Depolarization-induced calcium release from sarcoplasmic reticulum fragments. II. Release of calcium incorporated with ATP.

/ . Biochem., 79, 1067-1076 (1976)

Sarcoplasmic Reticulum Fragments II. Release of Calcium Incorporated without ATP Michiki KASAI and Hiroshi MIYAMOTO Department of Biophysical Engineering, Faculty of Engineering Science, Osaka University, Toyonaka, Osaka 560 Received for publication, October 23, 1975

Ca1+ incorporated in vesicles of sarcoplasmic reticulum fragments (SRF) by diffusion could be released rapidly by changing the ionic environment, by dilution from methanesulfonate (MS) to chloride. This ion exchange is considered to make the membrane potential of SRF inside-negative. Much faster release of Ca2+ was also observed upon osmotic change from high to low. These responses were very similar to the Ca!+ release from SRF after take up using ATP, but the release rate was slow in the case of anion exchange. The behavior of K + , Na+, sucrose, and inulin incorporated in SRF was followed upon similar treatment. These ions and molecules were not released upon ion exchange, but were immediately released by osmotic treatment. Therefore, the Ca+ release upon anion exchange was not due to the bursting of SRF, but to a direct effect such as a membrane potential change of the SRF. The behavior of anions such as Cl~ and propionate could not be followed by the same method because of the large permeability of these anions. It was also shown that Ca+ release upon ion exchange was not a direct effect of pH change. Liver microsomes did not show Ca+ release upon the same treatment as SRF.

In the previous paper (1), we examined Ca1+ to chloride by releasing Ca I+ . This anion exrelease from sarcoplasmic reticulum fragments change was considered to cause depolarization (SRF) after it had been taken up actively us- of the SRF membrane. This paper deals with ing ATP and showed that SRF responded to the release of Ca*+ from SRF, after its inanion replacement from methanesulfonate (MS) icorporated by diffusion without ATP. The purpose of this experiment was to Abbreviations: SR, sarcoplasmic reticulum; SRF, 'answer the following questions. Firstly, does sarcoplasmic reticulum fragments; MS, methanesul- Ca*+ incorporated by diffusion respond to exfonate; KMS, potassium methanesulfonate; EGTA, 'change of the ionic environment similarly to ethyleneglycol bis(2-aminoethyl)ether N.N.N'.N'- CaJ+ taken up by active transport? Secondly, tetraacetate. does anion exchange-induced Ca1+ release reVol. 79, No. 5, 1976

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Depolarization-induced Calcium Release from

M. KASAI and H. MIYAMOTO

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MATERIALS AND METHODS SRF was isolated by the method described in the previous paper (7). In this paper, almost all the experiments were carried out without ATP. All CaI+ in SRF was incorporated by diffusion unless otherwise specified. SRF was incubated at 4° overnight in a solution containing 0.3 M of various salts with 20 mM Tris-maleate (pH 6.5), 2 mM MgCl,, and 1 mM "CaCli+CaCU unless otherwise specified. The concentration of SRF was 15—30 mg protein/ml. The time course of Ca1+ release was determined as follows. The incubated samples were diluted 10- to 100-fold into a solution containing 0.3 M of various salts or no salt with 20 mM Tris-maleate (pH 6.5) and 2 mM MgClj. The final concentration of SRF was 0.2 mg/ml. After dilution, 1 ml aliquots were filtered through a Millipore filter and washed with 3 ml of the solution used for dilution. The amount of CaI+ left on the filter was determined by counting the radioactivity as described in the previous paper ( / ) . In order to determine the permeability of various ions or molecules such as Na+, K+, Cl", propionate", sucrose, and inulin, concentrated SRF was incubated with labeled compounds instead of Ca1+ and the time course of release of these substances was followed after dilution by the procedure used in the case of Ca I+ . The rate of_incorporation of CaI+ was also

