Carbonyl Cyanide M-chlorophenylhydrazone Treatment Research Articles

CHCHD2 and CHCHD10 regulate mitochondrial dynamics and integrated stress response

Mitochondrial dysfunction is becoming one of the main pathology factors involved in the etiology of neurological disorders. Recently, mutations of the coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) and 10 (CHCHD10) which encode two homologous proteins that belong to the mitochondrial CHCH domain protein family, are linked to Parkinson’s disease and amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD), respectively. However, the physiological and pathological roles of these twin proteins have not been well elaborated. Here, we show that, in physiological conditions, CHCHD2 and CHCHD10 interact with OMA1 and suppress its enzyme activity, which not only restrains the initiation of the mitochondrial integrated response stress (mtISR), but also suppresses the processing of OPA1 for mitochondrial fusion. Further, during mitochondria stress-induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment, CHCHD2 and CHCHD10 translocate to the cytosol and interacte with eIF2a, which attenuates mtISR overactivation by suppressing eIF2a phosphorylation and its downstream response. As such, knockdown of CHCHD2 and CHCHD10 triggers mitochondrial ISR, and such cellular response is enhanced by CCCP treatment. Therefore, our findings demonstrate the first “mtISR suppressor” localized in mitochondria for regulating stress responses in mammalian cells, which has a profound pathological impact on the CHCH2/CHCH10-linked neurodegenerative disorder.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Regulation of Parkin-dependent mitophagy by Bcl-2-associated athanogene (BAG) family members.

Quantitative Mitochondrial Proteomics Reveals ANXA7 as a Crucial Factor in Mitophagy.

Mitochondria are involved in many crucial cellular processes. Maintaining healthy mitochondria is essential for cellular homeostasis. Parkin-dependent mitophagy plays an important role in selectively eliminating damaged mitochondria in mammalian cells. However, mechanisms of Parkin-dependent mitophagy remain elusive. In this research, we performed data-independent acquisition-based quantitative mitochondrial proteomics to study the proteomic alterations of carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced Parkin-mediated mitophagy. We identified 222 differentially expressed proteins, with 76 upregulations and 146 downregulations, which were potentially involved in mitophagy. We then demonstrated that annexin A7 (ANXA7), a calcium-dependent phospholipid-binding protein, can translocate to impaired mitochondria upon CCCP treatment, where it played a pivotal part in the process of Parkin-dependent mitophagy via interacting with BASP1. As a mitochondrial uncoupling agent, CCCP indirectly regulated ANXA7 and BASP1 to induce Parkin-dependent mitophagy.

PGAM5 regulates PINK1/Parkin-mediated mitophagy via DRP1 in CCCP-induced mitochondrial dysfunction

Mitochondrial dynamics and mitophagy are critical processes for regulating mitochondrial homeostasis. Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein that plays crucial roles in apoptosis and necroptosis, but the roles of PGAM5 in mitochondrial dynamics and mitophagy remain unclear. In this study, we investigated the role of PGAM5 in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced mitochondrial damage and the correlation between mitochondrial dynamics and mitophagy using SH-SY5Y cells. We found that CCCP decreased mitochondrial membrane potential, resulting in mitochondrial dysfunction. CCCP increased PGAM5, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expression of the mitochondrial fraction in a time-dependent manner. Knockdown of PGAM5 inhibited DRP1 translocation without a change in OPA1 expression in CCCP-treated cells. Furthermore, knockdown of PGAM5 and DRP1 significantly blocked the increase of PTEN-induced putative protein kinase 1 (PINK1) and Parkin expression in the mitochondrial fraction of CCCP-treated cells. Interestingly, CCCP did not alter PINK1/Parkin expression in the mitochondrial fraction of OPA1 knockdown cells. Inhibiting mitophagy by PGAM5 knockdown accelerated CCCP-induced apoptosis. CCCP treatment also results in PINK1 stabilization on the mitochondrial membrane, which subsequently increases Parkin recruitment from the cytosol to abnormal mitochondria. In addition, we found that CCCP increased the level of mitochondrial LC3II, indicating that Parkin recruitment of PINK1 is a result of mitophagy. We propose that activation of PGAM5 is associated with DRP1 recruitment and PINK1 stabilization, which contribute to the modulation of mitophagy in CCCP-treated cells with mitochondrial dysfunction. In conclusion, we demonstrated that PGAM5 regulates PINK1-Parkin-mediated mitophagy, which can exert a neuroprotective effect against CCCP-induced apoptosis.

