InsP3 to recover 50% of their amplitude was 6.1 k 1.5 (mean f SEM) after a first injection of InsP, and 4.6 + 2.5 after a first injection of calcium. Control experiments were performed by replacing the calcium aspartate with carrier solution alone. Injection of carrier solution did not result in any appreciable inhibition of the response to subsequent injections of 100 PM InsP3. In seven cells at 20°C, injections of InsP3 caused depolarizations of 32 f 4 mV when they were delivered 1 s after injections of carrier solution. Control injections of InsP,, delivered without any prior injection, caused depolarizations of 33 f 5 mV. To investigate the effect of the counterion to calcium, 1 mM CaC12 was substituted for calcium aspartate. CaClz also inhibited the response to subsequent injections of 100 FM InsPX. Note that in many of the traces in Figures 1 and 2 discrete waves of depolarization caused by stray light or occurring spontaneously (Yeandle and Spiegler, 1973) are observed even during periods when the cell is desensitized to injections of InsP,. We attribute this lack of inhibition of the discrete waves to the localized nature within the R lobe of the elevation of [Ca*+], and consequent desensitization caused by injections of InsP, or calcium (Fein and Lisman, 1975; Fein et al., 1984; Payne and Fein, 1987). Injection of InsP3 Greatly Reduces the Elevation of [Ca*+]i Caused by Subsequent Injections of InsP3 In addition to a double-barreled micropipette containing 100 uM InsP3 in one barrel and 1 mM calcium

a

b

c

cl

e

) 20 mV

ILL IP3

IP3 2s

L-1 IP3 20s

Figure 3. Prior Injection quent InsP, injections Photoreceptor

IP3 104s

IP3

Ca 2s

Ca 20s

of InsP, Inhibits the Ability of Subseto Elevate [Ca*+], and Depolarize the

(a-c] Membrane potential (upper traces) and aequorin luminescence (middle traces) resulting from paired injections of 100 PM InsP? delivered 2 s apart (a) and from the second of a pair of injections of InsP, delivered 20 s (b) and 104 s apart (c). (d and e) Membrane potential and aequorin luminescence resulting from an injection of 100 RM InsP, followed 2 s later by an injection of 1 mM calcium (d). The response to an injection of 1 mM calcium delivered 20 s after an injection of InsP, is also shown (e). The injections of InsP, had a duration of 100 ms, pressure 40 psi. The injections of calcium had a duration of 400 ms, pressure 40 psi. The records of aequorin luminescence show the number of photon events detected during successive 20 ms counting periods.

aspartate in the other, some cells were also impaled with a second micropipette containing aequorin. Aequorin was injected into the dark-adapted cells by a series of five to ten pressure pulses, and the cells were held in the dark until the end of the experiment, when a bright-light flash was delivered (see below). Figures 3-6 illustrate data obtained from such a cell. Injection of InsP3 through one barrel greatly reduced not only the depolarization but also the peak aequorin luminescence evoked by a second injection of InsP3 delivered 2 s later (Figure 3a). Both the depolarization and the peak aequorin luminescence produced by the second injection recovered as the interval between the injections was increased (Figures 3b, 3c, and 4a). Recovery of the peak aequorin luminescence lagged somewhat behind the recovery of the depolarization (Figure 4a), but both were complete within 2 min. The time for full recovery from inhibition was generally somewhat longer for these cells than for those impaled with a double-barreled micropipette alone, possibly owing to damage incurred during the second impalement. The insensitivity of aequorin to small elevations of [Ca2+], (Brown and Blinks, 1974; Levy and Fein, 1985) and the steepness of the relationship between [Ca’+]i and the intensity of aequorin luminescence (Blinks et al., 1982) may explain why the recovery of the InsP?-

NeLlKJll 550

a

Injections 50

of

after

IP3

a

mV

counts/s

1

n

o0

5

10

b

Injections

of

I

20

15

time after

50-

IP3

injection

Ca

) 60

Jo

1 2000

I 1500

4o-Y

0

I

Figure 5. Prior Injectionof quent InsPj Injections Photoreceptor counts/s

5

10 15 20 time after injection (9

Figure 4. The Ability of InsP3 and Calcium Depolarize the Photoreceptor following InsPi

e

- 2000

IP3

mV

d

c

,20 rn”

(s)

after

b

:

;‘coo

L

!.

