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Fractional Ca2+Release from the Endoplasmic Reticulum Activates Ca2+Entry in Freshly Isolated Rabbit Aortic Endothelial Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
241, 471–475 (1997)
RC977844
Fractional Ca2/ Release from the Endoplasmic Reticulum Activates Ca2/ Entry in Freshly Isolated Rabbit Aortic Endothelial Cells Hisashi Sasajima,1 Xiaodong Wang, and Cornelis van Breemen2 Department of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Received November 10, 1997
The purpose of the present investigation was to examine the correlation between rates of endoplasmic reticulum (ER) depletion and activation of store operated channels (SOC) in freshly isolated rabbit aortic endothelial cells. We investigated the effects of 10mM cyclopiazonic acid (CPA), 10mM ryanodine, and 10mM caffeine on the rate of Ca2/ depletion from the ER and on Ca2/ and Mn2/ influx using fura-2 fluorescence. We observed that the spontaneous loss of the ACh-sensitive pool is slow. Activation of ryanodine receptors (caffeine, ryanodine) or inhibition of the ER Ca2/ pump (CPA) increased the rate of Ca2/ loss from the AChsensitive pool. CPA stimulated Mn2/ influx, while caffeine and ryanodine did not. Our results show non linear correlation between ER depletion and activation of divalent cation entry. In the case of CPA, less than 20% depletion of the ACh sensitive store was required for full activation of SOC, while caffeine and ryanodine deplete over 50% of the ACh sensitive store without activating any influx. These data suggest that only a small compartment of the ER is involved in regulation of Ca2/ entry. q 1997 Academic Press
It is generally accepted that endothelium forms a selective permeability barrier between the vascular and interstitial spaces and plays an important role in both short- and long-term regulation of vascular homeostasis and resistance. The vascular endothelial cells produce a variety of paracrine substances and ad1 Current address: Division of Cardiology, Department of Medicine, Wakayama Medical College, 7-27 Wakayama City, Japan 640. 2 To whom correspondence should be addressed at Department of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, B.C., Canada V6T 1Z3. Fax: /1-604-822-2281. E-mail: [email protected]. Abbreviations used: CPA, cyclopiazonic acid; ICRAC , Ca2/ release activated Ca2/ current; ER, endoplasmic reticulum; IP3 , inositol 1,4,5-trisphosphate; SOC, store operated channel.
hesion molecules which play an active role in inflammation, platelet aggregation, thrombosis, angiogenesis, fibrinolysis, and regulation of vascular tone. Agonist induced elevation of intracellular calcium concentration ([Ca2/]i) in vascular endothelial cells signals the production of nitric oxide and prostacyclin which inhibit smooth muscle tone. Previous studies have shown that Ca2/ channel activity in endothelial cells is regulated by agonists rather then by voltage. Ca2/ mobilizing agonists such as acetylcholine, histamine, bradykinin and ATP release intracellularly stored Ca2/ via inositol 1,4,5-trisphosphate (IP3) sensitive channels in the ER (1,2). We and others showed that the IP3 sensitive Ca2/ store is the major source for Ca2/ release and that refilling proceeds via CPA sensitive Ca2/ ATPase (3,4). Recent reports indicate the existence of functional ryanodine receptors in endothelial ER which appear to overlap with the IP3 sensitive store (3,5). Previous reports show that depletion of the intracellular Ca2/ store activates Ca2/ influx into non-excitable cells and suggest the existence of Ca2/ release activated Ca2/ current (ICRAC) in endothelial cells. Ca2/ store depletion using sarcoplasmic-endoplasmic reticulum Ca2/ ATPase (SERCA) inhibitors such as cyclopiazonic acid (CPA), thapsigargin and 2,5-di-t-butylhydroquinone (BHQ) activates Ca2/ influx in endothelial cells from human umbilical vein (6), bovine aorta (7) and bovine/calf pulmonary artery (8,9). It has been postulated that emptying of the intracellular Ca2/ store releases a small messenger molecule that stimulates Ca2/ influx (Ca2/ influx factor) which may be a cytochrome P450 product (10,11). Alternatively there may be a physical-chemical link between the peripheral ER and store operated channels (SOC) in the plasmalemma (12). In addition tyrosine phosphorylation may be involved in activation of SOC (13). At a more fundamental level little is known about the quantitative correlation between ER Ca2/ release and the initia-
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FIG. 1. Protocol for measuring the rate of Ca2/ depletion from the agonist sensitive intracellular Ca2/ store in freshly isolated rabbit aortic endothelial cells. (A) 10 mM ACh induces a [Ca2/]i transient in 0 Ca, PSS which depletes the Ca2/ store for the subsequent ACh response. (B) After the first ACh induced [Ca2/]i transient in 0 Ca, PSS the cells are repleted with Ca2/. They are subsequently incubated in 0 Ca, PSS for the experimental time period (in this case 300 seconds) before a second ACh application in the absence of extracellular Ca2/.