determined. In this case, concentrated SRF with no Cal+ was diluted into a solution containing 45Ca and various salts as used for the release experiments. The amount of 45Ca incorporated into SRF was determined by the method used in the case of Ca1+ release. The apparent volume (^1/mg protein) for ions and molecules was defined as follows: lO'xfl rflDD

where C is the concentration of membrane (mg protein/ml) before dilution, R is the radioactivity in SRF extrapolated to zero time as shown in Fig. 1, and Ro is the radioactivity of the diluted suspension before filtration through the Millipore filter. 45 Ca was obtained from International Chemical and Nuclear Corp., U.S.A.; "Na, [carboxylic acid - 14C]inulin, [1 - 14C]propionate (sodium salt), and MC1 were from the Radiochemical Centre, England: "K was from the Japan Atomic Energy Research Institute, Japan; [14C]sucrose was from the Commisariat a l'Energie Atomic, France. Other methods were the same as in the previous paper (7). RESULTS Transient Increase of Can Release Rate from SRF upon Ion Exchange—Isolated SRF was incubated overnight in a solution containing 0.3 M KMS or 0.3 M KCl with 20 mM Trismaleate (pH 6.5), 2 mM MgClj, and 1 mM "CaCl,+CaCl, at 4°. Experiments were started by dilution of this SRF suspension into two kinds of dilution media containing 0.3 M KMS or 0.3 M KCl with 20 mM Tris-maleate (pH 6.5) and 2 mM MgClt. The final concentration of SRF was 0.2 mg/ml. After dilution, 1 ml aliquots were taken and filtered through a Millipore filter at appropriate intervals and washed with 3 ml of the solution used as the dilution media in order to remove remaining free CaJ+. Next, the amount of CaI+ in SRF was determined. The results in four cases, dilution from KMS to KMS, KMS to KCl, KCl to KMS, and KCl to KCl, are shown in Fig. 1. In the case from KMS to KCl, a transient increase of the / . Biochem.


quire the presence of ATP? Thirdly, how do ions and molecules which cannot be actively transported in SRF behave during the exchange of ionic environment? CaI+ incorporated in SRF by diffusion was also released upon exchange of ionic environment. The response was very similar to the anion exchange-induced Ca8+ release from SRF which had taken up Cal+ actively. However, in some respects this Cat+ release was different from that of actively transported Ca2+, especially as regards the release rate. Finally, the effect of pH on Ca1+ release was extensively studied. It was shown that this anion exchange-induced Ca!+ release was not a direct effect of pH change.

Ca l+ RELEASE FROM SARCOPLASMIC RETICULUM.

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Incubation medium

5

10

15

TIME(min) Fig. 1. Time course of Ca'+ release from SRF under various conditions. SRF (20 mg/ml) was incubated for about 12 hr at 4° in a solution containing 0.3 M KMS or 0.3 M KC1 in addition to 1 IDM "CaCl,+CaCl,, 20 mM Tris-maleate (pH 6.5), and 2 mM MgCl,. Ca t+ release was followed after 100fold dilution with a solution containing 0.3 M KMS or 0.3 M KC1 in addition to 20 mM Tris-maleate (pH 6.5) and 2 mM MgCli. The remaining Ca1+ percent was plotted against the time after dilution. • , dilution from KMS to KMS; O, from KMStoKCl; A, from KC1 to KMS; A, from KC1 to KC1. The amount of Ca I+ retained in SRF at zero time was about 6.5 //mole/g SRF in each case.

rate of Ca2+ release was observed. This response was very similar to that of CaI+ taken up using ATP, as described in the previous paper ( / ) . In other cases, the release of Ca1+ from SRF was slow and exponential. The rate constant indicates the permeability of SRF for Ca1+. However, the rate of Ca1+ release was small, when compared with the anion exchangeinduced Ca1+ release of Ca t+ taken up using ATP. This was shown by the following washing method. SRF incubated in KMS was diluted with KMS solution and washed with various solutions after filtration through a Millipore filter. In this case, as shown in Table I, Ca t+ release was very small. This is significantly different from the case with Ca t+ taken up using ATP. Similar experiments were performed at a salt concentration of 0.15 M. AS shown in Fig. 2, the amount of Ca1+ released by anion Vol. 79, No. 5, 1976

Washing medium KMS

KC1

No salt

lmM CaCl,

100»

73, 92, 98t>

91, 98, 91»

50 mM CaCl,

100*

62

51

1

Remaining Ca1+ after the washing is expressed as a percentage of the value obtained in the case of KMS washing. b Various values were obtained from different preparations.

