Vol. 178, No. 2, 1991 July 31, 1991
BIOCHEMICAL
Ba2+ current
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 713-717
oscillation evoked by bradykinin ras-transformed fibroblasts
in
Haruhiro Higashidal, Naoto Hoshil, Minako HashiiI, Tao Fu2, Mami Nodal and Yoshinori Nozawa2 IDepartment of Biophysics, Neuroinformation ResearchInstitute, Kanazawa University School of Medicine, Kanazawa 920, Japan ZDepartment of Biochemistry, Gifu University School of Medicine, Gifu 500, Japan Received
June
21,
1991
SUMMARY : By voltage-clamp recording, we show a novel inward current which oscillates after activation with bradykinin or serum in v-Ki-rastransformed NIIW3T3 cells. The current oscillation was infrequently observed in control NIIW3T3 fibroblasts. The same stimulation evokes Ca2+ oscillations in the rus-transformed cells but not in parental cells (Fu et al., FEBS Lett. 281, 263-266, 1991). The results suggest that the oscillatory currents are generated by influxes of divalent cations to maintain Ca2+ oscillations in rar-transformed NIH/3T3 cells. @ 1991 Academic Press, Inc. Nonexcitable cells generaterepetitive transient increases in cytosolic free calcium concentration ([Caz+]i ), designatedas Ca2+oscillation, when stimulated with hormones or growth factors (l-9). Ca2+ oscillations are maintained by increased Ca2+ entry across the plasma membrane to refill the cytoplasm and Ca2+ stores (2-5). This inflow of Ca2+ is thought to occur through putative Ca2+ entry channels (lo- 12), whose electrophysiological properties are totally UllkllOWll.
Recently it has been reported that Caz+ oscillations are induced by bradykinin and bombesin or serum (13) in v-Ki-r-as-transformed NIN/3T3 cells (14), but not in control 3T3 fibroblast cells. Since the Ca2+ oscillation requires the presence of extracellular Ca2+, and since it is not inhibited by organic Ca2+ channel blockers (13), it is proposed that Ca2+influx occurs through Ca2+ entry channels rather than voltage-operated Ca2+ channels in ras-transformed cells. Here we describe oscillatory inward currents elicited by challenging to bradykinin or mitogenic agonists in rm-transformed fibroblasts. ABBREVIATION:
[CaZ+]i , cytosolic free calcium concentration.
713
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All righrs of reproduction in any form reserved.
Vol.
178,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
METHODS
NIH/3T3 fibroblast cells and Kirstein murine sarcoma virus-transformed NIH/3T3 (DT) cells (14) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal calf serum. Cells were then detached by exposure to Dulbecco’s phosphate buffered saline without Ca2+ or Mg2+ but containing 0.05% trypsin. Cells (2 x 105) were reseeded onto polyomithinecoated dishes (35~mm in diameter), and kept for 2-6 days before use. To measure Ca2+ currents, cells were incubated at 35 “C in the Baz+ solution (50 mM BaC12,30 mM NaCl, 5 mM CsCl, 0.8 mM MgCl2,25 mM glucose, 25 mM tetraethylammonium chloride, 0.1 nM tetrodotoxin and 5 mM Hepes, pH 7.3), whose components are the same as for measurement of L- and N-type Ca2+ currents in neuroblastoma hybrid cells reported previously (15). Solution for filling patch pipettes contained 150 mM CsCl, 1 mM MgC12, 10 mM EGTA (buffered by 1 M NaOH), 0.4 mM sodium ATP and 10 mM Hepes, pH 7.3, adjusted with NaOH. Cells were voltage-clamped by using patch-pipettes in whole-cell mode, and current was amplified by using single-electrode voltageclamp amplifiers (Nihonkoden CEZ-3 100 or Axoclamp-2A) in discontinuous mode, sampling at 3-6 kHz. Currents were filtered at 1 kHz and displayed on a Gould pen recorder. RESULTS
AND DISCUSSION
Fig. 1 shows whole cell Ca2+ currents recorded by a patch electrode in rus-transformed NIN/3T3 (DT) cells in perfusion medium containing 50 mM Ba2+ as a charge carrier (15) at holding membrane potentials of about -40 mV. Two types of inward current were evoked after stimulation with 100 nM bradykinin (Fig. la) : the initial slow inward current followed by repetitive transient inward currents. The latency time from the stimulation with bradykinin to the first transient inward current ranged from 0.3 to 9.8 min, with a mean value of 4.4 + 1.1 min (* s.e.m., n-10 cells). Once the first transient current was generated, inward currents recurred at various intervals for over 4 hrs. The initial slow inward current seen soon after application of bradykinin or fetal calf serum (10%) differed from repetitive transient currents in that it was Ni2+ resistant (1 mM) and associated with an increase of input conductance. This current seemsto be identical to the slow depolarization evoked by serum in neuroblastoma cells (16), and was not further characterized. The slow and recurrent inward currents correspond well in time with two types of [Caz+]i increase in DT cells reported previously (13): the first inositol trisphosphatedependent [Caz+li increase and subsequent Ca2+ oscillations which require extracellular Ca2+, respectively. Hereafter, we will designate the recurrent transient inward currents as Ba2+ current oscillations. As shown in Fig. lb and c, the frequency of Baz+ current oscillations changed as l-3 hrs passed after application of 10% fetal calf serum or bradykinin. The frequency grew from the mean value of 0.66 f 0.07 cycle per 714
Vol.
178, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a mV
Membrane
potential
(mV)
Fi e. 1 c Ba2+ current oscillations in DT cells. Q, Time course of current fluctuation recorded for 33 min from starting perfusion with 100 nM bradykinin (BK). Bradykinin produced an initial slow inward current (labelled with *) and recurrent transient inward currents. The first transient current is labelled with #. The cell was clamped at -41 mV. b and c, High frequency of Ba2+current oscillations in two other DT cells. Records were obtained 130 and 154 min after exposure to 10% fetal calf serum. Holding membrane potentials of -40 and -39 mV. d, Ba2+current oscillations recorded at various holding potentials in the fourth DT cell. Each example shows 2-3 oscillations. Holding potential is shown at the beginning of each example. Calibration of 1 nA for top two traces, and of 2 nA for the rest. e, Frequency of the Ba2+ current oscillation to voltage relationship is shown for the same cell in d . f, Currentvoltage relationships of the Ba2+ currentoscillation in the sameDT cell in d. The similar result in current-voltage relationships was obtained in 6 cells.
min (cpm) (n=38) to 10.4 * 1.4 cpm. The average amplitude decreased from 2.6 f 0.62 nA to 0.50 f 0.16 nA. Ba2+ current oscillations were observed in 85 out of 278 DT cells studied (3 1%) in responseto lo- 100 nM bradykinin, 100 nM bombesin or 10% fetal calf serum. In contrast, such current oscillations (with a mean frequency of 10.3 f 1.6 cpm) were observed in only 4 (3.5%) out of 120 control 3T3 cells and amplitudes were small (a mean value, 0.09 + 0.05 N. Ba2+ inward current oscillations were recorded at various holding potentials from -150 to +80 mV to show voltage-dependence(Fig. 1 d & f). At negative membrane potentials, amplitude was large and nearly constant, but at positive potentials amplitude was much smaller. However, the Ba2+ oscillatory inward current never reversed even at a membrane potential higher than +50 mV, unlike L-type Ca2+ currents (9). The frequency of the Ba2+ current oscillation was inversely correlated to its amplitude (Fig. le ) . The possibility that this Ba2+ inward current oscillation is the Na+/CaE+ exchange current (17) (the Na+/Ba2+ exchange (18) in this case) was ruled out, since Ba2+ current oscillations were evoked under the external Na+-free 715
Vol.
178,
No.
BIOCHEMICAL
2, 1991
[Ni*‘],
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
a
1000 Ni*+
[Nl*+l,
1
’ YpM)
C wwl#wwwJ: -6
SK&F 96365
1 mln
Pig. 2. Effect of Ni2+ and SK&F 96365 on Ba2+ inward current oscillation in two DT cells. a, One DT cell was continuously superfused with the 50 mM Baz+ solution containing 100 r&I bradykinin. The bar (Ni2+) indicates the time period (2 min) during which various concentrations of NiC12 (as shown at the beginning of each current trace) were added into the 50 mM Ba2+ solution. b, A plot of inhibition time of Ni2+ concentrationsin the same DT cell in Q. c, A current trace from another cell exposed to 3 uM SK&F 96365 during the time indicated by the bar (SK&F 96365).
