PKC Phosphorylation Research Articles

Protein Kinase C Downregulation Enhanced Extracellular Ca2+-Induced Relaxation of Isolated Mesenteric Arteries from Aged Dahl Salt-Sensitive Rats.

The Ca2+-sensing receptor (CaSR) detects small changes in extracellular calcium (Ca2+ e) concentration ([Ca2+]e) and transduces the signal into modulation of various signaling pathways. Ca2+-induced relaxation of isolated phenylephrine-contracted mesenteric arteries is mediated by the CaSR of the perivascular nerve. Elucidation of the regulatory mechanisms involved in vascular CaSR signaling may provide insights into the physiologic functions of the receptor and identify targets for the development of new treatments for cardiovascular pathologies such as hypertension. Protein kinase Cα (PKCα) is a critical regulator of multiple signaling pathways and can phosphorylate the CaSR leading to receptor desensitization. In this study, we used automated wire myography to investigate the effects of CaSR mutation and small-interfering RNA downregulation of PKCα on CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive (SS) rats on a low-salt diet. The data showed minimal relaxation responses of arteries to Ca2+ e in wild-type (SS) and CaSR mutant (SS-Casrem1Mcwi) rats. Mutation of the CaSR gene had no significant effect on relaxation. PKCα expression was similar in wild-type and mutant rats, and small-interfering RNA downregulation of PKCα and/or inhibition of PKC with the Ca2+-sensitive Gӧ 6976 resulted in a >80% increase in relaxation. Significant differences in EC50 values were observed between treated and untreated controls (P < 0.05 analysis of variance). The results indicate that PKCα plays an important role in the regulation of CaSR-mediated relaxation of mesenteric arteries, and its downregulation or pharmacological inhibition may lead to an increased Ca2+ sensitivity of the receptor and reversal of age-related changes in vascular tone. SIGNIFICANCE STATEMENT: G protein-coupled CaSR signaling leads to the regulation of vascular tone and may, therefore, play a vital role in blood pressure regulation. The receptor has several PKC phosphorylation sites in the C-terminal intracellular tail that mediate desensitization. We have previously shown that activation of the CaSR in neuronal cells leads to PKC phosphorylation, indicating that protein kinase C is an important regulator of CaSR function. Therefore, PKC in the CaSR signaling pathway in mesenteric arteries is a potential target for the development of new therapeutic approaches to treat hypertension and age-related vascular dysfunction. The present studies show that small-interfering RNA downregulation of PKCα and pharmacological inhibition of PKC enhanced CaSR-mediated relaxation of phenylephrine-contracted mesenteric arteries from aged Dahl salt-sensitive rats.

Spinocerebellar ataxia type 14 caused by a nonsense mutation in the PRKCG gene

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disorder characterized by cerebellar ataxia with myoclonus, dystonia, spasticity, and rigidity. Although missense mutations and a deletion mutation have been found in the protein kinase C gamma (PRKCG) gene encoding protein kinase C γ (PKCγ) in SCA14 families, a nonsense mutation has not been reported. The patho-mechanisms underlying SCA14 remain poorly understood. However, gain-of-function mechanisms and loss-of-function mechanisms, but not dominant negative mechanisms, were reported the patho-mechanism of SCA14. We identified the c.226C>T mutation of PRKCG, which caused the p.R76X in PKCγ by whole-exome sequencing in patients presenting cerebellar atrophy with cognitive and hearing impairment. To investigate the patho-mechanism of our case, we studied aggregation formation, cell death, and PKC inhibitory effect by confocal microscopy, western blotting with cleaved caspase 3, and pSer PKC motif antibodies, respectively.PKCγ(R76X)-GFP have aggregations the same as wild-type (WT) PKCγ-GFP. The PKCγ(R76X)-GFP inhibited PKC phosphorylation activity more than GFP alone. It also induced more apoptosis in COS7 and SH-SY5Y cells compared to WT-PKCγ-GFP and GFP.We first reported SCA14 patients with p.R76X in PKCγ who have cerebellar atrophy with cognitive and hearing impairment. Our results suggest that a dominant negative mechanism due to truncated peptides produced by p.R76X may be at least partially responsible for the cerebellar atrophy.