100

TIME (mln)

15

Fig. 2. Effect of salt concentration on Ca1+ release from SRF. (A) SRF (22 mg/ml) was incubated on 0 . 1 5 M KMS, lmM "CaCl,+CaCl,, 20 mM Trismaleate (pH 6.5), and 2mM MgCl,. Ca t+ release was followed after dilution with 0 . 1 5 M KMS ( • ) , 0 . 1 5 M KC1 (O), or no salt ( x ) together with 20 mM Tris-maleate (pH 6.5) and 2mM MgCl,. The final concentration of SRF was 0.2 mg/ml. Other conditions were the same as in Fig. 1. (B) The same experiment as (A) except that the salt concentration of the incubation and dilution media was 0.3 M.


TABLE I. Ca1+ release from incubated SRF by washing. SRF was incubated in 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), 2 mM MgCl,, and various concentration of CaCl,. At 30 sec after dilution in 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), and 2 mM MgClt, a 1 ml aliquot was filtered through a Millipore filter and washed with 3 ml of the solution 0.3 M KMS, 0.3 M KC1, or no salt together with 20 mM Tris-maleate (pH 6.5) and 2 mM MgCl,.

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/ . Biochem.


exchange was smaller than in the previous case (0.3 M); however, the response was qualitatively the same. Therefore, 0.3 M solution was used to investigate various characteristics in more detail, though this differs somewhat from the physiological state. Ca1+ was also released from SRF by osmotic change. SRF was incubated in a solution containing 0.3 M KMS as before, and diluted with a solution containing only 20 mM Tris-maleate and 2 mM MgCl2. A rapid and transient release of CaJ+ was observed, as in the case of anion exchange, as shown in Fig. 2. The amount of CaI+ released by this treatment was smaller than that upon anion exchange. The small release of CaI+ upon osmotic change as compared with the Ca I+ release reported in the previous paper ( / ), may be due mainly to the difference of Ca*+ content in SRF. This 10 10 0 TIME (mln) point will be discussed in the following section. On the other hand, the rate of Ca1+ re- Fig. 3. Release of various ions and molecule from lease upon osmotic change is as rapid as that SRF. In each experiment, SRF (15-20 mg/ml) was found in the previous paper ( / ) . When SRF incubated in 0.3 M KMS, 20 mM Tris-maleate (pH was incubated in a solution in which the Ca!+ 6.5), and 2 mM MgCl, in addition to the labelled concentration was less than 1 mM, Ca2+ was ions or molecules. The release was followed after sometimes not released from SRF upon os- dilution with 0.3 M KMS ( • ) , 0.3 M KC1 (O), or no motic change, contrary to the case of Fig. 2. salt (X) together with 20mM Tris-maleate (pH 6.5) of SRF It appeared to depend upon some unknown and 2 mM MgCl,. The final concentration [was 0.2mg/ml. (A) release of K+. (B) release of factors in the preparation procedure of the Na + ; Na+ concentration during incubation was 10 sample. mM. (C) release of sucrose; sucrose concentration Figure 2 also shows that the Ca t+ perme- during incubation was 10 mM. (D) release of inulin ; ability decreased upon decreasing the salt con- inulin concentration during incubation was about centration. The slow Ca2+ leakage after the 1 mM. Before incubation, the mixture of SRF suspension and inulin was homogenized few min rapid release in salt-free media corresponds to in order to permit entry of inulin into the SRF, but the extreme case. the apparent volume for inulin was only 0.45 /*l/mg. Release of Inulin, Sucrose, Na+, and K+ from SRF— In order to elucidate the characteristics of Ca2+ release upon anion exchange, maleate (pH 6.5) and 2 mM MgCl,. The re+ + the permeation of various ions and molecules sults are shown in Fig. 3. K and Na bethrough SRF membrane was studied when the haved similarly. The molecules or ions were ionic environment or osmotic pressure was not released upon anion exchange from MS" changed. SRF incubated in a solution contain- to Cl", but almost all the radioactive substances ing 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), were abruptly released upon osmotic change. and 2 mM MgClj in addition to radioactive However, the amounts of radioactive molecules [14C]inulin, [uC]sucrose, MNa, or "K, was di- released upon osmotic change were small comluted into 50—100 volumes of diluted media. pared to those of the ions. We cannot exThe time courses of release of these molecules plain this difference at present. and ions were followed. Three kinds of diluAs a whole, contrary to the case with Ca1+, tion media were used; (1) 0.3 M KMS, (2) 0.3 neither ions nor molecules were released upon M KC1, (3) no salt, all containing 20 mM Tris- anion exchange, but they were abruptly re-

