Calmodulin-dependent Protein Kinase Activity Research Articles

The CaMK Family Differentially Promotes Necroptosis and Mouse Cardiac Graft Injury and Rejection.

Organ transplantation is associated with various forms of programmed cell death which can accelerate transplant injury and rejection. Targeting cell death in donor organs may represent a novel strategy for preventing allograft injury. We have previously demonstrated that necroptosis plays a key role in promoting transplant injury. Recently, we have found that mitochondria function is linked to necroptosis. However, it remains unknown how necroptosis signaling pathways regulate mitochondrial function during necroptosis. In this study, we investigated the receptor-interacting protein kinase 3 (RIPK3) mediated mitochondrial dysfunction and necroptosis. We demonstrate that the calmodulin-dependent protein kinase (CaMK) family members CaMK1, 2, and 4 form a complex with RIPK3 in mouse cardiac endothelial cells, to promote trans-phosphorylation during necroptosis. CaMK1 and 4 directly activated the dynamin-related protein-1 (Drp1), while CaMK2 indirectly activated Drp1 via the phosphoglycerate mutase 5 (PGAM5). The inhibition of CaMKs restored mitochondrial function and effectively prevented endothelial cell death. CaMKs inhibition inhibited activation of CaMKs and Drp1, and cell death and heart tissue injury (n = 6/group, p < 0.01) in a murine model of cardiac transplantation. Importantly, the inhibition of CaMKs greatly prolonged heart graft survival (n = 8/group, p < 0.01). In conclusion, CaMK family members orchestrate cell death in two different pathways and may be potential therapeutic targets in preventing cell death and transplant injury.

Neurotensin contributes to cholestatic liver disease potentially modulating matrix metalloprotease-7

The diagnosis and treatment of biliary atresia pose challenges due to the absence of reliable biomarkers and limited understanding of its etiology. The plasma and liver of patients with biliary atresia exhibit elevated levels of neurotensin. To investigate the specific role of neurotensin in the progression of biliary atresia, the patient's liver pathological section was employed. Biliary organoids, cultured biliary cells, and a mouse model were employed to elucidate both the potential diagnostic significance of neurotensin and its underlying mechanistic pathway. In patients’ blood, the levels of neurotensin were positively correlated with matrix metalloprotease-7, interleukin-8, and liver function enzymes. Neurotensin and neurotensin receptors were mainly expressed in the intrahepatic biliary cells and were stimulated by bile acids. Neurotensin suppressed the growth and increased expression of matrix metalloprotease-7 in biliary organoids. Neurotensin inhibited mitochondrial respiration, oxidative phosphorylation, and attenuated the activation of calmodulin-dependent kinase kinase 2-adenosine monophosphate-activated protein kinase (CaMKK2-AMPK) signaling in cultured biliary cells. The stimulation of neurotensin in mice and cultured cholangiocytes resulted in the upregulation of matrix metalloprotease-7 expression through binding to its receptors, namely neurotensin receptors 1/3, thereby attenuating the activation of the CaMKK2-AMPK pathway. In conclusion, these findings revealed the changes of neurotensin in patients with cholestatic liver disease and its mechanism in the progression of the disease, providing a new understanding of the complex mechanism of hepatobiliary injury in children with biliary atresia.

Activation of NMDA receptors in brain endothelial cells increases transcellular permeability

Neurovascular coupling is a precise mechanism that induces increased blood flow to activated brain regions, thereby providing oxygen and glucose. In this study, we hypothesized that N-methyl-D-aspartate (NMDA) receptor signaling, the most well characterized neurotransmitter signaling system which regulates delivery of essential molecules through the blood–brain barrier (BBB). Upon application of NMDA in both in vitro and in vivo models, increased delivery of bioactive molecules that was mediated through modulation of molecules involved in molecular delivery, including clathrin and caveolin were observed. Also, NMDA activation induced structural changes in the BBB and increased transcellular permeability that showed regional heterogeneity in its responses. Moreover, NMDA receptor activation increased endosomal trafficking and facilitated inactivation of lysosomal pathways and consequently increased molecular delivery mediated by activation of calmodulin-dependent protein kinase II (CaMKII) and RhoA/protein kinase C (PKC). Subsequent in vivo experiments using mice specifically lacking NMDA receptor subunit 1 in endothelial cells showed decreased neuronal density in the brain cortex, suggesting that a deficiency in NMDA receptor signaling in brain endothelial cells induces neuronal losses. Together, these results highlight the importance of NMDA-receptor-mediated signaling in the regulation of BBB permeability that surprisingly also affected CD31 staining.

