Protection of differentiated neuronal NG108-15 cells from P2X7 receptor-mediated toxicity by taurine

BackgroundStrong P2X7 receptor (P2X7R) activation causes Ca²⁺ overload and consequent cell death. We previously showed that depletion of Ca²⁺ stores and endoplasmic reticulum (ER) stress in differentiated NG108-15 neuronal cells contributed to P2X7R-mediated cytotoxicity. In this work, we assessed whether taurine (2-aminoethanesulfonic acid) could prevent this P2X7R-mediated cytotoxicity in this neuronal cell line.MethodsCytotoxicity markers were assessed by MTT assay and Western blotting. Cytosolic Ca²⁺ and mitochondrial Ca²⁺ concentrations were measured microfluorimetrically using fura-2 and rhod-2, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2′,7′-dichlorodihydrofluorescein diacetate.ResultsSelective P2X7R agonist BzATP treatment causes neuronal cell death by causing cytosolic Ca²⁺ overload, depletion of Ca²⁺ stores, endoplasmic reticulum (ER) stress, and caspase-3 activation (cleaved caspase 3). Remarkably, taurine (10 mM) pretreatment could prevent P2X7R-mediated neuronal cell death by blocking BzATP-mediated ER stress as determined by phosphorylated eukaryotic translation initiation factor 2α (peIF2α) and C/EBP-homologous protein (CHOP). However, taurine did not block BzATP-induced Ca²⁺ overload and depletion of ER Ca²⁺ stores. Interestingly, P2X7R activation did not result in mitochondrial Ca²⁺ overload, nor did it affect mitochondrial membrane potential. BzATP-induced generation of intracellular reactive oxygen species (ROS) was prevented by taurine.ConclusionsThe neuroprotective effect by taurine is attributed to the suppression of P2X7R-mediated ER stress and ROS formation.     

Endoplasmic reticulum calcium release potentiates the ER stress and cell death caused by an oxidative stress in MCF-7 cells

Increase in cytosolic calcium concentration ([Ca²⁺]_c), release of endoplasmic reticulum (ER) calcium ([Ca²⁺]_er) and ER stress have been proposed to be involved in oxidative toxicity. Nevertheless, their relative involvements in the processes leading to cell death are not well defined. In this study, we investigated whether oxidative stress generated during ascorbate-driven menadione redox cycling (Asc/Men) could trigger these three events, and, if so, whether they contributed to Asc/Men cytoxicity in MCF-7 cells. Using microspectrofluorimetry, we demonstrated that Asc/Men-generated oxidative stress was associated with a slow and moderate increase in [Ca²⁺]_c, largely preceding permeation of propidium iodide, and thus cell death. Asc/Men treatment was shown to partially deplete ER calcium stores after 90 min (decrease by 45% compared to control). This event was associated with ER stress activation, as shown by analysis of eIF2 phosphorylation and expression of the molecular chaperone GRP94. Thapsigargin (TG) was then used to study the effect of complete [Ca²⁺]_er emptying during the oxidative stress generated by Asc/Men. Surprisingly, the combination of TG and Asc/Men increased ER stress to a level considerably higher than that observed for either treatment alone, suggesting that [Ca²⁺]_er release alone is not sufficient to explain ER stress activation during oxidative stress. Finally, TG-mediated [Ca²⁺]_er release largely potentiated ER stress, DNA fragmentation and cell death caused by Asc/Men, supporting a role of ER stress in the process of Asc/Men cytotoxicity. Taken together, our results highlight the involvement of ER stress and [Ca²⁺]_er decrease in the process of oxidative stress-induced cell death in MCF-7 cells.     

Modulation of intracellular Ca2+ signalling in HeLa cells by the apoptotic cell death enhancer PK11195

1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is a proven enhancer of apoptotic cell death in a variety of cellular models. This effect is independent of its established cellular target, the mitochondrial benzodiazepine receptor (mBzR), since it is able to promote cell death also in mBzR knockout cells. Thus recently it was suggested that PK11195 might exert its effect by modulating the expression and function of the oncogene Bcl-2. We have previously demonstrated that Bcl-2 modulates cellular Ca²⁺ homeostasis as its overexpression reduces the Ca²⁺ concentration in the endoplasmic reticulum (ER) ([Ca²⁺]_er), impairing mitochondrial and cytosolic Ca²⁺ overload during cellular stress and therefore inhibiting the induction of the apoptotic cascade. Here, using ER, mitochondria and cytosolic targeted aequorin probes, we show that cellular treatment with PK11195 induces opposite changes in cellular Ca²⁺ homeostasis, increasing the [Ca²⁺]_er and amplifying IP₃ induced Ca²⁺ transients in mitochondria ([Ca²⁺]_m) and cytosol ([Ca²⁺]_c). This work provides evidence for a novel pharmacological effect of PK11195 on Ca²⁺ signalling which may be linked to its effect on Bcl-2 and account for its role in apoptotic cell death.     