Ca I+ RELEASE FROM SARCOPLASMIC RETICULUM.

3000

10 TIME ( m l n )

Fig. 4. Incorporation of Ca ! t into SRF under various conditions. SRF (20 mg/ml) in 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), and 2mM MgCl, was diluted 10-fold with 0 . 3 M KMS ( • ) , 0 . 3 M KC1 (O), or no salt (X) in 20 mM Tris-maleate (pH 6.5), 2 mM MgCl,, and 1 mM '•CaCl.+CaCl,. 0.1ml aliquots were filtered through a Millipore filter and washed with 3 ml of 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), 2 mM MgCl,, and 1 mM EGTA. Other conditions were the same as in Fig. 1.

Vol. 79, No. 5, 1976

1071

course of Ca!+ influx was followed after dilution into three kinds of dilution media: (1) 0.3 M KMS, (2) 0.3 M KC1, (3) no salt. As shown in Fig. 4, a transient increase of Ca I+ influx was observed when MS" was replaced by Cl". The result was quite similar to the case of efflux upon anion exchange but the direction of CaI+ movement was reversed. Thus, it was concluded that this ion exchange caused an increase of the Ca2+ permeability of the SRF membrane in both directions. Ca£+ influx was also increased upon osmotic change. This is different from the case of efflux. It can be considered that SRF vesicles burst during osmotic change and then closed rapidly after the influx of Ca !+ . Dependence of Ca2+ Release upon Ca2+ Concentration in Incubation Media—The release of Ca2+ from SRF incubated in solutions containing various concentrations of Ca1+ was followed after 50- to 100-fold dilution into the dilution medium used in Fig. 1. As shown in Fig. 5, when the Ca!+ concentration was increased, anion exchange-induced Ca I+ release was reduced. Below 1 mM CaJ+, the response did not increase. On the other hand, the amount of CaI+ released upon osmotic change increased with increasing concentration of Ca I+ in the incubating medium. As an adsorption equilibrium between SRF and Ca2+ was attained during incubation, the internal free Cal+ concentration of SRF vesicles was considered to be approximately equal to the external one. Thus, on increasing the CaI+ concentration, the percentage of bound Ca l+ to total CaI+ incorporated must decrease, so the increase of Ca2+ release by osmotic response must be due to the increase of free Ca2+ in SRF. Therefore, it can be supposed that, in anion exchange-induced Ca2+ release, the SRF membrane does not release internal free Cal+ but some kind of bound Ca1+. Effect of External Ca2+ Concentration in the Dilution Medium on the Response to Anion Exchange—In the above experiments, the Ca I+ concentration of the dilution medium (outside of SRF) was not controlled. Thus, about 10 fiM Ca l+ was present outside the SRF when SRF incubated in 1 mM CaCU was diluted 100-fold. In this section the effect of the Ca1+