ACh signaling modulates activity of the GABAergic signaling network in the basolateral amygdala and behavior in stress-relevant paradigms.

The balance between excitatory and inhibitory (E/I) signaling is important for maintaining homeostatic function in the brain. Indeed, dysregulation of inhibitory GABA interneurons in the amygdala has been implicated in human mood disorders. We hypothesized that acetylcholine (ACh) signaling in the basolateral amygdala (BLA) might alter E/I balance resulting in changes in stress-sensitive behaviors. We therefore measured ACh release as well as activity of calmodulin-dependent protein kinase II (CAMKII)-, parvalbumin (PV)-, somatostatin (SOM)- and vasoactive intestinal protein (VIP)-expressing neurons in the BLA of awake, behaving male mice. ACh levels and activity of both excitatory and inhibitory BLA neurons increased when animals were actively coping, and decreased during passive coping, in the light-dark box, tail suspension and social defeat. Changes in neuronal activity preceded behavioral state transitions, suggesting that BLA activity may drive the shift in coping strategy. In contrast to exposure to escapable stressors, prolonging ACh signaling with a cholinesterase antagonist changed the balance of activity among BLA cell types, significantly increasing activity of VIP neurons and decreasing activity of SOM cells, with little effect on CaMKII or PV neurons. Knockdown of α7 or β2-containing nAChR subtypes in PV and SOM, but not CaMKII or VIP, BLA neurons altered behavioral responses to stressors, suggesting that ACh signaling through nAChRs on GABA neuron subtypes contributes to stress-induced changes in behavior. These studies show that ACh modulates the GABAergic signaling network in the BLA, shifting the balance between SOM, PV, VIP and CaMKII neurons, which are normally activated coordinately during active coping in response to stress. Thus, prolonging ACh signaling, as occurs in response to chronic stress, may contribute to maladaptive behaviors by shifting the balance of inhibitory signaling in the BLA.

Proteomic Analysis Reveals the Vital Role of Synaptic Plasticity in the Pathogenesis of Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE) is a chronic neurological disorder that is often resistant to antiepileptic drugs. The pathogenesis of TLE is extremely complicated and remains elusive. Understanding the molecular mechanisms underlying TLE is crucial for its diagnosis and treatment. In the present study, a lithium-pilocarpine-induced TLE model was employed to reveal the pathological changes of hippocampus in rats. Hippocampal samples were taken for proteomic analysis at 2 weeks after the onset of spontaneous seizure (a chronic stage of epileptogenesis). Isobaric tag for relative and absolute quantization (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC–MS/MS) technique was applied for proteomic analysis of hippocampus. A total of 4173 proteins were identified from the hippocampi of epileptic rats and its control, of which 27 differentially expressed proteins (DEPs) were obtained with a fold change > 1.5 and P < 0.05. Bioinformatics analysis indicated 27 DEPs were mainly enriched in “regulation of synaptic plasticity and structure” and “calmodulin-dependent protein kinase activity,” which implicate synaptic remodeling may play a vital role in the pathogenesis of TLE. Consequently, the synaptic plasticity-related proteins and synaptic structure were investigated to verify it. It has been demonstrated that CaMKII-α, CaMKII-β, and GFAP were significant upregulated coincidently with proteomic analysis in the hippocampus of TLE rats. Moreover, the increased dendritic spines and hippocampal sclerosis further proved that synaptic plasticity involves in the development of TLE. The present study may help to understand the molecular mechanisms underlying epileptogenesis and provide a basis for further studies on synaptic plasticity in TLE.

