- Email: [email protected]
Phosphatidylinositol 4,5-bisphosphate enhances calcium release from sarcoplasmic reticulum of skeletal muscle
Vol. 163, No. 3, 1989 September 29, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1487-1491
PHOSPHATIDYLINOSITOL
4,CBISPHOSPHATE ENHANCES CALCIUM RELEASE
FROM SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE Masaki Kobayashi*, Akiko Muroyama and Yasushi Ohizumi Mitsubishi Kasei Institute of Life Sciences Minamiooya, Machida, Tokyo 194, Japan Received
August
16,
1989
SUMMARX In the course of our study on the function of sarcoplasmic reticulum (SR) in skeletal muscle, the stimulatory action of phosphatidylinositol 4,5bisphosphate (PIP2) on the Ca2+ release from SR was demonstrated by using chemically skinned fibers and fragmented SR vesicles. PIP2 induced a tension spike followed by sustained contraction in skinned fibers. PIP2 enhanced the caffeineinduced Ca2+ release from SR vesicles at low concentrations and triggered Ca2+ release by itself at high concentrations. PIP2 also enhanced 45Ca2+ efflux from SR vesicles. However, inositol 1,4,5-triphosphate never produced these effects. The Ca2+-releasing action of PIP2 was only weakly affected by ruthenium red or procaine. These observations suggest that PIP2 activates an SR Ca2+ release channel whose properties are different from those of the Ca2+-induced Ca2+ release channel. 0 1989 Academic Press, Inc.
The Ca2+ release from sarcoplasmic reticulum (SR) plays a key role in the contraction
of skeletal muscle (1,2), but the identity of physiological SR Ca2+ release channel has yet been unresolved (3). Electrophysiological experiments using the reconstituted system have shown the presence of at least two types of Ca2+ release channels in the SR membrane (4,s).
One type, which has been identified as
the functional Ca2*-induced Ca2+ release channel, has been purified and extensively characterized
(6-8), whereas the other types of channels have hardly been charac-
terized. In various cell types, inositol 1,4,5-trisphosphate (IP3) has been proposed as the second messenger causing Ca2+ release from intracellular
stores (9).
Also in
skeletal muscle, IP3 was proposed to be a possible missing link between the transverse tubular membrane excitation appeared conflicting
results relative
and Ca2+ release from SR, but there have to this idea (10,ll).
In the course of our
studies on the SR Ca2+ releaser (12), the effects of IP3 and related compounds on *Correspondence should be addressed to: Masaki Kobayashi, Ph.D., Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194, Japan. Abbreviations: SR, sarcoplasmic reticulum ; IP3, inositol 1,4,5-t&phosphate ; PIP2, phosphatidylinositol 4,5-bisphosphate.
1487
0006-291x/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 163, No. 3, 1989
BIOCHEMICAL
AND 8lOPHYSlCAL
RESEARCH COMMUNICATIONS
Ca2+ release have been examined. Only a preliminary communication has appeared on the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on frog SR vesicles (13). Here we present the first report indicating that not IP3 but its precursor PIP2 enhances or induces Ca2+ release from mammalian skeletal muscle SR to cause skinned fiber contraction through a novel mechanism. MA-
AND
rbErHoDs
Isometric tension of chemically skinned fibers prepared from guinea-pig psoas muscle was measured as described previously (12). A small bundle of fibers was connected to a strain gage transducer (SensoNor AF801) and treated at 20°C for 30 min with 50 &ml saponin in a relaxing solution containing 90 mM K-methanesulfonate, 5 mM MgS04, 4 mM ATP, 2 mM EGTA and 50 mM MOPS (pH 7.0). After washing out saponin, Ca2+-loading and subsequent incubation in an EGTA-free solution caused spontaneous1 repeated contractions. Extravesicular CaS+ concentration of fragmented SR prepared from rabbit white muscle was measured at 30°C with a Ca2+ electrode (12,14). The assay mixture contained 0.05 mM CaC12, 90 mM KCI, 3 mM MgC12, 2 mM NaN3, 1 mM ATP, 5 mM creatine phosphate, 0.1 mg/ml creatine kinase, 1.5 mg/ml SR and 50 mM MOPS (pH 7.0). Ca2+ uptake was initiated by a simultaneous addition of ATP and creatine kinase. 