Arsenite‐induced endoplasmic reticulum‐dependent apoptosis through disturbance of calcium homeostasis in HBE cell line

Calcium (Ca²⁺) is a ubiquitous cell signal responsible for multiple fundamental cellular functions, including apoptosis. Whether the homeostasis of Ca²⁺ is involved in arsenite‐induced apoptosis remains unclear. In this study, we observed that arsenite significantly elevated the intracellular Ca²⁺ concentration in a dose‐ and time‐dependent manner. By using the Ca²⁺‐ATPase inhibitor, thapsigargin, and the inositol 1,4,5‐ trisphosphate receptors (IP3Rs) inhibitor, heparin, we further confirmed that the disturbance of endoplasmic reticulum (ER) Ca²⁺ homeostasis caused Ca²⁺ overload in the cells. Moreover, loss of ER Ca²⁺ homeostasis also led to ER stress, mitochondrial dysfunction, and NF‐κB activation. Importantly, pretreatment of cells with heparin remarkably attenuated the elevated cell apoptosis induced by arsenite, but inhibition of ER Ca²⁺ uptake with thapsigargin exacerbated arsenite‐induced cell damage significantly. Together, we demonstrated for the first time that arsenite disturbed the Ca²⁺ homeostasis in ER, which subsequently led to ER stress, mitochondrial dysfunction, and NF‐κB nuclear translocation, and thus consequently triggering cell apoptosis. Our findings indicate regulation of disrupted Ca²⁺ homeostasis in ER may be a potential strategy for prevention of arsenite toxicity. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 197–216, 2017.     

Loss of endoplasmic reticulum Ca2+ homeostasis: contribution to neuronal cell death during cerebral ischemia

The loss of Ca²⁺ homeostasis during cerebral ischemia is a hallmark of impending neuronal demise. Accordingly, considerable cellular resources are expended in maintaining low resting cytosolic levels of Ca²⁺. These include contributions by a host of proteins involved in the sequestration and transport of Ca²⁺, many of which are expressed within intracellular organelles, including lysosomes, mitochondria as well as the endoplasmic reticulum (ER). Ca²⁺ sequestration by the ER contributes to cytosolic Ca²⁺ dynamics and homeostasis. Furthermore, within the ER Ca²⁺ plays a central role in regulating a host of physiological processes. Conversely, impaired ER Ca²⁺ homeostasis is an important trigger of pathological processes. Here we review a growing body of evidence suggesting that ER dysfunction is an important factor contributing to neuronal injury and loss post-ischemia. Specifically, the contribution of the ER to cytosolic Ca²⁺ elevations during ischemia will be considered, as will the signalling cascades recruited as a consequence of disrupting ER homeostasis and function.     

Cadmium perturbs calcium homeostasis in rat osteosarcoma (ros 17/2.8) cells; a possible role for protein kinase c

The mechanism of the toxic effects of Cd²⁺ on bone cell function is not completely understood at this time. This study was designed to characterize the effect of Cd²⁺ on Ca²⁺ metabolism in ROS 17/2.8 cells. Cells were labeled with ⁴⁵Ca (1.87 mM Ca) for 20 h in the presence of 0.01, 0.1, or 1.0 μM Cd²⁺ and kinetic parameters were determined from ⁴⁵a efflux curves. Three kinetic compartments described the intracellular metabolism of ⁴⁵Ca. Cd²⁺ (0.01 μM) caused an approximate 9 × increase in Ca²⁺ flux across the plasma membrane and a decrease in the most rapidly exchanging intracellular Ca²¹ compartment (S₁). However, there was no change in total cell Ca²⁺, indicating an increased cycling of Ca²⁺ across the plasma membrane. Flux between S₁, and the intermediate Ca²⁺ compartment (83) was also increased and S₂ increased significantly. All Cd²⁺ induced changes in Ca²⁺ homeostasis were obliterated by concurrent treatment with 0.1 μM calphostin C (CC), a potent protein kinase C (PKC) inhibitor. This data suggests that Cd²⁺ perturbs Ca²⁺ metabolism via a PKC dependent process.     