) 60

to Elevate [Ca*+], and a Prior Injection of

(a) The peak depolarization (circles) and peak aequorin luminescence (triangles) resulting from injections of 100 PM InsP, are plotted against the time that had elapsed after a prior injection of InsP,. (b) The peak depolarization (circles) and peak aequorin luminescence (triangles) resulting from injections of 1 mM calcium are plotted against the time that had elapsed after a prior injection of 100 PM InsP,. The isolated points at the extreme right-hand side of each plot are the peak depolarization and aequorin luminescence resulting from injections delivered more than 60 s after any prior injection. All data were obtained from the experiment of Figure 3.

induced aequorin luminescence lagged behind that of the depolarization and why, at early stages during recovery, small InsP,-induced depolarizations, which might be caused by small elevations of [Ca*+]i, were not accompanied by detectable aequorin luminescence. For the same reason, we were unable to detect a residual elevation of [Caz+], after injections of calcium or InsP,, the decline of which might correlate with the recovery of sensitivity to InsP3. Thus we are unable to determine whether the inhibition of InsP3-induced calcium release requires the continuous presence of elevated [Ca*+]i. In contrast to the effect on responses to injections of InsP,, the depolarization and peak aequorin Iuminescence resulting from injection of calcium were little affected by an injection of InsP, delivered 2 s

to

Calcium Elevate

InhibitstheAbilityof Subse[Ca*+], and Depolarize the

(a-c) Membrane potential (upper traces) and aequorin luminescence (middle traces) resulting from injections of 1 mM calcium followed 2 s later by an injection of 100 PM InsP3 (a). Responses to injections of InsP? delivered 20 s (b) and 74 s (c) after an injection of calcium are also shown. (d and e) Membrane potential and aequorin luminescence resulting from paired injections of 1 mM calcium delivered 2 s apart (d) and from the second of a pair of injections of calcium delivered 20 s after the first (e). (f) Depolarization and aequorin luminescence resulting from a diffuse light flash, intensity -1 log unit, duration 20 ms. The shutter covering the aperture of the photomultiplier tube was opened IO ms after the light flash. The data were collected from the same ceil used for Figure 3. The duration and pressure of all the injections are the same as for Figure3. The records of aequorin luminescence show the number of photon events detected during successive 20 ms counting periods.

earlier (Figures 3d, 3e, and 4b). The lack of a reduction in the peak aequorin luminescence resulting from the injection of calcium shows that the prior injection of InsP, did not appreciably deplete active aequorin in the neighborhood of the injection site. The lack of a reduction in the depolarization resulting from the injection of calcium suggests that neither InsP3 itself nor the resulting elevation of [Ca*+]i inhibited the ability of calcium to depolarize the photoreceptor. Similar results obtained from other cells are compiled in Table 1. injection of Calcium Greatly Reduces the Elevation of [Ca’+]i Caused by Subsequent Injections of lnsPJ Prior injection of calcium into the same cell used for the experiments of Figures 3 and 4 greatly reduced the depolarization and aequorin luminescence resulting from an injection of InsP3 that was delivered 2 s later (Figure 5a). The amplitude of both the depolarization and the aequorin luminescence resulting from the InsP, injection recovered over the next 2 min (Figures 5b, 5c, and 6a). Note that the peak aequorin luminescence produced by the injection of calcium (Figure 5a) is similar to that produced by the first of the paired injections of InsPI shown in Figure 3a. In contrast to the effect on responses to injections of InsP,, the depolarization and peak aequorin Iu-