Maternal aging affects oocyte resilience to carbonyl cyanide-m-chlorophenylhydrazone -induced mitochondrial dysfunction in cows

Mitochondrial quality control is important for maintaining cellular and oocyte viability. In addition, aging affects mitochondrial quality in many cell types. In the present study, we examined how aging affects oocyte mitochondrial biogenesis and degeneration in response to induced mitochondrial dysfunction. Cumulus oocyte complexes were harvested from the ovaries of young (21‒45 months) and aged (≥120 months) cows and treated for 2 hours with 10 μM carbonyl cyanide-m- chlorophenylhydrazone (CCCP), or a vehicle control, after which cumulus oocyte complexes were subjected to in vitro fertilization and culture. CCCP treatment reduced ATP content and increased reactive oxygen species (ROS) levels in the oocytes of both young and aged cows. When CCCP-treated cumulus oocyte complexes were subsequently cultured for 19 hours and/or subjected to fertilization, high ROS levels in oocytes and a low rate of blastocyst development was observed in oocytes derived from aged cows. In addition, we observed differential responses in mitochondrial biogenesis to CCCP treatment between young and aged cows. CCCP treatment enhanced mitochondrial biogenesis concomitant with upregulation of SIRT1 expression in oocytes of young, but not aged, cows. In conclusion, aging affects mitochondrial quality control and recuperation of oocytes following CCCP-induced mitochondrial dysfunction.

Phospholamban Is Downregulated by pVHL-Mediated Degradation through Oxidative Stress in Failing Heart.

The E3 ubiquitin ligase, von Hippel–Lindau (VHL), regulates protein expression by polyubiquitination. Although the protein VHL (pVHL) was reported to be involved in the heart function, the underlying mechanism is unclear. Here, we show that pVHL was upregulated in hearts from two types of genetically dilated cardiomyopathy (DCM) mice models. In comparison with the wild-type mouse, both DCM mice models showed a significant reduction in the expression of phospholamban (PLN), a potent inhibitor of sarco(endo)plasmic reticulum Ca2+-ATPase, and enhanced interaction between pVHL and PLN. To clarify whether pVHL is involved in PLN degradation in failing hearts, we used carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial membrane potential (MMP)-lowering reagent, to mimic the heart failure condition in PLN-expressing HEK293 cells and found that CCCP treatment resulted in PLN degradation and increased interaction between PLN and pVHL. However, these effects were reversed with the addition of N-acetyl-l-cysteine. Furthermore, the co-transfection of VHL and PLN in HEK293 cells decreased PLN expression under oxidative stress, whereas knockdown of VHL increased PLN expression both under normal and oxidative stress conditions. Together, we propose that oxidative stress upregulates pVHL expression to induce PLN degradation in failing hearts.

Superoxide drives progression of Parkin/PINK1-dependent mitophagy following translocation of Parkin to mitochondria

Reactive oxygen species (ROS) and mitophagy are profoundly implicated in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). Several studies have suggested that ROS are not involved in mitochondrial translocation of Parkin which primes mitochondria for autophagic elimination. However, whether ROS play a role in the execution of mitophagy is unknown. In the present study, we show that carbonyl cyanide m-chlorophenylhydrazone (CCCP) treatment induced both mitochondrial depolarization and generation of ROS that were needed for the mitophagy process. Cells failed to proceed to complete mitophagy if CCCP treatment was discontinued even after recruitment of Parkin and autophagy machinery to mitochondria. Notably, treatment of pro-oxidant was able to replace CCCP treatment to take mitophagy forward, while it alone was insufficient to induce translocation of Parkin to mitochondria or autophagic clearance of mitochondria. In addition, an SOD mimetic that attenuated the superoxide level suppressed mitophagy, while an SOD inhibitor accumulated cellular superoxide and promoted mitophagy. Furthermore, blockage of the p38 signaling pathway inhibited mitophagy induced by ROS, suggesting that it may contribute to the activation of ROS-mediated mitophagy. Together, our study sheds light on the link between ROS and mitophagy at a molecular level, and suggests the therapeutic potential of regulating mitophagy through the superoxide–p38–mitophagy axis.