45Ca2+ efflux from SR vesicles passively preloaded with 45Ca2+ was measured as described previously (15). The 45Ca2+-preloaded SR suspensionwas diluted loo-fold with a reaction medium containing 90 mM KCl, 0.4 mM CaCl2, 1.9 mM EGTA and 50 mM MOPS (pH 7.0). The aliquots of the diluted suspension were filtered through Millipore filters and washed three times with a La3+,Mg2+-containing solution. The radioactivity of 45Ca remaining in SR vesicles was measured with a scintillation counter. RESULTS Chemically skinned skeletal muscle fibers retaining Ca2+-mobilizing activities of SR showed spontaneous and oscillatory contractions in solutions containing submicromolar Ca2+ (Fig. 1). The application of PIP2 (3-30 1~M) just after relaxation
* w
from spontaneous contraction induced a prompt tension spike followed by sustained contraction (Fig. la), but IP3 (up to 100 uM) never caused this contractile response. On the other hand, the addition of 0.4 mM caffeine,
a
10 min 5
an activator
Caffeine u Contraction of chemically skinned skeletal muscle fibers by phosphatidylinosltol 4,5-btsphosphate (PIPS) (a) or caffeine (b). Skinned fibers retaining sarcoplasmic reticulum (SR) function were prepared from guinea pig psoas muscle by treatment with saponln (50 ug/ml) for 30 min. PIP2 (30 PM) or caffeine (0.4 mh4) was applied just after relaxation from a spontaneous contraction (asterisk).
1488
of SR Ca2+-
BIOCHEMICAL
Vol. 163, No. 3, 1989
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3P
Hs
r
2 min CK
1
a
0.5 pea unit
Ca 1
F_ig. Cal+ release induced by PIP2 from fragmented SR vesicles prepared from rabbit skeletal muscle. Ths heavy (a-c) or light (d,e) fraction of SR was actively loaded with Ca + by adding ATP, creatijt phosphate (CP) and creatine kinase (CKk The extravesicular Ca concentration was monitored with a Ca + electrode. Treatment: a, caffeine (Caff, 0.4 mM); b, PIP2 (IO PM) plus caffeine (0.4 mM); c, PIP2 (50 PM); d, PIP2 (200 PM); e, A23187 (2 PM).
induced Ca2+ release channel, produced a tension spike followed by frequently repeated tension oscillation (Fig. lb). The Ca2+-releasing activity of fragmented SR was directly visualized by monitoring extravesicular Ca2+ concentration of SR with a sensitive Ca2+ electrode (Fig. 2). Upon the addition of ATP, free Ca2+ concentration decreased due to the active Ca2+ uptake by the heavy fraction of SR to reach an almost steady level. Under these conditions the application of caffeine (0.4 mM) caused a prompt Ca2+ release followed by a rapid Ca2+ reuptake (Fig. 2a), and the Ca2+-releasing action of caffeine was abolished by pretreatment with ruthenium red (0.3 PM) or additional Mg2+ (5 mM).
Pretreatment with PIP2 (above 5 PM) enhanced the caffeine-induced
P f i B Ic >
25
20
6 .E
0
1 Time
2 after
3 Dilution,
4
5 min
&& Effects of PIP2 or IP3 on the 45Ca2+ efflux from fragmented SR. The time course of the decrease in 45Ca content in SR vedcles was measured at 0°C after lOO-fold dilution of SR passively preloaded with 4%2+ into a control assay medium ( o ) or that containing 10 or M PIP2
( 0 ) or
100 L(M IP3
( A ).
1489
Vol. 163, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Accelerating effect of PIP2 on the 45Ca2+ efflux from the heavy fraction of fragmented SR for 1 min after dilution 45Ca2+ efflux (nmol/mg)
Treatment None PIP? PIP; PIP2 PIP2 PIPZ PIP2
5.4 8.7 Il.3 13.5 9.3 9.9 4.6
(1 IJM) (10 uM) (30 PM) (IO ~JM) + Ruthenium red (‘2 PM) (IO NM) + Procaine (10 mM) (10 uM) + MgC12 (5 mM)
Each value 1s the mean f standard
error
* f f * f f *
0.6 0.0 0.8 1.1 0.8 0.9 0.4
for 1 mln (%) 100 162 209 250 172 183 85
of mean (n=4).
