Azelnidipine protects HL-1 cardiomyocytes from hypoxia/reoxygenation injury by enhancement of NO production independently of effects on gene expression.

It remains to be elucidated whether Ca2+ antagonists induce pharmacological preconditioning to protect the heart against ischemia/reperfusion injury. The aim of this study was to determine whether and how pretreatment with a Ca2+ antagonist, azelnidipine, could protect cardiomyocytes against hypoxia/reoxygenation (H/R) injury in vitro. Using HL-1 cardiomyocytes, we studied effects of azelnidipine on NO synthase (NOS) expression, NO production, cell death and apoptosis during H/R. Action potential durations (APDs) were determined by the whole-cell patch-clamp technique. Azelnidipine enhanced endothelial NOS phosphorylation and NO production in HL-1 cells under normoxia, which was abolished by a heat shock protein 90 inhibitor, geldanamycin, and an antioxidant, N-acetylcysteine. Pretreatment with azelnidipine reduced cell death and shortened APDs during H/R. These effects of azelnidipine were diminished by a NOS inhibitor, L-NAME, but were influenced by neither a T-type Ca2+ channel inhibitor, NiCl2, nor a N-type Ca2+ channel inhibitor, ω-conotoxin. The azelnidipine-induced reduction in cell death was not significantly enhanced by either additional azelnidipine treatment during H/R or increasing extracellular Ca2+ concentrations. RNA sequence (RNA-seq) data indicated that azelnidipine-induced attenuation of cell death, which depended on enhanced NO production, did not involve any significant modifications of gene expression responsible for the NO/cGMP/PKG pathway. We conclude that pretreatment with azelnidipine protects HL-1 cardiomyocytes against H/R injury via NO-dependent APD shortening and L-type Ca2+ channel blockade independently of effects on gene expression.

Mechanisms for hypothermia during anaphylactic hypotension in awake rats.

Hypothermia develops during systemic anaphylaxis in rodents. The aim of this study was to elucidate the mechanism for the hypothermia by assessing the roles of locomotor activity, tail heat dissipation, heat production in the brown adipose tissue (BAT) activity, and chemical mediators during ovalbumin-induced anaphylactic hypotension in awake rats. We measured the core body temperature (Tcore) and mean blood pressure (MBP), along with the surface temperature of the interscapular region (TiScap), an indirect measure of BAT activity, and the tail (Ttail). During anaphylaxis, MBP decreased to the nadir of 53 ± 2 mmHg at 8 min with recovery toward baseline. Tcore began to decrease at 7.5 min with the nadir of 36.1 ± 0.2°C at 30 min from the baseline of 38.0 ± 0.1°C. TiScap also significantly decreased, but its onset was preceded by that of Tcore. Ttail decreased after antigen, suggesting the absence of increased heat dissipation from the tail. The physical activity, as evaluated by moved distances, did not decrease until 20 min after antigen, followed by a progressive decrease. Reduced movement using a restraint maneuver not only reduced Tcore in nonsensitized rats but also augmented the anaphylactic hypothermia in the early phase (1.5-18 min) in sensitized rats. Combined antagonism against platelet-activating factor (PAF) and histamine H1 receptors abolished antigen-induced hypotension but only attenuated hypothermia. In conclusion, decreased locomotor activity, but not tail heat dissipation or decreased BAT activity, may at least in part contribute to this hypothermia. PAF and histamine are involved mainly in hypotension but only partly in hypothermia during rat anaphylaxis.NEW & NOTEWORTHY Anaphylactic shock is a life-threatening systemic hypotension. Hypothermia is observed during systemic anaphylaxis of rats. We determined the mechanism as follows: decreased locomotor activity, but not tail heat dissipation or decreased BAT activity, may at least in part contribute to this hypothermia. PAF and histamine are involved mainly in hypotension, but only partly in hypothermia during rat anaphylaxis.

Mechanisms of fever-induced QT prolongation and torsades de pointes in patients with KCNH2 mutation.

AIMS: Patients with particular mutations of type-2 long QT syndrome (LQT2) are at an increased risk for malignant arrhythmia during fever. This study aimed to determine the mechanism by which KCNH2 mutations cause fever-induced QT prolongation and torsades de pointes (TdP). METHODS AND RESULTS: We evaluated three KCNH2 mutations, G584S, D609G, and T613M, in the Kv11.1 S5-pore region, identified in patients with marked QT prolongation and TdP during fever. We also evaluated KCNH2 M124T and R269W, which are not associated with fever-induced QT prolongation. We characterized the temperature-dependent changes in the electrophysiological properties of the mutant Kv11.1 channels by patch-clamp recording and computer simulation. The average tail current densities (TCDs) at 35°C for G584S, WT+D609G, and WT+T613M were significantly smaller and less increased with rising temperature from 35°C to 40°C than those for WT, M124T, and R269W. The ratios of the TCDs at 40°C to 35°C for G584S, WT+D609G, and WT+T613M were significantly smaller than for WT, M124T, and R269W. The voltage dependence of the steady-state inactivation curve for WT, M124T, and R269W showed a significant positive shift with increasing temperature; however, that for G584S, WT+D609G, and WT+T613M showed no significant change. Computer simulation demonstrated that G584S, WT+D609G, and WT+T613M caused prolonged action potential durations and early afterdepolarization formation at 40°C. CONCLUSION: These findings indicate that KCNH2 G584S, D609G, and T613M in the S5-pore region reduce the temperature-dependent increase in TCDs through an enhanced inactivation, resulting in QT prolongation and TdP at a febrile state in patients with LQT2.

Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes.

BACKGROUND: Gout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1. METHODS AND RESULTS: Macrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1ß secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1ß. MSU reduced intracellular K+ concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1ß, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1ß and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5. CONCLUSIONS: Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF.

Esm1 and Stc1 as Angiogenic Factors Responsible for Protective Actions of Adipose-Derived Stem Cell Sheets on Chronic Heart Failure After Rat Myocardial Infarction.

BACKGROUND: Although adipose-derived stem cell (ADSC) sheets improve the cardiac function after myocardial infarction (MI), underlying mechanisms remain to be elucidated. The aim of this study was to determine the fate of transplanted ADSC sheets and candidate angiogenic factors released from ADSCs for their cardiac protective actions.Methods and Results:MI was induced by ligation of the left anterior descending coronary artery. Sheets of transgenic (Tg)-ADSCs expressing green fluorescence protein (GFP) and luciferase or wild-type (WT)-ADSCs were transplanted 1 week after MI. Both WT- and Tg-ADSC sheets improved cardiac functions evaluated by echocardiography at 3 and 5 weeks after MI. Histological examination at 5 weeks after MI demonstrated that either sheet suppressed fibrosis and increased vasculogenesis. Luciferase signals from Tg-ADSC sheets were detected at 1 and 2 weeks, but not at 4 weeks, after transplantation. RNA sequencing of PKH (yellow-orange fluorescent dye with long aliphatic tails)-labeled Tg-ADSCs identified mRNAs of 4 molecules related to angiogenesis, including those of Esm1 and Stc1 that increased under hypoxia. Administration of Esm1 or Stc1 promoted tube formation by human umbilical vein endothelial cells. CONCLUSIONS: ADSC sheets improved cardiac contractile functions after MI by suppressing cardiac fibrosis and enhancing neovascularization. Transplanted ADSCs existed for >2 weeks on MI hearts and produced the angiogenic factors Esm1 and Stc1, which may improve cardiac functions after MI.

Anaphylaxis stimulates afferent vagal nerve activity and efferent sympathetic nerve activity in the stomach of anesthetized rats.

Systemic anaphylaxis is a life-threatening and allergic reaction that affects various organs. We previously reported that, in the stomach, gastric vasoconstriction occurring at the late phase (15-55 min after injection of ovalbumin antigen) was observed in anesthetized rats sensitized with ovalbumin. In addition, anaphylaxis enhances gastric motility and delays emptying. However, the role of extrinsic autonomic nervous system on antigen-induced gastric alterations was not known. Thus, using the same rat anaphylaxis model, we aimed to determine the changes in the efferent and afferent autonomic nerve activities in the stomach during anaphylactic hypotension. The findings showed that injection of ovalbumin antigen caused substantial systemic hypotension in all sensitized rats. The efferent gastric sympathetic nerve activity (ef-GSNA), but not the efferent vagal nerve activity, increased only at the early phase (1-10 min after injection of ovalbumin antigen) and showed baroreceptor reflex, as evidenced by a stimulatory response to sodium nitroprusside-induced hypotension. In general, excitation of ef-GSNA could induce pylorus sphincter contraction and gastric vasoconstriction. In the present study, we found that sympathectomy attenuated the anaphylaxis-induced decrease in gastric flux but not the increase in gastric vascular resistance. Thus, the increase in ef-GSNA may cause anaphylactic pylorus sphincter contraction but not anaphylactic gastric vasoconstriction. On the other hand, the afferent gastric vagal nerve activity, but not the afferent sympathetic nerve activity, increased during the early phase of anaphylactic hypotension. However, vagotomy produced no effects on the anaphylactic gastric dysfunction. In conclusion, the gastric sympathetic nerves partly modulate stomach function during systemic anaphylaxis.

ß-Adrenergic Blocker, Carvedilol, Abolishes Ameliorating Actions of Adipose-Derived Stem Cell Sheets on Cardiac Dysfunction and Remodeling After Myocardial Infarction.

