Ca entry at rest and during prolonged depolarization in dialyzed squid axons

Cell Calcium 3:

Ca ENTRY DIALYZED

19-41, 1982

AT REST AND DURING SQUID AXONS.

PROLONGED

DEPOLARIZATION

IN

DiPolo, R., Rojas, H. and Beaugg: L. Centro de Biofisica y Bioquimica, IVIC, Apartado 1827, Caracas lOlOA, Venezuela. *Institute M. y M. Ferreyra, CSrdoba, Argentina. (Reprint requests to R. DiPolo)

ABSTRACT

Ca influx has been studied in squid axons under inter nal dialysis control. In axons dialyzed with "normal" physiological conditions (Nai = 40-50 mM, Cal+ = 0.06-0.1 PM, ATP = 2 mM, Ki = 310 mM), 70% of the resting Ca influx is sensitive to external TTX (Ko.s = 5 nM), 20% of it can be accounted by the reversal of the Na-Ca exchange, and the remaining fraction (10%) is insensitive to TTX, D-600, and Nai. The Ca antagonic drug D-600 (50-100 PM) has an inhibi tory effect on the resting Ca influx. This coumpound was found to affect both the TTX sensitive and the Nai-dependent Ca influx components. In the presence of Nai and ATP, CaZ+ activates the carrier mediated Ca entry (Nai-dependent Ca influx). Most of the activation occurs in the submicromolar range of Cai+ concentrations (K0.a ~‘10.6 PM). In the absence of Nai and/or ATP, no activation of Ca influx by Ca!+ was found up to about 5 PM Ca?+. Prolonged depolariza tibn with high K, causes an increaie in Ca influx sustained for long time (minutes). Depolarizing the axons by removing This depolarization-induced Ca Ki causes the same effect. entry was only observed in axons containing Na.. In the absence of Nai, Ca influx decreases with increising K,. The activation of the carrier mediated Ca entry (electrogenic NafCa exchange) by membrane depolarization was found to be markedly dependent on the ICa*+li. Increasing the JCa*+li from 0.1 to 0.6 PM causes a ten fold increase in the extra Ca influx induced by a K-depolarization.

19

INTRODUCTION

It is normally held that, in many cell types, the "passive" influx of Ca down its electrochemical gradient occurs through specific gates or channels. However, the exact mechanism by which Ca crosses the membrane at rest is not well understood. A detailed analysis of Ca entry is of singular importance. First, because its magnitude represents the dissipation of the physiological Ca gradient which must be balanced exactly by an active outward movement of Ca (Ca pump), and secondly, because variations in the "passive" may induce important modifications in the level Ca of entry;aZ+, the i which is known to have considerable consequences on several cellular functions, (muscle contraction, neurosecretion, etc.). In principle, Ca may reach the intracellular medium via multiple pathways or channels: the existence of fast Ca channels (TTX sensitive) in excitable cells (l-2) suggest that at least part of the Ca "leak" could occur through "resting" Na channels. The presence of voltage sensitive, slow or late Ca channels (D-600, Co'+, and Mn2+ sensitive) to the resting Ca movement. (3-4), could also contribute Similarly, a reversed Na/Ca exchange could be part of the resting Ca influx (5-6-7). Reliable information on the relative contributions of each one of these pathways for Ca entry has been difficult to obtain in small cells, primarily because Ca influx is influenced by several factors including membrane potential, ionic and metabolic conditions (4-7-8). Furthermore, studies on the effect of membrane depolarization on Ca entry may be complicated by possible ionic redistributions (Naf, Cai2+, Kf) which could have side effects on Ca entry not related to membrane depolarization per se. In view of this, we have attempted to analyze Ca entry using squid axons under internal dialysis conditions which allow a more precise control of the intracellular medium, thus overcoming the above mentioned difficulties. The results obtained indicate that, in squid axons dialyzed with an internal solution closely resembling the physiological medium, between 65 to 70% of the resting Ca influx occurs via TTX sensitive channels, about 20% can be accounted for by the reversal of the Na/Ca exchange (Naidependent Ca influx) and the remaining fraction (lo%), penetrates by a route insensitive to Nai, Cat+, ATP, TTX and D-600 (basal Ca "leak"). The relative magnitude of these components was found to depend markedly on the composition of the internal of ATP, medium. At a constant Nai, and in the presence

20

increasing the ICi+l. try through the Na/C$

increases the magnitude exchange system.

of the Ca en-

Prolonged depolarization with Ko-rich or KimlOW solutions causes an increase in the Ca influx which is sustained This increase in Ca over long periods of time (minutes). entry was found to be completely depen'dent on the presence The extra Ca influx during prolonged memof internal Na. brane depolarization was found to be largely affected by the level of ICa'+li. This finding might have important physiological implications as a system which promotes a Ca< induced Ca entry.

