Development of systemic immunity to glioblastoma multiforme using tumor cells genetically engineered to express the membrane-associated isoform of macrophage colony-stimulating factor.

We investigated the ability of Fischer rat T9 glioblastoma cells transduced with cDNA genes for the secreted (s) or membrane-associated (m) isoform of M-CSF to elicit an antitumor response when implanted into syngeneic animals. Intracranial (i.c.) implantation of 1 x 10(5) T9 cells expressing mM-CSF (T9/mM-CSF) resulted in 80% tumor rejection. Electron microscopy of the T9/mM-CSF tumor site, 2-4 days postimplantation, showed marked infiltration by macrophages, many of which were in physical contact with the T9/mM-CSF cells. Animals that rejected T9/mM-CSF cells were resistant to i.c. rechallenge with T9 cells, but not syngeneic MadB106 breast adenocarcinoma cells, suggesting that T9-specific immunity can be generated within the brain via the endogenous APCs. Intracranial injection of parental T9, vector control (T9/LXSN), or T9 cells secreting M-CSF (T9/sM-CSF) was 100% fatal. Subcutaneous injection of 1 x 10(7) T9/sM-CSF, T9/LXSN, or parental T9 cells resulted in progressive tumors. In contrast, T9/mM-CSF cells injected s.c. were destroyed in 7-10 days and animals developed systemic immunity to parental T9 cells. Passive transfer of CD3+ T cells from the spleens of immune rats into naive recipients transferred T9 glioma-specific immunity. In vitro, splenocytes from T9/mM-CSF-immunized rats specifically proliferated in response to various syngeneic glioma stimulator cells. However, only marginal T cell-mediated cytotoxicity was observed by these splenocytes in a CTL assay against T9 target cells, regardless of restimulation with T9 cells. Subcutaneous immunization with viable T9/mM-CSF cells was effective in eradicating i.c. T9 tumors.     

T9 glioma cells expressing membrane-macrophage colony stimulating factor produce CD4+ T cell-associated protective immunity against T9 intracranial gliomas and systemic immunity against different syngeneic gliomas

Cloned T9 glioma cells (T9-C2) expressing the membrane form of macrophage colony stimulating factor (mM-CSF) inoculated subcutaneously into rats do not grow and glioma-specific immunity is stimulated. Immunotherapy experiments showed that intracranial T9 tumors present for one to four days could be successfully eradicated by peripheral vaccination with T9-C2 cells. CD4+ and CD8+ T splenocytes from immunized rats, when restimulated in vitro with T9 cells, produced interleukin-2 and -4. Protective immunity against intracranial T9 gliomas could only be adoptively transferred into naive rats by the CD4+ splenocytes obtained from T9-C2 immunized rats. Rats immunized by the T9-C2 tumor cells also resisted two different syngeneic gliomas (RT2 and F98) but allowed a syngeneic NUTU-19 ovarian cancer to grow. Such cross-protective immunity against unrelated gliomas suggests that mM-CSF transfected tumor cells have immunotherapeutic potential for use as an allogeneic tumor vaccine.     

BK channels are linked to inositol 1,4,5-triphosphate receptors via lipid rafts: a novel mechanism for coupling [Ca(2+)](i) to ion channel activation

Glioma cells prominently express a unique splice variant of a large conductance, calcium-activated potassium channel (BK channel). These channels transduce changes in intracellular calcium to changes of K(+) conductance in the cells and have been implicated in growth control of normal and malignant cells. The Ca(2+) increase that facilitates channel activation is thought to occur via activation of intracellular calcium release pathways or influx of calcium through Ca(2+)-permeable ion channels. We show here that BK channel activation involves the activation of inositol 1,4,5-triphosphate receptors (IP(3)R), which localize near BK channels in specialized membrane domains called lipid rafts. Disruption of lipid rafts with methyl-beta-cyclodextrin disrupts the functional association of BK channel and calcium source resulting in a >50% reduction in K(+) conductance mediated by BK channels. The reduction of BK current by lipid raft disruption was overcome by the global elevation of intracellular calcium through inclusion of 750 nm Ca(2+) in the pipette solution, indicating that neither the calcium sensitivity of the channel nor their overall number was altered. Additionally, pretreatment of glioma cells with 2-aminoethoxydiphenyl borate to inhibit IP(3)Rs negated the effect of methyl-beta-cyclodextrin, providing further support that IP(3)Rs are the calcium source for BK channels. Taken together, these data suggest a privileged association of BK channels in lipid raft domains and provide evidence for a novel coupling of these Ca(2+)-sensitive channels to their second messenger source.     