condition in 10 cells tested (data not shown), suggesting that the oscillatory inward current results primarily from an inflow of Ba2+. Inhibitors of receptor-mediated Ca2+ entry, Ni2+ (10) and SK&F 96365 (19), were examined. Fig. 2a shows Baz+ current oscillation before, during and after a 120 set exposure to various concentrations of Ni2+. Application of more than 20-50 uM Ni2+ abruptly terminated Ba2+ current oscillations (n= 14 cells). On removal of Ni2+, the oscillations resumed. The higher the concentration of Ni2+, the longer the inhibition continued (Fig. 2b). We also observed the inhibition of Ba2+ current oscillations by other inorganic divalent cations: 0.1 mM Cd2+ (3 cells); 0.05 or 0.55 mM Mn2+ (2 cells). Fig. 2c illustrates one typical example of an inhibitory effect of 3 uM SK&F 96365 on Ba2+ current oscillation (n=5 cells). The present investigation has revealed an oscillatory influx of Ba2+ in rastransformed NIW3T3 cells stimulated with bradykinin or mitogenic agonists. Use of Ba2+ as a charge carrier allowed the first and direct measurement of Ca2+ entry currents to confirm and extend previous deductions based on measurements of Sr2+-, Ba2+- or Mn2+-influxes that induce increases or quench of fura- fluorescence (10,l I). The question whether Ba2+ current oscillations are synchronized with Ca2+ oscillations remains to be solved. However, parallels in onset or cessation of Ba2+ current oscillations and Ca2+ oscillations (13) suggest close correlation between them. In addition to the known functions of ras proteins (see Ref. 20), we therefore suggest that ras proteins, once 716
Vol.
178,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
activated by agonists that engage the phosphoinositide pathway, constantly transduce signals on Ca2+ entry channels in the plasma membrane of transformed cells. Acknowledgments
We thank Dr. Y. Sugimoto for cells and Dr. J. E. Merritt for SK&F 96365. This investigation was supported in part by research grants from the Ministry of Education, Science and Culture of Japan. REFERENCES
1. Berridge, M.J. & Irvine, R. (1989) Nature 34 1, 197-205. 2. Berridge, M.J. (1990) J. Biol. Chem. 265, 9583-9586. 3. Jacob, R., Merritt, J. E., Hallam, T. J. & Rink, T. J. (1988) Nature 335, 40-45. 4. Harootunian, A. T., Kao, J. P. Y., Paranjape, S. & Tsien, R. Y. (1991) Science 25 1,75-78. 5. Wakui, M., Osipchuk, Y. V. & Petersen,0. H. (1991) Cell 63, 1025-1032. 6. Loessberg, P. A., Zhao, H. & Muallem, S. (1991) J. Biol. Chem. 26 6, 1363-1366. 7. Zhao, H., Loessberg, P. A., Sachs, G. & Muallem, S. (1990) J. Biol. Chem. 2 65,20856-20862.
8, Malgaroli, A., Fesce, R. & Meldolesi, J. (1990) J. Biol. Chem. 2 6 5, 30053008. 9. Tsien, R. W. & Tsien R. Y. (1990) Ann. Rev. Cell Biol. 6, 715-760. 10. Jacob, R. (1990) J. Physiol. 421, 55-77. 11. Kwan, C.-Y. & Putney, J. W. Jr. (1990) J. Biol. Chem. 265, 678-684. 12. Putney, J. W. Jr. (1990) Cell Calcium 11, 611-624. 13. Fu, T., Sugimoto, Y., Oki, T., Murakami, S., Okano. Y. & Nozawa, Y. (1991) FEBS Letters 281,263-266. 14. Noda, M., Selinger, Z., Scohrick, E. M. & Bassin, R.H. (1983) Proc. Nat. Acad. Sci. USA 80, 5602-5606. 15. Higashida, H., Hashii, M., Fukuda, K., Caulfield, M. P., Numa, S. & Brown, D. A. (1990) Proc. R.. Sot. London B 242,68-74. 16. Moolenaar, W. H., Mummery, C. L., Van de Saag,P. T. & de Laat, S. W. (1981) Cel123, 789-798. 17. Lipp, P. & Pott, L. (1988) J. Physiol. 397, 601-630. 18. Potreau, D., Richard, S., Nargeot, J. & Raymond, G. (1987) Pfhigers Archiv 410,326-334.
19. Merritt, J.E., Armstrong, W.P., Benham, C.D., Hallam, T.J., Jacob, R., Jaxa-Chamiec, A., Leigh, B.K., McCarthy, S.A., Moores, K.E. Jz Rink, T.J. (1990) Biochem. J. 271, 515-522. 20. Boume, H. R., Sanders,D. A. & McCormick, F. (1990) Nature 34 9, 117-127.