Biased signaling of Ca2+-sensing receptors in cardiac myocytes regulates GIRK channel activity

Ca2+-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca2+. CaSRs display biased G protein signaling by coupling to different classes of heterotrimeric G proteins depending on agonist and cell type. In this study we used fluorescent biosensors to directly analyze G protein coupling to CaSRs and downstream signaling in living cells. In HEK 293 cells, CaSRs displayed biased signaling: elevation of extracellular Ca2+ or application of the alternative agonist spermine caused activation of Gi- and Gq-proteins. Adult cardiac myocytes express endogenous CaSRs, which have been implicated in regulating Ca2+ signaling and contractility. Biased signaling of CaSRs has not been investigated in these cells. To evaluate efficiencies of Gi- and Gq-signaling via CaSRs in rat atrial myocytes, we measured G protein-activated K+ (GIRK) channels. Activation of GIRK requires binding of Gβγ subunits released from Gi proteins, whereas Gq-signaling results in inhibition of GIRK channel activity. Stimulation of CaSRs by Ca2+ or spermine failed to directly activate Gi and GIRK channels. When GIRK channels were pre-activated via endogenous M2 receptors, stimulation of CaSRs caused pronounced inhibition of GIRK currents. This effect was specific to CaSR activation: GIRK current inhibition was sensitive to NPS-2143, a negative allosteric modulator of CaSRs, and abrogated by FR900359, a direct inhibitor of Gq. GIRK current inhibition was also sensitive to the PKC inhibitor chelerythrine, suggesting that following activation of CaSR and Gq, GIRK currents are modulated by PKC phosphorylation. We conclude from this data that cardiac CaSRs do not activate Gi and affect GIRK currents preferentially via the Gq/PKC pathway.

Role of the putative PKC phosphorylation sites of the type IIc sodium-dependent phosphate transporter in parathyroid hormone regulation.

Injection of parathyroid hormone (PTH) rapidly stimulates renal Pi excretion, in part by downregulating NaPi-IIa (Npt2a/SLC34A1) and NaPi-IIc (Npt2c/SLC34A3) transporters. The mechanisms underlying the effects of PTH on NaPi-IIc are not fully elucidated. We analyzed the effect of PTH on inorganic phosphate (Pi) reabsorption in Npt2a-/- mice to eliminate the influence of Npt2a on renal Pi reabsorption. In opossum kidney (OK) cells and Xenopus oocytes, we investigated the effect of NaPi-IIc transporter phosphorylation. Studies of mice with mutations of NaPi-IIc protein in which serine and threonine were replaced with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated NaPi-IIc, were also performed to evaluate the involvement of phosphorylation in the regulation of transport function. The Npt2a-/- experiments showed that PTH administration rapidly inactivated NaPi-IIc function in the apical membrane of proximal tubular cells. Analysis of mutant proteins (S71, S138, T151, S174, T583) at putative protein kinase C sites, revealed that S138 markedly suppressed the function and cellular expression of mouse NaPi-IIc in Xenopus oocytes and OK cells. In addition, 138D had a short half-life compared with wild-type protein. The present study suggests that acute regulation of NaPi-IIc protein by PTH is involved in the inactivation of Na+-dependent Pi cotransporter activity and that phosphorylation of the transporter is involved in the rapid modification.

KCNE1 Subunit Phosphorylation Leads to IKs Internalization in Response to Chronic Calcium-Dependent PKC Activation

Decrease of the cardiac slowly activating delayed rectified K+ current (IKs), formed by KCNQ1 and KCNE1 subunits, is observed in a number of diseases states such as heart failure and diabetes and is associated with increased risk for cardiac arrhythmias and sudden death. Ca2+-dependent PKC isoforms (cPKC) are chronically activated in many pathological conditions. Recently, we found that sustained cPKC stimulation leads to KCNQ1 subunit internalization and decrease of IKs channel activity. Here, we show that KCNE1 is internalized together with KCNQ1 in response to sustained cPKC stimulus using immunostaining and confocal imaging. In addition, we show that KCNE1 is necessary for channel internalization independently of subunit stoichiometry both in heterologous expression systems and cardiomyocytes. Mutation of a PKC phosphorylation site shown to be important to cPKC acute regulation of the channel (KCNE1-S102A) was necessary to channel internalization. Altogether, our results suggest that KCNE1(S102) phosphorylation by cPKC leads to KCNQ1/KCNE1 internalization in response to sustained cPKC stimulus. Our results suggest that KCNE1(S102) is an important target for anti-arrhythmic drugs that would prevent IKs pathological remodeling leading to cardiac arrhythmias.