tion of Ca2/ entry. One study(24) suggested that these two events are directly proportional; however in this communication we show that less then 20% depletion of the acetylcholine sensitive Ca2/ store is sufficient for the activation of store operated channels. Furthermore activation of divalent cation influx depends on the mechanism of Ca2/ release. MATERIALS AND METHODS Isolation of the rabbit endothelial cells. Endothelial cells were obtained from the thoracic aorta of New Zealand White rabbits (2-
2.5 kg) as previously described (3,15). The rabbit was killed with CO2 asphyxiation and exsanguinated. The thoracic aorta was rapidly removed and freed of the surrounding fat and connective tissue under a dissecting microscope, the aorta was placed in a test tube containing Ca2/-free PSS with 0.1 mg/ml collagenase, 0.1 % elastase and 1mg/ml bovine serum albumin for 40 minutes at 377C. Endothelial cells were dispersed by use of a Pasteur pipette. The endothelial cells were seeded on a glass coverslip precoated with poly-D-lysine at room temperature before the experiment and used within 5 hours. [Ca2/]i measurement using fura-2 fluorescence. [Ca2/]i was measured using a microscope-based fluorometer from Photon Technology International, London, Canada. The endothelial cells on the coverslip were loaded with 1mM fura-2/AM (acetoxymethylester) in normal
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FIG. 2. Effects of the various agents on the rate of Ca2/ loss from the ACh sensitive store in freshly isolated rabbit aortic endothelial cells. Each data point represents the [Ca2/]i transient after the indicated time in 0 Ca,PSS plus respective agent as a percentage of the initial control response (see protocol in Fig. 1B). The rate of spontaneous loss of the ACh sensitive store in Ca2/ free solution is slow. In contrast, the rates of loss of CPA, ryanodine, and caffeine sensitive pools were faster than the spontaneous loss of the ACh sensitive store.
PSS for 30 min at room temperature. The coverslip was mounted on a Nikon inverted microscope (Nikon, Diaphot). The cells were excited alternately at 340 and 380 nm and the light collected at 510nm. The ratio of the two intensities at 340 or 380 nm constitutes a relative measure of the free [Ca2/]i . Mn2/ quenching experiment. The Mn2/ quenching method was used for direct measurement of the divalent cation entry into the endothelial cells. 200 mM MnCl2 was added to the Ca2/-free bathing solution. The cells were excited at 360 nm, which is the isosbestic point for fura-2 Ca2/ fluorescence. Mn2/ binds to the intracellular dye and quenches its fluorescence after it enters the cells. The slope of the fluorescence intensity curve reflects the rate of Mn2/ entry. Chemicals and solutions. Fura-2 AM was purchased from Molecular Probe (Eugene, Ore., USA). All other materials were purchased from Sigma Chemicals (St. Louis, Mo., USA). The normal PSS solution contained (mM) NaCl 126, KCl 5, CaCl2 1, MgCl2 1.2, D-glucose 11, HEPES 10. 0mM Ca2/ PSS (0Ca PSS) solution contained (mM) NaCl 126, KCl 5, MgCl2 1.2, D-glucose 11, HEPES 10. All solutions were adjusted to pH 7.4. The solutions were superfused into the experimental chamber and a vacuum suction pump was used in order to keep a constant fluid level. Experiments were carried out at room temperature. Data from multiple experiments are given as mean { SEM.