leased by osmotic change. This release was considered to be due to the bursting of SRF membrane. On the basis of these results, the replacement of MS~ by Cl~ caused an increase of permeability of CaI+ only through the SRF membrane. In addition, these results clearly show that anion exchange did not cause the bursting of SRF vesicles. Therefore it is clear that anion exchange-induced CaJ+ release is quite different from the osmotic change response, and its CaI+ specificity suggests that it may have physiological significance. Increase of Ca'+ Influx Rate upon Anion Exchange—All the experiments so far carried out dealt with the efflux of Ca!+ from SRF. Next, the influx of CaI+ into SRF during ion exchange and osmotic change was examined. SRF (20 mg/ml) was incubated in 0.3 M KMS, 20 mM Tris-maleate, and 2 mM MgClj, in the absence of Ca1+ at 4° for several hr. The time

II


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( m l n )

5OO0 •

5 10 DIVALENT CATION (mM)

Fig. 6. Effect of divalent cations on Cal+ release from SRF. SRF (14 mg/ml) was incubated in 0.3 M KMS, 20mM Tris-maleate (pH 6.5), 2mM MgCl,, and 1 mM 4*CaClf+CaCl|. After dilution with various concentrations of divalent cations in 0.3 M KC1, 20 mM Tris-maleate (pH 6.5), and 2mM MgCl,, the time course of Ca1+ release was followed. In the case of MnCl,, about 15 /IM Cal+ was always present. Zero concentration of divalent cations was obtained in 1 mM EGTA. The ordinate shows the Ca t+ remaining in SRF 2 min after dilution. ( • ) MnCl,; (O) CaCl,; the dotted line is a control obtained by dilution with KMS solution.

concentration outside the SRF is described. When SRF was incubated in 0.3 M KMS containing 1 mM or 10 mM CaClj, the CaI+ release rate in KMS (control experiment) was not modified when the CaJ+ concentration outside the SRF was changed from 10 ftM to 10 mM. However, the rate of Ca l+ release in KC1 was very much affected. As shown in Fig. 6, a very small amount of Ca t+ increased the rate, but higher concentrations reduced it. To clarify this acceleration effect due to external Cas+, the release experiment was carried out in the presence of EGTA. The release in KMS was not affected by EGTA, whereas Ca1+ release in KC1 was almost completely inhibited. These data are shown in Fig. 6. Rapid Ca l+ release in KC1 required the presence of a small amount of outside Ca1+, however, this is also different from the case of release of Ca1+ from SRF after take-up using ATP. In the latter case, Ca I+ was released by ion exchange even in the presence of EGTA. Inhibition of Anton Exchange-induced Ca2* Release—The effect of calcium spike inhibitors J.

Biochem.


TIME

Fig. 5. Effect of Cat+ concentration during incubation on CaI+ release. SRF (14 mg/ml) was incubated in 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), 2 mM MgCl,, and various concentrations of 45CaCI,+CaCl,. After dilution with 0.3 M KMS ( • ) , 0.3 M KC1 (O), or no salt ( x ) in 20 mM Tris-maleate (pH 6.5) and 2 mM MgCl, (the final concentration of SRF was 0.2 mg/ml), the time course of CaJ+ release was followed as in Fig. 1. Cal+ concentration during incubation was: (A) 1 mM; (B) 10 mM; (C) 100 mM. The total radioactivity of each sample was constant, so Ca1+ content at zero time corresponds to the apparent volume for Ca I+ .