Exendin-4 prevented pancreatic beta cells from apoptosis in (Type I) diabetic mouse via keap1-Nrf2 signaling.

Nrf2 is an essential part of the defense mechanism of vertebrates and protects them from surrounding stress via participation in stimulated expression of detoxification as well as antioxidant enzymes. It also exerts a role in defending hosts from different stress in the environment, including reactive oxygen species. Our study investigates the role of exendin-4 on Nrf2 pathway as well as cell death in pancreatic β-cell and in non-obese diabetic mice. Result of study indicates exendin-4 mediates activation of Keap1-Nrf2-ARE pathway and may serve as a potential agent to treat type I diabetes mellitus. In our research, we observed excessive reactive oxygen species production, low level of cell death, and PKC phosphorylation on exendine-4 treatment. Nrf2 knockdown led to suppression of reactive oxygen species generation as well as increasing apoptosis. Moreover, siRNA-mediated Nrf2 down-regulation attenuated the suppressive effect of exendin-4 in pancreatic β-cell viability, via modulating apoptosis promoting- and counteracting-proteins, Bax, and Bcl-2.

Increased expression of ryanodine receptor type-2 during atrial fibrillation by miR-106-25 cluster independent mechanism

Atrial fibrillation (AF), the most frequently encountered cardiac arrhythmia in the clinical setting and the foremost cause of stroke, results from a progressive decrease in atrial refractoriness. In addition, defective calcium signaling has been shown to play a central role in AF pathogenesis. Recently it was shown that the miR-106b-25 cluster is suppressed in patients with AF, which increased ryanodine receptor 2 (RyR2) expression. Expression of the miR-106b-25 cluster and RyR2 protein were determined in our institutional series of patients with AF. Hemodynamic properties, RyR2 binding, suppression of ATP2A2 (encoding ATPase sarcoplasmic/endoplasmic reticulum Ca2 + transporting 2) were also determined. We found that all patients had elevated RyR2 protein expression; however, a cohort of patients with AF had high miR-93, miR-106b, and miR-25 expression. There was no difference in hemodynamic properties, RyR2 binding, or suppression of ATP2A2 in either cohort of patients with AF when compared to patients with normal sinus rhythm (NSR). Immunoblot assay showed hyperactive Akt, S6K, and S6 kinases in patients with AF as compared to patients with NSR. Protein kinase C activation, as measured by PKC phosphorylation, was also hyperactive in patients with AF. Cumulatively, our findings show that RyR2 expression is regulated by multiple mechanisms including the miR-106b-25, and that PKC activation might provide novel clues to increased intracellular calcium levels during AF pathogenesis.

Structure⁻Activity Relationship Study of Newly Synthesized Iridium-III Complexes as Potential Series for Treating Thrombotic Diseases.

Platelets play a major role in hemostatic events and are associated with various pathological events, such as arterial thrombosis and atherosclerosis. Iridium (Ir) compounds are potential alternatives to platinum compounds, since they exert promising anticancer effects without cellular toxicity. Our recent studies found that Ir compounds show potent antiplatelet properties. In this study, we evaluated the in vitro antiplatelet, in vivo antithrombotic and structure–activity relationship (SAR) of newly synthesized Ir complexes, Ir-1, Ir-2 and Ir-4, in agonists-induced human platelets. Among the tested compounds, Ir-1 was active in inhibiting platelet aggregation induced by collagen; however, Ir-2 and Ir-4 had no effects even at their maximum concentrations of 50 μM against collagen and 500 μM against U46619-induced aggregation. Similarly, Ir-1 was potently inhibiting of adenosine triphosphate (ATP) release, calcium mobilization ([Ca2+]i) and P-selectin expression induced by collagen-induced without cytotoxicity. Likewise, Ir-1 expressively suppressed collagen-induced Akt, PKC, p38MAPKs and JNK phosphorylation. Interestingly, Ir-2 and Ir-4 had no effect on platelet function analyzer (PFA-100) collagen-adenosine diphosphate (C-ADP) and collagen-epinephrine (C-EPI) induced closure times in mice, but Ir-1 caused a significant increase when using C-ADP stimulation. Other in vivo studies revealed that Ir-1 significantly prolonged the platelet plug formation, increased tail bleeding times and reduced the mortality of adenosine diphosphate (ADP)-induced acute pulmonary thromboembolism in mice. Ir-1 has no substitution on its phenyl group, a water molecule (like cisplatin) can replace its chloride ion and, hence, the rate of hydrolysis might be tuned by the substituent on the ligand system. These features might have played a role for the observed effects of Ir-1. These results indicate that Ir-1 may be a lead compound to design new antiplatelet drugs for the treatment of thromboembolic diseases.