Neuroprotective effects of adenosine isolated from Cordyceps cicadae against oxidative and ER stress damages induced by glutamate in PC12 cells

Glutamate has been proven to induce oxidative stress through the formation of reactive oxygen species (ROS) and increased calcium overload which results in neuronal injury, development of neurodegenerative diseases and death. Adenosine is one of the bioactive nucleosides found in Cordyceps cicadae and it has displayed several pharmacological activities including neuroprotection. In this study, the protective effects of adenosine from C. cicadae against glutamate-induce oxidative stress in PC12 cells were evaluated. The exposure of PC12 cells to glutamate (5 mM) induced the formation of ROS, increased Ca²⁺ influx, endoplasmic reticulum (ER) stress and up regulated the expression of pro-apoptotic factor Bax. However, pretreatment with adenosine markedly increased cell viability, decreased the elevated levels of ROS and Ca²⁺ induced by glutamate. Furthermore adenosine increased the activities of GSH-Px and SOD, as well as retained mitochondria membrane potential (MMP), increased Bcl-2/Bax ratio, and reduced the expression of ERK, p38, and JNK. Overall, our results suggest that adenosine may be a promising potential therapeutic agent for the prevention and treatment of neurodegenerative disorders.     

Induction of Suicidal Erythrocyte Death by Nelfinavir

The HIV protease inhibitor, nelfinavir, primarily used for the treatment of HIV infections, has later been shown to be effective in various infectious diseases including malaria. Nelfinavir may trigger mitochondria-independent cell death. Erythrocytes may undergo eryptosis, a mitochondria-independent suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). During malaria, accelerated death of infected erythrocytes may decrease parasitemia and thus favorably influence the clinical course of the disease. In the present study, phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2'',7''-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca²⁺]_i from Fluo3-fluorescence. A 48 h treatment of human erythrocytes with nelfinavir significantly increased the percentage of annexin-V-binding cells (≥5µg/mL), significantly decreased forward scatter (≥2.5µg/mL), significantly increased ROS abundance (10 µg/mL), and significantly increased [Ca²⁺]_i (≥5 µg/mL). The up-regulation of annexin-V-binding following nelfinavir treatment was significantly blunted, but not abolished by either addition of the antioxidant N-acetylcysteine (1 mM) or removal of extracellular Ca²⁺. In conclusion, exposure of erythrocytes to nelfinavir induces oxidative stress and Ca²⁺ entry, thus leading to suicidal erythrocyte death characterized by erythrocyte shrinkage and erythrocyte membrane scrambling.     

Novel roles for ceramides, calpains and caspases in kidney proximal tubule cell apoptosis: lessons from in vitro cadmium toxicity studies

Apoptosis is a tightly regulated physiological process, which can be initiated by toxic stimuli, such as cadmium (Cd²⁺). Cd²⁺ (10-50 μM) induces a rapid increase in reactive oxygen species (ROS) (≥30 min) in a cell line derived from the S1 segment of rat kidney proximal tubule, without any apparent mitochondrial dysfunction. The sphingolipid ceramide is an important second messenger in apoptosis. Short exposure to Cd²⁺ (3 h) causes an increase in ceramides, which occurs downstream of ROS formation, and may interact with cellular components, such as endoplasmic reticulum and mitochondria. Following apoptosis initiation, execution must take place. The classical executioners of apoptosis are caspases, a family of cysteine proteases. However, increasing studies report caspase-independent apoptosis, which questions the essentiality of caspases for apoptosis implementation. With low micromolar Cd²⁺ concentrations (<10 μM), caspases are only activated after 24 h and not at earlier time points, which supports the notion of caspase-independent apoptosis. Due to increased cytosolic Ca²⁺ under pathological conditions, a role for the Ca²⁺-dependent proteases, calpains, has emerged. Calpain activation by Cd²⁺ (3-6 h) seems to be regulated by ceramide levels, in order to induce apoptosis. Calpain and caspase substrates overlap but yield different fragments, which may explain their diverse downstream targets. Furthermore, calpains and caspases may interact with one another to enhance, as seen by Cd²⁺, or diminish apoptosis.In this review, we discuss novel roles for ceramides, calpains and caspases as part of Cd²⁺-induced apoptotic signalling pathways in the kidney proximal tubule and their in vivo relevance to Cd²⁺-induced nephrotoxicity.     