Ca2+ release from SR in a concentration-dependent
manner (Fig.
2b).
The
administration of a high concentration (40-100 uM) of PIP2 alone produced a Ca2+ release from SR, followed by a gradual Ca2+ reuptake (Fig. SC). The PIP2 (50 uM)induced Ca2+ release was partially suppressedby 1 ~.IM ruthenium red, and abolished by 2 PM ruthenium red or 5 mM Mg2+.
Heparin, a potent inhibitor of specific IP3
binding (16) and IP3-induced Ca2+ release (17), (lOug/ml)
had no effect on the Ca2+
release by PIP2. An important observation is that in the light fraction of fragmented SR, PIP2 (up to 200 uM) never produced a prompt Ca2+ release (Fig. 2d), whereas a Ca2+ ionophore A23187 (2 uM) caused a sustained Ca2+ leakage (Fig. 2e) which was unaffected by ruthenium red (2 PM) or Mg2+ (5 mM). Furthermore, a high concentration (200 PM) of phosphatidylinositol 4-phosphate also induced Ca2+ release from the heavy fraction of SR (data not shown).
However, other related
compounds including IP3 (500 pM), phosphatidylinositol (200 uM), phosphatidic acid (200 uM) and myo-inositol (1 mM) failed to enhance or cause SR Ca2+ release. In order to examine quantitatively the Ca2+-releasing activity of PIP2, the 45Ca2+ efflux from the heavy fraction of fragmented SR was measured. PIP2 (10 !JM) markedly accelerated the 45Ca2+ efflux from SR, whereas IP3 (100 PM) caused no significant effect, or rather a slight decrease, in the 45Ca2+ efflux rate (Fig. 3). As shown in Table 1, PIP2 (l-30 LIM) enhanced the 45Ca2+ efflux for 1 min after dilution in a concentration-dependent manner. The PIP2 (10 PM)-induced increase in 45Ca2+ efflux rate was not greatly affected by ruthenium red (2 IJM) or procaine (10 mM), but was abolished by Mg2+ (5mM). DISCUSSION During recent years there has been a growing awareness that IP3 generated by PIP2 hydrolysis acts as the second messenger of cellular signal transduction in a variety of cell types (9). For skeletal muscle, however, numerous discrepant results have appeared and the basic problem of whether or not IP3 induces Ca2+ release from SR still awaits a definitive answer (10,ll). In the present experiments, it was reproducibly observed that IP3 caused no apparent effect on skeletal muscle prepaInstead, a water-soluble phospholipid PIP2 enhanced the contractility of rations. skinned fibers from mammalian skeletal muscle by stimulating SR Ca2+ release. 1490
Vol. 163, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The effect of PIP2 was rather resistant to ruthenium red in comparison with that of caffeine, and the PIPp-caused contractile response of skinned fibers was clearly different from that caused by caffeine. These observations are consistent with the most likely idea that PIP2 activates a novel type of SR Ca2+ release channel whose properties are different from those of well-known Ca2+-induced Ca2+ release channel. In contrast with a Ca2+ ionophore A23187, PIP2 never induced Ca2+ release from the light fraction of SR, demonstrating that PIP2 has no ionophoretic activity in the SR membrane. This is the first publication other than an abstract (13) reporting the Ca2+ -releasing activity of PIP2 from SR or SR vesicles. It is of great interest to study whether or not the small-conductance Ca2+ release channel (4,5) is the same as the PIP2-sensitive channel. The results described here, together with a highly water-soluble property of PIP2 at physiological pH (18), suggest an attractive idea that PIP2 may play an important role in the intracellular signal transduction through Ca2+ movements under certain conditions. REFERENCES ;:
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Martonosi, A. N. (1984) Physiol. Rev. 64, 1240-1320. Endo, M. (1977) Physiol. Rev. 57, 71-108. Riiegg, J. C. (1986) Calcium in Muscle Activation, pp. 29-58, Springer-Verlag, Berlin. Smith, J. S., Coronado, R., and Meissner, G. (1986) Biophys. J. 50, 921-928. Suirez-Isla, B. A., Orozco, C., Heller, P. F., and Froehlich, J. P. (1986) Proc. Natl. Acad. Sci. USA 83, 7741-7745. Smith, J. S., Imagawa, T., Ma, J., Fill, M., Campbell, K. P., and Coronado, R. (1988) J. Gen. Physiol. 92, l-26. Lai, F. A., Erickson, H. P., Rousseau, E, Liu, Q.-Y., and Meissner, G. (1988) Nature 331, 315-319. Wagenknecht, T., Grassucci, R., Frank, J., Saito, A., Inui, M., and Fleischer, S. (1989) Nature 338, 167-170. Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315- 321. Volpe, P., Salviati, G., DiVirgilio, F., and Pozzan, T. (1985) Nature 316, 347349. Ehrlich, B. E., and Watras, J. (1988) Nature 336, 583-586. Nakamura, Y., Kobayashi, J., Gilmore, J., Mascal, M., Rinehart, K. L., Jr., Nakamura, H., and Ohizumi, Y. (1986) J. Biol. Chem. 261, 4139-4142. Ogawa, Y., and Harafuji, H. (1987) Proceedings of the 10th International Congress of Pharmacology, P 1062 (abstract). Kobayashi, M., Ishida, Y., Shoji, N., and Ohizumi, Y. (1988) J. Pharmacol. Exp. Ther. 246, 667-673. Kobayashi, M., Shoji, N., and Ohizumi, Y. (1987) Biochim. Biophys. Acta 903, 96-102. Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136. Cullen, P. J., Comerford, J. G., and Dawson, A. P. (1988) FEBS Lett. 228, 5759. Schacht, J. (1981) Methods Enzymol. 72, 626-631.
1491
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 1487-1491
PHOSPHATIDYLINOSITOL
4,CBISPHOSPHATE ENHANCES CALCIUM RELEASE
FROM SARCOPLASMIC RETICULUM OF SKELETAL MUSCLE Masaki Kobayashi*, Akiko Muroyama and Yasushi Ohizumi Mitsubishi Kasei Institute of Life Sciences Minamiooya, Machida, Tokyo 194, Japan Received
August
16,
1989
SUMMARX In the course of our study on the function of sarcoplasmic reticulum (SR) in skeletal muscle, the stimulatory action of phosphatidylinositol 4,5bisphosphate (PIP2) on the Ca2+ release from SR was demonstrated by using chemically skinned fibers and fragmented SR vesicles. PIP2 induced a tension spike followed by sustained contraction in skinned fibers. PIP2 enhanced the caffeineinduced Ca2+ release from SR vesicles at low concentrations and triggered Ca2+ release by itself at high concentrations. PIP2 also enhanced 45Ca2+ efflux from SR vesicles. However, inositol 1,4,5-triphosphate never produced these effects. The Ca2+-releasing action of PIP2 was only weakly affected by ruthenium red or procaine. These observations suggest that PIP2 activates an SR Ca2+ release channel whose properties are different from those of the Ca2+-induced Ca2+ release channel. 0 1989 Academic Press, Inc.
The Ca2+ release from sarcoplasmic reticulum (SR) plays a key role in the contraction
of skeletal muscle (1,2), but the identity of physiological SR Ca2+ release channel has yet been unresolved (3). Electrophysiological experiments using the reconstituted system have shown the presence of at least two types of Ca2+ release channels in the SR membrane (4,s).
One type, which has been identified as
the functional Ca2*-induced Ca2+ release channel, has been purified and extensively characterized
(6-8), whereas the other types of channels have hardly been charac-
terized. In various cell types, inositol 1,4,5-trisphosphate (IP3) has been proposed as the second messenger causing Ca2+ release from intracellular
stores (9).
Also in
skeletal muscle, IP3 was proposed to be a possible missing link between the transverse tubular membrane excitation appeared conflicting
results relative
and Ca2+ release from SR, but there have to this idea (10,ll).