717
BIOCHEMICAL
Ba2+ current
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 713-717
oscillation evoked by bradykinin ras-transformed fibroblasts
in
Haruhiro Higashidal, Naoto Hoshil, Minako HashiiI, Tao Fu2, Mami Nodal and Yoshinori Nozawa2 IDepartment of Biophysics, Neuroinformation ResearchInstitute, Kanazawa University School of Medicine, Kanazawa 920, Japan ZDepartment of Biochemistry, Gifu University School of Medicine, Gifu 500, Japan Received
June
21,
1991
SUMMARY : By voltage-clamp recording, we show a novel inward current which oscillates after activation with bradykinin or serum in v-Ki-rastransformed NIIW3T3 cells. The current oscillation was infrequently observed in control NIIW3T3 fibroblasts. The same stimulation evokes Ca2+ oscillations in the rus-transformed cells but not in parental cells (Fu et al., FEBS Lett. 281, 263-266, 1991). The results suggest that the oscillatory currents are generated by influxes of divalent cations to maintain Ca2+ oscillations in rar-transformed NIH/3T3 cells. @ 1991 Academic Press, Inc. Nonexcitable cells generaterepetitive transient increases in cytosolic free calcium concentration ([Caz+]i ), designatedas Ca2+oscillation, when stimulated with hormones or growth factors (l-9). Ca2+ oscillations are maintained by increased Ca2+ entry across the plasma membrane to refill the cytoplasm and Ca2+ stores (2-5). This inflow of Ca2+ is thought to occur through putative Ca2+ entry channels (lo- 12), whose electrophysiological properties are totally UllkllOWll.
Recently it has been reported that Caz+ oscillations are induced by bradykinin and bombesin or serum (13) in v-Ki-r-as-transformed NIN/3T3 cells (14), but not in control 3T3 fibroblast cells. Since the Ca2+ oscillation requires the presence of extracellular Ca2+, and since it is not inhibited by organic Ca2+ channel blockers (13), it is proposed that Ca2+influx occurs through Ca2+ entry channels rather than voltage-operated Ca2+ channels in ras-transformed cells. Here we describe oscillatory inward currents elicited by challenging to bradykinin or mitogenic agonists in rm-transformed fibroblasts. ABBREVIATION:
[CaZ+]i , cytosolic free calcium concentration.
713
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All righrs of reproduction in any form reserved.
Vol.
178,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
METHODS
NIH/3T3 fibroblast cells and Kirstein murine sarcoma virus-transformed NIH/3T3 (DT) cells (14) were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% fetal calf serum. Cells were then detached by exposure to Dulbecco’s phosphate buffered saline without Ca2+ or Mg2+ but containing 0.05% trypsin. Cells (2 x 105) were reseeded onto polyomithinecoated dishes (35~mm in diameter), and kept for 2-6 days before use. To measure Ca2+ currents, cells were incubated at 35 “C in the Baz+ solution (50 mM BaC12,30 mM NaCl, 5 mM CsCl, 0.8 mM MgCl2,25 mM glucose, 25 mM tetraethylammonium chloride, 0.1 nM tetrodotoxin and 5 mM Hepes, pH 7.3), whose components are the same as for measurement of L- and N-type Ca2+ currents in neuroblastoma hybrid cells reported previously (15). Solution for filling patch pipettes contained 150 mM CsCl, 1 mM MgC12, 10 mM EGTA (buffered by 1 M NaOH), 0.4 mM sodium ATP and 10 mM Hepes, pH 7.3, adjusted with NaOH. Cells were voltage-clamped by using patch-pipettes in whole-cell mode, and current was amplified by using single-electrode voltageclamp amplifiers (Nihonkoden CEZ-3 100 or Axoclamp-2A) in discontinuous mode, sampling at 3-6 kHz. Currents were filtered at 1 kHz and displayed on a Gould pen recorder. RESULTS
AND DISCUSSION
Fig. 1 shows whole cell Ca2+ currents recorded by a patch electrode in rus-transformed NIN/3T3 (DT) cells in perfusion medium containing 50 mM Ba2+ as a charge carrier (15) at holding membrane potentials of about -40 mV. Two types of inward current were evoked after stimulation with 100 nM bradykinin (Fig. la) : the initial slow inward current followed by repetitive transient inward currents. The latency time from the stimulation with bradykinin to the first transient inward current ranged from 0.3 to 9.8 min, with a mean value of 4.4 + 1.1 min (* s.e.m., n-10 cells). Once the first transient current was generated, inward currents recurred at various intervals for over 4 hrs. The initial slow inward current seen soon after application of bradykinin or fetal calf serum (10%) differed from repetitive transient currents in that it was Ni2+ resistant (1 mM) and associated with an increase of input conductance. This current seemsto be identical to the slow depolarization evoked by serum in neuroblastoma cells (16), and was not further characterized. The slow and recurrent inward currents correspond well in time with two types of [Caz+]i increase in DT cells reported previously (13): the first inositol trisphosphatedependent [Caz+li increase and subsequent Ca2+ oscillations which require extracellular Ca2+, respectively. Hereafter, we will designate the recurrent transient inward currents as Ba2+ current oscillations. As shown in Fig. lb and c, the frequency of Baz+ current oscillations changed as l-3 hrs passed after application of 10% fetal calf serum or bradykinin. The frequency grew from the mean value of 0.66 f 0.07 cycle per 714
Vol.