Reactive oxygen species trigger Parkin/PINK1 pathway–dependent mitophagy by inducing mitochondrial recruitment of Parkin

Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria.

53BP1 contributes to regulation of autophagic clearance of mitochondria

Autophagy, the primary recycling pathway within cells, plays a critical role in mitochondrial quality control under normal growth conditions and in the cellular response to stress. Here we provide evidence that 53BP1, a DNA damage response protein, is involved in regulating mitochondrial clearance from the cell via a type of autophagy termed mitophagy. We found that when either human or mouse cells were 53BP1-deficient, there was an increase in mitochondrial abnormalities, as observed through staining intensity, aggregation, and increased mass. Moreover, a 53BP1-depleted cell population included an increased number of cells with a high mitochondrial membrane potential (ΔΨm) relative to controls, suggesting that the loss of 53BP1 prevents initiation of mitophagy thereby leading to the accumulation of damaged mitochondria. Indeed, both 53BP1 and the mitophagy-associated protein LC3 translocated to mitochondria in response to damage induced by the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). The recruitment of parkin, an E3-ubiquitin ligase, to mitochondria in response to CCCP treatment was significantly decreased in 53BP1-deficient cells. And lastly, using p53-deficient H1299 cells, we confirmed that the role of 53BP1 in mitophagy is independent of p53. These data support a model in which 53BP1 plays an important role in modulating mitochondrial homeostasis and in the clearance of damaged mitochondria.

Interaction between RING1 (R1) and the Ubiquitin-like (UBL) Domains Is Critical for the Regulation of Parkin Activity

Parkin is an E3 ligase that contains a ubiquitin-like (UBL) domain in the N terminus and an R1-in-between-ring-RING2 motif in the C terminus. We showed that the UBL domain specifically interacts with the R1 domain and negatively regulates Parkin E3 ligase activity, Parkin-dependent mitophagy, and Parkin translocation to the mitochondria. The binding between the UBL domain and the R1 domain was suppressed by carbonyl cyanide m-chlorophenyl hydrazone treatment or by expression of PTEN-induced putative kinase 1 (PINK1), an upstream kinase that phosphorylates Parkin at the Ser-65 residue of the UBL domain. Moreover, we demonstrated that phosphorylation of the UBL domain at Ser-65 prevents its binding to the R1 domain and promotes Parkin activities. We further showed that mitochondrial translocation of Parkin, which depends on phosphorylation at Ser-65, and interaction between the R1 domain and a mitochondrial outer membrane protein, VDAC1, are suppressed by binding of the UBL domain to the R1 domain. Interestingly, Parkin with missense mutations associated with Parkinson disease (PD) in the UBL domain, such as K27N, R33Q, and A46P, did not translocate to the mitochondria and induce E3 ligase activity by m-chlorophenyl hydrazone treatment, which correlated with the interaction between the R1 domain and the UBL domain with those PD mutations. These findings provide a molecular mechanism of how Parkin recruitment to the mitochondria and Parkin activation as an E3 ubiquitin ligase are regulated by PINK1 and explain the previously unknown mechanism of how Parkin mutations in the UBL domain cause PD pathogenesis.

Involvement of inhibitory PAS domain protein in neuronal cell death in Parkinson's disease.