RESULTS Ca2/ depletion rate of endoplasmic reticulum. Figure 1 illustrates the experimental approach for measuring the rate of Ca2/ loss from the ER. Figure 1-A shows that the first exposure of ACh in Ca2/-free solution induced a transient [Ca2/]i increase which depletes the
agonist sensitive store since a second exposure to ACh in 0Ca PSS induced no increase of [Ca2/]i . Figure 1-B illustrates the protocol for measuring the spontaneous loss of the ACh sensitive pool in Ca2/ free solution. 10mM ACh induced a transient [Ca2/]i increase in 0Ca PSS after exposure to 0Ca PSS for 50 sec. After the first ACh exposure, the cells were superfused with PSS for 300 sec in order to allow refilling of the intracellular Ca2/ store and a second ACh response was measured after an experimental time period in 0Ca PSS. Plotting the ratio of the peak [Ca2/]i in response to the second ACh exposure over that of the first against time in 0Ca solution provides a measure of the depletion rate of the ACh sensitive store in Ca2/ free solution. By using the same protocol but adding 10mM CPA, 10mM ryanodine, or 10 mM caffeine to the second 0Ca PSS perfusion period, we measured the effects of these agents on the rate of ER Ca2/ depletion. Figure 2 shows that the spontaneous rate of ER Ca2/ loss is slow(90.51%/5.01 at 300 sec). The various agents tested increased the rate of Ca2/ loss from the ACh sensitive pool in the following order(nÅ4, at 300sec): caffeine(59.9%/2.04) ú ryanodine(64.54%/2.79) ú CPA(70.85%/1.76). Measurement of divalent cation entry using Mn2/ quenching. In attempt to correlate the effects of CPA, ryanodine and caffeine on ER Ca2/ depletion with possible activation of the Ca2/ influx pathway, we mea-
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FIG. 3. Effect of 10 mM cyclopiazonic acid (CPA) on the Ca2/ influx pathway in freshly isolated rabbit aortic endothelial cells. The rate of fura-2 quenching by Mn2/ influx. Addition of Mn2/ increased the slope of the quenching curve and 100 seconds after addition of CPA the slope showed a further increase. This is a typical trace of 6 experiments.
sured Mn2/ quenching of fura-2 fluorescence under comparable conditions. The fura-2 signal was quenched by adding 200mM Mn2/ to the nominally Ca2/ free solution. The slope represents basal Mn2/ entry. Figure 3 shows that addition of 200mM Mn2/ increases the slope of fura-2 quenching and that addition of CPA further increases Mn2/ entry after a delay 80-100sec. In contrast, ryanodine and caffeine did not activate Mn2/ influx even until 600 seconds, when the ACh store was depleted by 50% (data not shown). DISCUSSION In this study we examined the effects of 10 mM CPA, 10 mM ryanodine and 10 mM caffeine on the ACh sensitive Ca2/ store in the ER of the endothelial cells using fura-2 fluorescence microscopy. Although it has been reported that SERCA blockers, ACh, ryanodine and caffeine deplete the internal Ca2/ store, the relative rates of depletion have not been investigated. We observed (as shown in Figure 1-A) that ACh rapidly depletes the Ca2/ store while the spontaneous loss of the ACh sensitive pools in Ca2/ free solution is slow. This confirms the results of our previous study(3). CPA, caffeine and ryanodine all enhance the rate of Ca2/ loss. Among them caffeine has the greatest effect on the rate of Ca2/ depletion. 10mM CPA was used in this study because its effect is reversible and it is reported
that CPA inhibits both isoforms of ER ATPase (14), while thapsigargin and BHQ are more specific. To correlate the magnitude of Ca2/ loss from the ACh sensitive store with the time of the onset of divalent cation influx through SOC, we measured divalent cation entry using Mn2/ quenching of fura-2 fluorescence. CPA stimulates Mn2/ influx. In contrast, ryanodine and caffeine did not stimulate Mn2/ influx, but rather had a small effect of reducing the basal rate of Mn2/ entry. ACh as well CPA have been shown to activate Ca2/ influx in this cell preparation(15). Our previous report describing the effects of several Ca2/ entry blockers has suggested that the Ca2/ influxes caused by the two different modes of activation (ACh or CPA) were indistinguishable from each and suggested that ACh may activate influx in parallel to CPA without obligatory depletion of the store. This raises the question whether depletion of the intracellular store is the necessary step in activating Ca2/ influx as suggested by the ‘‘capacitative’’ theory(16). The most cogent evidence for depletion activated influx is that SERCA blockers initiate Ca2/ influx. CPA, thapsigargin and BHQ deplete internal Ca2/ stores without apparent activation of G-proteins and phospholipase C (17-19) and many studies have shown that these compounds stimulate Ca2/ influx in various nonexcitable cells (18-20). Although the precise relation-