METHODS

Most experiments were performed with live specimens of the tropical squid Dorvteuthis plei at IVIC-Caracas-Venezue A few experiments were performed on the species Loligo la. pealei at the Marine Biological Laboratory Woods Hole, USA. the giant axons of the squid were disAfter decapitation, sected from the mantle in artificial sea water. After the axons were mounted horizontally in carefully cleansing, a dialysis chamber. The general dialysis technique for influx (7) measure ments has already been described in detail. Porous glass capillaries were used to dialyze axons from Loligo (mean (D.plei) diameter 600 urn). For axons of smaller diameter constitu(mean diameter 410 pm), control of the axoplasmic ents could be achieved with porous plastic capillaries (9). In evaluating the performance of the dialysis technique, the extra Ca influx during electrical stimulation was deter mined in dialyzed axons of both L.pealei and D.plei and compared to those measured in intact axons from the same In dialyzed L.pealei axons, the extra Ca influx, species. average 6.7 + 1.9 f.mole.cm-?imoulse" due to stimulation. (n=4) from 10 mM Ca, (20';). This value, agrees rather well with those reported by Hodgkin and Keynes (1) in L. forbesi at the same temperature, and, when interpolated-to from that reported by Brinley 16°C (l), it is not different In dialyzed D.plei axons, the et al. (10) in L.pealei. was 6.2 If.1.5 f.mole. average Ca entry during stimulation (n=4) from 10 mM Ca, (19'C). This can be cm -2.impulse-' compared with a value of 9.3 f 2.4 f.mole.cm-*.impulse-' (n=5) measured in the same species using the axoplasmic In all dialyzed experiments in extrusion technique (11). which measurements of Ca entry during stimulation were made, the total jEGTA/ range from l-2 mM (free EGTA always >300 It is important to stress that no significant differuM). ence in the Ca transport properties were found between the two squid species used in the present work.

21

The standard dialysis solution had the following composition (mM): K, 310; Na, 40-50 (D.plei), SO-60 (L.pealei); Mg, 4 in excess of the ATP concentration; TRIS, 30; Cl, '98, Aspartate, 310; EGTA, l-2; Glycine, 330; pH (19'C) 7.3. Replace ment of Na, K or Mg was compensated with equiosmolar amounts of TRIS. The osmolarity was adjusted to 980 milliosmols. All internal solutions contained 10 pg/ml oligomycin. The ionized Ca concentrations were calculated using a Ca EGTA dissociation constant (KD), of 0.15 uM (0.3 ionic strength, PH 7.3) (12). Since the measured KD may be subject to errors inherent in the Arsenazo method, we have recently redetermined it (Vergara and DiPolo, unpublished results) using the method described by Bers (13) employing Ca selective electrodes (Neutral ligand Ca electrode). This procedure allows to determine the KA for Ca EGTA and the free Ca '+ for the actu al conditions used in our dialysis experiments. The KD value obtained by this method was 0.196 pM. Since the actual KD measured by this method is only 30% higher than the one reported previously, we have used the original figure of 0.15 pM to calculate the Ca2+ concentration of our dialysis solution. The standard artificial sea water (ASW) had the following composition (mM): K, 10; Na, 440; Mg, 50; Ca, 10; Cl, 580; EDTA, 0.1, pH (19'C) 7.6. The changes in K were made by simultaneous addition or removal of similar amounts of NaCl. For the experiments in which the external Ca concentration was varied, both the Na and Mg concentrations were kept constant at 300 and 20 mM respectively and TRIS used to adjust the osmolarity. The osmolarity of all external solutions was set to 1000 milliosmols. NaCN (1 mM) were always included in the external medium. All reagents used in the present work were analytical Radioactive solutions were made by adding solid grade. New England nuclear) directly to 45-CaC12 (15-30 mCi/mg. ATP (Vanadium the external solutions. (3 x lo* cpm/ml). free) was obtained from Sigma as TRIS salt, neutralized TTX with TRISOH and stored at -20°C as a 250 mM solution. was obtained from Sigma in 1 mg vials with citrate buffer. 101 uM stock solutions in distilled water were stored at D-600 (a kind gift of 5°C and used for several months. Professor Oberdorf of Knoll Ag. Ludwigshafer to Dr. Carlo Caputo) was made in ethanol (26 mg/ml) and diluted with The concentration of ethanol the experimental solution. was always less than 0.1% (v/v).

