Dec 24, 1991 - and §Amylin Pharmaceuticals Inc., 9373 Towne Center Drive,. Suite 250, San Diego, CA 92121, U.S.A. do not question that this channel can be ...
343
BJ Letters
dependent mechanism. Further work is needed to elucidate at least one additional pathway for Ca2+ entry, which may be Mn2+ impermeant, and which stopped-flow fluorimetry indicates is activated prior to the release of Ca2+ from intracellular stores by agonists such as thrombin. Stewart 0. SAGE,* Paul SARGEANT,* Janet E. MERRITT,t Martyn P. MAHAUT-SMITHt and Timothy J. RINK§ *The Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, U.K., tSmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts. AL6 9AR, U.K., tDepartment of Cellular and Molecular Medicine, Howard Hughes Medical Institute, School of Medicine, University of California, San Diego, La Jolla, CA 92093, U.S.A., and §Amylin Pharmaceuticals Inc., 9373 Towne Center Drive, Suite 250, San Diego, CA 92121, U.S.A. 1. Alonso, M. T., Alvarez, J., Montero, M., Sanchez, A. & GarciaSancho, J. (1991) Biochem. J. 280, 783-789 2. Alvarez, J., Montero, M. & Garcia-Sancho, J. (1991) Biochem. J. 274, 193-197 3. Montero, M., Alvarez, J. & Garcia-Sancho, J. (1991) Biochem. J. 277, 73-79 4. Sage, S. O., Reast, R. & Rink, T. J. (1990) Biochem. J. 265, 675-680 5. Mahaut-Smith, M. P., Sage, S. 0. & Rink, T. J. (1990) J. Biol. Chem. 265, 10479-10483 6. Mahaut-Smith, M. P., Rink, T. J. & Sage, S. 0. (1990) J. Physiol. (London) 434, 38P 7. Sage, S. O., Merritt, J. E., Hallam, T. J. & Rink, T. J. (1989) Biochem. J. 258, 923-926 8. Sargeant, P., Clarkson, W. D., Sage, S. 0. & Heemskerk, J. W. M. (1992) J. Physiol. (London), in the press 9. Sage, S. 0. & Rink, T. J. (1990) Biochem. J. 265, 306-307 10. Sage, S. 0. & Rink, T. J. (1987) J. Biol. Chem. 262, 16364-16369 11. Mertz, L. M., Baum, B. J. & Ambuakar, I. S. (1990) J. Biol. Chem. 265, 15010-15014 12. Merritt, J. E. & Hallam, T. J. (1988) J. Biol. Chem. 263, 6161-6164
do not question that this channel can be permeated by Ca2+, as stated by Sage et al. [1], but feel that "it is not clear whether it could explain the Ca2` influx observed in intact cells" [2]. For example, Penner et al. [6] described a similar cation channel activated by agonists in mast cells, but they felt it could not explain the agonist-induced Ca2+ influx observed in intact cells. This view proved to be correct, as Hoth & Penner [7] found later a different channel (which, incidentally, was activated by emptying the Ca2+ stores) that better fitted this role. The experimental support for the involvement of mechanism (ii) in thrombin-induced Ca2+ entry was the observation that the lag for the [Ca2+]1 increase induced by thrombin was 26 % longer in Ca2+-free medium than in Ca2+-containing medium [3,8]. This compares to a 1000-2000 % increase of the lag time observed for ADP on Ca2+ removal [3,8]. The delay observed for thrombin on (a) Ca2+-free medium
Ca2+-containing medium 12 0.9
Influx
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1 mM-EGTA
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Received 24 December 1991
5
1 min
Agonist-evoked Ca2+ entry in human platelets: a reply Sage et al. [1] claim that agonists such as thrombin or PAF evoke Ca2+ entry in platelets by mechanisms not dependent on the emptying of the intracellular Ca2+ stores and report that the effects of the cytochrome P-450 inhibitors econazole and miconazole on agonist-evoked entry of Ca2+ or Mn2+ are only marginal. This controverts our recent proposal that agonistinduced Ca2+ entry in platelets is secondary to the emptying of intracellular Ca2+ stores and that a cytochrome P-450 may be involved in coupling the stores to the plasma membrane channels [2]. On the basis of data obtained with stop-flow fluorimetry Sage et al. [3] had proposed that agonist-induced Ca2+ entry may take place by "three different mechanisms .: (i) a close coupling of ADP receptors to Ca2+ entry..., for which Mn2+ is an effective substitute; (ii) an early phase of thrombin-stimulated entry, possibly activated by diffusible second messengers, that passes Mn2+ only poorly; (iii) a later phase of thrombin-evoked entry that is promoted by emptying of the dischargeable intracellular Ca2+ pool". Mn2+ is also an effective substitute for mechanism (iii), which was proposed to apply also for a delayed (200 ms lag) response to ADP [4]. Non-selective cation channels, identified later in platelet membrane patches [5], were proposed to mediate mechanism (i). We ..