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ship between the Ca2/ influx pathway activated by depletion of internal Ca2/ stores and that activated by Ca2/ mobilizing agonists have not been clear, a number of models have been proposed to explain how the internal stores can regulate the calcium release activated channels (CRAC) in the plasma membrane. Some models propose the existence of messenger molecules and consider that the depleted ER releases a factor which then diffuses to the membrane to open CRAC (21,22). Alternatively others consider that information may be transferred more directly through protein-protein interaction (12,23). The most interesting result in this study is that although caffeine and ryanodine deplete the ACh sensitive store at a faster rate then CPA they do not activate Ca2/ influx. We reported previously that caffeine failed to cause transient Ca2/ release in this native endothelial cell preparation but did slowly deplete the IP3 sensitive store (3). Our previous paper also showed that although caffeine was able to inhibit IP3 sensitive Ca2/ release induced by ACh(3,15), it did not inhibit the ACh induced Ca2/ influx (15). Our data related to CPA induced Ca2/ loss demonstrate that less than 20% depletion of the ACh sensitive store is required for the activation of SOC (Fig. 2). In contrast 50 % depletion of the ACh sensitive Ca2/ store by either caffeine or ryanodine did not activate Mn2/ entry. These results in contrast to an other study (24) show an imperfect correlation between ER depletion and activation of Mn2/ influx. We conclude therefore that if depletion of the intracellular Ca2/ store is a necessary step in activating Ca2/ influx as suggested by capacitative theory, then most likely only a distinct Ca2/ store of limited size is linked to SOC. Our data indicate that this compartment is depleted more rapidly that the bulk of the ER and therefore may be located peripherally. This compartment would further distinguish itself by a lack of ryanodine receptors. Alternatively it is also possible that store depletion is not an obligatory step in the activation process of agonist stimulated influx (15) and the influx caused by SERCA blockade is based on other unknown mechanism involving a caveoli like Ca2/ store attached to the membrane as has been recently suggested (25).
REFERENCES 1. Freay, A., Johns, A., Adams, D. J., Ryan, U. S., and van Breemen, C. (1989) Pflug Arch - Eur. J. Physiol. 414, 377–384. 2. Lambert, T. L., Kent, R. S., and Whorton, A. R. (1986) J. Biol. Chem. 261, 15288–15293. 3. Wang, X., Lau, F., Li, L., Yoshikawa, A., and van Breemen, C. (1995) Circ. Res. 77, 37–42. 4. Li, L., and van Breemen, C. (1996) Am. J. Physiol. 270, H837– H848. 5. Lesh, R. E., Marks, A. R., Somlyo, A. V., Fleischer, S., and Somlyo, A. P. (1993) Circ. Res. 72, 481–488. 6. Oike, M., Gericke, M., Droogmans, G., and Nilius, B. (1994) Cell Calcium 16, 367–376. 7. Vaca, L., and Kunze, D. L. (1994) Am. J. Physiol. 267, C920– C925. 8. Pasyk, E., Inazu, M., and Daniel, E. E. (1995) Am. J. Physiol. 268, H138–H146. 9. Schilling, W. P., Cabello, O. A., and Rajan, L. (1992) Biochem. J. 284, 521–530. 10. Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809– 814. 11. Graier, W. F., Simecek, S., and Sturek, M. (1995) J. Physiol. 482, 259–274. 12. Berridge, M. J. (1995) Biochem. J. 312, 1–11. 13. Sharma, N. R., and Davis, M. J. (1996) Am. J. Physiol. 270, H267–H274. 14. Papp, B., Paszty, K., Kovacs, T., Sarkadi, B., Gardos, G., Enouf, J., and Enyedi, A. (1993) Cell Calcium 14, 531–538. 15. Wang, X., and van Breemen, C. (1997) J. Vas. Res. 34, 196–207. 16. Putney, J. W., Jr., and Bird, G. S. (1993) Cell 75, 199–201. 17. Oldershaw, K. A., and Taylor, C. W. (1990) FEBS Lett. 274, 214– 216. 18. Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W., Jr. (1989) J. Biol. Chem. 264, 12266–12271. 19. Foder, B., Scharff, O., and Thastrup, O. (1989) Cell Calcium 10, 477–490. 20. Mason, M. J., Mahaut-Smith, M. P., and Grinstein, S. (1991) J. Biol. Chem. 266, 10872–10879. 21. Parekh, A. B., Terlau, H., and Stuhmer, W. (1993) Nature 364, 814–818. 22. Davies, E. V., and Hallett, M. B. (1995) Biochem. Biophys. Res. Comm. 206, 348–354. 23. Fasolato, C., Innocenti, B., and Pozzan, T. (1994) Trends Pharmacol. Sci. 15, 77–83. 24. Jacob, R. (1990) J. Physiol. 421, 55–77. 25. Bode, H. P., and Netter, K. J. (1996) Biochem. Pharmacol. 51, 993–1001.