Fisetin‐induced apoptosis of human oral cancer SCC‐4 cells through reactive oxygen species production,endoplasmic reticulum stress,caspase‐, and mitochondria‐dependent signaling pathways

Oral cancer is one of the cancer‐related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin‐induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin‐induced cell death and associated signal pathways on human oral cancer SCC‐4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca²⁺, mitochondria membrane potential (ΔΨ_m), and caspase‐8, ‐9, and ‐3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca²⁺ production, and decreased the level of ΔΨ_m and increased caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin‐induced cell apoptosis in SCC‐4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl‐2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC‐4 cells. We also used ATF‐6α, ATF‐6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria‐, and caspase‐dependent pathways.     

Palmitic acid-induced lipotoxicity and protection by (+)-catechin in rat cortical astrocytes

BackgroundAstrocytes do not only maintain homeostasis of the extracellular milieu of the neurons, but also play an active role in modulating synaptic transmission. Palmitic acid (PA) is a saturated fatty acid which, when being excessive, is a significant risk factor for lipotoxicity. Activation of astrocytes by PA has been shown to cause neuronal inflammation and demyelination. However, direct damage by PA to astrocytes is relatively unexplored. The aim of this study was to identify the mechanism(s) of PA-induced cytotoxicity in rat cortical astrocytes and possible protection by (+)-catechin.MethodsCytotoxicity and endoplasmic reticulum (ER) markers were assessed by MTT assay and Western blotting, respectively. Cytosolic Ca²⁺ and mitochondrial membrane potential (MMP) were measured microfluorimetrically using fura-2 and rhodamine 123, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2′-7′-dichlorodihydrofluorescein diacetate.ResultsExposure of astrocytes to 100 μM PA for 24 h resulted in apoptotic cell death. Whilst PA-induced cell death appeared to be unrelated to ER stress and perturbation in cytosolic Ca²⁺ signaling, it was likely a result of ROS production and subsequent MMP collapse, since ascorbic acid (anti-oxidant, 100 μM) prevented PA-induced MMP collapse and cell death. Co-treatment of astrocytes with (+)-catechin (300 μM), an anti-oxidant found abundantly in green tea, significantly prevented PA-induced ROS production, MMP collapse and cell death.ConclusionOur results suggest that PA-induced cytotoxicity in astrocytes may involve ROS generation and MMP collapse, which can be prevented by (+)-catechin.     

Cadmium stimulates mouse skin fibroblast apoptosis by affecting intracellular homeostasis

Context: Cadmium (Cd²⁺) is an important industrial and environmental pollutant and has been shown to induce apoptosis in a variety of cell types and tissues. Objective: To assess the specific effects of low-dose Cd^2+?on the skin. This organ is easily exposed to Cd²⁺, but how it damages cells is not fully understood. Materials and methods: Mouse skin fibroblasts were treated with low doses of Cd^2+?(0.4, 0.8 or 1.6?μM) for 12–48?h, and we observed cell morphological alterations, measured DNA damage and quantified cell viability changes. Results: Cd²⁺-treated fibroblasts exhibited morphological changes and evidence of DNA damage, as well as higher numbers of apoptotic and necrotic cells. There were increased caspase ?3, ?8 and ?9 activities when fibroblasts were treated with 0.4, 0.8 and 1.6?μM CdCl₂ for 24?h. Higher intracellular calcium (Ca²⁺) and reactive oxygen species (ROS) levels, and enhanced efflux of extracellular Ca^2+?and potassium (K⁺). The mitochondrial membrane potential was lowered in treated cells, and the cell cycle arrested in the G0/G1 phase. Bax and Fas gene expression increased and Bcl-2 gene expression decreased. Discussion: The results demonstrate that Cd^2+?exerts typical apoptotic effects in mouse skin fibroblasts. It strongly inhibited proliferation and induced apoptosis in a dose- and duration-dependent manner. Ca^2+?homeostasis was disturbed by oxidative stress, mitochondrial dysfunction and caspase-mediated apoptosis. Conclusion: K^+?efflux and Bax, Bcl-2 and Fas gene expression regulation play important roles in Cd²⁺-induced dysfunction by disrupting intracellular homeostasis in mouse skin fibroblasts.     

