Simultaneous measurement of cytosolic and mitochondrial Ca(2+) during ischemia in mice whole-brain slice preparation and its application to drug evaluation.

We developed a conventional imaging method to measure Ca(2+) concentration in cytosol (using FuraRed as an indicator) and mitochondria (using Rhod-2 as an indicator), simultaneously, by alternative excitation with specific wave length. After confirming the availability of the method in Hela cells, we applied it to mouse whole-brain slice -preparation, which was exposed to oxygen- and glucose-deprived artificial cerebrospinal fluid (ischemic ACSF) for 12 min. The fluorescence (>570 nm) at the cerebral cortex and hippocampus due to FuraRed (excited by 480 ± 10 nm) decreased (indicating the increase in cytosolic Ca(2+)-concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca(2+) concentration) during exposure to ischemic conditions. We found the characteristic protective effects of cyclosporine A (10(-6) M), a known blocker for mitochondrial permeability transition, and SEA0400 (10(-6) M), a blocker for Na(+)/Ca(2+) exchanger, on the abnormal Ca(2+) increase in cytosol. We confirmed that the present method will be useful for future pathological and pharmacological studies on ischemia-induced brain damage.

Evaluation of the protective effects of cyclosporin a and FK506 on abnormal cytosolic and mitochondrial Ca²âº dynamics during ischemia and exposure to high glutamate concentration in mouse brain slice preparations.

We examined the protective effects of the immunosuppressants cyclosporin A (CsA) and FK506 on abnormal cytosolic Ca²âº ([Ca²âº]c) and mitochondrial Ca²âº concentration ([Ca²âº]m) dynamics induced by ischemia or high L-glutamate concentration in mouse brain slice preparations. We used fura-4F and rhod-2 as indicators for [Ca²âº]c and [Ca²âº]m, respectively, in their acetoxymethylester form. Slice preparations loaded with either of these two indicators were exposed to ischemic artificial cerebrospinal fluid (oxygen- and glucose-deprived medium) for 12 min or to aerobic medium with high L-glutamate concentration (isotonic 20 mM L-glutamate) for 5 min. CsA (1 - 10 µM) showed significant protective effects on the maximum increase in ischemia-induced [Ca²âº]c and [Ca²âº]m. FK506 (10 µM) showed significant protective effects on the [Ca²âº]m increase, but not on the ischemia-induced [Ca²âº]c increase. Both immunosuppressants showed almost equal protective effects on the [Ca²âº]c and [Ca²âº]m increases induced by high L-glutamate concentration. These results suggest that the protective effects of CsA and FK506 on Ca²âº overloading may be dependent upon the common pharmacological sites of actions relating to their effects as immunosuppressants. The small, but significant depressant effects of these drugs could give us important clues for rescuing critical brain damage induced by Ca²âº overloading.

Evaluation of putative inhibitors of mitochondrial permeability transition for brain disorders--specificity vs. toxicity.

Inhibition of mitochondrial permeability transition (mPT) has emerged as a promising approach for neuroprotection and development of well-tolerated mPT inhibitors with favorable blood-brain barrier penetration is highly warranted. In a recent study, 28 clinically available drugs with a common heterocyclic structure were identified as mPT inhibitors e.g. trifluoperazine, promethazine and nortriptyline. In addition, neuroprotection by structurally unrelated drugs e.g. neurosteroids, 4-hydroxy-tamoxifen and trimetazidine has been attributed to direct inhibition of mPT. The regulation of mPT is complex and highly dependent on the prevailing experimental conditions. Several features of mPT, such as swelling, depolarization or NADH oxidation, can also occur independently of the mPT phenomenon. Here, in isolated rodent brain-derived and human liver mitochondria, we re-evaluate drugs promoted as potent mPT inhibitors. We address the definition of an mPT inhibitor and present strategies to reliably detect mPT inhibition in vitro. Surprisingly, none of the 12 compounds tested displayed convincing mPT inhibition or effects comparable to cyclophilin D inhibition by the non-immunosuppressive cyclophilin inhibitor D-MeAla(3)-EtVal(4)-Cyclosporin (Debio 025). Propofol and 2-aminoethoxydiphenyl borate (2-APB) inhibited swelling in de-energized mitochondria but did not increase calcium retention capacity (CRC). Progesterone, trifluoperazine, allopregnanolone and 4-hydroxy-tamoxifen dose-dependently reduced CRC and respiratory control and were thus toxic rather than beneficial to mitochondrial function. Interestingly, topiramate increased CRC at high concentrations likely by a mechanism separate from direct mPT inhibition. We conclude that a clinically relevant mPT inhibitor should have a mitochondrial target and increase mitochondrial calcium retention at concentrations which can be translated to human use.