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.     

Ca2+-activated K channels in parotid acinar cells: The functional basis for the hyperpolarized activation of BK channels

Fluid secretion relies on a close interplay between Ca²⁺-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca²⁺ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca²⁺ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca²⁺ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca²⁺ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca²⁺. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes.Key words: ion channels, Ca²⁺-activated K channels, maxi-K channels, IK1 channels     

Cytotoxic T cell immunity against the non-immunogenic, murine, hepatocellular carcinoma Hepa1-6 is directed towards the novel alternative form of macrophage colony stimulating factor

Mouse Hepa1-6 hepatocellular carcinoma (HCC) cells were transduced with the membrane form of macrophage colony stimulating factor (mM-CSF). When mM-CSF transduced Hepa1-6 cells were injected subcutaneously into mice, these cells did not form tumors. The spleens of these immunized mice contained cytotoxic CD8⁺ T lymphocytes (CTL) that killed the unmodified Hepa1-6 cells. We show that the alternative form of macrophage colony stimulating factor (altM-CSF) induced CTL-mediated immunity against Hepa1-6 cells. AltM-CSF is restricted to the H-2D^b allele. CTLs killed RMA-S cells loaded with exogenous altM-CSF peptide. Vaccination of mice with dendritic cells pulsed with the altM-CSF peptide stimulated anti-Hepa1-6 CTLs. Hyper-immunization of mice with mM-CSF Hepa1-6 cells showed inflammation of the liver and kidneys. Although altM-CSF was expressed within liver and kidney cells, its intensity was lower than Hepa1-6 cells. AltM-CSF was detected within the human HepG2 cell line. These studies suggest that altM-CSF may be a tumor antigen for HCC.     

Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells

Curcumin is considered a pharmacologically safe agent that may be useful in cancer chemoprevention and therapy. Here, we show for the first time that curcumin effectively induces paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation that results from swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis by cycloheximide blocked curcumin-induced vacuolation and subsequent cell death, indicating that protein synthesis is required for this process. The levels of AIP-1/Alix protein, a known inhibitor protein of paraptosis, were progressively downregulated in curcumin-treated malignant breast cancer cells, and AIP-1/Alix overexpression attenuated curcumin-induced death in these cells. ERK2 and JNK activation were positively associated with curcumin-induced cell death. Mitochondrial superoxide was shown to act as a critical early signal in curcumin-induced paraptosis, whereas proteasomal dysfunction was mainly responsible for the paraptotic changes associated with ER dilation. Notably, curcumin-induced paraptotic events were not observed in normal breast cells, including mammary epithelial cells and MCF-10A cells. Taken together, our findings on curcumin-induced paraptosis may provide novel insights into the mechanisms underlying the selective anti-cancer effects of curcumin against malignant cancer cells.     

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl^–, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl^– concentration [Cl^–]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl^–]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents. heat shock; geldanamycin; cellular stress; channel trafficking; transepithelial chloride transport     

Current Transients Associated with BK Channels in Human Glioma Cells

We have previously demonstrated the expression of BK channels in human glioma cells. There was a curious feature to the whole-cell currents of glioma cells seen during whole-cell patch-clamp: large, outward current transients accompanied repolarization of the cell membrane following an activating voltage step. This transient current, I _transient, activated and inactivated rapidly (1 ms). The I-V relationship of I _transient had features that were inconsistent with simple ionic current through open ion channels: (i) I _transient amplitude peaked with a –80 mV voltage change and was invariant over a 200 mV range, and (ii) I _transient remained large and outward at –140 mV. We provide evidence for a direct relationship of I _transient to glioma BK currents. They had an identical time course of activation, identical pharmacology, identical voltage-dependence, and small, random variations in the amplitude of the steady-state BK current and I _transient seen over time were often perfectly in phase. Substituting intracellular K⁺ with Cs⁺, Li⁺, or Na ⁺ ions reversibly reduced I _transient and BK currents. I _transient was not observed in recordings of other BK currents (hbr5 expressed in HEK cells and BK currents in rat neurons), suggesting I _transient is unique to BK currents in human glioma cells. We conclude that I _transient is generated by a mechanism related to the deactivation, and level of prior activation, of glioma BK channels. To account for these findings we propose that K⁺ ions are trapped within glioma BK channels during deactivation and are forced to exit to the extracellular side in a manner independent of membrane potential.     