22

RESULTS

The effect of Nail of the resting Ca influx. and D-600. The mean resting Ca influx measured under nearly physiological conditions (Nat = 40 mM, Cai+ = 0.1 DM, ATP = 2 mM) was 117.3 f 5.5 f.mole.cm-2.s-' (f/CS) (sEM of n=25) from This value is similar to the values reported 10 mM Ca,. previously for dialyzed (7) and intact axons (l-5-7). Components

TTX

Figure 1 shows an experiment designed to explore the possible components of the resting Ca entry in axons dialyzed with a standard dialysis medium. After beginning the Ca influx increases slowly with time until it dialysis, The removal reaches a steady state level of about 113 f/CS.

310K-0.06yM

INT

4ONo

EGTA-2ATP

ONo IOCo - ASW IOOnM-TTX

IO Co-ASW

EXT

Co2+-I

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120l

T *

-

l

d E 0 al&? f 5

l

0

c4Q X 0

_e ,D-600

X

340IL zs

.P

,

O0

l

l

The effect of Nai, TTX and D-600 on the Ca influx Figure 1. in an axon dialyzed with near phy;io+ogical conditions. Ca influx in f.mole.cm .S- . Abscissa: time in Ordinate: The horizontal lines represent the different steadyhours. states of Ca influx obtained under various experimental conUnless otherwise stated, all solute concentrations ditions. Axon diameter 420 urn (D.plei). are in mM. 23

15% reduction of this value. of internal sodium produces Interestingly, addition of 100 nM TTX to the external radioactive medium causes a decrease in Ca influx to a value near 15 f/CS. This basal level of influx was not modified by addition of D-600 (75 uM) which is known to block the voltage sensitive slow or "late Ca channel" (4 >. As clearly shown in Fig. 1, the large inhibitory effect of TTX on the resting Ca influx is totally reversible. This effect, also occurs in the presence of Nai since, in four different experiments in which the effect of higher TTX cop centrations were tested, Ca influx decreased from a mean of 125 f/CS to 43 f/CS which represents an inhibition of 65%. It could be argued that the marked inhibition of the resting In order Ca influx by TTX is only seen in dialyzed axons. to examine this possibility, Ca influx was measured in intact axons both in the presence, and in the absence, of TTX. In the presence of external TTX (300 nM), Ca influx decreases from 103.7 f 6.2 f/CS (n=8) to 46 + 5.1 (n=4), thus reproducing the observation in dialyzed axons. Figure 2 shows a dose response in an axon dialyzed free of internal

3lOK-0

INT

IpMCd+-I

curve for TTX obtained Na. TTX, at a concen-

EGTA-2ATP

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2

3

4

TIME

(hr)

The effect of different TTX concentrations on Ca Figure 2. In order to eliminate the Nai-dependent component, influx. Nai2was substituted by TRIS. Ordinate: Ca influx in f.mole. Abscissa: time in hours. .s-? The Ko.5 is close to imnM. Axon diameter 400 pm (D.plei). 24

of the Ca tration of 5 nM, produces nearly 50% inhibition 50 nM TTX is able to produce nearly, maximal inhiinflux. in the TTX concentration bition, since further increases (100 and 300 nM) have no effect on the basal level of the influx. As shown at the end of the experiment, addition of 90 mM Na to the internal medium produces an increment in the Ca influx similar in magnitude to that found in the absence of TTX (7), thus indicating that TTX has apparently no effect on the Nai-dependent Ca influx. Figure 3 shows an experiment in which the effect of D-600 (50 uM) was examined in an axon superfused without TTX and internally perfused with standard media containing first ONa, and then 90 mM Na. In the absence of internal

310 K- 0 06 yM Ca2+ I EGTA-2

INT

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2

3

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4

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TIME (hr)

Figure 3. The effect of D-600 on both TTX sensitive and Ca influx components. Nai- dependent Ordinate: Ca influx in f.mole.cm-2.s-'. Abscissa: time in hours. Observed that D-600 partially inhibits both components of Ca entry. Unless otherwise stated, all solute concentrations are in mM. Axon diamter 410 pm (D.plei).

