Mutant ryanodine receptors in catecholaminergic polymorphic ventricular tachycardia generate delayed afterdepolarizations due to increased propensity to Ca2+ waves.

AIMS: Mutations in cardiac ryanodine receptors (RyR2s) are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), characterized by risk of polymorphic ventricular tachyarrhythmias and sudden death during exercise. Arrhythmias are caused by gain-of-function defects in RyR2, but cellular arrhythmogenesis remains elusive. METHODS AND RESULTS: We recorded endocardial monophasic action potentials (MAPs) at right ventricular septum in 15 CPVT patients with a RyR2 mutation (P2,328S, Q4,201R, and V4,653F) and in 12 control subjects both at baseline and during epinephrine infusion (0.05 microg/kg/min). At baseline 3 and during epinephrine infusion, four CPVT patients, but none of the control subjects, showed delayed afterdepolarizations (DADs) occasionally coinciding with ventricular premature complexes. In order to study the underlying mechanisms, we expressed two types of mutant RyR2 (P2,328S and V4,653F) causing CPVT as well as wild-type RyR2 in HEK 293 cells. Confocal microscopy of Fluo-3 loaded cells transfected with any of the three RyR2s showed no spontaneous subcellular Ca(2+) release events at baseline. Membrane permeable cAMP analogue (Dioctanoyl-cAMP) triggered subcellular Ca(2+) release events as Ca(2+) sparks and waves. Cells expressing mutant RyR2s showed spontaneous Ca(2+) release events at lower concentrations of cAMP than cells transfected with wild-type RyR2. CONCLUSION: CPVT patients show DADs coinciding with premature action potentials in MAP recordings. Expression studies suggest that DADs are caused by increased propensity of abnormal RyR2s to generate spontaneous Ca(2+) waves in response to cAMP stimulation. Increased sensitivity of mutant RyR2s to cAMP may explain the occurrence of arrhythmias during exercise or emotional stress in CPVT.     

An endogenous A2B adenosine receptor coupled to cyclic AMP generation in human embryonic kidney (HEK 293) cells

1. Cyclic AMP generation by adenosine analogues was examined in human embryonic kidney (HEK 293) cells by use of a [³H]-adenine pre-labelling methodology.
2. Adenosine analogues showed the following rank order of potency (pD₂ value): 5′-N-ethylcarboxamidoadenosine (NECA, 5.24)>2-chloroadenosine (4.41) ⩾ adenosine (4.19)=N⁶-(2-(4-aminophenyl)-ethylamino)adenosine (APNEA, 4.11). The A_2A-selective agonist {"type":"entrez-protein","attrs":{"text":"CGS21680","term_id":"878113053","term_text":"CGS21680"}}CGS21680 failed to elicit a significant stimulation of cyclic AMP generation at concentrations below 30 μM.
3. Of these agents, NECA was observed to exhibit the greatest intrinsic activity, while in comparison maximal responses to adenosine (76±8% NECA response), 2-chloroadenosine (70±6%) and APNEA (40±3%) were significantly reduced.
4. Antagonists of the NECA-evoked cyclic AMP generation showed the rank order of apparent affinity (apparent pA₂ value): CGS 15943 (7.79)=XAC (7.74)>DPCPX (7.01)=PD115199 (6.93)=8FB-PTP (6.80)>KF 17837 (5.98)>3-propylxanthine (5.13).
5. Agarose gel electrophoresis of the products of the polymerase chain reaction, with cDNA generated from HEK 293 cell total RNA showed virtually identical patterns and nucleotide sizes in comparison with the vector for the full length human brain A_2B adenosine receptor.
6. We concluded that HEK 293 cells express an endogenous adenosine receptor coupled to cyclic AMP generation which is of the A_2B subtype.

     

Analysis of receptor-G protein interactions in permeabilized cells

Receptor-induced binding of the stable GTP analogue, guanosine 5-[-thio]triphosphate (GTP [S]), to guanine nucleotide-binding regulatory proteins (G proteins) was measured in various permeabilized cells. In myeloid differentiated human leukemia (HL-60) cells, permeabilized with either digitonin, streptolysin O or Staphylococcus aureus -toxin, binding of GTP [S] induced by three distinct chemoattractant receptors was observed. The extent of receptor-stimulated GTP [S] binding (maximally about 2-fold) was independent of the type of permeabilizing agent used. In human erythroleukemia cells permeabilized with digitonin, agonist activation of thrombin and neuropeptide Y receptors increased GTP [S] binding by 1.8- and 1.5-fold, respectively. Finally, in adherently grown human embryonic kidney cells permeabilized with digitonin, activation of the stably expressed human muscarinic m3 receptor increased GTP[S] binding by about 1.6-fold. In digitonin-permeabilized HL-60 cells, a quantitative analysis of formyl peptide receptors and interacting G proteins was performed. About 50,000 formyl peptide receptors per cell were detected. Agonist binding to these receptors was fully sensitive to regulation by guanine nucleotides and pertussis toxin. The number of high-affinity GTP [S] binding sites, most likely representing heterotrimeric G proteins, was calculated to be about 670,000 per cell. Stimulation of formyl peptide receptors led to the activation of about 130,000 of high-affinity GTP [S] binding sites, indicating a ratio of about three activated G proteins per one agonist-activated receptor.Overall, this study indicates that receptor-stimulated GTP [S] binding to G proteins in permeabilized cells is a sensitive and rapid method for analyzing receptor-G protein interactions, which can be applied to a variety of cultured cells and for various receptor systems.     