BACKGROUND: Treatment of myocardial infarction (MI) includes inhibition of the sympathetic nervous system (SNS). Cell-based therapy using adipose-derived stem cells (ASCs) has emerged as a novel therapeutic approach to treat heart failure in MI. The purpose of this study was to determine whether a combination of ASC transplantation and SNS inhibition synergistically improves cardiac functions after MI.Methods and Results:ASCs were isolated from fat tissues of Lewis rats. In in vitro studies using cultured ASC cells, mRNA levels of angiogenic factors under normoxia or hypoxia, and the effects of norepinephrine and a ß-blocker, carvedilol, on the mRNA levels were determined. Hypoxia increased vascular endothelial growth factor (VEGF) mRNA in ASCs. Norepinephrine further increased VEGF mRNA; this effect was unaffected by carvedilol. VEGF promoted VEGF receptor phosphorylation and tube formation of human umbilical vein endothelial cells, which were inhibited by carvedilol. In in vivo studies using a rat MI model, transplanted ASC sheets improved contractile functions of MI hearts; they also facilitated neovascularization and suppressed fibrosis after MI. These beneficial effects of ASC sheets were abolished by carvedilol. The effects of ASC sheets and carvedilol on MI heart functions were confirmed by Langendorff perfusion experiments using isolated hearts. CONCLUSIONS: ASC sheets prevented cardiac dysfunctions and remodeling after MI in a rat model via VEGF secretion. Inhibition of VEGF effects by carvedilol abolished their beneficial effects.

Uric Acid-Induced Enhancements of Kv1.5 Protein Expression and Channel Activity via the Akt-HSF1-Hsp70 Pathway in HL-1 Atrial Myocytes.

BACKGROUND: Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents. CONCLUSIONS: Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.

Restoration of mutant hERG stability by inhibition of HDAC6.

The human ether-a-go-go-related gene (hERG) encodes the α subunit of a rapidly activating delayed-rectifier potassium (IKr) channel. Mutations of the hERG cause long QT syndrome type 2 (LQT2). Acetylation of lysine residues occurs in a subset of non-histone proteins and this modification is controlled by both histone acetyltransferases and deacetylases (HDACs). The aim of this study was to clarify effects of HDAC(s) on wild-type (WT) and mutant hERG proteins. WThERG and two trafficking-defective mutants (G601S and R752W) were transiently expressed in HEK293 cells, which were treated with a pan-HDAC inhibitor Trichostatin A (TSA) or an isoform-selective HDAC6 inhibitor Tubastatin A (TBA). Both TSA and TBA increased protein levels of WThERG and induced expression of mature forms of the two mutants. Immunoprecipitation showed an interaction between HDAC6 and immature forms of hERG. Coexpression of HDAC6 decreased acetylation and, reciprocally, increased ubiquitination of hERG, resulting in its decreased expression. siRNA against HDAC6, as well as TBA, exerted opposite effects. Immunochemistry revealed that HDAC6 knockdown increased expression of the WThERG and two mutants both in the endoplasmic reticulum and on the cell surface. Electrophysiology showed that HDAC6 knockdown or TBA treatment increased the hERG channel current corresponding to the rapidly activating delayed-rectifier potassium current (IKr) in HEK293 cells stably expressing the WT or mutants. Three lysine residues (K116, K495 and K757) of hERG were predicted to be acetylated. Substitution of these lysine residues with arginine eliminated HDAC6 effects. In HL-1 mouse cardiomyocytes, TBA enhanced endogenous ERG expression, increased IKr, and shortened action potential duration. These results indicate that hERG is a substrate of HDAC6. HDAC6 inhibition induced acetylation of hERG which counteracted ubiquitination leading its stabilization. HDAC6 inhibition may be a novel therapeutic option for LQT2.

Anaphylactic hypotension causes renal and adrenal sympathoexcitaion and induces c-fos in the hypothalamus and medulla oblongata.