25

The adNa, Ca influx reaches a steady value of 60 f/CS. dition of D-600 causes a small (17%) drop in the Ca influx. Interestingly, addition of 90 mM Na to the dialysis medium in the presence of D-600 causes only a partial activation of Ca influx, since removal of the drug from the external medium reactivates the Ca influx to a much higher value. At the end of the experiment, 300 nM TTX were added externally producing the described inhibitory effect. It should be pointed out that, in these experiments D-600 has been used at concentrations similar to those used by other authors to inhibit the Ca entry in other preparations (14-15). In experiments not shown, higher concentrations of D-600 did cause greater inhibition of Ca influx in the absence of TTX. However, at these higher concentrations, D-600 also caused progressive inhibition of the action potential. It seems likely, therefore, that the inhibition of Ca influx by D-600 has an "unspecific" effect on both, TTX sensitive channels, and Na/Ca exchange mechanism. In support of this view, is the finding (Fig. 1) that, in the presence of external TTX and in the absence of internal Na, D-600 causes no further inhibition of the Ca influx.

The

activation

of Ca influx

by

Ca'+ 1

A recent finding related to the effect of internal cal cium on Ca fluxes in squid axons, is that the level of Ca2+ k not only control the magnitude of the components of the outward movements of Ca (Ca "pump" and Na/Ca exchange) (16,17) but it also affect the magnitude of the Ca influx (Nai- dependent component) which occurs through the NafCa exchange mechanism (7). In the experiment of Figure 4, the effect of different lCa'+li on Ca influx was explored in an axon dialyzed with, and without, ATP. Prior to the beginning of the dialysis, the axon was incubated in artificial sea water containing 1 mM NaCN. This treatment is known to reduce the ATP levels to a value close to 100 ~.IM(18). With no ATP in the dialysis medium, increasing the ionized Cai up to 0.6 pM causes This behaviour no effect on the steady level of Ca influx. contrasts to that found in the nresence of ATP. At 0.3 ~_IM Ca influx increases by a factor of two from the Ca?+ stkad; value obtained at 0 Cal+, and by a factor of 4 when the Cal+ is rised to 0.6 PM. The result of several experiments designed to explore the dependance of the Ca influx on jCa!+\, in the presence Up to 0.1 pM Cal+ and absence of ATP, is shown in Fig. 51 there is little activation of the Ca influx by Caf+. Clearly most of the increase in Ca?+occurs in the submicroThe activamolar Ca concentration range (Ko.5 2 0.6 PM).

26

I

2

3

1

1

I

1

4

5

6

7

TIME ( hr)

Figure 4. The effect of ICa*+/' on Ca influx. The axon was preincubated in ASW containing 1 mM NaCN. Ordinate: Ca influx in f.mole.cm-2.s-'. Abscissa: time in hours. In the first part of the experiment, the effect of inis ex lored in the absence creasing the (Ca'+li on Cainflux s Note the lack of effect of Cai+ on the influx of ATP. level. In the second art (Note the change in scale) of the experiment, different PCa'+j i were tested in the presence of 2 mM ATP. Axon diameter 575 pM (L.pealei).

tion of the Ca influx by Ca.2+ observed in the presence of ATP, is totally dependent 0: ;he presence of Nai ions in the dialysis medium (7), thus showing that this effect is mediated through the Nai-dependent Ca influx component. The possibility that the activation of the Nai-dependent Ca influx by Calf is due to an inhibitory effect of free ECTA is unlikely, since, in dialyzed axons, an increase in the free /EGTA[ at constant ICa2+li causes no effect on Ca influx Figure 5 also shows that, in the absence of ATP, (7). increasing the Cal+ up to about 5-10 pM Ca2+ causes no changes in the Ca influx. The increase in Ca influx observed under conditions of very high Caz+ (> 10 PM), and in

27

the absence of ATP, is not apparently related to the presence of Nai, since its removal (experiments not shown) causes an increase rather than a decrease in the Ca influx. A possible explanation for this effect is that this component could represent the manifestation of a Ca/Ca exchange which is known to be present under conditions of elevated ICa2+(i (19).

6oo-

~Col,

_ [Noli=

q

IO mM

+ ATP

5OmM

7 f u 400-

I

I

I

I

0.1

I

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I

100

I

loo0

(#A)

Ca influx as a function of ICa 2+ I. in axons Figure 5. Ordinate: Calinflux in dialyzed with and without ATP. Ionized Ca concentrations in f.mole.cm'2.s-'. Abscissa: Black dots (o), the dialysis fluid (logarithmic scale). Open steady-state Ca influx in the presence of 2 mM ATP. circles (o), steady state Ca influx in the absence of ATP. The number in parenthesis refers to different experiments. The bars represent the standard error of the mean (SEM). The value was 50 mM, The Na. for all of these experiments of Ca influx in the nominal absence of Ca?+. both in the toithe closed presence or absence of ATP, correspond circle (0) on the ordinate line.