The mitochondrial calcium uniporter promotes arrhythmias caused by high-fat diet

Obesity and diabetes increase the risk of arrhythmia and sudden cardiac death. However, the molecular mechanisms of arrhythmia caused by metabolic abnormalities are not well understood. We hypothesized that mitochondrial dysfunction caused by high fat diet (HFD) promotes ventricular arrhythmia. Based on our previous work showing that saturated fat causes calcium handling abnormalities in cardiomyocytes, we hypothesized that mitochondrial calcium uptake contributes to HFD-induced mitochondrial dysfunction and arrhythmic events. For experiments, we used mice with conditional cardiac-specific deletion of the mitochondrial calcium uniporter (Mcu), which is required for mitochondrial calcium uptake, and littermate controls. Mice were used for in vivo heart rhythm monitoring, perfused heart experiments, and isolated cardiomyocyte experiments. MCU KO mice are protected from HFD-induced long QT, inducible ventricular tachycardia, and abnormal ventricular repolarization. Abnormal repolarization may be due, at least in part, to a reduction in protein levels of voltage gated potassium channels. Furthermore, isolated cardiomyocytes from MCU KO mice exposed to saturated fat are protected from increased reactive oxygen species (ROS), mitochondrial dysfunction, and abnormal calcium handling. Activation of calmodulin-dependent protein kinase (CaMKII) corresponds with the increase in arrhythmias in vivo. Additional experiments showed that CaMKII inhibition protects cardiomyocytes from the mitochondrial dysfunction caused by saturated fat. Hearts from transgenic CaMKII inhibitor mice were protected from inducible ventricular tachycardia after HFD. These studies identify mitochondrial dysfunction caused by calcium overload as a key mechanism of arrhythmia during HFD. This work indicates that MCU and CaMKII could be therapeutic targets for arrhythmia caused by metabolic abnormalities.

Alteration of calcium signalling in cardiomyocyte induced by simulated microgravity and hypergravity.

ObjectivesCardiac Ca2+ signalling plays an essential role in regulating excitation‐contraction coupling and cardiac remodelling. However, the response of cardiomyocytes to simulated microgravity and hypergravity and the effects on Ca2+ signalling remain unknown. Here, we elucidate the mechanisms underlying the proliferation and remodelling of HL‐1 cardiomyocytes subjected to rotation‐simulated microgravity and 4G hypergravity.Materials and MethodsThe cardiomyocyte cell line HL‐1 was used in this study. A clinostat and centrifuge were used to study the effects of microgravity and hypergravity, respectively, on cells. Calcium signalling was detected with laser scanning confocal microscopy. Protein and mRNA levels were detected by Western blotting and real‐time PCR, respectively. Wheat germ agglutinin (WGA) staining was used to analyse cell size.ResultsOur data showed that spontaneous calcium oscillations and cytosolic calcium concentration are both increased in HL‐1 cells after simulated microgravity and 4G hypergravity. Increased cytosolic calcium leads to activation of calmodulin‐dependent protein kinase II/histone deacetylase 4 (CaMKII/HDAC4) signalling and upregulation of the foetal genes ANP and BNP, indicating cardiac remodelling. WGA staining indicated that cell size was decreased following rotation‐simulated microgravity and increased following 4G hypergravity. Moreover, HL‐1 cell proliferation was increased significantly under hypergravity but not rotation‐simulated microgravity.ConclusionsOur study demonstrates for the first time that Ca2+/CaMKII/HDAC4 signalling plays a pivotal role in myocardial remodelling under rotation‐simulated microgravity and hypergravity.

Highlights From the Circulation Family of Journals.

Highlights From the Circulation Family of Journals.

Melatonin Promotes Nerve Regeneration Following End-to-Side Neurorrhaphy by Accelerating Cytoskeletal Remodeling via the Melatonin Receptor-dependent Pathway

Acceleration of cytoskeletal remodeling in regenerated axons is crucial for a fully functional recovery following peripheral nerve injury (PNI). Melatonin plays important roles in cell differentiation and protection of cytoskeleton stability, thus, the present study aimed to investigate whether melatonin can enhance neurite outgrowth and promote cytoskeletal remodeling in a PNI animal model and in differentiated neurons.End-to-side neurorrhaphy (ESN) rat model was used for assessing cytoskeletal rearrangement in regenerated axon. Subject rats received 1 mg/kg/day melatonin injection for one month. The amplitude of compound muscle action potentials and the number of re-innervated motor end plates on target muscles were assessed to represent the functional recovery after ESN.Melatonin treatment enhanced functional recovery after ESN, compared to the saline treated group. Additionally, in spinal cord and peripheral nerve tissue, animals receiving melatonin displayed enhanced expression of GAP43 and β3-tubulin one month after ESN, and an increased number of re-innervated motor end plates on their target muscle. In vitro analysis revealed that melatonin treatment significantly promoted neurite outgrowth, and increased expression of melatonin receptors as well as β3-tubulin in mouse neuroblastoma Neuro-2a (N2a) cells. Treatment with a melatonin receptor antagonist, luzindole, significantly suppressed melatonin receptors and β3-tubulin expression.Importantly, we found that melatonin treatment suppressed activation of calmodulin-dependent protein kinase II (CaMKII) in vitro and in vivo, suggesting that the β3-tubulin remodeling may occur via CaMKII-mediated Ca2+ signaling. These results suggested that melatonin may promote functional recovery after PNI by accelerating cytoskeletal remodeling through the melatonin receptor-dependent pathway.

