Proc Natl Acad Sci U S A 1997 94:9434-9439
Presenter: Y.M. Leung
Agonist-triggered Ca entry is important in a number of physiological processes such as smooth muscle contraction, mast cell secretion and cell growth. One of the means by which agonists activate Ca entry is by the depletion of intracellular non-mitochondrial Ca stores (part of sarcoplasmic reticulum) as a consequence of inositol 1,4,5-trisphosphate-induced Ca release. Such store-operated Ca entry mechanism has also been known as capacitative Ca entry (CCE). Intracellular Ca stores could be discharged artificially by intracellular Ca pump inhibitors such as thapsigargin or cyclopiazonic acid. The latter agents could be shown to induce Ca store depletion and subsequent Ca entry without any production of second messengers.
There has been a continuous search for blockers of the CCE during the past few years. Several putative blockers of CCE have been found: SKF 96365, LOE908, LU52396 and tetrandrine. However, these agents are not quite selective. For example, most of them block voltage-operated Ca channels (VOCC). The use of them to selectively block CCE is therefore limited to non-excitable cells which lack VOCC.
LU52396 had its debut in 1995 (Eur. J. Pharmacol. 289: 23-31) and was shown to inhibit CCE in Hela cells with an IC50 = 2 uM. Even at 10 uM, LU52396 did not significantly perturb intracellular Ca stores or the membrane potential. Another attractive aspect of LU52396 was that it inhibited second-messenger-operated Ca channel with remarkably lower potency (IC50 = 40 uM). One pitfall of LU52396 is that it blocked VOCC at low micromolar level (Cell Calcium 1996 19: 269-279). This limits its use as a CCE blocker to non-excitable cells.
The sustained phase of Ca entry triggered by OKT3 (antibody against CD3) in Jurkat T cells, which was triggered by the capacitative mechanism, could be substantially inhibited by some known Ca channel blockers such as SKF 96365, nitrendipine (high concentration: 35 microM, therefore non-specific) and Zn. Such Ca entry was not affected by LU 52396 (even at high concentration). Similarly, the sustained phase of Ca entry triggered by thapsigargin was also insensitive to LU52396. The latter agent was only effective in blocking CCE when added before OKT3 and thapsigargin.
The small Ca-elevating effect of LU52396 (eg. Fig. 4) should be investigated in more detail There is an unstable baseline in Ca-free experiments (Fig. 5). Also, in this experiment, a control should be included to show the Ca entry in the absence of LU52396 and OKT3. In disagreement with the author's description, I think the inhibition of Ca entry by LU52396 (Fig. 5C) is still substantial (not "only a slight inhibition") when compared to the corresponding control in 5A. Again in Fig. 5, whether LU52396 affected the Ca release by OKT3 was uncertain. Quantitative results are needed. If LU52396 significantly inhibited Ca release, of course it would affect the subsequent Ca entry. "Similar results were obtained for thapsigargin (data not shown)". (Second last paragraph of the Results). I think since thapsigargin experiments would be the most crucial ones in the present context, such results should be shown, in both qualitative and quantitative manners. The authors should not compare the present results, in a seemingly quantitative manner, with those obtained in a previous publication (p. 96, left column, lines 17-22).
The single most important finding in this paper is that LU52396 appears to inhibit the CCE channel only in its closed state. What advantages (or disadvantages) could there be when this drug is applied for experimental and (potentially) therapeutic purposes? The authors should have some reflection about this in the discussion.
Hitherto there are only a few organic compounds that could block CCE. LU 52396 is one of these compounds and so far there are only two papers about this compound. Therefore, more thorough investigation of the properties of this compound is expected in the future. The selectivity and usefulness of LU52396 has been described above in the background. The present paper describes a very interesting and unique feature of LU52396: the blockade by this drug of the CCE channel was only observed when the latter is in the closed state. No such "non-use- dependence" has ever been reported with other CCE blockers. In fact, for example, tetrandrine blocks CCE channel regardless of its open states (Br. J. Pharmacol. 1994 113: 767-774). Therefore, LU52396 could possibly be employed to study the activation/inactivation kinetics of the CCE channel.
The pharmacological properties of the recently described antagonist for capacitative Ca entry LU52396 were investigated and compared to known Ca antagonists in Jurkat T lymphocytes. In the first set of experiments, cells were stimulated with the anti-CD3 monoclonal antibody OKT3 and, subsequently, Ca antagonists were added. Under such conditions SKF 96365, econazole, nitrendipine and ZnCl2 dose-dependently antagonized Ca signalling, whereas LU52396 in concentrations up to 100 microM did not. In contrast, when LU52396 was added a few minutes before OKT3, a dose-dependent inhibition of the OKT3-stimulated Ca signals by LU 52396 was observed. Likewise, by prior addition of LU52396 to thapsigargin-stimulated Jurkat T cells, a dose-dependent inhibition of Ca signals was achieved. The IC50 value of LU52396 for both agonists was about 5 microM. LU52396 also inhibited Jurkat T cell proliferation, but showed cytotoxic effects at concentrations > 50 uM. Our data indicate that, in contrast to the other Ca antagonists SKF 96365, econazole, nitrendipine and ZnCl2, LU52396 recognized the channel for capacitative Ca entry only when intracellular Ca was low and the channel was in its closed state.