In the course of our
studies on the SR Ca2+ releaser (12), the effects of IP3 and related compounds on *Correspondence should be addressed to: Masaki Kobayashi, Ph.D., Mitsubishi Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194, Japan. Abbreviations: SR, sarcoplasmic reticulum ; IP3, inositol 1,4,5-t&phosphate ; PIP2, phosphatidylinositol 4,5-bisphosphate.
1487
0006-291x/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 163, No. 3, 1989
BIOCHEMICAL
AND 8lOPHYSlCAL
RESEARCH COMMUNICATIONS
Ca2+ release have been examined. Only a preliminary communication has appeared on the effect of phosphatidylinositol 4,5-bisphosphate (PIP2) on frog SR vesicles (13). Here we present the first report indicating that not IP3 but its precursor PIP2 enhances or induces Ca2+ release from mammalian skeletal muscle SR to cause skinned fiber contraction through a novel mechanism. MA-
AND
rbErHoDs
Isometric tension of chemically skinned fibers prepared from guinea-pig psoas muscle was measured as described previously (12). A small bundle of fibers was connected to a strain gage transducer (SensoNor AF801) and treated at 20°C for 30 min with 50 &ml saponin in a relaxing solution containing 90 mM K-methanesulfonate, 5 mM MgS04, 4 mM ATP, 2 mM EGTA and 50 mM MOPS (pH 7.0). After washing out saponin, Ca2+-loading and subsequent incubation in an EGTA-free solution caused spontaneous1 repeated contractions. Extravesicular CaS+ concentration of fragmented SR prepared from rabbit white muscle was measured at 30°C with a Ca2+ electrode (12,14). The assay mixture contained 0.05 mM CaC12, 90 mM KCI, 3 mM MgC12, 2 mM NaN3, 1 mM ATP, 5 mM creatine phosphate, 0.1 mg/ml creatine kinase, 1.5 mg/ml SR and 50 mM MOPS (pH 7.0). Ca2+ uptake was initiated by a simultaneous addition of ATP and creatine kinase. 45Ca2+ efflux from SR vesicles passively preloaded with 45Ca2+ was measured as described previously (15). The 45Ca2+-preloaded SR suspensionwas diluted loo-fold with a reaction medium containing 90 mM KCl, 0.4 mM CaCl2, 1.9 mM EGTA and 50 mM MOPS (pH 7.0). The aliquots of the diluted suspension were filtered through Millipore filters and washed three times with a La3+,Mg2+-containing solution. The radioactivity of 45Ca remaining in SR vesicles was measured with a scintillation counter. RESULTS Chemically skinned skeletal muscle fibers retaining Ca2+-mobilizing activities of SR showed spontaneous and oscillatory contractions in solutions containing submicromolar Ca2+ (Fig. 1). The application of PIP2 (3-30 1~M) just after relaxation
* w
from spontaneous contraction induced a prompt tension spike followed by sustained contraction (Fig. la), but IP3 (up to 100 uM) never caused this contractile response. On the other hand, the addition of 0.4 mM caffeine,
a
10 min 5
an activator
Caffeine u Contraction of chemically skinned skeletal muscle fibers by phosphatidylinosltol 4,5-btsphosphate (PIPS) (a) or caffeine (b). Skinned fibers retaining sarcoplasmic reticulum (SR) function were prepared from guinea pig psoas muscle by treatment with saponln (50 ug/ml) for 30 min. PIP2 (30 PM) or caffeine (0.4 mh4) was applied just after relaxation from a spontaneous contraction (asterisk).