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Fractional Ca2/ Release from the Endoplasmic Reticulum Activates Ca2/ Entry in Freshly Isolated Rabbit Aortic Endothelial Cells Hisashi Sasajima,1 Xiaodong Wang, and Cornelis van Breemen2 Department of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Received November 10, 1997
The purpose of the present investigation was to examine the correlation between rates of endoplasmic reticulum (ER) depletion and activation of store operated channels (SOC) in freshly isolated rabbit aortic endothelial cells. We investigated the effects of 10mM cyclopiazonic acid (CPA), 10mM ryanodine, and 10mM caffeine on the rate of Ca2/ depletion from the ER and on Ca2/ and Mn2/ influx using fura-2 fluorescence. We observed that the spontaneous loss of the ACh-sensitive pool is slow. Activation of ryanodine receptors (caffeine, ryanodine) or inhibition of the ER Ca2/ pump (CPA) increased the rate of Ca2/ loss from the AChsensitive pool. CPA stimulated Mn2/ influx, while caffeine and ryanodine did not. Our results show non linear correlation between ER depletion and activation of divalent cation entry. In the case of CPA, less than 20% depletion of the ACh sensitive store was required for full activation of SOC, while caffeine and ryanodine deplete over 50% of the ACh sensitive store without activating any influx. These data suggest that only a small compartment of the ER is involved in regulation of Ca2/ entry. q 1997 Academic Press
It is generally accepted that endothelium forms a selective permeability barrier between the vascular and interstitial spaces and plays an important role in both short- and long-term regulation of vascular homeostasis and resistance. The vascular endothelial cells produce a variety of paracrine substances and ad1 Current address: Division of Cardiology, Department of Medicine, Wakayama Medical College, 7-27 Wakayama City, Japan 640. 2 To whom correspondence should be addressed at Department of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall, Vancouver, B.C., Canada V6T 1Z3. Fax: /1-604-822-2281. E-mail: [email protected]. Abbreviations used: CPA, cyclopiazonic acid; ICRAC , Ca2/ release activated Ca2/ current; ER, endoplasmic reticulum; IP3 , inositol 1,4,5-trisphosphate; SOC, store operated channel.
hesion molecules which play an active role in inflammation, platelet aggregation, thrombosis, angiogenesis, fibrinolysis, and regulation of vascular tone. Agonist induced elevation of intracellular calcium concentration ([Ca2/]i) in vascular endothelial cells signals the production of nitric oxide and prostacyclin which inhibit smooth muscle tone. Previous studies have shown that Ca2/ channel activity in endothelial cells is regulated by agonists rather then by voltage. Ca2/ mobilizing agonists such as acetylcholine, histamine, bradykinin and ATP release intracellularly stored Ca2/ via inositol 1,4,5-trisphosphate (IP3) sensitive channels in the ER (1,2). We and others showed that the IP3 sensitive Ca2/ store is the major source for Ca2/ release and that refilling proceeds via CPA sensitive Ca2/ ATPase (3,4). Recent reports indicate the existence of functional ryanodine receptors in endothelial ER which appear to overlap with the IP3 sensitive store (3,5). Previous reports show that depletion of the intracellular Ca2/ store activates Ca2/ influx into non-excitable cells and suggest the existence of Ca2/ release activated Ca2/ current (ICRAC) in endothelial cells. Ca2/ store depletion using sarcoplasmic-endoplasmic reticulum Ca2/ ATPase (SERCA) inhibitors such as cyclopiazonic acid (CPA), thapsigargin and 2,5-di-t-butylhydroquinone (BHQ) activates Ca2/ influx in endothelial cells from human umbilical vein (6), bovine aorta (7) and bovine/calf pulmonary artery (8,9). It has been postulated that emptying of the intracellular Ca2/ store releases a small messenger molecule that stimulates Ca2/ influx (Ca2/ influx factor) which may be a cytochrome P450 product (10,11). Alternatively there may be a physical-chemical link between the peripheral ER and store operated channels (SOC) in the plasmalemma (12). In addition tyrosine phosphorylation may be involved in activation of SOC (13). At a more fundamental level little is known about the quantitative correlation between ER Ca2/ release and the initia-
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FIG. 1. Protocol for measuring the rate of Ca2/ depletion from the agonist sensitive intracellular Ca2/ store in freshly isolated rabbit aortic endothelial cells. (A) 10 mM ACh induces a [Ca2/]i transient in 0 Ca, PSS which depletes the Ca2/ store for the subsequent ACh response. (B) After the first ACh induced [Ca2/]i transient in 0 Ca, PSS the cells are repleted with Ca2/. They are subsequently incubated in 0 Ca, PSS for the experimental time period (in this case 300 seconds) before a second ACh application in the absence of extracellular Ca2/.