Feedback 551

Inhibition

of Calcium

a

Injections 50

Release

of

IP3

after

Ca

mV

counts/s

r

resulting from the second injection was actually larger (Figures 5d and 6b). A similar increase was observed in three other cells (Table 1). The greater elevation of [Ca2+l, following the second calcium injection might be due to a localized saturation of the cell’s calciumbuffering capacity by the first injection. Whatever the explanation, the lack of any reduction in the peak aequorin luminescence resulting from the second injection of calcium suggests that the first injection did not appreciably deplete active aequorin in the neighborhood of the injection site. Also, the elevation of [Ca2+]i caused by the first injection of calcium did not appear to interfere greatly with the ability of a subsequent injection to depolarize the photoreceptor. Similar results obtained from other cells are compiled in Table 1. At the end of the experiment, we delivered a brief, diffuse flash of light and recorded the accompanying depolarization and aequorin luminescence (Figure 50. The peak aequorin luminescence caused by this flash was approximately 7 times greater than that seen after calcium or InsP3 injections. This indicates that the total amount of calcium that bright light can release inside the photoreceptor is larger than the amount injected during calcium injections or released following InsP3 injections. This accords with the ability of bright flashes to completely suppress the response to subsequent injections of InsP, (Brown et al., 1984; Fein et al., 1984) and suggests that the inhibition of InsP,-induced calcium release that we have studied here is of physiological importance.

2000 1

40!: Q

1500

30

-1000

20

10

i 500

1

oi,i 0

1

5

b

Injections

of Ca

after

Ca

counts/s

mV

13500

5or

0 0

lo

) 60

10 15 20 time after injection (9)

5

L 10 15 20 time after injection (9)

Figure 6. The Ability of InsP, Depolarize the Photoreceptor Calcium

and Calcium following

-0

) 60

to Elevate [Ca*+], and a Prior Injection of

The Recovery of Sensitivity to a Second Injection of InsP3 Is Not Slowed When Extracellular Calcium Is Removed We wished to determinewhether the inhibition of the response to the second of a pair of InsP, injections is caused by calcium released from intracellular stores or by calcium that enters the cytoplasm through the ion channels that mediate the InsP3-induced depolarization. We also wished to determine whether the refilling of depleted calcium stores from the extracellular space played any role in the recovery of sensitivity to InsP3. We therefore investigated the time course of the recovery of sensitivity to InsP3 in cells that were bathed in seawater that had a reduced calcium concentration. The data in Figure 7 have appeared previously (Payne et al., 1988) and have been included to facilitate comparison with the data in Figures l-6. As in Figure la, paired injections of InsP3 were deliv-

(a) The peak depolarization (circles) and aequorin luminescence (triangles) resulting from injections of 100 PM InsP, are plotted against the time that had elapsed after a prior injection of 1 mM calcium. (b) The peak depolarization (circles) and aequorin luminescence (triangles) resulting from injections of 1 mM calcium are plotted against the time that had elapsed after a prior injection of calcium. The isolated points at the extreme right-hand side of each plot are the peak depolarization and aequorin luminescence resulting from injections delivered more than 60 s after any prior injection. All data were obtained from the experiment of Figure 5.

minescence resulting from a second injection of calcium were not greatly diminished by a prior calcium injection delivered 2 s earlier (Figures 5d, 5e, and 6b). On the contrary, at times less than 10 s after the first calcium injection, the peak aequorin luminescence

Table 1. Peak Aequorin of a Pair of Injections Paired

Luminescence and of InsP3 or Calcium

Injections

Peak depolarization (% control) Peak aequorin luminescence (% control) Number of cells injected