Tributyltin-induced endoplasmic reticulum stress and its Ca-mediated mechanism

Organotin compounds, especially tributyltin chloride (TBT), have been widely used in antifouling paints for marine vessels, but exhibit various toxicities in mammals. The endoplasmic reticulum (ER) is a multifunctional organelle that controls post-translational modification and intracellular Ca^2 + signaling. When the capacity of the quality control system of ER is exceeded under stress including ER Ca^2 + homeostasis disruption, ER functions are impaired and unfolded proteins are accumulated in ER lumen, which is called ER stress. Here, we examined whether TBT causes ER stress in human neuroblastoma SH-SY5Y cells. We found that 700 nM TBT induced ER stress markers such as CHOP, GRP78, spliced XBP1 mRNA and phosphorylated eIF2α. TBT also decreased the cell viability both concentration- and time-dependently. Dibutyltin and monobutyltin did not induce ER stress markers. We hypothesized that TBT induces ER stress via Ca^2 + depletion, and to test this idea, we examined the effect of TBT on intracellular Ca^2 + concentration using fura-2 AM, a Ca^2 + fluorescent probe. TBT increased intracellular Ca^2 + concentration in a TBT-concentration-dependent manner, and Ca^2 + increase in 700 nM TBT was mainly blocked by 50 μM dantrolene, a ryanodine receptor antagonist (about 70% inhibition). Dantrolene also partially but significantly inhibited TBT-induced GRP78 expression and cell death. These results suggest that TBT increases intracellular Ca^2 + concentration by releasing Ca^2 + from ER, thereby causing ER stress.     

Mitochondrial Dysfunction and Ca2+ Overload in Injured Sertoli Cells Exposed to Bisphenol A

Bisphenol‐A (BPA) is well known as one of endocrine‐disrupting chemicals and testicular toxicant. In this present study, we determined whether BPA caused cell injury through mitochondria impairment and ROS overproduction. The cellular ROS production, mitochondrial ATP synthetase activity and Ca²⁺ concentration were examined. We have found BPA caused the cellular mitochondria dysfunction and followed by cell death in Sertoli cells. Moreover cytoplasm Ca²⁺ overload was also involved. Furthermore, pretreatment with N‐acetyl‐L‐cysteine (NAC) could alleviate the damage by causing a remarkable decrease in ROS production and mitochondrial dysfunction. Collectively, our results showed that BPA exposure induced Sertoli cell apoptosis because of excessive ROS generation and mitochondrial dysfunction. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 823–831, 2017.     

Tributyltin chloride induced testicular toxicity by JNK and p38 activation,redox imbalance and cell death in sertoli-germ cell co-culture

The widespread use of tributyltin (TBT) as biocides in antifouling paints and agricultural chemicals has led to environmental and marine pollution. Human exposure occurs mainly through TBT contaminated seafood and drinking water. It is a well known endocrine disruptor in mammals, but its molecular mechanism in testicular damage is largely unexplored. This study was therefore, designed to ascertain effects of tributyltin chloride (TBTC) on sertoli-germ cell co-culture in ex-vivo and in the testicular tissue in-vivo conditions. An initial Ca²⁺ rise followed by ROS generation and glutathione depletion resulted in oxidative damage and cell death. We observed p38 and JNK phosphorylation, stress proteins (Nrf2, MT and GST) induction and mitochondrial depolarization leading to caspase-3 activation. Prevention of TBTC reduced cell survival and cell death by Ca²⁺ inhibitors and free radical scavengers specify definitive role of Ca²⁺ and ROS. Sertoli cells were found to be more severely affected which in turn can hamper germ cells functionality. TBTC exposure in-vivo resulted in increased tin content in the testis with enhanced Evans blue leakage into the testicular tissue indicating blood–testis barrier disruption. Tesmin levels were significantly diminished and histopathological studies revealed marked tissue damage. Our data collectively indicates the toxic manifestations of TBTC on the male reproductive system and the mechanisms involved.     