178, No. 2, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
a mV
Membrane
potential
(mV)
Fi e. 1 c Ba2+ current oscillations in DT cells. Q, Time course of current fluctuation recorded for 33 min from starting perfusion with 100 nM bradykinin (BK). Bradykinin produced an initial slow inward current (labelled with *) and recurrent transient inward currents. The first transient current is labelled with #. The cell was clamped at -41 mV. b and c, High frequency of Ba2+current oscillations in two other DT cells. Records were obtained 130 and 154 min after exposure to 10% fetal calf serum. Holding membrane potentials of -40 and -39 mV. d, Ba2+current oscillations recorded at various holding potentials in the fourth DT cell. Each example shows 2-3 oscillations. Holding potential is shown at the beginning of each example. Calibration of 1 nA for top two traces, and of 2 nA for the rest. e, Frequency of the Ba2+ current oscillation to voltage relationship is shown for the same cell in d . f, Currentvoltage relationships of the Ba2+ currentoscillation in the sameDT cell in d. The similar result in current-voltage relationships was obtained in 6 cells.
min (cpm) (n=38) to 10.4 * 1.4 cpm. The average amplitude decreased from 2.6 f 0.62 nA to 0.50 f 0.16 nA. Ba2+ current oscillations were observed in 85 out of 278 DT cells studied (3 1%) in responseto lo- 100 nM bradykinin, 100 nM bombesin or 10% fetal calf serum. In contrast, such current oscillations (with a mean frequency of 10.3 f 1.6 cpm) were observed in only 4 (3.5%) out of 120 control 3T3 cells and amplitudes were small (a mean value, 0.09 + 0.05 N. Ba2+ inward current oscillations were recorded at various holding potentials from -150 to +80 mV to show voltage-dependence(Fig. 1 d & f). At negative membrane potentials, amplitude was large and nearly constant, but at positive potentials amplitude was much smaller. However, the Ba2+ oscillatory inward current never reversed even at a membrane potential higher than +50 mV, unlike L-type Ca2+ currents (9). The frequency of the Ba2+ current oscillation was inversely correlated to its amplitude (Fig. le ) . The possibility that this Ba2+ inward current oscillation is the Na+/CaE+ exchange current (17) (the Na+/Ba2+ exchange (18) in this case) was ruled out, since Ba2+ current oscillations were evoked under the external Na+-free 715
Vol.
178,
No.
BIOCHEMICAL
2, 1991
[Ni*‘],
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
b
a
1000 Ni*+
[Nl*+l,
1
’ YpM)
C wwl#wwwJ: -6
SK&F 96365
1 mln
Pig. 2. Effect of Ni2+ and SK&F 96365 on Ba2+ inward current oscillation in two DT cells. a, One DT cell was continuously superfused with the 50 mM Baz+ solution containing 100 r&I bradykinin. The bar (Ni2+) indicates the time period (2 min) during which various concentrations of NiC12 (as shown at the beginning of each current trace) were added into the 50 mM Ba2+ solution. b, A plot of inhibition time of Ni2+ concentrationsin the same DT cell in Q. c, A current trace from another cell exposed to 3 uM SK&F 96365 during the time indicated by the bar (SK&F 96365).
