Joanna E. Kowalczyk

Identification of a voltage-gated potassium channel in gerbil hippocampal mitochondria

Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.

Enantiomeric composition of terpenic hydrocarbons in essential oils from Juniperus communis L.

Abstract α-Cyclodextrin complexation under appropriate conditions of partition gas chromatography has been applied for chiral discrimination of the hydrocarbon fraction of juniper oils of various origins. The essential oils under investigation were obtained from crushed juniper berries and leaves cultivated on plantations in Austria, Italy and Poland. It was found that the main component of the hydrocarbon fraction of almost all the juniper oils under investigation was a laevorotatory enantiomer, S(−)-α-pinene, and that the ratio of its S to R enantiomer may vary greatly (from 5 to 1) depending on the origin of the material, i.e. the part of the plant (berries or leaves), and the place of its cultivation.

Approach to direct chiral recognition of some terpenic hydrocarbon constituents of essential oils by gas chromatography systems via α-cyclodextrin complexation

Abstract The application of a very selective chiral stationary phase (Celite coated with α-cyclodextrin in formamide solution) in gas chromatography is described. Using this phase it is possible to separate enantiomeric mixtures of α-pinene, β-pinene, limonene and camphene. The separation of these compounds in essential oils is also demonstrated.

Association of protein kinase C delta and phospholipid scramblase 3 in hippocampal mitochondria correlates with neuronal vulnerability to brain ischemia

Recent findings support the idea that mitochondrial integrity plays an important role in the propagation of excitotoxic ischemic signal and PKC is implicated in the regulation of mitochondrial membranes properties. One of the targets of PKC delta is phospholipid scramblase 3 (PLSCR3), an enzyme responsible for cardiolipin translocation from the inner to outer mitochondrial membrane. To get an insight into in vivo mechanism by which PKC delta mediates ischemia/reperfusion injury of hippocampal neurons, we examined the effects of transient brain ischemia in gerbil on association of PKC delta with mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant regions, and interactions between PKC delta and PLSCR3. Postischemic, biphasic and brain region-specific translocation of PKC delta to mitochondria was observed. First peak was at 30-60 min of reperfusion and the second was observed after 72-96 h following ischemia. PKC delta was translocated to mitochondria only in CA1 region. The PLSCR3 mRNA and protein was detected in brain by RT-PCR and sequence analysis, Western blotting and immunocytochemistry in electron microscopy (EM). Co-immunoprecipitation and double-labeled immuno-EM showed association of PKC delta and PLSCR3 in postischemic CA1 mitochondria. Additionally, the amount of tBid associated with mitochondria was elevated 96 h following ischemia. Our data suggest that in the postischemic brain PKC delta co-localizes with PLSCR3 in mitochondria and this event might influence the mitochondrial membranes architecture and delayed neurons degeneration.

Cytochrome c binds to inositol (1,4,5) trisphosphate and ryanodine receptors in vivo after transient brain ischemia in gerbils

Previously we have shown that the biphasic efflux of mitochondrial protein cytochrome c to cytoplasm is one of the important events of the delayed postichemic neuronal death. We concluded that early and transient appearance of cytochrome c in cytoplasm of cells recovering after ischemia was decisive for initiation of the pathological signaling cascade leading to neuronal death, but the precise mechanism remained unknown. In vitro cytochrome c was identified as a messenger that coordinates mitochondrial-endoplasmatic reticulum interactions that drive apoptosis. Here we show that in vivo cytochrome c interacts with inositol (1,4,5) trisphosphate receptor type 1 in gerbil hippocampus subjected to transient brain ischemia and short reperfusion. Moreover, cytochrome c binds also to ryanodine receptor type 2, the role of which in postischemic neuronal death is suggested. The complexes could be coimmunoprecipitated by antibodies against any of the two proteins. Our data verified that the mechanism observed in vitro applies to the pathological in vivo situation.

Chromatographic studies of the enantiomeric composition of some therapeutic compositions applied in the treatment of liver and kidney diseases

A gas-liquid chromatographic system with alpha-cyclodextrin in formamide medium (coated on Chromosorb) was used for the separation of enantiomers of alpha-pinene, beta-pinene, limonene and camphene in medicines applied in the therapy of liver and kidney diseases. The drugs under investigation were produced in Poland (Terpichol and Terpinex), in Germany (Rowachol and Rowatinex) and in Slovenia (Uroterp). It was found that, depending on the manufacturer, medicines possessing similar chemical compositions differ considerably from one another regarding the content of enantiomers, mainly those of alpha-pinene.

Protein kinase C beta in postischemic brain mitochondria.

PKC is implicated in the regulation of mitochondrial metabolism. We examined the association of PKCβ with mitochondria and followed postischemic changes in its amount in mitochondria isolated from ischemia-vulnerable (CA1) and ischemia-resistant (CA2-4,DG) hippocampus in gerbil model of transient brain ischemia. Our observations suggest that transient ischemic episode induces a significant, rapid and long lasting increase of PKCβ in mitochondria in CA2-4,DG, which may bespeak neuroprotection. In organotypic hippocampal culture (OHC) model of neurodegeneration, PKCβ inhibition imposed over NMDA toxicity extended the death area beyond the CA1. These results suggest that PKCβ might have a protective effect against excitotoxic damage in rat OHC. The pull-down method and LC-MS/MS analysis revealed mitochondrial proteins that can bind directly with PKCβΙ. The proteins were parts of i) mitochondrial redox carriers forming the electron transport chain including ATP synthase and ii) MPTP: ANT and creatine kinase. PKCβ acting through mitochondrial proteins could play a role in protecting the cells from death by e.g. influencing ROS and ATP production after ischemia in CA2-4,DG region of the hippocampus.

Chromatographic analysis of some commercial samples of camphene via cyclodextrin inclusion processes

A gas—liquid chromatographic system containing α-cyclodextrin in formamide medium (coated on Celite) was found to be useful for the separation of camphene enentiomers, with a separation factor α [t′R(-)/t′R(+)] equal to 3.6 (at 30°C). The packing was applied in the analytical mode for a study of the composition of five commercial camphenes, and in the micropreparative mode to establish the enantiomeric composition of separated camphenes.