Vol. 285
10 25 [SK&F 96365] (#M)
0
Ot
N3
25
(C)
OCH3
CK OCH3 SK&F 96365
CI Econazole
Fig. 1. Effects of cytochrome P450 inhibitors on agonist-induced Call entry in human platelets (a) Effects of econazole on the increase of [Ca2"], of fura-2-loaded human platelets stimulated with thrombin in the presence and in the absence of external Ca2l (1 mM-CaCl2 or 1 mM-CaCl2 + 2 mMEGTA). Econazole (10 /sM; Eco) was added 2 min before thrombin (1 unit/ml; Thr). [Ca2"], was estimated from the ratio of the fluorescences excited at 340 and at 380 nm. The difference in peak height of the control responses (with no added drug) measured in the presence or absence of external Ca2+ is attributed to Ca21 influx. (b) Typical raw fluorescence traces (with scale converted to [Ca21]i) of quin2-loaded platelets stimulated with ADP (20 Mm) in the presence or in the absence of external Ca2+ (I mM-CaCl2 or I mM-EGTA). SK&F 96365 (at the concentrations shown) was added 2 min before ADP. Reproduced from Merritt et al. [3]. (c) Comparison of the structures of SK&F 96365 and econazole.
BJ Letters
344 removal of extracellular Ca2+ might have alternative explanations, for example slight slowing of the kinetics of thrombin binding to the receptor in the absence of external Ca2 . Mechanism (ii) was not detected in Mn2+ uptake experiments, so that it was proposed to be selective for Ca2+ over Mn2+ [3]. In any case, Sage et al. [1] are right in pointing out that mechanism (ii) would have not been detected in our experiments with Mn2' and that "experiments measuring agonist-evoked rises in [Ca2+]i in the presence of external Ca2+...would be useful to judge how important any store-regulated or cytochrome-dependent entry pathway is in platelets" [1]. The results of such experiments are shown in Fig. 1. Fig. l(a) illustrates the effects of econazole, a cytochrome P-450 inhibitor, on the increases of [Ca2+]1 induced by thrombin in Ca2+containing and in Ca2+-free medium. Econazole prevented most of the extra effect observed with thrombin in Ca2+-containing medium and had little effect in Ca2+-free medium. These results indicate that most of the entry of Ca2+ induced by thrombin takes place through the cytochrome-dependent pathway and suggests that the contribution of mechanism (ii) to the increase of [Ca2+]i is a minor one. Similar evidence for ADP-induced Ca2+ entry was incidentally provided by Merritt et al. [9] using compound SK&F 96365. The chemical structures of SK&F 96365 and econazole are very similar, as illustrated in Fig. l(c). In addition, we have shown that compound SK&F 96365 is a cytochrome P-450 inhibitor with an IC50 of 5-10 /M [2,10]. Fig. l(b) reproduces the results of Merritt et al. (Fig. 2a of [9]). Compound SK&F 96365, at 25 /M, reduced the increase of [Ca2+]1 observed in Ca2+-containing medium to essentially the same value found in Ca2+-free medium. By contrast, it caused very little decrease in the signal observed in Ca2+-free medium. These observations suggest that, as with thrombin, most ADPinduced Ca2+ influx takes place through the cytochromedependent pathway and are consistent with our results on Mn2+ entry [2]. Sage et al. [1] report only minor