28

Ca influx in response rich solutions.

to prolonged

depolarizations

with

K,-

Squid axons, as well as other preparations, show an increase in Ca entry in response to prolonged depolarizations induced by K ions (2). Since exposure for long pe(several minutes) to K-rich solutions might riods of time induce secondary changes in the intracellular ionic composi tion, it could be thought that the net Ca uptake observed under this conditions might not only reflect changes in mem brane potential, but also variations in the intracellular medium. In order to examine the factors involved in Ca permeability increase during prolonged depolarizations, we have studied the effect of internal and external K ions on Ca influx under conditions of internal control of NaZ, Cai+ and ATP. In few experiments, the effect of K, on membrane potential was measured in dialyzed axons. No significant differences were found from those previously reported in intact axons from the same species (20). Figure 6 shows the effect of 100 mM K, on Ca influx Increas in an axon dialyzed with 0.1 pM Cazf and 2 mM ATP. ing the K, from 10 to 100 mM (Na was substituted by K) promotes an increase in the Ca influx from 115 f/CS to a steady value of 190 f/CS. This effect of K, is partially reversi ble. The Ca influx stimulation by K, is most probably dueto an effect of membrane depolarization and not to a reduction of Na, since, in control experiments, Ca influx was not affected by reducing the Na, from 440 to 300 mM. In fact, Requena et al. (21) found no effect of Na, on Ca entry The upon decreasing its concentration from 440 to 180 mM. fact that prolonged depolarization induced by removal of Ki (experiments not shown) also caused an increase in Ca influx, strongly suggests an effect of membrane potential on the extra Ca influx observed in axons superfused with high Ko. A rather unexpected but interesting finding, is the progressive decrease in Ca influx observed after prolonged depolarization in axons dialysis without internal Na. This effect is illustrated in Fig. 7. After starting the dialysis, Ca influx increases to a steady level of about 83 f/CS in the absence of Nai. Changing the external medium to one containing 100 K causes a small transient increase in the Ca influx, followed by a decrease in the influx level to about 50 f/CS. Further increases in the external K+ concen tration, first to 200 and then to 450 mM, causes the Ca in-flux to decrease progressively to 35 and 20 f/CS respectively. In experiments in which small increases in K, (2550 mM) were tested on Ca influx, it was found that, in the presence of Nai, this treatment always induced an increase in Ca influx. However, in axons dialyzed without Nai, the effect was rather variable.

29

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g = 410JJm

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(hr)

Figure 6. The effect of prolonged K, -depolarization on the Ordinate: Ca influx in the presence of internal Na ions. Ca influx in f.mole.cm-2.s-'. Abscissa: time in hours. The Note the changes in the external solution. arrowsindicate the reversibility of the K effect. Axon diameter 370 uM (D.plei).

The effect of prolonged depolarization with K, on Ca in axons influx in the absence of Nai was also reproduced in which depolarization was achieved by decreasing the IK(i* This is shown in Fig. 8 and strongly points to an effect of rather than to an effect of K+ membrane depolarization, per se. Experiments were also carried out to explore whether the level of intracellular Ca2+ affect the Na.-dependent Fig. 9 extra Ca influx caused by membrane depolariza *-Ion. shows an experiment in which the effect of 100 mM K, upon the Ca influx was explored in an axon dialyzed with two different jCa2+j.. In order to enlarge the Nai-dependent Ca influx compon$nt, the internal sodium was purposely raised

30

INT

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The effect of prolonged K depolarization on the Figure 7. Ordinate: Ca influx in the absence of internal Na ions. time in hours. The Abscissa: Ca influx in f.mole.tm-2s". arrow indicates the changes in the external solution. Internal sodium was renlaced isosmoticallv bv TRIS ions. are in mM. Unless otherwise stated, all concentrations Axon diameter 410 uM (D.plei).

After the Ca influx had reached a steady state to 90 mM. of 135 f/CS in the presence of 0.1 yM Ca?+, increasing the external K+ to 100 mM caused a 2-3 fold kncrease in the Ca Similar to the experiment of Fig. 7, this effect influx. was reversible after restablishing the normal lOK-ASW. In the second half of the experiment, a similar protocol was repeated but in the presence of a higher Ca.+ concentration (0.6 PM). Increasing the internal Ca from 6.1 to 0.6 PM, in the presence of lOK-ASW, causes the expected activation Under of the Nai- dependent Ca influx (see also Fig, 4). these conditions, depolarization with 100 K, caused a much larger increase in the Ca influx to a value almost 15 times higher than that obtained in the presence of 0.1 PM Ca2+

31

(lOK-ASW). This experiment indicates that the magnitude of the extra Ca entry, induced by a prolon ed depolarization, is clearly affected by the level of ICa ri +'I-, In fact, the of O.luM extra Ca influx, induced by 100 K, in the $resence Cal+, was only 165 f/CS as compared with 1400 f/CS seen at This activating effect of ICa'+li on the ex0.6 UM Ca?+. induced by membrane depolarization, is only tra Ca in*lux that this observed in axons containing Nai, thus indicating phenomena occurs mainly through the reversal of the Na/Ca exchange mechanism.