tion of Ca2/ entry. One study(24) suggested that these two events are directly proportional; however in this communication we show that less then 20% depletion of the acetylcholine sensitive Ca2/ store is sufficient for the activation of store operated channels. Furthermore activation of divalent cation influx depends on the mechanism of Ca2/ release. MATERIALS AND METHODS Isolation of the rabbit endothelial cells. Endothelial cells were obtained from the thoracic aorta of New Zealand White rabbits (2-
2.5 kg) as previously described (3,15). The rabbit was killed with CO2 asphyxiation and exsanguinated. The thoracic aorta was rapidly removed and freed of the surrounding fat and connective tissue under a dissecting microscope, the aorta was placed in a test tube containing Ca2/-free PSS with 0.1 mg/ml collagenase, 0.1 % elastase and 1mg/ml bovine serum albumin for 40 minutes at 377C. Endothelial cells were dispersed by use of a Pasteur pipette. The endothelial cells were seeded on a glass coverslip precoated with poly-D-lysine at room temperature before the experiment and used within 5 hours. [Ca2/]i measurement using fura-2 fluorescence. [Ca2/]i was measured using a microscope-based fluorometer from Photon Technology International, London, Canada. The endothelial cells on the coverslip were loaded with 1mM fura-2/AM (acetoxymethylester) in normal
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FIG. 2. Effects of the various agents on the rate of Ca2/ loss from the ACh sensitive store in freshly isolated rabbit aortic endothelial cells. Each data point represents the [Ca2/]i transient after the indicated time in 0 Ca,PSS plus respective agent as a percentage of the initial control response (see protocol in Fig. 1B). The rate of spontaneous loss of the ACh sensitive store in Ca2/ free solution is slow. In contrast, the rates of loss of CPA, ryanodine, and caffeine sensitive pools were faster than the spontaneous loss of the ACh sensitive store.
PSS for 30 min at room temperature. The coverslip was mounted on a Nikon inverted microscope (Nikon, Diaphot). The cells were excited alternately at 340 and 380 nm and the light collected at 510nm. The ratio of the two intensities at 340 or 380 nm constitutes a relative measure of the free [Ca2/]i . Mn2/ quenching experiment. The Mn2/ quenching method was used for direct measurement of the divalent cation entry into the endothelial cells. 200 mM MnCl2 was added to the Ca2/-free bathing solution. The cells were excited at 360 nm, which is the isosbestic point for fura-2 Ca2/ fluorescence. Mn2/ binds to the intracellular dye and quenches its fluorescence after it enters the cells. The slope of the fluorescence intensity curve reflects the rate of Mn2/ entry. Chemicals and solutions. Fura-2 AM was purchased from Molecular Probe (Eugene, Ore., USA). All other materials were purchased from Sigma Chemicals (St. Louis, Mo., USA). The normal PSS solution contained (mM) NaCl 126, KCl 5, CaCl2 1, MgCl2 1.2, D-glucose 11, HEPES 10. 0mM Ca2/ PSS (0Ca PSS) solution contained (mM) NaCl 126, KCl 5, MgCl2 1.2, D-glucose 11, HEPES 10. All solutions were adjusted to pH 7.4. The solutions were superfused into the experimental chamber and a vacuum suction pump was used in order to keep a constant fluid level. Experiments were carried out at room temperature. Data from multiple experiments are given as mean { SEM.