Intracellular influx of calcium induced by quartz particles in alveolar macrophages

Historical studies report that cellular injury and silicosis are related to cytosolic free calcium (Ca²⁺). Moreover, reactive oxygen species (ROS) have been linked to cellular injury. However, the detail mechanism of the increase in [Ca²⁺]_i and the relationship between [Ca²⁺]_i and ROS production remains unknown. Quartz particle has been found to increase [Ca²⁺]_i and activate the generation of ROS. Our hypothesis is that [Ca²⁺]_i increase induced by quartz particle is from extracellular Ca²⁺ through the Ca²⁺ channel, and [Ca²⁺]_i increase is believed to activate ROS production. In order to examine this hypothesis, we treated rat alveolar macrophages with quartz (SiO₂) particles and used laser scanning confocal microscopy to measure [Ca²⁺]_i and the fluorescence intensity of ROS. Time- and dose-dependent increases in [Ca²⁺]_I and ROS in macrophages as well as cell viability were observed. Through chelating extracellular Ca²⁺ with ethylene glycol tetraacetic acid and releasing intracellular Ca²⁺ with thapsigargin, we found that 72.7% of the [Ca²⁺]_i increase was due to the influx of Ca²⁺ from the extracellular environment, via Ca²⁺ channels in the plasma membrane. By adding mannitol to scavenge hydroxyl radicals (OH·), and removing surface iron from the quartz particles to reduce OH· generation, we observed a reduced level of ROS generation, whereas the increase in [Ca²⁺]_i was unaffected. When using EGTA to reduce [Ca²⁺]_i, we observed a decrease in ROS production. This study suggests that the [Ca²⁺]_i influx was independent of OH· production, and the [Ca²⁺]_i increase resulted in ROS production. These results further indicate that there is a strong relationship between cytosolic free Ca²⁺ content and cellular injury as well as silica exposure.     

Calmodulin-mediated cadmium inhibition of phosphodiesterase activity,in vitro

Ion-stripped bovine brain calmodulin (CaM) binds 4 moles Cd²⁺ as well as 4 moles Ca²⁺ per mole protein, with similar affinity; in the presence of 1 mM Mg²⁺ the molar binding ratio of CaM for Ca²⁺ decreased to 3, the apparent K_0.5 for Ca²⁺ nearly doubled, but the binding characteristics of CaM for Cd²⁺ were not changed. Saturating concentrations Ca²⁺ did not affect the molar binding ratio of CaM for Cd²⁺, but increased the apparent K_0.5 for Cd²⁺; vice versa, saturating concentrations Cd²⁺ decreased the molar binding ratio for Ca²⁺ to 2 without affecting the apparent K_0.5 for Ca²⁺.CaM-independent phosphodiesterase (PDE) activity was inhibited at [Cd²⁺]>10^–5 M. Cd²⁺-CaM as well as Ca²⁺-CaM activated PDE. However, the Cd²⁺-CaM complex is less effective than the Ca²⁺-CaM complex in stimulating CaM-dependent enzyme activities. Cd²⁺ inhibits Ca²⁺- and CaM-dependent PDE in a competitive way. Introduction of Cd²⁺ in a medium containing Ca²⁺ and CaM may, therefore, result in a reduction of CaM-dependent enzyme stimulation.By its interference with Ca²⁺- and CaM- dependent PDE activity, Cd²⁺ could upset the catabolic pathway of cellular cyclic nucleotide metabolism.     

Ca2+/calmodulin dependent kinase II: A critical mediator in determining reperfusion outcomes in the heart?

Ischaemic heart disease is a major cause of death and disability in the Western world, and a substantial health burden. Cardiomyocyte Ca²⁺ overload is known to significantly contribute to contractile dysfunction and myocyte death in ischaemia and reperfusion, and significant advancements have been made in identifying the downstream mediators and cellular origins of this Ca²⁺ mismanagement. Ca²⁺/calmodulin‐dependent kinase II (CaMKII) is recognized as an important mediator linking pathological changes in subcellular environments to modifications in cardiomyocyte Ca²⁺ handling. Activated in response to fluctuations in cellular Ca²⁺ and to various post‐translational modifications, CaMKII targets numerous Ca²⁺ channels/transporters involved in Ca²⁺ handling and contractile function regulation. CaMKII is activated early in reperfusion, where it exacerbates Ca²⁺ leak from the sarcoplasmic reticulum and promotes the onset of ventricular arrhythmias. Inhibiting CaMKII can increase functional recovery in reperfusion and reduce apoptotic/necrotic death, at least partly through indirect and direct influences on mitochondrial Ca²⁺ levels and function. Yet, CaMKII can also have beneficial actions in ischaemia and reperfusion, in part by providing inotropic support for the stunned myocardium and contributing as an intermediate to cardioprotective preconditioning signalling cascades. There is considerable potential in targeting CaMKII as a part of a surgical reperfusion strategy, though further mechanistic understanding of the relationship between CaMKII activation status and the extent of ischaemia/reperfusion injury are required to fully establish an optimal pharmacological approach.