ONo-0.06yM

INT

3lOK

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1

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-

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3

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Effect of removing internal K on Ca ingluxlin Figure 8. f.mole.cm.s- . Ordenate: the absence of internal Na. The arrows indicate changes in time in hours. Abscissa: Internal K ions were replaced isosthe internal medium. Axon diameter 400 uM (D.plei). motically by TRIS.

32

3lOK-90Na-

INT EXT

O.lyM ASW IO K

I

I EGTA-2ATP

Ca2+

ASW IOOK

0.6yM ASW IO K

I

Ca2+ ASW ’ IOOK

1 ASW IOK

Figure 9. The effect of ICa2+li on the K, -stimulated Ca f.mole.cm'2.s'1. influx. Ordinate: Abscissa: time in Note the large extra Ca influx in 0.6 ~.IMCa?+, as hours. compared to that at 0.1 PM in the presence of lOOK$: Axon diameter 373 Urn (D.plei).

DISCUSSION

The

resting

Ca influx

In this work,

we have

studied

33

the properties

of the

resting or "passive" Ca influx in squid axons under proper control of the intracellular medium. A mean resting Ca influx of 117 f/CS from axons bathed in 10 mM Cao and dialyzed under "normal" physiological conditions (Nai = 40 mM; Cal+= 0.1 PM; Ki = 310 mM; ATP = 2 mM) is in remarkable agreement with previous reported values for both intact (1,5,7) and dialyzed axons (7). An interesting finding is that between 60 to 70% of the resting Ca influx in squid axons goes through a "channel" which is sensitive to the presence of external TTX. This contrasts with previous reports on squid axons (22,23), according to which TTX did not appreciably change the level of Ca influx. A likely explanation for this apparent discrepancy might lie in the much higher reso lution in Ca influx obtained by the presence technique (7). The fact that TTX also blocks about 50% of the "resting" Na influx (24,25,DiPolo and Beaugb, unpublished) strongly suggests that a "resting" common channel is shared by both Na+ and Ca2+ to enter the axon. The remarkable similarity in the Ko.5 (5 nM) for the inhibition of both resting and voltage activated Na channels (26) by TTX, suggests that these two operational pathways might, indeed, represent the same molecular entity. On the of both resting and activated other hand, the selectivity Na channels for Na and Ca ions appears to be somewhat difCa influx, measured for In fact, the TTX sensitive ferent. an ASW containing 10 mM Ca,, is about 0.075 p.mole.cm-*.s-' (P/CS) (see Fig. 1, and text), which is about 0.0075 P/CS This may be compared with the TTX per mM-Ca outside. conresting Na influx of about 16.4 P/CS from a solution to about 0.036 P/CS taining 450 mM Na, (23), corresponding Taking into account that the inward per mM-Na outside. force exerted by the membrane potential on a Ca ion is twice as great as that for a sodium ion, from the constant to calculate the apfield equation (27,28), it is possible parent permeability coefficients for these two ions across the TTX sensitive channel (for this computation a resting The calculated membrane potential of -60 mV was used). channel turns out to be 0.11. It PCa/PNa for the "resting" that for the case of activated is interesting to notice, sodium channels,the PCa/PNa reported are: 0.1 for voltage clamped perfused squid axons (29), and 0.01 for aequorin Although perfused axons may behave injected axons (2). differently from intact injected axons, it is worth pointdialyzed and perfused axons ing that the similarity between since in our work we measured restmay be only fortuitous ing permeability ratios while Meves and Vogel (29) measured The difpermeability ratios of activated Na channels. ference in selectivity between perfused and injected has been explained in terms of differences in axons time of measurements of the reversal potenionic strengh, tial for sodium and methods used in the calculation of the At present we have no data on permeability ratios (29). axons for the activated the PCa/PNa ratio in dialyzed