Scorpion alpha and alpha-like toxins differentially interact with sodium channels in mammalian CNS and periphery

Scorpion alpha-toxins from Leiurus quinquestriatus hebraeus, LqhII and LqhIII, are similarly toxic to mice when administered by a subcutaneous route, but in mouse brain LqhII is 25-fold more toxic. Examination of the two toxins effects in central nervous system (CNS), peripheral preparations and expressed sodium channels revealed the basis for their differential toxicity. In rat brain synaptosomes, LqhII binds with high affinity, whereas LqhIII competes only at high concentration for LqhII-binding sites in a voltage-dependent manner. LqhII strongly inhibits sodium current inactivation of brain rBII subtype expressed in HEK293 cells, whereas LqhIII is weakly active at 2 microM, suggesting that LqhIII affects sodium channel subtypes other than rBII in the brain. In the periphery, both toxins inhibit tetrodotoxin-sensitive sodium current inactivation in dorsal root ganglion neurons, and are strongly active directly on the muscle and on expressed muI channels. Only LqhII, however, induced repetitive end-plate potentials in mouse phrenic nerve-hemidiaphragm muscle preparation by direct effect on the motor nerve. Thus, rBII and sodium channel subtypes expressed in peripheral nervous system (PNS) serve as the main targets for LqhII but are mostly not sensitive to LqhIII. Toxicity of both toxins in periphery may be attributed to the direct effect on muscle. Our data elucidate, for the first time, how different toxins affect mammalian central and peripheral excitable cells, and reveal unexpected subtype specificity of toxins that interact with receptor site 3.     

hHCN2基因的表达和电生理记录

目的将hHCN2基因有效转染到HEK293细胞，建立一种研究心肌离子通道的有效模型。方法采用Lipofactamine2000脂质体将hHCN2基因转染HEK293细胞。运用全细胞膜片钳技术检测克隆hHCN2基因表达的情况。结果pcDNA3-hHCN4真核表达载体转染HEK293细胞3d后，记录到转染成功的hHCN2基因编码的通道电流。结论该实验采用的技术稳定可靠，为开展克隆离子通道结构和功能关系的研究奠定了基础。     

Cisplatin toxicity reduced in human cultured renal tubular cells by oxygen pretreatment

Abstract

Cisplatin is an effective and widely used chemotherapy agent and its side effects, particularly nephrotoxicity, limit its usage and related platinum-based drugs. Cisplatin nephrotoxicity is mainly due to extremely increase in reactive oxygen species (ROS) generation leading to kidney tubular cell death. Preconditioning with oxidative stress has been demonstrated to stimulate the cellular adaptation to subsequent severe oxidative stress. Short term oxygen pre-exposure as a mild oxidative stress may enhance some endogenous defense mechanisms, so its effect on Cisplatin induced cell death was investigated in present research. We studied the effects of hyperoxic environment pre-exposure on Cisplatin toxicity in an in-vitro model of cultured human embryonic tubular epithelial cells (AD293). Viability of AD293 cells, as evaluated by MTT-assay, was affected by Cisplatin in a time (1–4?h) dependent model. Biochemical markers of cell apoptosis were evaluated using immunoblotting. Pretreatment with nearly pure oxygen (≥90%) for 2?h significantly reduced the level of cell damage. Activated caspase 3 and Bax/Bcl-2 ratio were significantly increased in Cisplatin-treated cells. Oxygen pretreatment inhibited caspase 3 activation and decreased Bax/Bcl-2 ratio. Oxygen pre-treatment itself not showed any cytotoxicity in exposure times up to 3?h. Our data indicate that hyperoxic preconditioning reduces Cisplatin toxicity in cultured human tubular epithelial cells. The exact mechanism of protection is unclear, though enhancement of some endogenous defense mechanisms and subsequently scavenging of free oxygen radicals may play an important role.     