Hsp72 functions as a natural inhibitory protein of c-Jun N-terminal kinase

Hsp72, a major inducible member of the heat shock protein family, can protect cells against many cellular stresses including heat shock. In our present study, we observed that pretreatment of NIH 3T3 cells with mild heat shock (43 degrees C for 20 min) suppressed UV-stimulated c-Jun N-terminal kinase 1 (JNK1) activity. Constitutively overexpressed Hsp72 also inhibited JNK1 activation in NIH 3T3 cells, whereas it did not affect either SEK1 or MEKK1 activity. Both in vitro binding and kinase studies indicated that Hsp72 bound to JNK1 and that the peptide binding domain of Hsp72 was important to the binding and inhibition of JNK1. In vivo binding between endogenous Hsp72 and JNK1 in NIH 3T3 cells was confirmed by co-immunoprecipitation. Hsp72 also inhibited JNK-dependent apoptosis. Hsp72 antisense oligonucleotides blocked Hsp72 production in NIH 3T3 cells in response to mild heat shock and concomitantly abolished the suppressive effect of mild heat shock on UV-induced JNK activation and apoptosis. Collectively, our data suggest strongly that Hsp72 can modulate stress-activated signaling by directly inhibiting JNK.     

A catalytically inactive mutant of type I cGMP-dependent protein kinase prevents enhancement of large conductance, calcium-sensitive K+ channels by sodium nitroprusside and cGMP

The activation of large conductance, calcium-sensitive K(+) (BK(Ca)) channels by the nitric oxide (NO)/cyclic GMP (cGMP) signaling pathway appears to be an important cellular mechanism contributing to the relaxation of smooth muscle. In HEK 293 cells transiently transfected with BK(Ca) channels, we observed that the NO donor sodium nitroprusside and the membrane-permeable analog of cGMP, dibutyryl cGMP, were both able to enhance BK(Ca) channel activity 4-5-fold in cell-attached membrane patches. This enhancement correlated with an endogenous cGMP-dependent protein kinase activity and the presence of the alpha isoform of type I cGMP-dependent protein kinase (cGKI). We observed that co-transfection of cells with BK(Ca) channels and a catalytically inactive ("dead") mutant of human cGKIalpha prevented enhancement of BK(Ca) channel in response to either sodium nitroprusside or dibutyryl cGMP in a dominant negative fashion. In contrast, expression of wild-type cGKIalpha supported enhancement of channel activity by these two agents. Importantly, both endogenous and expressed forms of cGKIalpha were found to associate with BK(Ca) channel protein, as demonstrated by a reciprocal co-immunoprecipitation strategy. In vitro, cGKIalpha was able to directly phosphorylate immunoprecipitated BK(Ca) channels, suggesting that cGKIalpha-dependent phosphorylation of BK(Ca) channels in situ may be responsible for the observed enhancement of channel activity. In summary, our data demonstrate that cGKIalpha alone is sufficient to promote the enhancement of BK(Ca) channels in situ after activation of the NO/cGMP signaling pathway.     