condition in 10 cells tested (data not shown), suggesting that the oscillatory inward current results primarily from an inflow of Ba2+. Inhibitors of receptor-mediated Ca2+ entry, Ni2+ (10) and SK&F 96365 (19), were examined. Fig. 2a shows Baz+ current oscillation before, during and after a 120 set exposure to various concentrations of Ni2+. Application of more than 20-50 uM Ni2+ abruptly terminated Ba2+ current oscillations (n= 14 cells). On removal of Ni2+, the oscillations resumed. The higher the concentration of Ni2+, the longer the inhibition continued (Fig. 2b). We also observed the inhibition of Ba2+ current oscillations by other inorganic divalent cations: 0.1 mM Cd2+ (3 cells); 0.05 or 0.55 mM Mn2+ (2 cells). Fig. 2c illustrates one typical example of an inhibitory effect of 3 uM SK&F 96365 on Ba2+ current oscillation (n=5 cells). The present investigation has revealed an oscillatory influx of Ba2+ in rastransformed NIW3T3 cells stimulated with bradykinin or mitogenic agonists. Use of Ba2+ as a charge carrier allowed the first and direct measurement of Ca2+ entry currents to confirm and extend previous deductions based on measurements of Sr2+-, Ba2+- or Mn2+-influxes that induce increases or quench of fura- fluorescence (10,l I). The question whether Ba2+ current oscillations are synchronized with Ca2+ oscillations remains to be solved. However, parallels in onset or cessation of Ba2+ current oscillations and Ca2+ oscillations (13) suggest close correlation between them. In addition to the known functions of ras proteins (see Ref. 20), we therefore suggest that ras proteins, once 716
Vol.
178,
No.
2, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
activated by agonists that engage the phosphoinositide pathway, constantly transduce signals on Ca2+ entry channels in the plasma membrane of transformed cells. Acknowledgments
We thank Dr. Y. Sugimoto for cells and Dr. J. E. Merritt for SK&F 96365. This investigation was supported in part by research grants from the Ministry of Education, Science and Culture of Japan. REFERENCES
1. Berridge, M.J. & Irvine, R. (1989) Nature 34 1, 197-205. 2. Berridge, M.J. (1990) J. Biol. Chem. 265, 9583-9586. 3. Jacob, R., Merritt, J. E., Hallam, T. J. & Rink, T. J. (1988) Nature 335, 40-45. 4. Harootunian, A. T., Kao, J. P. Y., Paranjape, S. & Tsien, R. Y. (1991) Science 25 1,75-78. 5. Wakui, M., Osipchuk, Y. V. & Petersen,0. H. (1991) Cell 63, 1025-1032. 6. Loessberg, P. A., Zhao, H. & Muallem, S. (1991) J. Biol. Chem. 26 6, 1363-1366. 7. Zhao, H., Loessberg, P. A., Sachs, G. & Muallem, S. (1990) J. Biol. Chem. 2 65,20856-20862.
8, Malgaroli, A., Fesce, R. & Meldolesi, J. (1990) J. Biol. Chem. 2 6 5, 30053008. 9. Tsien, R. W. & Tsien R. Y. (1990) Ann. Rev. Cell Biol. 6, 715-760. 10. Jacob, R. (1990) J. Physiol. 421, 55-77. 11. Kwan, C.-Y. & Putney, J. W. Jr. (1990) J. Biol. Chem. 265, 678-684. 12. Putney, J. W. Jr. (1990) Cell Calcium 11, 611-624. 13. Fu, T., Sugimoto, Y., Oki, T., Murakami, S., Okano. Y. & Nozawa, Y. (1991) FEBS Letters 281,263-266. 14. Noda, M., Selinger, Z., Scohrick, E. M. & Bassin, R.H. (1983) Proc. Nat. Acad. Sci. USA 80, 5602-5606. 15. Higashida, H., Hashii, M., Fukuda, K., Caulfield, M. P., Numa, S. & Brown, D. A. (1990) Proc. R.. Sot. London B 242,68-74. 16. Moolenaar, W. H., Mummery, C. L., Van de Saag,P. T. & de Laat, S. W. (1981) Cel123, 789-798. 17. Lipp, P. & Pott, L. (1988) J. Physiol. 397, 601-630. 18. Potreau, D., Richard, S., Nargeot, J. & Raymond, G. (1987) Pfhigers Archiv 410,326-334.
19. Merritt, J.E., Armstrong, W.P., Benham, C.D., Hallam, T.J., Jacob, R., Jaxa-Chamiec, A., Leigh, B.K., McCarthy, S.A., Moores, K.E. Jz Rink, T.J. (1990) Biochem. J. 271, 515-522. 20. Boume, H. R., Sanders,D. A. & McCormick, F. (1990) Nature 34 9, 117-127.
717