Dietary Saturated Fat Promotes Arrhythmia by Activating NOX2 (NADPH Oxidase 2).

Obesity and diets high in saturated fat increase the risk of arrhythmias and sudden cardiac death. However, the molecular mechanisms are not well understood. We hypothesized that an increase in dietary saturated fat could lead to abnormalities of calcium homeostasis and heart rhythm by a NOX2 (NADPH oxidase 2)-dependent mechanism. We investigated this hypothesis by feeding mice high-fat diets. In vivo heart rhythm telemetry, optical mapping, and isolated cardiac myocyte imaging were used to quantify arrhythmias, repolarization, calcium transients, and intracellular calcium sparks. We found that saturated fat activates NOX (NADPH oxidase), whereas polyunsaturated fat does not. The high saturated fat diet increased repolarization heterogeneity and ventricular tachycardia inducibility in perfused hearts. Pharmacological inhibition or genetic deletion of NOX2 prevented arrhythmogenic abnormalities in vivo during high statured fat diet and resulted in less inducible ventricular tachycardia. High saturated fat diet activates CaMK (Ca2+/calmodulin-dependent protein kinase) in the heart, which contributes to abnormal calcium handling, promoting arrhythmia. We conclude that NOX2 deletion or pharmacological inhibition prevents the arrhythmogenic effects of a high saturated fat diet, in part mediated by activation of CaMK. This work reveals a molecular mechanism linking cardiac metabolism to arrhythmia and suggests that NOX2 inhibitors could be a novel therapy for heart rhythm abnormalities caused by cardiac lipid overload.

Cue-induced reinstatement of alcohol-seeking behavior is associated with increased CaMKII T286 phosphorylation in the reward pathway of mice

Cue-induced reinstatement of alcohol-seeking is a hallmark behavioral pathology of addiction. Evidence suggests that reinstatement (e.g., relapse), may be regulated by cell signaling systems that underlie neuroplasticity. A variety of plasticity events require activation of calcium calmodulin-dependent protein kinase II (CaMKII) in components of the reward pathway, such as the nucleus accumbens and amygdala. We sought to determine if cue-induced reinstatement of alcohol-seeking behavior is associated with changes in the activation state (e.g., phosphorylation) of CaMKII-T286. Male C57BL/6J mice (n=14) were trained to lever press on a fixed-ratio-4 schedule of sweetened alcohol (2% sucrose+9% EtOH) reinforcement. After 14-d of extinction (no cues or reinforcers), mice underwent a response-contingent reinstatement (n=7) vs. an additional day of extinction (n=7). Brains were removed immediately after the test and processed for evaluation of pCaMKII-T286 immunoreactivity (IR). Number of pCaMKII-T286 positive cells/mm2 was quantified from coronal brain sections using Bioquant Image Analysis software. Mice emitted significantly more responses on the alcohol vs. the inactive lever throughout the baseline phase with average alcohol intake of 1.1±0.03g/kg/1-h. During extinction, responses on the alcohol lever decreased to inactive lever levels by day 7. Significant cue-induced reinstatement of alcohol-seeking was observed during a single test with no effects on the inactive lever. Reinstatement was associated with increased pCaMKII-T286 IR specifically in amygdala (LA and BLA), nucleus accumbens (AcbSh), lateral septum, mediodorsal thalamus, and piriform cortex as compared to extinction control. Brain regions showing no change included the dorsal striatum, medial septum, cingulate cortex, habenula, paraventricular thalamus, and ventral hypothalamus. These results show response contingent cue-induced reinstatement of alcohol-seeking behavior is associated with selective increases in pCaMKII-T286 in specific reward- and memory-related brain regions of male C57BL/6J mice. Primary molecular mechanisms of associative learning and memory may regulate relapse in alcohol addiction.