effects of econazole and miconazole on the agonist-evoked Mn2+ entry and no effect of 3 ,uM-miconazole on the increase of [Ca2+1] induced by ADP. Certainly, these findings are difficult to reconcile with ours [2], and even with their previous results with SK&F 96365 ([9]; see Fig. lb here). Similarly, Sage et al. [1] report no effects of econazole on the early time courses of ADP-evoked rises of [Ca2+]1 or ADP-evoked Mn2+ entry measured by stopped-flow fluorimetry. This, again, contrasts with previously detected effects of SK&F 96365 in the same parameters measured by the same procedure [4]. WE do not have a definite explanation for these discrepancies. Perhaps the concentration of econazole and miconazole used by Sage et al. (3 #M) was too small. We report an
IC50 value of 3 /M in platelets [2] and it is possible that a fraction of these strongly apolar drugs might have bound to the stop-flow apparatus or to contaminating plasma proteins. Finally, Sage et al. [1] report that imidazole antimycotics at concentrations higher than 3 4uM produce an increase of [Ca2+]1 in platelets, "indicating non-specific effects". We also find a slight increase of [Ca2+]1 in some platelet batches (see for example Fig. lb), although at higher concentrations (5-10 tM), and specific for Ca2` over Ni2+. In our hands, this side effect did not pose serious problems. Sage et al. [1] are right, however, in cautioning the use of these inhibitors, since the useful concentration range is very narrow and varies with cell type. "High" concentrations permeabilize the plasma membrane to Ni2+, and this is an useful test which should be performed in control experiments. In summary, we believe that the store-regulated Ca2+/Mn2+ entry pathway involving cytochrome P-450 can explain most of the Ca2+ entry induced by agonists such as thrombin, PAF and ADP in platelets. Of course, contributions from other mechanisms cannot be excluded, but, in our hands, they are quantitatively minor. Javier GARCIA-SANCHO, Javier ALVAREZ, Mayte MONTERO, Maria Teresa ALONSO and Ana SANCHEZ
Departamento de Bioquimica, Biologia Molecular y Fisiologia, Facultad de Medicina, Universidad de Valladolid, 47005 Valladolid, Spain 1. Sage, S. O., Sargeant, P., Merritt, J. E., Mahaut-Smith, M. P. & Rink, T. J. (1992) Biochem. J. 285, 341-343 2. Alonso, M. T., Alvarez, J., Montero, M., Sinchez, A. & GarciaSancho, J. (1991) Biochem. J. 280, 783-789 3. Sage, S. O., Merritt, J. E., Hallam, T. J. & Rink, T. J. (1989) Biochem. J. 258, 923-926 4. Sage, S. O., Reast, R. & Rink, T. J. (1990) Biochem. J. 265, 675-680 5. Mahaut-Smith, M. P., Sage, S. 0. & Rink, T. J. (1990) J. Biol. Chem. 265, 10479-10483 6. Penner, R., Matthews, G. & Neher, E. (1988) Nature (London) 334, 499-504 7. Hoth, M. & Penner, R. (1992) Nature (London) 355, 353-356 8. Sage, S. 0. & Rink, T. J. (1987) J. Biol. Chem. 262, 16364-16369 9. Merritt, J. E., Armstrong, P., Benham, C. D., Hallam, T. J., Jacob, R., Jaxa-Chamiec, A., Leigh, B. K., McCarthy, S. A., Moores, K. E. & Rink, T. J. (1990) Biochem. J. 271, 515-522 10. Garcia-Sancho, J., Alvarez, J., Montero, M. & Villalobos, C. (1992) Trends Pharmacol. Sci. 13, 12-13
Received 19 March 1992
1992