34

sodium channel. It is interesting to point out that the activation of TTX sensitive channels by certain toxins (30) which hitherto have been considered to represent the voltage sensitive Na channels, showed a similar selectivity between Na and Ca as that found here. If the TTX sensitive resting channels are responsible for most of the Ca entry during physiological conditions, then alterations in their permeability would certainly unbalance Ca "passive" leak and active Ca extrusion, thereby leading to changes in the level of the ICa'+li. For instance, the antiepilectic drug diphenylhydantoin is known to block the resting Na channels in squid axons in the same way as TTX does (31). It is therefore conceivable that a decrease of Ca influx across this pathway induce by this drug (DiPolo, unpublished) could explain some of its pharmacological effects. Similarly Honerjager et al., (32) have shown that the cardiotonic drug AR-L57 decreases the light emitted by aequorin injected squid axons. Since this drug also inhibits the veratridine induced TTX sensitive Na efflux (same authors), it is likely that the inhibition of "resting" TTX sensitive Ca channels could partially contribute to the observed decrease in the ICa'+li. The finding that only about 20% of the resting Ca entry, measured under nearly physiological conditions, is carried-mediated (reverse of the Na/Ca exchange), is consistent with the low activati n of this mechanism in axons 4 in which the intracellular Cai + is near 0.1 UM (16,17).The remaining fraction of the resting influx (lo%), insensitive to changes in ionic and metabolic conditions as well as to the Ca antagonic drug D-600, most likely represents an "unspecific" leakage pathway. at present, the However, existance of other specific routes for Ca entry cannot be discarded. The experiments with the Ca antagonic agent D-600 are of special interest, since there seems to be little doubt that the slow "late Ca channel", does not seem to serve as a resting "passive" pathway for Ca entry. In fact, in the presence of TTX and in the absence of internal Na, this drug has no effect on the Ca influx. As already mentioned, the partial inhibitory effects of D-600 on both the TTX sensitive Ca entry and the Nai-dependent Ca influx, could probably be attributable to an "unspecific" anesthetic effect of the drug observed at the concentrations used in these experiments (50-100 uM). Nevertheless, the inhibitory effect of D-600 on Ca influx should be taken into account when searching for the existence of Ca "channels" in preparations in which no control of the internal medium can be achieved.

35

Intracellular system.

Ca

2+

activates

Ca entry

by

the Na/Ca

exchange

With regard to the Na/Ca exchange mechanism, a classical carrier mediated Ca counter transport system could in theory be able to cause: Na,-dependent Ca efflux, Ca,dependent Ca efflux, Nai-dependent Ca influx and Ca,-depend ent Ca influx. The way in which Cai+ affect these Ca movements is of interest, since several Ca mediated processes are accompained by a rise in the Cat+ concentration. The experiments of Figs. 4 and 5 confirm and extend the finding (7) that the level of ICa'+li modulates the magnitude of the Nai-dependent Ca influx. An interesting property of this transconcentration effect of Caf+ (Transactivation) is that it is only observed in axons dialyzed with Nai and ATP. A similar metabolic dependence has been observed for the ouabain insensitive Ca,-dependent Na efThe fact that the Cai-stimulated Ca influx flux (5,33). is dependent entirely on Nai and ATP, strongly indicates that this phenomena is intimately related to a membrane and not a side effect of the rise in the transport process, Ca"+ in the cytosol (see also 7). As already mentioned, the small but significant increase in the Ca influx observed in the absence of ATP and at extremely high Cat+ (see Fig. due to a small Ca-Ca exchange component, 5), is most probably since removal of Nai causes an increased in Ca influx. From the relationship between the Nai-dependent Ca influx and ICa'+li, it is apparent that most of the activating effect of Cai'+ occurs in the range of submicromolar Ca?+ concentrations. The Ko.5 for this gffect (0.6 PM) di?Ffers significantly from that of the Ca.-k activated Na,dependent Ca efflux (15 PM) under similariexperimental conditions (17). This finding, and the fact that the Naidependent Ca influx is totally dependent on ATP (7), makes deviate significantly from this component of the Ca influx the classical reverse of a counter transport mechanism. These additional features of the Ca entry in exchange for when developing Nai should be take into consideration models for the Na-Ca exchange mechanism. The activation of the Ca entry (Nai-dependent Ca inconsequences on flux) by ICa*+(i might also have important the net Ca extrusion via the NafCa exchange system. In at high ICa*+(i (+lO PM), the magnitude of fact, although Ca efflux is much larger than that of the the Na, -dependent at submicromolar ICa*+li the Na.-dependent Ca influx, (17) re !t ative magnitude of both unfdirectional Ca fluxes will certainly tend to decrease the net Ca extrusion by the This consideration further supcoupled Na-Ca mechanism. ports the idea that the Na/Ca exchange mechanism is mostly effective as a Ca extrusion system when the ICa*+li reached micromolar concentrations (17).