Inhibitory PAS domain protein (IPAS), a repressor of hypoxia-inducible factor-dependent transcription under hypoxia, was found to exert pro-apoptotic activity in oxidative stress-induced cell death. However, physiological and pathological processes associated with this activity are not known. Here we show that IPAS is a key molecule involved in neuronal cell death in Parkinson’s disease (PD). IPAS was ubiquitinated by Parkin for proteasomal degradation following carbonyl cyanide m-chlorophenyl hydrazone treatment. Phosphorylation of IPAS at Thr12 by PTEN-induced putative kinase 1 (PINK1) was required for ubiquitination to occur. Activation of the PINK1–Parkin pathway attenuated IPAS-dependent apoptosis. IPAS was markedly induced in the midbrain following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, and IPAS-deficient mice showed resistance to MPTP-induced degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc). A significant increase in IPAS expression was found in SNpc neurons in patients with sporadic PD. These results indicate a mechanism of neurodegeneration in PD.

Parkin-induced ubiquitination of Mff promotes its association with p62/SQSTM1 during mitochondrial depolarization.

The ubiquitin ligase Parkin and autophagic adapter protein p62 are known to function in a common pathway controlling mitochondrial autophagy (mitophagy). However, the evidence supporting that p62 is directly recruited by ubiquitinated proteins remains undetermined. Here, we demonstrate that mitochondrial fission factor (Mff) associates with Parkin and carbonyl cyanide m-chlorophenyl hydrazone treatment significantly increases the affinity of Parkin with Mff. After recruitment to depolarized mitochondria, Parkin mediates poly-ubiquitination of Mff at lysine 251. Replacement of lysine 251 by arginine (K251R) totally abrogates Parkin-stimulated ubiquitination of Mff. Subsequently, the ubiquitinated Mff promotes its association with p62. Mff knockout interferes with p62 translocation to damaged mitochondria. Only re-transfection of Mff WT, but not K251R mutant, rescues this phenotype. Furthermore, loss of Mff results in failure of Parkin translocation and final clearance of damaged mitochondria. Thus, our data reveal functional links among Mff, p62, and the selective autophagy of mitochondria, which are implicated in the pathogenesis of neurodegeneration diseases.

Mitophagy is primarily due to alternative autophagy and requires the MAPK1 and MAPK14 signaling pathways

In cultured cells, not many mitochondria are degraded by mitophagy induced by physiological cellular stress. We observed mitophagy in HeLa cells using a method that relies on the pH-sensitive fluorescent protein Keima. With this approach, we found that mitophagy was barely induced by carbonyl cyanide m-chlorophenyl hydrazone treatment, which is widely used as an inducer of PARK2/Parkin-related mitophagy, whereas a small but modest amount of mitochondria were degraded by mitophagy under conditions of starvation or hypoxia. Mitophagy induced by starvation or hypoxia was marginally suppressed by knockdown of ATG7 and ATG12, or MAP1LC3B, which are essential for conventional macroautophagy. In addition, mitophagy was efficiently induced in Atg5 knockout mouse embryonic fibroblasts. However, knockdown of RAB9A and RAB9B, which are essential for alternative autophagy, but not conventional macroautophagy, severely suppressed mitophagy. Finally, we found that the MAPKs MAPK1/ERK2 and MAPK14/p38 were required for mitophagy. Based on these findings, we conclude that mitophagy in mammalian cells predominantly occurs through an alternative autophagy pathway, requiring the MAPK1 and MAPK14 signaling pathways.

CCCP-Induced LC3 lipidation depends on Atg9 whereas FIP200/Atg13 and Beclin 1/Atg14 are dispensable

Treatment of cells with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial proton gradient uncoupler, can result in mitochondrial damage and autophagy activation, which in turn eliminates the injured mitochondria in a Parkin-dependent way. How CCCP mobilizes the autophagy machinery is not fully understood. By analyzing a key autophagy step, LC3 lipidation, we examined the roles of two kinase complexes typically involved in the initiation and nucleation phases of autophagy, namely the ULK kinase complex (UKC) and the Beclin 1/Atg14 complex. We found that CCCP-induced LC3 lipidation could be independent of Beclin 1 and Atg14. In addition, deletion or knockdown of the UKC component FIP200 or Atg13 only led to a partial reduction in LC3 lipidation, indicating that UKC could be also dispensable for this step during CCCP treatment. In contrast, Atg9, which is important for transporting vesicles to early autophagosomal structure, was required for CCCP-induced LC3 lipidation. Taken together, these data suggest that CCCP-induced autophagy and mitophagy depends more critically on Atg9 vesicles than on UKC and Beclin 1/Atg14 complex.