Honokiol induces caspase-independent paraptosis via reactive oxygen species production that is accompanied by apoptosis in leukemia cells

Our previous report has shown that honokiol (HNK), a constituent of Magnolia officinalis, induces a novel form of non-apoptotic programmed cell death in human leukemia NB4 and K562 cells. In this study, we further explored the relationship between the cell death pathway and cytoplasmic vacuolization and studied the underlying mechanism of leukemia cell death mediated by honokiol. The results showed that low concentrations of honokiol activated an novel alternative cell death fitted the criteria of paraptosis, such as cytoplasmic vacuolization derived from endoplasmic reticulum swelling, lack of caspase activation, and lack of apoptotic morphology. Results further indicated that the cell death was time- and concentration-dependent. In addition, honokiol-induced paraptosis did not involve membrane blebbing, chromatin condensation and phosphatidylserine exposure at the outer of the plasma membrane. The mechanism of the cell death may be associated, at least in part, with the increased generation of reactive oxygen species. Further analysis showed that honokiol induces cell death predominantly via paraptosis and to a certain extent via apoptosis in NB4 cells, and predominantly via apoptosis and to a certain extent via paraptosis in K562 cells. These observations suggest that cell death occurs via more than one pathway in leukemia cells and targeting paraptosis may be an alternative and promising avenue for honokiol in leukemia therapy.     

Suppression of NF-kappaB activation by Entamoeba histolytica in intestinal epithelial cells is mediated by heat shock protein 27

Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) 27 and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp27 and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-kappaB activation induced by interleukin-1beta; specifically, the NF-kappaB-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of IkappaB-alpha were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp27 in suppressing NF-kappaB activation at IkappaB kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp27 interacts with IKK-alpha and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp27 amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp27 abrogated SAP-induced NF-kappaB inhibition. In conditioned epithelial cells, Hsp27 was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.     

The Membrane-Associated Transient Receptor Potential Vanilloid Channel Is the Central Heat Shock Receptor Controlling the Cellular Heat Shock Response in Epithelial Cells

The heat shock response (HSR) is a highly conserved molecular response to various types of stresses, including heat shock, during which heat-shock proteins (Hsps) are produced to prevent and repair damages in labile proteins and membranes. In cells, protein unfolding in the cytoplasm is thought to directly enable the activation of the heat shock factor 1 (HSF-1), however, recent work supports the activation of the HSR via an increase in the fluidity of specific membrane domains, leading to activation of heat-shock genes. Our findings support the existence of a plasma membrane-dependent mechanism of HSF-1 activation in animal cells, which is initiated by a membrane-associated transient receptor potential vanilloid receptor (TRPV). We found in various non-cancerous and cancerous mammalian epithelial cells that the TRPV1 agonists, capsaicin and resiniferatoxin (RTX), upregulated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70 and Hsp90 respectively, while the TRPV1 antagonists, capsazepine and AMG-9810, attenuated the accumulation of Hsp70, Hsp90 and Hsp27 and Hsp70, Hsp90, respectively. Capsaicin was also shown to activate HSF-1. These findings suggest that heat-sensing and signaling in mammalian cells is dependent on TRPV channels in the plasma membrane. Thus, TRPV channels may be important drug targets to inhibit or restore the cellular stress response in diseases with defective cellular proteins, such as cancer, inflammation and aging.     

Generation of human innate immune responses towards membrane macrophage colony stimulating factor (mM-CSF) expressing U251 glioma cells within immunodeficient (NIH-nu/beige/xid) mice

The response of human peripheral blood mononuclear cells (PBMC) to cloned human HLA-A2+ U251 glioma cells (U251-2F11/TK) expressing membrane macrophage colony stimulating factor (mM-CSF) was investigated in vitro and in vivo. Enriched human monocytes derived from cancer patients produced a respiratory burst following 20min of interaction with mM-CSF expressing U251 glioma cells. This respiratory burst response was not observed in the enriched human monocytes following similar exposure to the viral vector control U251 (U251-VV) cells. Reactive oxygen species such as H(2)O(2) and HOCl produced death of the U251 cells. The U251-2F11/TK cells failed to grow in severely compromised combined immunodeficient (NIH-bg-nu-xidBR) mice that were depleted of murine monocyte/macrophages then reconstituted with human HLA-A2+ PBMC. Reactive oxygen species (ROS) were produced by PBMC, both in vitro and in vivo in response tomM-CSF expressing U251 cells. U251-2F11/TK cells failed to form subcutaneous tumors in macrophage depleted mice reconstituted with human PBMC; whereas, progressive growth of such tumors was observed with the U251-VV cells. U251-2F11/TK tumors formed if the initial inoculums of PBMC were depleted of monocytes. From this work it can be concluded that mM-CSF transduced U251-2F11/TK glioma cells can safely stimulate human innate immune responses in vivo.     