Peak

Depolarization

IP, after 35 f 20 10 f 7 5

IP,

Resulting IP, after 32 + 16 18 + 15 5

from

Calcium

the

Second Calcium a2 f 13 110 f 7 2

after

IP,

Calcium 83 f 134 f 4

after

Calcium

19 15

Peak depolarization and peak aequorin luminescence (mean f SD) were caused by an injection of either 1 mM calcium aspartate or 100 PM InsPj delivered 2 s after a prior injection of either calcium or InsP 3, as indicated in the top row of the table. The values without a prior injection. are expressed as a percentage of those caused by control injections of calcium or InsP3, which were delivered

mV 40 30

r -

20

-

0 0 A

* . 0

10

-

* . r .,

1 Figure7. Response Calcium

I

I

I

I

2

3

4

5

IIll

’ 16

I

17

,

18 s

TheTimeCourseoftheRecoveryfrom lnhibitionofthe to InsP, Is Unaffected by the Removal of Extracellular

The peak depolarization resulting from the second of a pair of injections of InsP3 is plotted against the time interval between the paired injections. Responses were recorded in calcium-free ECXA-ASW (squares) and in normal ASW, before (inverted triangles) and after (triangles) exposure to calcium-free ECXA-ASW.

ered, and a curve was obtained as in Figure 2a that described the recovery of the depolarization caused by the second injection (Figure 7). The artificial seawater (ASW) bathing the preparation was then switched to one in which calcium was replaced with 1 mM ECTA. After 25 min in darkness, paired injections of InsP3 were again delivered. The depolarization caused by the first injection increased from 30 to 36 mV (Figure 7), consistent with previous findings (Payne et al., 1986a). However, neither the extent of the inhibition of the response to the second injection nor the time taken for recovery was appreciably affected. Similar results were obtained in experiments on three other cells. We therefore find no evidence for the involvement of extracellular calcium in the inhibition of the response to InsP, or the recovery of sensitivity. Discussion The results provide direct evidence for the proposal that InsP3-induced calcium release is inhibited by prior elevation of [Ca2+]i (Payne et al., 1988), so that the calcium released by InsP3 in ventral photoreceptors feeds back to prevent further calcium release. Injections of InsP, and of calcium that elevate [Ca2+]i to a similar extent also similarly inhibit calcium release by subsequent injections of InsP3 (Figures 3-6). This result indicates that prior elevation of [Caz+]i is sufficient, by itself, to account for the extent and time course of inhibition. Depletion of InsP,-sensitive calcium stores need not be invoked to account for the observed inhibition. The time course of recovery of sensitivity to InsP3 and the dependence of the rate of recovery on temperature are similar after injections of calcium and InsP3 (Figure 2) and are unaffected by the removal of extracellular calcium (Figure 7). The latter result shows that calcium released from intracellular stores by the first injection of InsP3 is sufficient to inhibit the response to subsequent injections of InsP3.

The aequorin luminescence did not detect a small residual elevation of [Ca2+], that might have lingered after injections of calcium or InsP3, during the period in which lnsP,-induced calcium release was inhibited. It is possible that the large transient elevation of [Ca2+]r immediately following the injections triggered an inhibitory mechanism that did not require a residual elevation of [Ca2+]i to sustain its action. The calcium-induced phosphorylation of a protein, for example, might inhibit calcium release. The time course of inhibition would then be determined by the rate of dephosphoryiation, which might be independent of [Ca’“]i. Alternatively, the aequorin method might simply have been too insensitive to detect a residual elevation of [Ca2+]i. Both possibilities are consistent with the high sensitivity of the recovery rate to temperature. Both the active removal of calcium from the cytosol by Ca2+/Mg2+ ATPase activity (Rega and Garrahan, 1986) and the relaxation of some inhibitory mechanism might be highly dependent on temperature. Further studies using calcium-sensitive ion-selective micropipettes may resolve this issue. Feedback Inhibition May Terminate the Response to Injection of InsP3 and Cause Oscillatory Bursts of Calcium Release Inhibition of the response to injected InsP3 is rapid, being complete in the time that it takes for the depolarization caused by an injection of InsP3 to decay to baseline (Figures 1 and 2). It is possible, therefore, that inhibition, rather than metabolism of InsP,, limits the duration of the response to InsP,. Metabolism of InsP3 to inactive products might then occur more slowly, during the period of inhibition of the response to InsP,. If, however, metabolism of InsP, is incomplete for some reason, and the InsP3 concentration remains elevated after sensitivity returns, then further cycles of calcium release and desensitization might occur. This may explain the oscillatory bursts of depolarization that can continue for tens of seconds after large injections of lnsP3 into ventral photoreceptors (Corson and Fein, 1987; Payne et al., 1988) and which can continue for tens of minutes following the injection of inositol trisphosphorothioate (InsP&), an analog of InsPa that is resistant to metabolism (Taylor et al., 1989; Payne and Potter, 1990, Biophys. J., abstract). The 3 s to 10 s period between the bursts of depolarization is similar to the time taken for recovery from inhibition (Payne et al., 1988). Inhibition of InsP3-Induced Calcium Release May Prevent Depletion of Calcium Stores during Phototransduction Biochemical studies performed on other cell types suggest several sites at which calcium might act to inhibit InsP,-induced calcium release. The binding of InsP, to a receptor might be inhibited by calcium, as has been observed in microsomes from rat cerebellum (Worley et al., 1987), or calcium might block a calcium channel in the endoplasmic reticulum that is