ROS-induced mitochondrial depolarization initiates PARK2/PARKIN-dependent mitochondrial degradation by autophagy

Reactive oxygen species (ROS) have been implicated as a signal for general autophagy. Both mitochondrial-produced and exogenous ROS induce autophagosome formation. However, it is unclear whether ROS are required for the selective autophagic degradation of mitochondria, a process called mitophagy. Recent work using carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial-uncoupling reagent, has been shown to induce mitophagy. However, CCCP treatment may not be biologically relevant since it causes the depolarization of the entire mitochondrial network. Since mitochondria are the main ROS production sites in mammalian cells, we propose that short bursts of ROS produced within mitochondria may be involved in the signaling for mitophagy. To test this hypothesis, we induced an acute burst of ROS within mitochondria using a mitochondrial-targeted photosensitizer, mitochondrial KillerRed (mtKR). Using mtKR, we increased ROS levels in the mitochondrial matrix, which resulted in the loss of membrane potential and the subsequent activation of PARK2-dependent mitophagy. Importantly, we showed that overexpression of the mitochondrial antioxidant protein, superoxide dismutase-2, can squelch mtKR-induced mitophagy, demonstrating that mitochondrial ROS are responsible for mitophagy activation. Using this assay, we examined the impact of mitochondrial morphology on mitophagy. It was shown recently that elongated mitochondria are more resistant to mitophagy through unknown mechanisms. Here, we show that elongated mitochondria are more resistant to ROS-induced damage and mitophagy compared with fragmented mitochondria, suggesting that mitochondrial morphology has an important role in regulating ROS and mitophagy. Together, our results suggest that ROS-induced mitochondrial damage may be an important upstream activator of mitophagy.

Mitochondria regulate autophagy by conserved signalling pathways

Autophagy is a conserved degradative process that is crucial for cellular homeostasis and cellular quality control via the selective removal of subcellular structures such as mitochondria. We demonstrate that a regulatory link exists between mitochondrial function and autophagy in Saccharomyces cerevisiae. During amino-acid starvation, the autophagic response consists of two independent regulatory arms-autophagy gene induction and autophagic flux-and our analysis indicates that mitochondrial respiratory deficiency severely compromises both. We show that the evolutionarily conserved protein kinases Atg1, target of rapamycin kinase complex I, and protein kinase A (PKA) regulate autophagic flux, whereas autophagy gene induction depends solely on PKA. Within this regulatory network, mitochondrial respiratory deficiency suppresses autophagic flux, autophagy gene induction, and recruitment of the Atg1-Atg13 kinase complex to the pre-autophagosomal structure by stimulating PKA activity. Our findings indicate an interrelation of two common risk factors-mitochondrial dysfunction and autophagy inhibition-for ageing, cancerogenesis, and neurodegeneration.

Slow non-specific accumulation of 2'-deoxy and 2'-O-methyl oligonucleotide probes at mitochondria in live cells.