Ca1+ RELEASE FROM SARCOPLASMIC RETICULUM. II

Effect of pH Change on Ca2+ Release—The following model of Cal+ release from SR or SRF has been proposed (3, 4). Membrane potential change causes the pH change of the vesicle; depolarization causes an increase of

1/2 to 1 pH unit under physiological conditions. At higher pH, the CaI+ permeability is larger and Ca1+ is released. This CaI+ release was sufficient to initiate muscle contraction. In this section, the effect of pH will be described in detail. First, the permeability to Ca1+ at various pH's was examined. SRF incubated at various pH's (5.5—8.5) under physiological salt conditions in the presence of Cal+ was diluted with a solution of the same pH. As shown in Fig. 7A, at high pH the Cal+ flux was very large, and slowdown of CaE+ release following initial rapid Ca£+ release was observed. Next, the effect of pH jump was examined. Figure 7B shows the results when SRF incubated at pH 6.5 was diluted with solutions of various pH's of the same salt compositions. It is evident that the time courses of CaI+ release in Fig. 7A are similar to those in Fig. 7B. This shows that pH jump has no effect, that is, the Ca2+ release profile seems to be determined by the pH at which Ca1+ release takes place, independently of the pH of the initial medium. This was confirmed when a sample solution incubated at high pH was diluted to different solutions of lower pH.

10 0 TIME ( m i n )

Fig. 7. Effect of pH on Ca1+ release from SRF. (A) No pH jump. SRF (23.8 mg/ml) incubated in 0.15 M KC1, 20 mM Tris-maleate, 2 mM MgCl,, and 1 mM 4aCaCl,+CaCl, of various pH's was diluted with 0.15 M KC1, 20 mM Tris-maleate, and 2 mM MgCl, of the same pH and Cal+ release was followed. The final concentration of SRF was 0.2 mg/ml. The pH values in the experiments are shown in the figure. (B) pH jump. The same SRF as in (A) was incubated medium at pH 6.5 and diluted with solutions of 0.15 M KC1, 20 mM Tris-maleate, and 2 mM MgCl, at various pH's, and Ca t+ release was followed as in (A). Vol. 79, No. 5, 1976


such as Co*+ and Mn t+ ( 2 ) on CaI+ release by ion exchange was examined. SRF incubated in a solution containing KMS was diluted with solutions containing various concentrations of Mn1+. The time course of CaI+ release was followed. The release of CaI+ in KMS was not affected by Mn1+, but that in KC1 was injhibited. In Fig. 6, the CaI+ radioactivity ref maining in SRF at 2 min after dilution in KC1 is plotted against the concentration of Mn t+ . 1 mM Mn*+ showed 50% inhibition of this response. Co1* was also inhibitory in the same concentration range. This effect of divalent cations is very similar to calcium spike inhibition. However, the presence of sucrose either in the incubation medium or in the dilution medium did not inhibit Ca2+ release. The effect of these two kinds of inhibitors on Ca1+ release from incubated SRF was different from that on CaI+ release from SRF which had taken up Ca1+ using ATP.

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1074

TABLE II. Ca 1+ release due to pH jump. A : Ca t+ was taken up under the conditions shown in Fig. 8. One minute after the initiation of Ca I + uptake the suspension was diluted 11-fold in 0.15 M KC1, 20 mM Tris-maleate (pH 6.5), and 2 mM MgCl. 1 ml aliquots were filtered through a Millipore filter and washed with solutions of various pH's of the same salt composition. B : Incubated SRF was diluted in pH 6.5 solution as shown in Fig. 7. 30 sec after dilution, 1 ml aliquots were filtered through a Millipore filter and washed with solutions of various pH's.

Ca2+ Release from Liver Microsomes—As in the previous paper ( / ) , the effect of ion exchange on liver microsomes was examined. Liver microsomes incubated with Ca£+ in KMS solution were diluted in various solutions and Ca1+ release followed. As shown in Fig. 9, Ca !+ was not released upon ion exchange from

100 -

Tl ME ( m l n )

Fig. 8. Effect of pH change on CaI+ release from SRF which had taken up Ca2+ using ATP. Cal+ was taken up in 0.15 M KC1, 20 mM Tris-maleate (pH 6.5), 300 fiM 4SCaCI,+CaCl2, 1 mM ATP, and 2.2 mg/ml SRF. One min after the initiation of Ca2+ uptake, the suspension was diluted 11-fold with solutions of various pH's and CaI+ release was followed as in Fig. 7.