Role of viral RNA and lipid in the adverse events associated with the 2010 Southern Hemisphere trivalent influenza vaccine

In Australia, during the 2010 Southern Hemisphere (SH) influenza season, there was an unexpected increase in post-marketing adverse event reports of febrile seizures (FS) in children under 5 years of age shortly after vaccination with the CSL 2010 SH trivalent influenza vaccine (CSL 2010 SH TIV) compared to previous CSL TIVs and other licensed 2010 SH TIVs. In an accompanying study, we described the contribution to these adverse events of the 2010 SH influenza strains as expressed in the CSL 2010 SH TIV using in vitro cytokine/chemokine secretion from whole blood cells and induction of NF-κB activation in HEK293 reporter cells. The aim of the present study was to identify the root cause components that elicited the elevated cytokine/chemokine and NF-κB signature. Our studies demonstrated that the pyrogenic signal was associated with a heat-labile, viral-derived component(s) in the CSL 2010 SH TIV. Further, it was found that viral lipid-mediated delivery of short, fragmented viral RNA was the key trigger for the increased cytokine/chemokine secretion and NF-κB activation. It is likely that the FS reported in children <5 years were due to a combination of the new influenza strains included in the 2010 SH TIV and the CSL standard method of manufacture preserving strain-specific viral components of the new influenza strains (particularly B/Brisbane/60/2008 and to a lesser extent H1N1 A/California/07/2009). These combined to heighten immune activation of innate immune cells, which in a small proportion of children <5 years of age is associated with the occurrence of FS. The data also demonstrates that CSL TIVs formulated with increased levels of splitting agent (TDOC) for the B/Brisbane/60/2008 strain can attenuate the pro-inflammatory signals in vitro, identifying a potential path forward for generating a CSL TIV indicated for use in children <5 years.     

SIRT4 inhibits cigarette smoke extracts-induced mononuclear cell adhesion to human pulmonary microvascular endothelial cells via regulating NF-κB activity

Cigarette smoking is an important risk factor for chronic obstructive pulmonary disease (COPD), yet its pathogenic mechanisms are not yet fully understood. Endothelial dysfunction is known to be involved in the pathogenesis of COPD. A detailed understanding of the mechanism involved in its progression would have a substantial impact on the optimization and development of treatment strategies. Here, we report that the expression of SIRT4, a mitochondrial sirtuin, is markedly down-regulated in cigarette smoke extract (CSE)-treated human pulmonary microvascular endothelial cells (HPMECs). Overexpression of SIRT4 significantly inhibits CSE-induced mononuclear cell adhesion to HPMECs. Consistently, we found that overexpression of SIRT4 attenuates the induction of vascular cell adhesion molecule 1 (VCAM-1) and E-selectin. Importantly, SIRT4 was found to negatively regulate CSE-induced NF-κB activation via inhibiting the degradation of IκBα. Moreover, we also found that proinflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6, the downstream target genes of NF-κB, are also inhibited by overexpression of SIRT4. These results suggest that SIRT4 protects HPMECs exposed to CSE stress via a mechanism that may involve the NF-κB pathway. Strategies based on the enhancement of SIRT4 may prove to be beneficial in the treatment of cigarette smoking caused COPD.     

Orexin A decreases lipid peroxidation and apoptosis in a novel hypothalamic cell model

Current data support the idea that hypothalamic neuropeptide orexin A (OxA; hypocretin 1) mediates resistance to high fat diet-induced obesity. We previously demonstrated that OxA elevates spontaneous physical activity (SPA), that rodents with high SPA have higher endogenous orexin sensitivity, and that OxA-induced SPA contributes to obesity resistance in rodents. Recent reports show that OxA can confer neuroprotection against ischemic damage, and may decrease lipid peroxidation. This is noteworthy as independent lines of evidence indicate that diets high in saturated fats can decrease SPA, increase hypothalamic apoptosis, and lead to obesity. Together data suggest OxA may protect against obesity both by inducing SPA and by modulation of anti-apoptotic mechanisms. While OxA effects on SPA are well characterized, little is known about the short- and long-term effects of hypothalamic OxA signaling on intracellular neuronal metabolic status, or the physiological relevance of such signaling to SPA. To address this issue, we evaluated the neuroprotective effects of OxA in a novel immortalized primary embryonic rat hypothalamic cell line. We demonstrate for the first time that OxA increases cell viability during hydrogen peroxide challenge, decreases hydrogen peroxide-induced lipid peroxidative stress, and decreases caspase 3/7 induced apoptosis in an in vitro hypothalamic model. Our data support the hypothesis that OxA may promote obesity resistance both by increasing SPA, and by influencing survival of OxA-responsive hypothalamic neurons. Further identification of the individual mediators of the anti-apoptotic and peroxidative effects of OxA on target neurons could lead to therapies designed to maintain elevated SPA and increase obesity resistance.     

Ca2+-dependent modulation of single human cardiac L-type calcium channels by the calcineurin inhibitor cyclosporine

OBJECTIVE: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. METHODS: Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. RESULTS: A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. CONCLUSIONS: We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.