Melatonin synthesis in the human ciliary body triggered by TRPV4 activation: Involvement of AANAT phosphorylation

Melatonin is a substance synthesized in the pineal gland as well as in other organs. This substance is involved in many ocular functions, giving its synthesis in numerous eye structures. Melatonin is synthesized from serotonin through two enzymes, the first limiting step into the synthesis of melatonin being aralkylamine N-acetyltransferase (AANAT). In this current study, AANAT phosphorylation after the activation of TRPV4 was studied using human non-pigmented epithelial ciliary body cells. Firstly, it was necessary to determine the adequate time and dose of the TRPV4 agonist GSK1016790A to reach the maximal phosphorylation of AANAT. An increase of 72% was observed after 5 min incubation with 10 nM GSK (**p < 0.05, n = 6) with a concomitant rise in N-acetyl serotonin and melatonin synthesis. The involvement of a TRPV4 channel in melatonin synthesis was verified by antagonist and siRNA studies as a previous step to studying intracellular signalling. Studies performed on the second messengers involved in GSK induced AANAT phosphorylation were carried out by inhibiting several pathways. In conclusion, the activation of calmodulin and calmodulin-dependent protein kinase II was confirmed, as shown by the cascade seen in AANAT phosphorylation (***p < 0.001, n = 4). This mechanism was also established by measuring N-acetyl serotonin and melatonin levels. In conclusion, the activation of a TRPV4 present in human ciliary body epithelial cells produced an increase in AANAT phosphorylation and a further melatonin increase by a mechanism in which Ca-calmodulin and the calmodulin-dependent protein kinase II are involved.

Calcium/Calmodulin Protein Kinase II-Dependent Ryanodine Receptor Phosphorylation Mediates Cardiac Contractile Dysfunction Associated With Sepsis.

Sepsis is associated with cardiac contractile dysfunction attributed to alterations in Ca handling. We examined the subcellular mechanisms involved in sarcoplasmic reticulum Ca loss that mediate altered Ca handling and contractile dysfunction associated with sepsis. Randomized controlled trial. Research laboratorySUBJECTS:: Male wild type and transgenic miceINTERVENTIONS:: We induced sepsis in mice using the colon ascendens stent peritonitis model. Twenty-four hours after colon ascendens stent peritonitis surgery, we observed that wild type mice had significantly elevated proinflammatory cytokine levels, reduced ejection fraction, and fractional shortening (ejection fraction %, 54.76 ± 0.67; fractional shortening %, 27.53 ± 0.50) compared with sham controls (ejection fraction %, 73.57 ± 0.20; fractional shortening %, 46.75 ± 0.38). At the cardiac myocyte level, colon ascendens stent peritonitis cells showed reduced cell shortening, Ca transient amplitude and sarcoplasmic reticulum Ca content compared with sham cardiomyocytes. Colon ascendens stent peritonitis hearts showed a significant increase in oxidation-dependent calcium and calmodulin-dependent protein kinase II activity, which could be prevented by pretreating animals with the antioxidant tempol. Pharmacologic inhibition of calcium and calmodulin-dependent protein kinase II with 2.5 µM of KN93 prevented the decrease in cell shortening, Ca transient amplitude, and sarcoplasmic reticulum Ca content in colon ascendens stent peritonitis myocytes. Contractile function was also preserved in colon ascendens stent peritonitis myocytes isolated from transgenic mice expressing a calcium and calmodulin-dependent protein kinase II inhibitory peptide (AC3-I) and in colon ascendens stent peritonitis myocytes isolated from mutant mice that have the ryanodine receptor 2 calcium and calmodulin-dependent protein kinase II-dependent phosphorylation site (serine 2814) mutated to alanine (S2814A). Furthermore, colon ascendens stent peritonitis S2814A mice showed preserved ejection fraction and fractional shortening (ejection fraction %, 73.06 ± 6.31; fractional shortening %, 42.33 ± 5.70) compared with sham S2814A mice (ejection fraction %, 71.60 ± 4.02; fractional shortening %, 39.63 ± 3.23). Results indicate that oxidation and subsequent activation of calcium and calmodulin-dependent protein kinase II has a causal role in the contractile dysfunction associated with sepsis. Calcium and calmodulin-dependent protein kinase II, through phosphorylation of the ryanodine receptor would lead to Ca leak from the sarcoplasmic reticulum, reducing sarcoplasmic reticulum Ca content, Ca transient amplitude and contractility. Development of organ-specific calcium and calmodulin-dependent protein kinase II inhibitors may result in a beneficial therapeutic strategy to ameliorate contractile dysfunction associated with sepsis.