RESULTS Ca2/ depletion rate of endoplasmic reticulum. Figure 1 illustrates the experimental approach for measuring the rate of Ca2/ loss from the ER. Figure 1-A shows that the first exposure of ACh in Ca2/-free solution induced a transient [Ca2/]i increase which depletes the
agonist sensitive store since a second exposure to ACh in 0Ca PSS induced no increase of [Ca2/]i . Figure 1-B illustrates the protocol for measuring the spontaneous loss of the ACh sensitive pool in Ca2/ free solution. 10mM ACh induced a transient [Ca2/]i increase in 0Ca PSS after exposure to 0Ca PSS for 50 sec. After the first ACh exposure, the cells were superfused with PSS for 300 sec in order to allow refilling of the intracellular Ca2/ store and a second ACh response was measured after an experimental time period in 0Ca PSS. Plotting the ratio of the peak [Ca2/]i in response to the second ACh exposure over that of the first against time in 0Ca solution provides a measure of the depletion rate of the ACh sensitive store in Ca2/ free solution. By using the same protocol but adding 10mM CPA, 10mM ryanodine, or 10 mM caffeine to the second 0Ca PSS perfusion period, we measured the effects of these agents on the rate of ER Ca2/ depletion. Figure 2 shows that the spontaneous rate of ER Ca2/ loss is slow(90.51%/5.01 at 300 sec). The various agents tested increased the rate of Ca2/ loss from the ACh sensitive pool in the following order(nÅ4, at 300sec): caffeine(59.9%/2.04) ú ryanodine(64.54%/2.79) ú CPA(70.85%/1.76). Measurement of divalent cation entry using Mn2/ quenching. In attempt to correlate the effects of CPA, ryanodine and caffeine on ER Ca2/ depletion with possible activation of the Ca2/ influx pathway, we mea-
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FIG. 3. Effect of 10 mM cyclopiazonic acid (CPA) on the Ca2/ influx pathway in freshly isolated rabbit aortic endothelial cells. The rate of fura-2 quenching by Mn2/ influx. Addition of Mn2/ increased the slope of the quenching curve and 100 seconds after addition of CPA the slope showed a further increase. This is a typical trace of 6 experiments.
sured Mn2/ quenching of fura-2 fluorescence under comparable conditions. The fura-2 signal was quenched by adding 200mM Mn2/ to the nominally Ca2/ free solution. The slope represents basal Mn2/ entry. Figure 3 shows that addition of 200mM Mn2/ increases the slope of fura-2 quenching and that addition of CPA further increases Mn2/ entry after a delay 80-100sec. In contrast, ryanodine and caffeine did not activate Mn2/ influx even until 600 seconds, when the ACh store was depleted by 50% (data not shown). DISCUSSION In this study we examined the effects of 10 mM CPA, 10 mM ryanodine and 10 mM caffeine on the ACh sensitive Ca2/ store in the ER of the endothelial cells using fura-2 fluorescence microscopy. Although it has been reported that SERCA blockers, ACh, ryanodine and caffeine deplete the internal Ca2/ store, the relative rates of depletion have not been investigated. We observed (as shown in Figure 1-A) that ACh rapidly depletes the Ca2/ store while the spontaneous loss of the ACh sensitive pools in Ca2/ free solution is slow. This confirms the results of our previous study(3). CPA, caffeine and ryanodine all enhance the rate of Ca2/ loss. Among them caffeine has the greatest effect on the rate of Ca2/ depletion. 10mM CPA was used in this study because its effect is reversible and it is reported
that CPA inhibits both isoforms of ER ATPase (14), while thapsigargin and BHQ are more specific. To correlate the magnitude of Ca2/ loss from the ACh sensitive store with the time of the onset of divalent cation influx through SOC, we measured divalent cation entry using Mn2/ quenching of fura-2 fluorescence. CPA stimulates Mn2/ influx. In contrast, ryanodine and caffeine did not stimulate Mn2/ influx, but rather had a small effect of reducing the basal rate of Mn2/ entry. ACh as well CPA have been shown to activate Ca2/ influx in this cell preparation(15). Our previous report describing the effects of several Ca2/ entry blockers has suggested that the Ca2/ influxes caused by the two different modes of activation (ACh or CPA) were indistinguishable from each and suggested that ACh may activate influx in parallel to CPA without obligatory depletion of the store. This raises the question whether depletion of the intracellular store is the necessary step in activating Ca2/ influx as suggested by the ‘‘capacitative’’ theory(16). The most cogent evidence for depletion activated influx is that SERCA blockers initiate Ca2/ influx. CPA, thapsigargin and BHQ deplete internal Ca2/ stores without apparent activation of G-proteins and phospholipase C (17-19) and many studies have shown that these compounds stimulate Ca2/ influx in various nonexcitable cells (18-20). Although the precise relation-