100

pH of washing medium

10

Experiment •

a

TIME (mln)

5.5

6.5

7.5

8.5

A

102

100*

97

92

B

101

lOO*

95

92

Remaining Ca l+ content after washing is expressed as a percentage of the value obtained at pH 6.5.

Fig. 9. Ca*+ release from liver microsomes. Liver microsomes (21.3 mg/ml) were incubated for about 12 hr at 0° in 0.3 M KMS, 20 mM Tris-maleate (pH 6.5), 2mM MgCl,, and 1 mM "CaCI,+CaCI,. After dilution with 0.3M KMS ( • ) , 0.3 M KC1 (O), or no salt (x) in 20 mM Tris-maleate (pH 6.5), and 2mM MgCl,, Cal+ release was followed. J. Biockem.


Next, the rate of CaI+ release was estimated by the washing method, as in the previous section. SRF incubated at pH 6.5 was diluted with a solution of the same pH. After filtration through a Millipore filter, the filter was washed with the solution of different pH. Upon this treatment, only a small amount of Ca2+ was released, as shown in Table II. That is, the rate of Cal+ release was not as fast as the anion exchange-induced Ca t+ release described in the previous paper (1). Finally, Ca I+ release from SRF upon pH change after take-up using ATP was examined. SRF which had taken up CaI+ at pH 6.5 was diluted with solutions of various pH's. As shown in Fig. 8, the time courses of Ca*+ release were very similar to those in Fig. 7. In this case, Ca*+ release was also examined by the washing method. As shown in Table II, the Ca I+ release was not very fast in this case too. Thus, when the pH was changed, rapid and transient Ca2+ release similar to anion exchange-induced Ca1+ release from SRF after take-up using ATP was not observed. The release upon changing the pH was due to the high permeability for CaI+ at high pH. The Ca1+ release upon anion exchange was not directly caused by pH change.

Ca' + RELEASE FROM SARCOPLASMIC RETICULUM.

DISCUSSION In "RESULTS," it is shown that Ca t+ was released rapidly and transiently when the ionic environment of SRF was changed from KMS to KC1. This rapid release was due to the exchange of ionic environment because the CaI+ release rate in KC1 or KMS itself was slow. Before dilution the membrane potential can be considered to be zero if the Donnan potential due to the charges of SRF is neglected. Upon anion exchange, the membrane potential of SRF probably changes to become inside-negative, because the permeability of Cl" is considered to be larger than that of MS". It can be concluded that upon anion exchange Ca1+ was released from the bound state inside the SRF for the following reasons. When the osmotic pressure was decreased, many kinds of ions or molecules were released as a result of bursting of the membrane. These ions and molecules are considered to have been free in the vesicles. The release of CaI+ decreased when the CaI+ concentration was decreased. This may be due to a decrease in the ratio of free CaI+ to bound Ca t+ in SRF vesicles. This is consistent with the fact that vesicles release mainly free ions or molecules upon osmotic change. However, Ca t+ was specifically released when the ionic environment was changed. Moreover, the amount of CaI+ release increased with decreasing Ca1+ concentration when the ratio of bound Ca t+ was increased. These results are similar to the case of Ca I+ taken up using ATP, but a significant difference is the slow rate of CaI+ release. Many possible reasons Vol. 79, No. 5, 1976