Phosphorylation state of Ser165 in α-tubulin is a toggle switch that controls proliferating human breast tumors

Engineered overexpression of protein kinase Cα (PKCα) is known to phosphorylate Ser165 in α-tubulin resulting in stimulated microtubule dynamics and cell motility, and activation of an epithelial-mesenchymal transition (EMT) in non-transformed human breast cells. Here it is shown that endogenous phosphorylation of native α-tubulin in two metastatic breast cell lines, MDA-MB-231-LM2–4175 and MDA-MB-468 is detected at PKC phosphorylation sites. α-Tubulin mutants that simulated phosphorylated (S165D) or non-phosphorylated (S165 N) states were stably expressed in MDA-MB-231-LM2–4175 cells. The S165D-α-tubulin mutant engendered expression of the EMT biomarker N-cadherin, whereas S165 N-α-tubulin suppressed N-cadherin and induced E-cadherin expression, revealing a ‘cadherin switch’. S165 N-α-tubulin engendered more rapid passage through the cell cycle, induced shorter spindle fibers and exhibited more rapid proliferation. In nude mice injected with MDA-MB-231-LM2–4175 cells, cells expressing S165 N-α-tubulin (but not the S165D mutant) produced hyper-proliferative lung tumors with increased tumor incidence and higher Ki67 expression. These results implicate the phosphorylation state of Ser165 in α-tubulin as a PKC-regulated molecular switch that causes breast cells to exhibit either EMT characteristics or hyper-proliferation. Evaluation of genomic databases of human tumors strengthens the clinical significance of these findings.

Simvastatin Enhances Activity and Trafficking of α7 Nicotinic Acetylcholine Receptor in Hippocampal Neurons Through PKC and CaMKII Signaling Pathways.

Simvastatin (SV) enhances glutamate release and synaptic plasticity in hippocampal CA1 region upon activation of α7 nicotinic acetylcholine receptor (α7nAChR). In this study, we examined the effects of SV on the functional activity of α7nAChR on CA1 pyramidal cells using patch-clamp recording and explored the underlying mechanisms. We found that the treatment of hippocampal slices with SV for 2 h induced a dose-dependent increase in the amplitude of ACh-evoked inward currents (IACh) and the level of α7nAChR protein on the cell membrane without change in the level of α7nAChR phosphorylation. These SV-induced phenotypes were suppressed by addition of farnesol (FOH) that converts farnesyl pyrophosphate, but not geranylgeraniol. Similarly, the farnesyl transferase inhibitor FTI277 was able to increase the amplitude of IACh and enhance the trafficking of α7nAChR. The treatment with SV enhanced phosphorylation of CaMKII and PKC. The SV-enhanced phosphorylation of CaMKII rather than PKC was blocked by FOH, Src inhibitor PP2 or NMDA receptor antagonist MK801 and mimicked by FTI. The SV-enhanced phosphorylation of PKC was sensitive to the IP3R antagonist 2-APB. The SV-increased amplitude of IACh was suppressed by PKC inhibitor GF109203X and Go6983, or CaMKII inhibitor KN93. The SV- and FTI-enhanced trafficking of α7nAChR was sensitive to KN93, but not GF109203X or Go6983. The PKC activator PMA increased α7nAChR activity, but had no effect on trafficking of α7nAChR. Collectively, these results indicate that acute treatment with SV enhances the activity and trafficking of α7nAChR by increasing PKC phosphorylation and reducing farnesyl-pyrophosphate to trigger NMDA receptor-mediated CaMKII activation.

The receptor for activated C kinase 1 (RACK1) mediating immune response in thick shell mussel Mytilus coruscus

The receptor for activated C kinase 1 (RACK1) is a intracellular receptor for the protein kinase C family which mediates various biological processes. Here, a novel RACK1 gene termed Mc-RACK1 was identified from thick shell mussel, Mytilus coruscus. Mc-RACK1 shared typical RACK1 domains containing WD repeats, PKC phosphorylation sites, N-myristoylation sites, PKC activation sites, TK phosphorylation site and WD motifs. Mc-RACK1 was constitutively expressed in all examined tissues, and its expression in gills, haemocytes and digestive glands were significantly up-regulated upon LPS challenge. Mc-RACK1 showed a significantly down-regulated expression in gills and haemocytes at the early phase upon copper exposure. Mc-RACK1 in haemocytes was silenced after receiving its dsRNA, meanwhile, the increases of SOD and CAT activity were investigated. Further, Mc-RACK1 could activate the NF-κB and ISRE reporter in HEK-293T cells. These suggested that Mc-RACK1 had a deeper involvement in mollusc immunity, and played an important role in antioxidant system.