Molecular beacons (MBs) have the potential to provide a powerful tool for rapid RNA detection in living cells, as well as monitoring the dynamics of RNA expression in response to external stimuli. To exploit this potential, it is necessary to distinguish true signal from background signal due to non-specific interactions. Here, we show that, when cyanine-dye labeled 2′-deoxy and 2′-O-methyl oligonucleotide probes are inside living cells for >5 h, most of their signals co-localize with mitochondrial staining. These probes include random-sequence MB, dye-labeled single-strand linear oligonucleotide and dye-labeled double-stranded oligonucleotide. Using carbonyl cyanide m-chlorophenyl hydrazone treatment, we found that the non-specific accumulation of oligonucleotide probes at mitochondria was driven by mitochondrial membrane potential. We further demonstrated that the dye-labeled oligonucleotide probes were likely on/near the surface of mitochondria but not inside mitochondrial inner membrane. Interestingly, oligonucleotides probes labeled respectively with Alexa Fluor 488 and Alexa Fluor 546 did not accumulate at mitochondria, suggesting that the non-specific interaction between dye-labeled ODN probes and mitochondria is dye-specific. These results may help design and optimize fluorescence imaging probes for long-time RNA detection and monitoring in living cells.

Reactive Oxygen Species Regulate Caspase Activation in Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Resistant Human Colon Carcinoma Cell Lines

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Mitochondrial Ca2+ uptake is important over low [Ca2+]i range in arterial smooth muscle.

Mitochondrial Ca(2+) uptake is usually thought to occur only when intracellular Ca(2+) concentration ([Ca(2+)](i)) is high. We investigated whether mitochondrial Ca(2+) removal participates in shaping [Ca(2+)](i) signals in arterial smooth muscle over a low [Ca(2+)](i) range. [Ca(2+)](i) was measured using fura 2-loaded, voltage-clamped cells from rat femoral arteries. Both diazoxide and carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized the mitochondria. Diazoxide application increased resting [Ca(2+)](i), suggesting that Ca(2+) is sequestered in mitochondria. Over a low [Ca(2+)](i) range, diazoxide and CCCP slowed Ca(2+) removal rate, determined after a brief depolarization. When [Ca(2+)](i) was measured during sustained depolarization to -30 mV, CCCP application increased [Ca(2+)](i). When Ca(2+) transients were repeatedly evoked by caffeine applications, CCCP application elevated resting [Ca(2+)](i). Caffeine-induced Ca(2+) transients were compared before and after CCCP application using the half decay time, or time required to reduce increase in [Ca(2+)](i) by 50% (t((1/2))). CCCP treatment significantly increased t((1/2)). These results suggest that Ca(2+) removal to mitochondria in arterial smooth muscle cells may be important at a low [Ca(2+)](i).

Sustained elevation of calcium induces Ca 2+/calmodulin-dependent protein kinase II clusters in hippocampal neurons

Treatment of cultured hippocampal neurons with the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) in the absence of glucose mimics ischemic energy depletion and induces formation of Ca 2+/calmodulin-dependent protein kinase II (CaMKII) clusters, spherical structures with diameters of 75–175 nm [Dosemeci et al., J. Neurosci. 20 (2000) 3076–3084]. The demonstration that CaMKII clustering occurs in the intact, adult rat brain upon interruption of blood flow indicates that clustering is not confined to cell cultures. Application of N-methyl- D-aspartate (250 μM, 15 min) to hippocampal cultures also induces cluster formation, suggesting a role for Ca 2+. Indeed, intracellular Ca 2+ monitored with Fluo3-AM by confocal microscopy reaches a sustained high level within 5 min of CCCP treatment. The appearance of immunolabeled CaMKII clusters, detected by electron microscopy, follows the onset of the sustained increase in intracellular Ca 2+. Moreover, CaMKII does not cluster when the rise in intracellular Ca 2+ is prevented by the omission of extracellular Ca 2+ during CCCP treatment, confirming that clustering is Ca 2+-dependent. A lag period of 1–2 min between the onset of high intracellular Ca 2+ levels and the formation of CaMKII clusters suggests that a sustained increase in Ca 2+ level is necessary for the clustering. CaMKII clusters disappear within 2 h of returning the cultures to normal incubation conditions, at which time no significant cell death is detected. These results indicate that pathological conditions that promote sustained episodes of Ca 2+ overload result in a transitory clustering of CaMKII into spherical structures. CaMKII clustering may represent a cellular defense mechanism to sequester a portion of the CaMKII pool, thereby preventing excessive protein phosphorylation.