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can be considered, for example; 1) In the case of Ca1+ taken up using ATP, the membrane potential is inside-positive, so ion exchange from MS" to Cl~ may cause a potential change from positive to negative. However, in the case of incubated SRF, the potential change may be only from zero to negative. The original state of membrane potential may affect the function of CaI+ release. To clarify this point, SRF incubated in KC1 was diluted in KC1, then filtered on a Millipore filter, and washed twice with KMS and KC1. However, in the second washing, no marked release of CaI+ was observed. Thus, this possibility seems unlikely. 2) The state of Ca I+ taken up using ATP may be different from that of CaI+ incorporated without ATP. 3) The chemical state of the membrane may be affected by ATP hydrolysis, for example, the existence of E-P complex (5) in Ca1+transporting molecules. In any event, the anion exchange-induced Ca!+ release was also specific to SRF because, in the case of liver microsomes, Cas+ release due to anion exchange was not observed. The effect of pH was studied extensively. As a result, it was found that the CaI+ release upon ion exchange was not a direct result of pH change of the SRF membrane. From the experiment shown in Fig. 3, the permeability of many kinds of ions and molecules can be estimated. In Table III, the TABLE III. Apparent volume and permeability for various ions and molecules. All data were taken from Figs. 3 and 5. Species

Condit :ions ,

pl/mg)

(min)

Permeability ratio

K+

1.8

1.

Na +

2.6

0.7

0.71 0.87 Sucrose 1.36 Inulin 0.45 Ca 1+ l m M CaCl, 8.7 b 10 mM CaCl, 4.5 b b 100 mM CaCl, 2.2

30< 30< 10 17 13

0.06> 0.06> 0.18 0.11 0.14

1 Permeability for K + was taken as 1. b Large K»p P for Ca' + is due to the binding of Ca*+ to SRF.


KMS to KC1, but was released upon osmotic treatment. Large amounts of Ca l+ were released upon osmotic change compared with Figs. 2 (B) or 5 (A). The apparent volume of liver microsomes for CaI+ was 1.39 /*l/mg in this case. This value is very small compared with that of SRF under the same conditions (8.7 /il/mg in the case of Fig. 5 (A)). Thus, liver microsomes must contain a high ratio of free Ca1+ in the vesicles. This may be the reason why a large amount of CaJ+ was released upon osmotic change.

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Finally, we have no direct evidence of the membrane potential change. However, none of the experimental results rule out the possibility of membrane potential change. The authors wish to thank Prof. F. Oosawa of their laboratory for valuable suggestions and discussions, and also members of his laboratory for their support. This investigation was supported by a research grant from the Ministry of Education of Japan. REFERENCES 1. Kasai, M. & Miyamoto, H. (1976)./. Biochem. 79, 1053-1066 2. Hagiwara, S. & Nakajima, S. (1966) J. Gen. Physiol. 49, 793-806 3. Nakamura, Y. & Schwartz, A. (1970) Biochem., Biophys. Res. Commun. 41, 830-836 4. Nakamaru, Y. & Schwartz, A. (1972) / . Gen. Physiol. 59, 22-32 5. Tonomura, Y. (1972) Muscle Proteins, Muscle Contraction and Cation Transport pp. 305-356, University of Tokyo Press, Tokyo 6. Duggan, P.F. & Martonosi, A. (1970) / . Gen. Physiol. 56, 147-167 7. Nilsson, R., Peterson, E., & Dallner, G. (1973) / . Cell BM. 56, 762-776 8. Wieth, J.O. (1970) / . Physiol. 207, 581-609

/. Bicchem.


relative permeabipties and the apparent volumes are shown. The apparent volumes for cations were about 1 ^1/mg. This value is small compared with the volume expected from other measurements (6"). This point will be discussed in detail in subsequent papers (Miyamoto et al., in preparation). For anions such as Cl~ and propionate", the apparent volume was very small (0.05—0.2 ^1/mg) and the permeability could not be determined. This small volume may be attributed to impermeability of the SRF vesicles ( 7 ) or alternatively to very rapid release of anions (8). The first possibility is not reasonable in the light of measurements of the exclusion volume for these anions. For anions which cannot enter the vesicles, the exclusion volume must be large. However, the measured exclusion volumes were not distinguishable from those for cations, so the permeability for anions must be very large. It could not be measured by the method described in this paper even at low temperature. These results are consistent with the view that SRF is anion-selective. SRF may be very similar in this respect to red blood cell membrane (

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