Exendin-4 promotes pancreatic β-cell proliferation via inhibiting the expression of Wnt5a.

Exendin-4, a glucagon-like peptide-1 receptor agonist, is currently regarded as an effective therapeutic strategy for type-2 diabetes. Previous studies indicated that exendin-4 promoted β cell proliferation. However, the underlying mechanisms remain largely unknown. Recently it was reported that exendin-4 promoted pancreatic β cell proliferation by regulating the expression level of Wnt4. The present study was designed to investigate whether other Wnt isoforms take part in accommodation of β-cell proliferation. We found that exendin-4 promotes the proliferation and suppresses the expression of Wnt5a in INS-1 cell line and C57Bl/6 mouse pancreatic β-cells. Further mechanistic study demonstrated that exendin-4 promoted INS-1 cell proliferation partly through down-regulating the expression of Wnt5a. Furthermore, Wnt5a could induce the activation of calmodulin-dependent protein kinase II in INS-1 cells, thereby decreasing the cellular stable β-catenin and its nuclear translocation, and finally reduce the expression of cyclin D1. In addition, we also found that both of the receptors (Frz-2 and Ror-2) mediated the effect of Wnt5a on β cell line INS-1 proliferation. Taken together, this study suggests that Wnt5a plays a critical role in exendin-4-induced β-cell proliferation, indicating that Wnt5a might be a novel regulator in counterbalance of β cell mass.

Type III Transforming Growth Factor-β Receptor Drives Cardiac Hypertrophy Through β-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

The role of type III transforming growth factor-β receptor (TβRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TβRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TβRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TβRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TβRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TβRIII in vitro. Cardiac-specific transgenic expression of TβRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TβRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TβRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TβRIII in vivo. Our data suggest that TβRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of β-arrestin2 with both TβRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TβRIII expression results in cardiac hypertrophy through β-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-β and β-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac-specific transgenic expression of TβRIII mice. Our findings may provide a novel target for control of myocardial hypertrophy.

Neuroprotective Effect of Puerarin on Glutamate-Induced Cytotoxicity in Differentiated Y-79 Cells via Inhibition of ROS Generation and Ca(2+) Influx.

Glutamate toxicity is estimated to be the key cause of photoreceptor degeneration in the pathogenesis of retinal degenerative diseases. Oxidative stress and Ca2+ influx induced by glutamate are responsible for the apoptosis process of photoreceptor degeneration. Puerarin, a primary component of Kudzu root, has been widely used in the clinical treatment of retinal degenerative diseases in China for decades; however, the detailed molecular mechanism underlying this effect remains unclear. In this study, the neuroprotective effect of puerarin against glutamate-induced cytotoxicity in the differentiated Y-79 cells was first investigated through cytotoxicity assay. Then the molecular mechanism of this effect regarding anti-oxidative stress and Ca2+ hemostasis was further explored with indirect immunofluorescence, flow cytometric analysis and western blot analysis. Our study showed that glutamate induced cell viability loss, excessive reactive oxygen species (ROS) generation, calcium overload and up-regulated cell apoptosis in differentiated Y-79 cells, which effect was significantly attenuated with the pre-treatment of puerarin in a dose-dependent manner. Furthermore, our data indicated that the neuroprotective effect of puerarin was potentially mediated through the inhibition of glutamate-induced activation of mitochondrial-dependent signaling pathway and calmodulin-dependent protein kinase II (CaMKII)-dependent apoptosis signal-regulating kinase 1(ASK-1)/c-Jun N-terminal kinase (JNK)/p38 signaling pathway. The present study supports the notion that puerarin may be a promising neuroprotective agent in the prevention of retinal degenerative diseases.