1488
of SR Ca2+-
BIOCHEMICAL
Vol. 163, No. 3, 1989
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
3P
Hs
r
2 min CK
1
a
0.5 pea unit
Ca 1
F_ig. Cal+ release induced by PIP2 from fragmented SR vesicles prepared from rabbit skeletal muscle. Ths heavy (a-c) or light (d,e) fraction of SR was actively loaded with Ca + by adding ATP, creatijt phosphate (CP) and creatine kinase (CKk The extravesicular Ca concentration was monitored with a Ca + electrode. Treatment: a, caffeine (Caff, 0.4 mM); b, PIP2 (IO PM) plus caffeine (0.4 mM); c, PIP2 (50 PM); d, PIP2 (200 PM); e, A23187 (2 PM).
induced Ca2+ release channel, produced a tension spike followed by frequently repeated tension oscillation (Fig. lb). The Ca2+-releasing activity of fragmented SR was directly visualized by monitoring extravesicular Ca2+ concentration of SR with a sensitive Ca2+ electrode (Fig. 2). Upon the addition of ATP, free Ca2+ concentration decreased due to the active Ca2+ uptake by the heavy fraction of SR to reach an almost steady level. Under these conditions the application of caffeine (0.4 mM) caused a prompt Ca2+ release followed by a rapid Ca2+ reuptake (Fig. 2a), and the Ca2+-releasing action of caffeine was abolished by pretreatment with ruthenium red (0.3 PM) or additional Mg2+ (5 mM).
Pretreatment with PIP2 (above 5 PM) enhanced the caffeine-induced
P f i B Ic >
25
20
6 .E
0
1 Time
2 after
3 Dilution,
4
5 min
&& Effects of PIP2 or IP3 on the 45Ca2+ efflux from fragmented SR. The time course of the decrease in 45Ca content in SR vedcles was measured at 0°C after lOO-fold dilution of SR passively preloaded with 4%2+ into a control assay medium ( o ) or that containing 10 or M PIP2
( 0 ) or
100 L(M IP3
( A ).
1489
Vol. 163, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table 1 Accelerating effect of PIP2 on the 45Ca2+ efflux from the heavy fraction of fragmented SR for 1 min after dilution 45Ca2+ efflux (nmol/mg)
Treatment None PIP? PIP; PIP2 PIP2 PIPZ PIP2
5.4 8.7 Il.3 13.5 9.3 9.9 4.6
(1 IJM) (10 uM) (30 PM) (IO ~JM) + Ruthenium red (‘2 PM) (IO NM) + Procaine (10 mM) (10 uM) + MgC12 (5 mM)
Each value 1s the mean f standard
error
* f f * f f *
0.6 0.0 0.8 1.1 0.8 0.9 0.4
for 1 mln (%) 100 162 209 250 172 183 85
of mean (n=4).
Ca2+ release from SR in a concentration-dependent
manner (Fig.
2b).
The
administration of a high concentration (40-100 uM) of PIP2 alone produced a Ca2+ release from SR, followed by a gradual Ca2+ reuptake (Fig. SC). The PIP2 (50 uM)induced Ca2+ release was partially suppressedby 1 ~.IM ruthenium red, and abolished by 2 PM ruthenium red or 5 mM Mg2+.
Heparin, a potent inhibitor of specific IP3
binding (16) and IP3-induced Ca2+ release (17), (lOug/ml)
had no effect on the Ca2+
release by PIP2. An important observation is that in the light fraction of fragmented SR, PIP2 (up to 200 uM) never produced a prompt Ca2+ release (Fig. 2d), whereas a Ca2+ ionophore A23187 (2 uM) caused a sustained Ca2+ leakage (Fig. 2e) which was unaffected by ruthenium red (2 PM) or Mg2+ (5 mM). Furthermore, a high concentration (200 PM) of phosphatidylinositol 4-phosphate also induced Ca2+ release from the heavy fraction of SR (data not shown).