LPS enhances platelets aggregation via TLR4, which is related to mitochondria damage caused by intracellular ROS, but not extracellular ROS

Platelet is an important cell contributing to hemostasis and immunity. Bacterial lipopolysaccharide (LPS), mainly functioning by stimulating toll-like receptor 4 (TLR4), mediates platelet activation and sepsis. However, the inter-relationship between these players in sepsis remains unknown. We found that the aggregation of platelets was enhanced in complete blood of sepsis patients than that of healthy donors. PRP isolated from complete blood of healthy donors was used in the following study to filter out the interference of irrelevant cells. The results shown that the maximum aggregation rate (MAR) was significantly higher in LPS-challenged PRP model than that of controls, and administration of the specific TLR4 inhibitor, TAK242, reduced the MAR in this model. LPS promoted P-selectin expression and intracellular ROS production, and both TAK242 and N-acetyl-L-cysteine (NAC) could depressed the LPS-induced increase of P-selectin and intracellular ROS. H2O2 administration increased P-selectin expression partially but had little effect on intracellular ROS, thought it increased mitochondrial damage. In vivo, LPS increased both intracellular ROS and CD62P comparing with that of controls, effects that were prevented by TAK242. Furthermore, platelet aggregation through LPS-TLR4 pathway was involved in AKT, PKC and p38 phosphorylation but not cGMP/cAMP pathway. In conclusion, this study shows that intracellular ROS, not extracellular ROS such as H2O2, plays a crucial role in facilitating platelet aggregation via LPS/TLR4 pathway, and this process was involved in AKT, PKC and p38 phosphorylation but not cGMP/cAMP pathway. The results would helpful for understanding the role of intracellular ROS and LPS-TLR4 pathway in platelet aggregation.

MARCKS phosphorylation by PKC strongly impairs cell polarity in the chick neural plate.

Neurulation involves a complex coordination of cellular movements that are in great part based on the modulation of the actin cytoskeleton. MARCKS, an F-actin-binding protein and the major substrate for PKC, is necessary for gastrulation and neurulation morphogenetic movements in mice, frogs, and fish. We previously showed that this protein accumulates at the apical region of the closing neural plate in chick embryos, and here further explore its role in this process and how it is regulated by PKC phosphorylation. PKC activation by PMA caused extensive neural tube closure defects in cultured chick embryos, together with MARCKS phosphorylation and redistribution to the cytoplasm. This was concomitant with an evident disruption of neural plate cell polarity and extensive apical cell extrusion. This effect was not due to actomyosin hypercontractility, but it was reproduced upon MARCKS knockdown. Interestingly, the overexpression of a nonphosphorylatable form of MARCKS was able to revert the cellular defects observed in the neural plate after PKC activation. Altogether, these results suggest that MARCKS function during neurulation would be to maintain neuroepithelial polarity through the stabilization of subapical F-actin, a function that appears to be counteracted by PKC activation.

Contractile responses to endothelin-1 are regulated by PKC phosphorylation of cardiac myosin binding protein-C in rat ventricular myocytes