Calcium channels contribute to albiflorin-mediated antinociceptive effects in mouse model

Albiflorin (AF), one of important bioactive constituents of Paeonia lactiflora Radix, possesses neuro-protective effect. The present study aims to investigate the antinociceptive activities of AF and possible mechanisms. AF suppressed acetic acid-caused writhing, lengthened the latency period of mouse in hot plate test, and reduced the licking and biting response time of the injected mouse paw during phase I and phase II, and it suggested that AF exerted the antinociceptive activity mainly through central nervous system. Nimodipine, a commonly used calcium channels blocker, strongly lengthened AF-enhanced latency period of mouse in hot plate test. Compared with control group, AF reduced the levels of euronal nitric oxide synthase (nNOS), and enhanced the levels of serotonin (5-HT) in serum and/or hypothalamus before and after 30-s thermal stimuli. The reduced activation of calmodulin-dependent protein kinase II and c-Jun N-terminal kinase in hypothalamus was observed in AF-treated mice. Collectively, AF-mediated antinociceptive activities were, at least partially, related to calcium channels.

Long-Term Potentiation: From CaMKII to AMPA Receptor Trafficking.

For more than 20 years, we have known that Ca(2+)/calmodulin-dependent protein kinase (CaMKII) activation is both necessary and sufficient for the induction of long-term potentiation (LTP). During this time, tremendous effort has been spent in attempting to understand how CaMKII activation gives rise to this phenomenon. Despite such efforts, there is much to be learned about the molecular mechanisms involved in LTP induction downstream of CaMKII activation. In this review, we highlight recent developments that have shaped our current thinking about the molecular mechanisms underlying LTP and discuss important questions that remain in the field.

The role of CaMKII in diabetic heart dysfunction.

Diabetes mellitus (DM) is an increasing epidemic that places a significant burden on health services worldwide. The incidence of heart failure (HF) is significantly higher in diabetic patients compared to non-diabetic patients. One underlying mechanism proposed for the link between DM and HF is activation of calmodulin-dependent protein kinase (CaMKIIδ). CaMKIIδ mediates ion channel function and Ca(2+) handling during excitation-contraction and excitation-transcription coupling in the myocardium. CaMKIIδ activity is up-regulated in the myocardium of diabetic patients and mouse models of diabetes, where it promotes pathological signaling that includes hypertrophy, fibrosis and apoptosis. Pharmacological inhibition and knockout models of CaMKIIδ have shown some promise of a potential therapeutic benefit of CaMKIIδ inhibition, with protection against cardiac hypertrophy and apoptosis reported. This review will highlight the pathological role of CaMKIIδ in diabetes and discuss CaMKIIδ as a therapeutic target in DM, and also the effects of exercise on CaMKIIδ.

Reduced endoplasmic reticulum stress-induced apoptosis and impaired unfolded protein response in TRPC3-deficient M1 macrophages.

Endoplasmic reticulum (ER) stress is a prominent mechanism of macrophage apoptosis in advanced atherosclerotic lesions. Recent studies from our laboratory showed that advanced atherosclerotic plaques in Apoe(-/-) mice with bone marrow deficiency of the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and fewer apoptotic macrophages than animals transplanted with Trpc3(+/+) bone marrow. In vitro, proinflammatory M1 but not anti-inflammatory M2 macrophages derived from Trpc3(-/-)Apoe(-/-) animals exhibited reduced ER stress-induced apoptosis. However, whether this was due to a specific effect of TRPC3 deficiency on macrophage ER stress signaling remained to be determined. In the present work we used polarized macrophages derived from mice with macrophage-specific deficiency of TRPC3 to examine the expression level of ER stress markers and the activation status of some typical mediators of macrophage apoptosis. We found that the reduced susceptibility of TRPC3-deficient M1 macrophages to ER stress-induced apoptosis correlates with an impaired unfolded protein response (UPR), reduced mitochondrion-dependent apoptosis, and reduced activation of the proapoptotic molecules calmodulin-dependent protein kinase II and signal transducer and activator of transcription 1. Notably, none of these pathways was altered in TRPC3-deficient M2 macrophages. These findings show for the first time an obligatory requirement for a member of the TRPC family of cation channels in ER stress-induced apoptosis in macrophages, underscoring a rather selective role of the TRPC3 channel on mechanisms related to the UPR signaling in M1 macrophages.