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ship between the Ca2/ influx pathway activated by depletion of internal Ca2/ stores and that activated by Ca2/ mobilizing agonists have not been clear, a number of models have been proposed to explain how the internal stores can regulate the calcium release activated channels (CRAC) in the plasma membrane. Some models propose the existence of messenger molecules and consider that the depleted ER releases a factor which then diffuses to the membrane to open CRAC (21,22). Alternatively others consider that information may be transferred more directly through protein-protein interaction (12,23). The most interesting result in this study is that although caffeine and ryanodine deplete the ACh sensitive store at a faster rate then CPA they do not activate Ca2/ influx. We reported previously that caffeine failed to cause transient Ca2/ release in this native endothelial cell preparation but did slowly deplete the IP3 sensitive store (3). Our previous paper also showed that although caffeine was able to inhibit IP3 sensitive Ca2/ release induced by ACh(3,15), it did not inhibit the ACh induced Ca2/ influx (15). Our data related to CPA induced Ca2/ loss demonstrate that less than 20% depletion of the ACh sensitive store is required for the activation of SOC (Fig. 2). In contrast 50 % depletion of the ACh sensitive Ca2/ store by either caffeine or ryanodine did not activate Mn2/ entry. These results in contrast to an other study (24) show an imperfect correlation between ER depletion and activation of Mn2/ influx. We conclude therefore that if depletion of the intracellular Ca2/ store is a necessary step in activating Ca2/ influx as suggested by capacitative theory, then most likely only a distinct Ca2/ store of limited size is linked to SOC. Our data indicate that this compartment is depleted more rapidly that the bulk of the ER and therefore may be located peripherally. This compartment would further distinguish itself by a lack of ryanodine receptors. Alternatively it is also possible that store depletion is not an obligatory step in the activation process of agonist stimulated influx (15) and the influx caused by SERCA blockade is based on other unknown mechanism involving a caveoli like Ca2/ store attached to the membrane as has been recently suggested (25).
REFERENCES 1. Freay, A., Johns, A., Adams, D. J., Ryan, U. S., and van Breemen, C. (1989) Pflug Arch - Eur. J. Physiol. 414, 377–384. 2. Lambert, T. L., Kent, R. S., and Whorton, A. R. (1986) J. Biol. Chem. 261, 15288–15293. 3. Wang, X., Lau, F., Li, L., Yoshikawa, A., and van Breemen, C. (1995) Circ. Res. 77, 37–42. 4. Li, L., and van Breemen, C. (1996) Am. J. Physiol. 270, H837– H848. 5. Lesh, R. E., Marks, A. R., Somlyo, A. V., Fleischer, S., and Somlyo, A. P. (1993) Circ. Res. 72, 481–488. 6. Oike, M., Gericke, M., Droogmans, G., and Nilius, B. (1994) Cell Calcium 16, 367–376. 7. Vaca, L., and Kunze, D. L. (1994) Am. J. Physiol. 267, C920– C925. 8. Pasyk, E., Inazu, M., and Daniel, E. E. (1995) Am. J. Physiol. 268, H138–H146. 9. Schilling, W. P., Cabello, O. A., and Rajan, L. (1992) Biochem. J. 284, 521–530. 10. Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809– 814. 11. Graier, W. F., Simecek, S., and Sturek, M. (1995) J. Physiol. 482, 259–274. 12. Berridge, M. J. (1995) Biochem. J. 312, 1–11. 13. Sharma, N. R., and Davis, M. J. (1996) Am. J. Physiol. 270, H267–H274. 14. Papp, B., Paszty, K., Kovacs, T., Sarkadi, B., Gardos, G., Enouf, J., and Enyedi, A. (1993) Cell Calcium 14, 531–538. 15. Wang, X., and van Breemen, C. (1997) J. Vas. Res. 34, 196–207. 16. Putney, J. W., Jr., and Bird, G. S. (1993) Cell 75, 199–201. 17. Oldershaw, K. A., and Taylor, C. W. (1990) FEBS Lett. 274, 214– 216. 18. Takemura, H., Hughes, A. R., Thastrup, O., and Putney, J. W., Jr. (1989) J. Biol. Chem. 264, 12266–12271. 19. Foder, B., Scharff, O., and Thastrup, O. (1989) Cell Calcium 10, 477–490. 20. Mason, M. J., Mahaut-Smith, M. P., and Grinstein, S. (1991) J. Biol. Chem. 266, 10872–10879. 21. Parekh, A. B., Terlau, H., and Stuhmer, W. (1993) Nature 364, 814–818. 22. Davies, E. V., and Hallett, M. B. (1995) Biochem. Biophys. Res. Comm. 206, 348–354. 23. Fasolato, C., Innocenti, B., and Pozzan, T. (1994) Trends Pharmacol. Sci. 15, 77–83. 24. Jacob, R. (1990) J. Physiol. 421, 55–77. 25. Bode, H. P., and Netter, K. J. (1996) Biochem. Pharmacol. 51, 993–1001.
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