Irradiated tumor cell vaccine for treatment of an established glioma. I. Successful treatment with combined radiotherapy and cellular vaccination

Rats bearing a 5-day intracranial (i.c.) syngeneic glioma were treated with a subcutaneous (s.c.) vaccination consisting of irradiated glioma cells or a multimodality approach composed of radiotherapy plus s.c. vaccination. Vaccination of rats harboring a T9 glioma with 5 x 10(6) irradiated T9.F glioma cells (a clone derived from the T9 glioblastoma cell line) resulted in a marked enhancement of i.c. glioma growth and a significant decrease in survival. Histopathology of the tumors from vaccinated rats revealed a massive glioma composed of healthy tumor tissue lacking any marked inflammation, edema or hemorrhage. Analysis of the tumor-infiltrating mononuclear cells indicated that gliomas from vaccinated rats contained a 10-fold greater lymphoid infiltrate per milligram of tumor as compared to tumors from non-vaccinated rats, suggesting that the vaccination had induced immune cells to localize to the i.c. glioma. Combined treatment consisting of 15 Gy of whole head irradiation of the 5-day glioma followed by vaccination with T9.F cells resulted in a significant increase in survival compared to that of non-treated rats, 45% of which remained tumor-free. Microscopic evaluation in survivors of the tumor implantation site revealed the presence of hemosiderin-laden macrophages and other mononuclear cells, with the absence of tumor cells within the residual lesion. When survivors were challenged s.c. with viable T9.F glioma cells, a delayed-type hypersensitivity (DTH) reaction appeared at the challenge site. T cells purified from these rats proliferated and secreted Th(1)-associated cytokines when stimulated with irradiated T9.F glioma cells, and lysed T9.F target cells. In contrast, when these rats were challenged s.c. with the unrelated MadB106 adenocarcinoma, tumor formation was observed. These findings indicate that the treatment of an established i.c. glioma with a cellular vaccination alone may induce enhanced tumor growth; however, when the vaccination is combined with radiation therapy, the results are beneficial in terms of increased survival time or complete remission that is accompanied by the development of tumor-specific cellular immunity.     

Localization of heat shock proteins in cerebral cortical cultures following induction by celastrol

Hsp70, Hsp32, and Hsp27 were induced by celastrol in rat cerebral cortical cultures at dosages that did not affect cell viability. Pronounced differences were observed in the cellular localization of these heat shock proteins in cell types of cerebral cortical cultures. Celastrol-induced Hsp70 localized to the cell body and cellular processes of neurons that were identified by neuron-specific βIII-tubulin. Hsp70 was not detected in adjacent GFAP-positive glial cells that demonstrated a strong signal for Hsp27 and Hsp32 in both glial cell bodies and cellular processes. Cells in the cerebral cortex region of the brain are selectively impacted during the progression of Alzheimer’s disease which is a “protein misfolding disorder.” Heat shock proteins provide a line of defense against misfolded, aggregation-prone proteins. Celastrol is a potential agent to counter this neurodegenerative disorder as recent evidence indicates that in vivo administration of celastrol in a transgenic model of Alzheimer’s reduces an important neuropathological hallmark of this disease, namely, amyloid beta pathology that involves protein aggregation.     

Mitochondrial Ca2+-activated K+ channels more efficiently reduce mitochondrial Ca2+ overload in rat ventricular myocytes

We investigated the role of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel, the mitochondrial big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca(2+) in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca(2+), we pretreated cells with ouabain before applying mitochondrial K(ATP) or BK(Ca) channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 +/- 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 muM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K(ATP) channel by 5-hydroxydecanoate, the mitochondrial BK(Ca) channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca(2+) during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca(2+). These results suggest that although mitochondrial BK(Ca) and K(ATP) channels contribute to reduction of ouabain-induced mitochondrial Ca(2+) overload, activation of the mitochondrial BK(Ca) channel more efficiently reduces ouabain-induced mitochondrial Ca(2+) overload in our experimental model.