Feedback 553

Inhibition

of Calcium

Release

opened by InsP3. Alternatively, calcium might greatly accelerate the rate at which InsP, is metabolized, preventing injected InsP3 from reaching its site of action. Stimulation of both InsPs-5-phosphatase and InsP,-3-kinase by calcium in extracts of various tissues has been reported (Sasaguri et al., 1985; Biden and Wollheim, 1986). However, preliminary experiments performed on Limulus ventral photoreceptors show that depolarization by InsPsS, is also subject to inhibition by prior injection of calcium or by illumination (R. Payne and B. V. L. Potter, unpublished data). Since InsP& is resistant to metabolism, it is unlikely that calcium inhibits the response to InsP& by increasing the rate of its metabolism. If InsP, is the messenger that mediates light-induced release of calcium, then the negative feedback loop that we describe in this paper will function to protect the photoreceptor’s stores of calcium from complete depletion during bright illumination. The release of calcium by light is thought to play a role in both adaptation and possibly excitation in this photoreceptor (Fein and Payne, 1989). The calcium feedback mechanism may be essential to maintain transduction with reduced sensitivity during or after exposure to bright light. Biophysical evidence suggests that negative feedback controls the sensitivity of the ventral photoreceptor to light (Stieve et al., 1986; Grzywacz and Hillman, 1988). The present results suggest a molecular site of action of a negative feedback control on the production of one intracellular messenger, calcium. Whether similar controls act on the production of other putative messengers in the visual cascade, such as InsPs or cyclic guanosine monophosphate (Johnson et al., 1986), remains to be seen. Experimental

Procedures

Recording and Stimulation Conventional methods for intracellular recording and for stimulating ventral nerve photoreceptors were used, as described elsewhere (Fein and Charlton, 1977; Millecchia and Mauro, 1969). Cells were stimulated with white light from a IOOW quartzhalogen source (model 6333; Oriel Corp., Stratford, CT), which was passed through a heat filter (Schott KC3, Ealing Optics, South Natick, MA), neutral density (ND) filters, and shutters before being focused onto the specimen plane. The intensity of light at the specimen, with no intervening ND filters, was 80 mW/cma. Light intensities are quoted in this paper as log units of attenuation relative to this intensity. To view the preparation with an infrared-sensitive video camera, cells were also continuously illuminated by an infrared beam, created by passing a second beam of light from the quartz-halogen lamp through an infrared filter (Schott RGIOOO) before focusing it onto the specimen. Control and Measurement of Specimen Temperature The ventral nerves were pinned into a plexiglass chamber, volume 0.5 ml, and ASW was passed through the chamber at a rate of 5 ml/min. Prior to entering the chamber, the ASW was cooled with a Peltier device (Interconnection Products Inc., Pompano Beach, FL). A miniature copper-constantan thermocouple was placed as close to the nerve as possible, and a digital thermometer (model DP30, Omega Inc., Stamford, CT) was used to record the temperature within the ASW. Observation Light from