However, other related
compounds including IP3 (500 pM), phosphatidylinositol (200 uM), phosphatidic acid (200 uM) and myo-inositol (1 mM) failed to enhance or cause SR Ca2+ release. In order to examine quantitatively the Ca2+-releasing activity of PIP2, the 45Ca2+ efflux from the heavy fraction of fragmented SR was measured. PIP2 (10 !JM) markedly accelerated the 45Ca2+ efflux from SR, whereas IP3 (100 PM) caused no significant effect, or rather a slight decrease, in the 45Ca2+ efflux rate (Fig. 3). As shown in Table 1, PIP2 (l-30 LIM) enhanced the 45Ca2+ efflux for 1 min after dilution in a concentration-dependent manner. The PIP2 (10 PM)-induced increase in 45Ca2+ efflux rate was not greatly affected by ruthenium red (2 IJM) or procaine (10 mM), but was abolished by Mg2+ (5mM). DISCUSSION During recent years there has been a growing awareness that IP3 generated by PIP2 hydrolysis acts as the second messenger of cellular signal transduction in a variety of cell types (9). For skeletal muscle, however, numerous discrepant results have appeared and the basic problem of whether or not IP3 induces Ca2+ release from SR still awaits a definitive answer (10,ll). In the present experiments, it was reproducibly observed that IP3 caused no apparent effect on skeletal muscle prepaInstead, a water-soluble phospholipid PIP2 enhanced the contractility of rations. skinned fibers from mammalian skeletal muscle by stimulating SR Ca2+ release. 1490
Vol. 163, No. 3, 1989
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The effect of PIP2 was rather resistant to ruthenium red in comparison with that of caffeine, and the PIPp-caused contractile response of skinned fibers was clearly different from that caused by caffeine. These observations are consistent with the most likely idea that PIP2 activates a novel type of SR Ca2+ release channel whose properties are different from those of well-known Ca2+-induced Ca2+ release channel. In contrast with a Ca2+ ionophore A23187, PIP2 never induced Ca2+ release from the light fraction of SR, demonstrating that PIP2 has no ionophoretic activity in the SR membrane. This is the first publication other than an abstract (13) reporting the Ca2+ -releasing activity of PIP2 from SR or SR vesicles. It is of great interest to study whether or not the small-conductance Ca2+ release channel (4,5) is the same as the PIP2-sensitive channel. The results described here, together with a highly water-soluble property of PIP2 at physiological pH (18), suggest an attractive idea that PIP2 may play an important role in the intracellular signal transduction through Ca2+ movements under certain conditions. REFERENCES ;:
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Martonosi, A. N. (1984) Physiol. Rev. 64, 1240-1320. Endo, M. (1977) Physiol. Rev. 57, 71-108. Riiegg, J. C. (1986) Calcium in Muscle Activation, pp. 29-58, Springer-Verlag, Berlin. Smith, J. S., Coronado, R., and Meissner, G. (1986) Biophys. J. 50, 921-928. Suirez-Isla, B. A., Orozco, C., Heller, P. F., and Froehlich, J. P. (1986) Proc. Natl. Acad. Sci. USA 83, 7741-7745. Smith, J. S., Imagawa, T., Ma, J., Fill, M., Campbell, K. P., and Coronado, R. (1988) J. Gen. Physiol. 92, l-26. Lai, F. A., Erickson, H. P., Rousseau, E, Liu, Q.-Y., and Meissner, G. (1988) Nature 331, 315-319. Wagenknecht, T., Grassucci, R., Frank, J., Saito, A., Inui, M., and Fleischer, S. (1989) Nature 338, 167-170. Berridge, M. J., and Irvine, R. F. (1984) Nature 312, 315- 321. Volpe, P., Salviati, G., DiVirgilio, F., and Pozzan, T. (1985) Nature 316, 347349. Ehrlich, B. E., and Watras, J. (1988) Nature 336, 583-586. Nakamura, Y., Kobayashi, J., Gilmore, J., Mascal, M., Rinehart, K. L., Jr., Nakamura, H., and Ohizumi, Y. (1986) J. Biol. Chem. 261, 4139-4142. Ogawa, Y., and Harafuji, H. (1987) Proceedings of the 10th International Congress of Pharmacology, P 1062 (abstract). Kobayashi, M., Ishida, Y., Shoji, N., and Ohizumi, Y. (1988) J. Pharmacol. Exp. Ther. 246, 667-673. Kobayashi, M., Shoji, N., and Ohizumi, Y. (1987) Biochim. Biophys. Acta 903, 96-102. Worley, P. F., Baraban, J. M., Supattapone, S., Wilson, V. S., and Snyder, S. H. (1987) J. Biol. Chem. 262, 12132-12136. Cullen, P. J., Comerford, J. G., and Dawson, A. P. (1988) FEBS Lett. 228, 5759. Schacht, J. (1981) Methods Enzymol. 72, 626-631.
1491