The shortening of sarcomeres that co-ordinates the pump function of the heart is stimulated by electrically-mediated increases in [Ca2+]. This process of excitation-contraction coupling (ECC) is subject to modulation by neurohormonal mediators that tune the output of the heart to meet the needs of the organism. Endothelin-1 (ET-1) is a potent modulator of cardiac function with effects on contraction amplitude, chronotropy and automaticity. The actions of ET-1 are evident during normal adaptive physiological responses and increased under pathophysiological conditions, such as following myocardial infarction and during heart failure, where ET-1 levels are elevated. In myocytes, ET-1 acts through ETA- or ETB-G protein-coupled receptors (GPCRs). Although well studied in atrial myocytes, the influence and mechanisms of action of ET-1 upon ECC in ventricular myocytes are not fully resolved. We show in rat ventricular myocytes that ET-1 elicits a biphasic effect on fractional shortening (initial transient negative and sustained positive inotropy) and increases the peak amplitude of systolic Ca2+ transients in adult rat ventricular myocytes. The negative inotropic phase was ETB receptor-dependent, whereas the positive inotropic response and increase in peak amplitude of systolic Ca2+ transients required ETA receptor engagement. Both effects of ET-1 required phospholipase C (PLC)-activity, although distinct signalling pathways downstream of PLC elicited the effects of each ET receptor. The negative inotropic response involved inositol 1,4,5-trisphosphate (InsP3) signalling and protein kinase C epsilon (PKCε). The positive inotropic action and the enhancement in Ca2+ transient amplitude induced by ET-1 were independent of InsP3 signalling, but suppressed by PKCε. Serine 302 in cardiac myosin binding protein-C was identified as a PKCε substrate that when phosphorylated contributed to the suppression of contraction and Ca2+ transients by PKCε following ET-1 stimulation. Thus, our data provide a new role and mechanism of action for InsP3 and PKCε in mediating the negative inotropic response and in restraining the positive inotropy and enhancement in Ca2+ transients following ET-1 stimulation.

PKC Phosphorylation and mu-calpain Truncation of the C-Terminal End Segment of SM22alpha Regulates its F-Actin Binding and Mechanical Tension Modulated Degradation

PKC Phosphorylation and mu-calpain Truncation of the C-Terminal End Segment of SM22alpha Regulates its F-Actin Binding and Mechanical Tension Modulated Degradation

The Gq-GPCR Pathway Evokes Tightly Controlled TRPV1 Activation

The detection and propagation of a pain signal must be coupled with the regulation and control of such a signal. TRPV1 is well established as a major pain receptor, capable of detecting a multitude of stimuli, including endogenous inflammatory mediators such as arachidonic acid and anandamide, in addition to exogenous molecules such as the spicy chemical capsaicin. However, endogenous inflammatory mediators evoke noticeably weaker channel activation in comparison to exogenous molecules. Here, we employed pharmacological and molecular biology tools to dissect the regulated activation of TRPV1 in the inflammatory response via the Gq/GPCR pathway. By employing specific techniques such as perforated-patch recordings, the DREADD system, and calcium imaging of both neurons and a heterologous system, we worked to preserve the native intracellular cascades which are essential for Gq-mediated TRPV1 activation. By doing so, we found that two downstream pathways converge on TRPV1 to evoke a tightly regulated response. The first, the production of endo-vanilloids by lipoxygenases, are essential in producing the ligand that activates TRPV1 through the intracellularly located vanilloid binding site. The second, channel sensitization through PKC phosphorylation, is necessary for the metabolites to successfully activate the channel. We propose that through this dual cascade, a tightly regulated channel activation is achieved. Functionally, this regulation prevents the depolarization block associated with exogenous activation of TRPV1 by capsaicin, and ensures prolonged neuronal firing, as is found during nociceptor activation by inflammatory mediators. Overall, we propose that the requirement of multiple signalling events maintains a subdued TRPV1 activation in order to evoke regulated neuronal response during inflammation.

Chronic Exercise Increases Compliant Titin and Kettin Isoform Content in Cardiac Muscle of Rat and Drosophila Models

Reduced cardiac passive stiffness correlates with increased exercise tolerance in mammals. Titin, the giant elastic protein, decreases passive stiffness by expression of larger titin isoforms (increased N2BA:N2B ratio) or altered post-translational modifications (increased PKA, decreased PKC phosphorylation). Short-term and acute exercise has been previously shown to change post-translational modifications but not the N2BA:N2B. Kettin is an elastic protein in invertebrates that is also differentially spliced. Little is known about kettin isoform splicing in response to exercise. The purpose of our study was to evaluate changes in titin and kettin isoform content following chronic exercise or sedentary conditions. 4 week old male Sprague-Dawley rats were randomly assigned to cages with (exercise, n=18) and without (sedentary, n=17) running wheels. After 12 weeks, hearts were harvested and the left ventricle solubilized and proteins separated using SDS-VAGE. Titin N2BA:N2B ratio increased (p=0.047) in exercising animals with wheels compared to sedentary animals. Degraded titin to total titin ratio and total titin to myosin heavy chain content ratios were unchanged. Canton S flies (Drosophila) were exercised up to 3 hrs per day 5 times per week for 3 weeks using the PowerTower, a machine that repetitively induces negative geotaxis (climbing exercise). Abdominal segments from whole, flash frozen Drosophila were solubilized from 3 technical replicates containing 10 Drosophila each for both the exercised and un-exercised groups. Abdominal samples contain both cardiac and skeletal muscles, but are more enriched for cardiac muscle than thoraces. Three isoforms of kettin were consistently identified using western blot. Preliminary data showed an increased expression of larger kettin isoforms sizes in exercise versus un-exercised samples. These preliminary studies indicate that both mammalian and invertebrate cardiac tissues have increased compliant isoforms of titin and kettin, respectively.

Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Alterations in Ca2+ homeostasis affect neuronal survival. However, the identity of Ca2+ channels and the mechanisms underlying neurotoxin-induced neuronal degeneration are not well understood. In this study, the dopaminergic neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridium ions (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mimic Parkinson's disease (PD), induced neuronal degeneration by decreasing store-mediated Ca2+ entry. The function of the transient receptor potential canonical (TRPC)-1 channel was decreased upon exposure to the neurotoxins, followed by a decrease in TRPC1 expression. Similar to neurotoxins, samples from patients with PD exhibited attenuated TRPC1 expression, which was accompanied by a decrease in autophagic markers and a subsequent increase in apoptosis markers. Furthermore, exposure to neurotoxins attenuated PKC phosphorylation, decreased expression of autophagic markers, and increased apoptosis in SHSY-5Y neuroblastoma cells, which was again dependent on TRPC1. Prolonged neurotoxin treatment attenuated the binding of NF-κB to the TRPC1 promoter, which resulted in a decrease in TRPC1 expression, thereby attenuating autophagy and activating cell death. Restoration of TRPC1 expression rescued the effects of the dopaminergic neurotoxins in neuroblastoma cells by increasing Ca2+ entry, restoring NF-κB activity, and promoting autophagy. Overall, these results suggest that dopaminergic neurotoxins initially decreased Ca2+ entry, which inhibited the binding of NF-κB to the TRPC1 promoter, thereby inhibiting TRPC1 expression and resulting in cell death by preventing autophagy.-Sukumaran, P., Sun, Y., Antonson, N., Singh, B. B. Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K+ current (IKr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased IKr is involved in increased cardiac arrhythmogenicity. Stimulation of α1A -adrenoreceptors or angiotensin II AT1 receptors is known to inhibit IKr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of IKr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record IKr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α1A -adrenoreceptor or AT1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of IKr by the α1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of IKr by activation of AT1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of IKr by activation of α1A -adrenoreceptors or AT1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms.

Rapamycin inhibits ox-LDL-induced inflammation in human endothelial cells in vitro by inhibiting the mTORC2/PKC/c-Fos pathway.

Rapamycin and its derivative possess anti-atherosclerosis activity, but its effects on adhesion molecule expression and macrophage adhesion to endothelial cells during atherosclerosis remain unclear. In this study we explored the effects of rapamycin on ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells in vitro and the underlying mechanisms. Ox-LDL (6-48 μg/mL) dose-dependently increased the protein levels of two adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in human umbilical vein endothelial cells (HUVECs), whereas pretreatment with rapamycin (1-10 μmol/L) dose-dependently inhibited ox-LDL-induced increase in the adhesion molecule expression and macrophage adhesion to endothelial cells. Knockdown of mTOR or rictor, rather than raptor, mimicked the effects of rapamycin. Ox-LDL (100 μg/mL) time-dependently increased PKC phosphorylation in HUVECs, which was abolished by rapamycin or rictor siRNA. Pretreatment with PKC inhibitor staurosporine significantly reduced ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells, whereas pretreatment with PKC activator PMA/TPA attenuated the inhibitory effect of rapamycin on adhesion molecule expression. Ox-LDL (100 μg/mL) time-dependently increased c-Fos levels in HUVECs, and pretreatment with rapamycin or rictor siRNA significantly decreased expression of c-Fos. Knockdown of c-Fos antagonized ox-LDL-induced adhesion molecule expression and macrophage adhesion to endothelial cells. Our results demonstrate that rapamycin reduces ox-LDL-stimulated adhesion molecule expression and macrophage adhesion to endothelial cells by inhibiting mTORC2, but not mTORC1, and mTORC2 acts through the PKC/c-Fos signaling pathway.