of Aequorin Luminescence the preparation was collected

by an objective

lens

(L25xFL, 0.36 NA, Leitz, Wetzler, FRC) and projected onto a dichroic mirror (DC675LP, Omega Optical Inc., Brattelboro, VT) mounted at 45% to the light beam. Infrared light passed through the dichroic mirror and was focused onto the infrared-sensitive TV camera. Visible light including aequorin luminescence, which was reflected from the dichroic mirror, was focused onto the photocathode of a photomultiplier tube (R464, Hamamatsu Corp., Bridgewater, NJ). TTL pulses from the output of a photoncounting amplifier/discriminator (3470IAD6, Pacific Instruments, Concord, CA) were counted, placed into time bins, and continuously displayed by an IBM AT computer using ASYST software (ASYST Software Technologies, Rochester, NY) and a graphics accelerator card (CODASI, Dataq Instruments, Akron, OH). The membrane potential and stimulus monitors were also sampled by the same computer at a rate of 250 Hz and continuously displayed together with the photomultiplier trace. All data were stored after sampling onto the hard disc of the computer. Pressure Injection of Substances into Cells Rapid pressure injection of substances into cells through singlebarreled micropipettes was achieved as previously described (Corson and Fein, 1983). Double-barreled micropipettes were made from “theta” glass tubing (TGC 150-10, Clark Electromedical Instruments, Pangbourne, UK). After pulling the micropipette, the tips were broken under a microscope to obtain a tip diameter suitable for microinjection. The micropipettes were filled, and a fine platinum wire was inserted into the barrel containing InsP,, so as to record membrane potential. Polypropylene tubes were inserted into both barrels, and the tops of the barrels were sealed using low melting point wax. The polypropylene tubes were connected to pressure lines via electronically controlled valves for injection and the wire to an electrometer for electrical recording. Chemicals and Solutions All chemicals injected into the cells were dissolved in carrier solution (100 mM potassium aspartate, IO mM HEPES [pH %O]). Potassium aspartate and aspartic acid were obtained from Sigma Chemical Corp. (St. Louis, MO). All inorganic reagents were of analytical grade. lnsPl was obtained from Calbiochem (San Diego, CA). Calcium aspartate was made by the addition of stoichiometric amounts of Ca(OH)z to aspartic acid. The normal ASW contained 435 mM NaCI, IO mM CaCI,, 10 mM KCI, 20 mM MgC&, 25 mM MgS04, and 10 mM HEPES (pH 70). Calciumfree ECTA-ASW was made by replacing the CaCI, with 1 mM ECTA obtained from Sigma Chemical Corp. Recombinant aequorin was the generous gift of Drs. 0. Shimomura(Marine Biological laboratory, Woods Hole, MA), Dr. S. lnouye (Chisso Chemical Corp., Yokohama, Japan), and Dr. Y. Kishi (Dept. of Chemistry, Harvard University, Cambridge, MA). Recombinant aequorin was made by incubating recombinant apoaequorin (Inouye et al., 1985, 1989) with coelenterazine (Kishi et al., 1972; Musicki et al., 1986). For microinjection, the aequorin was dissolved at a concentration of 6.7 mg/ml in carrier solution containing 100 uM ECTA. Acknowledgments We thank Drs. 0. Shimomura, S. Inouye, and Y. Kishi for their generous gift of recombinant aequorin. We also thank Drs. Simon Levy and Laurinda Jaffe for their comments and Ms. Janice Briscoe for her proofreading. This work was supported by grants EY03793 and EY07743. R. P is an Alfred P. Sloan Research Fellow. Received

December

12, 1989;

revised

January

30, 1990.

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