36

Ca entry during prolonged depolarization It is well established that in several preparations the uptake of Ca is greatly increased in the presence of a Since high concentration of external potassium ions (4-8). this experimental manipulation has been widely used for the it in axons study of Ca entry, in this work we have analyzed in which proper control of the internal medium could be achieved by internal dialysis. The experiment of Figure 6 reproduces the common observation that K-depolarization increases Ca influx in squid This steady increase in Ca entry could be the axons (4). Ca channels which do result of: opening of voltage dependent Ca inward movement through the electrogenic not inactivate, Since the Na/Ca exchange system, or a combination of both. most commonly used Ca antagonist agent D-600 can affect both systems (3, see also Fig. 3), its used could lead to mislead ing information about the origin of the depolarization in- An easy way to avoid this difficulty is to duced Ca entry. eliminate completely the entry of Ca through the Na/Ca exchange by removing the Na ions from the internal dialysis medium. Figure 7 shows that under this condition, K-depolarization fail to induce the expected steady increase in Ca influx, thus indicating that most of the K-stimulated Ca entry, during a steady prolonged depolarization, occurs through the reversal of the Na/Ca countertransport system. This observation agrees with that of Mullins and Requena using aequorin inyected squid axons from L.pealei (32). An interesting finding, that could be related to the present work, is that depolarization (K ions) induced Ca uptake in isolated cardiac muscle membranes involves vesicles that have the ability to promote Na/Ca exchange (35). It is necessary to emphazise that the K,-depolarization dependent Ca entry studied in this work, only concerns to those mechanism able to maintain a steady Ca entry during prolonged depolarization. In fact, with the resolution of the present flux measurements (minutes), earlier events ("late Ca channels") cannot be visualized since they inactivate in few seconds (2,4,23). An interesting observation, is that in axons dialyzed without internal Na, increasing the external K or decreasing the internal K, causes a reduction in the Ca influx (see Figs. 7 and 8). Since, in the absence of Nai (absence of Nai-dependent Ca influx), nearly 80% of the remaining Ca influx is blocked by TTX, this finding could be explained by a decrease in the TTX sensitive resting Ca channel permeability, with increasing depolarization.

the is the and

In line with the observation that (Ca2+(. activates reversal of the Na/Ca exchange (Nai-dependekt Ca influx), the result of Fig. 9, which shows that the magnitude of K-stimulated Ca influx observed in the presence of Nai ATP, is markedly enhanced by increasing ICa2+li. In 37

fact, an increase in Ca.i2+ from 0.1 to 0.6 PM produces a ten fold increase in the extra Ca entry induced by K ions. This finding further su ports the idea put forward that the 1: on the entrance modulating effect of Cai+ of Ca is directly related to a process (Na/Ca exchange), occurring at the level of the membrane. This observation, might have important physiological implications. If the observed dependence of the Nai-Ca, exchange on Caiz+ occurs also in other excita ble preparations, such as cardiac muscle fibres, then Ca entry, via voltage dependent Ca channels (increased in Ca?+ ) could contribute to further activate Ca entry via the Na?Ca exchange during the plateau of the action potential. An interesting phenomenon which could be related to the Cai induced Ca entry is the result reported by Baker et al., from (2), in squid axons, that the rate of light emission aequorin in response to a constant depolarization increases slowly over a period of hours. A_possible_ex~~~n~ifo~n~or this effect is that the moderate increase in under those conditions might trigge: a INaIi occurring larger Ca entry through the Na/Ca exchange upon depolarizaSmith and Zucker (36) have given clear tion. Recently, evidences that although no facilitation of the inward Ca current occurs following a train of depolarizing pulses, there is a continuous rise in submembrane Cai2+ concentration. In the light of the present ex eriments one might speculate that the existence of a Caiz$ -induce Ca entry (Na/Ca exchange) although not manifest as an inward current from it could contribute to the increase in residual Cai+ This process might be imprevious activity (summation). portant for the mechanism of synaptic facilitation (37). further investigations are needed to estaNevertheless, blishe whether a Cai-induced Ca influx found in squid axons, Na/Ca is also present in other preparations exhibiting exchange. ACKNOWLEDGEMENT

We wish to thank C. Caputo for fruitful discussions and comments on the Manuscript. The expertise of Isabel Otaegui in preparing the manuscript is acknowledged. This work was partially supported by grant NSF-USA-BNS-80-25579 and PNUD/UNESCO RLA 78-024.

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The role of calcium Journal of Physiology.

Received 14.10.81 Revised version received and accepted 20.1.82

41

The 5,5-