NEW FINDINGS: What is the central question of this study? Whether anaphylaxis affects sympathetic outflows to the brown adipose tissue (BAT) and adrenal gland and whether anaphylaxis affects some brain areas in association with sympathetic regulation. What is the main finding and its importance? Sympathoexcitatory responses to anaphylaxis occurred regionally in the kidney and adrenal gland, but not in the thermogenesis-related BAT. Further, anaphylactic hypotension also caused increase in c-fos immunoreactivity in the hypothalamic and medullary areas. Moreover, catecholaminergic neurons of the brainstem cause adrenal sympathoexcitation in a baroreceptor-independent manner. ABSTRACT: We previously reported that sympathetic nerve activity (SNA) to the kidney and the hindlimb increases during anaphylactic hypotension in anaesthetized rats. Based on this evidence, we examined effects of anaphylactic hypotension on SNA to the brown adipose tissue (BAT), and the adrenal gland and kidney in anaesthetized rats. We demonstrated that adrenal and renal SNA, but not BAT-SNA, were stimulated. In addition, the effects of anaphylaxis on neural activities of the hypothalamic and medullary nuclei, which are candidates for relaying efferent SNA to the peripheral organs, were investigated via immunohistochemical staining of c-fos. Anaphylaxis increased c-fos expression in the neurons of the paraventricular nucleus (PVN) of the hypothalamus and in those of the nucleus tractus solitarii (NTS) and rostral ventrolateral medulla (RVLM) of the medulla oblongata; c-fos was expressed in γ-aminobutyric acid (GABA)-ergic neurons of the NTS and in the catecholaminergic neurons of the RVLM. In addition, c-fos expression in the rostral NTS and mid NTS during anaphylaxis was reduced by sinoaortic baroreceptor denervation; however, increased c-fos expression in the caudal NTS and RVLM or adrenal sympathoexcitation were not affected by sinoaortic baroreceptor denervation. These results indicated that anaphylactic hypotension activates the hypothalamic PVN and the medullary NTS and RVLM independently of the baroreflex pathway. Further, it stimulated efferent SNA to the adrenal gland and kidney to restore blood pressure.

Protective Effects of Topiroxostat on an Ischemia-Reperfusion Model of Rat Hearts.

BACKGROUND: Ischemia/reperfusion (I/R) injury triggers cardiac dysfunctions via creating reactive oxygen species (ROS). Because xanthine oxidase (XO) is one of the major enzymes that generate ROS, inhibition of XO is expected to suppress ROS-induced I/R injury. However, it remains unclear whether XO inhibition really yields cardioprotection during I/R. The protective effects of the XO inhibitors, topiroxostat and allopurinol, on cardiac I/R injury were evaluated.Methods and Results:Using isolated rat hearts, ventricular functions, occurrence of arrhythmias, XO activities and thiobarbituric acid reactive substances (TBARS) productions and myocardial levels of adenine nucleotides before and after I/R, and cardiomyocyte death markers during reperfusion, were evaluated. Topiroxostat prevented left ventricular dysfunctions and facilitated recovery from arrhythmias during I/R. Allopurinol and the antioxidant, N-acetylcysteine (NAC), exhibited similar effects at higher concentrations. Topiroxostat inhibited myocardial XO activities and TBARS productions after I/R. I/R decreased myocardial levels of ATP, ADP and AMP, but increased that of xanthine. While topiroxostat, allopurinol or NAC did not change myocardial levels of ATP, ADP or AMP after I/R, all of the agents decreased the level of xanthine. They also decreased releases of CPK and LDH during reperfusion. CONCLUSIONS: Topiroxostat showed protective effects against I/R injury with higher potency than allopurinol or NAC. It dramatically inhibited XO activity and TBARS production, suggesting suppression of ROS generation.

Dynamical mechanisms of phase-2 early afterdepolarizations in human ventricular myocytes: insights from bifurcation analyses of two mathematical models.

Early afterdepolarization (EAD) is known as a cause of ventricular arrhythmias in long QT syndromes. We theoretically investigated how the rapid (IKr) and slow (IKs) components of delayed-rectifier K+ channel currents, L-type Ca2+ channel current (ICaL), Na+/Ca2+ exchanger current (INCX), Na+-K+ pump current (INaK), intracellular Ca2+ (Cai) handling via sarcoplasmic reticulum (SR), and intracellular Na+ concentration (Nai) contribute to initiation, termination, and modulation of phase-2 EADs, using two human ventricular myocyte models. Bifurcation structures of dynamical behaviors in model cells were explored by calculating equilibrium points, limit cycles (LCs), and bifurcation points as functions of parameters. EADs were reproduced by numerical simulations. The results are summarized as follows: 1) decreasing IKs and/or IKr or increasing ICaL led to EAD generation, to which mid-myocardial cell models were especially susceptible; the parameter regions of EADs overlapped the regions of stable LCs. 2) Two types of EADs (termination mechanisms), IKs activation-dependent and ICaL inactivation-dependent EADs, were detected; IKs was not necessarily required for EAD formation. 3) Inhibiting INCX suppressed EADs via facilitating Ca2+-dependent ICaL inactivation. 4) Cai dynamics (SR Ca2+ handling) and Nai strongly affected bifurcations and EAD generation in model cells via modulating ICaL, INCX, and INaK Parameter regions of EADs, often overlapping those of stable LCs, shifted depending on Cai and Nai in stationary and dynamic states. 5) Bradycardia-related induction of EADs was mainly due to decreases in Nai at lower pacing rates. This study demonstrates that bifurcation analysis allows us to understand the dynamical mechanisms of EAD formation more profoundly. NEW & NOTEWORTHY: We investigated mechanisms of phase-2 early afterdepolarization (EAD) by bifurcation analyses of human ventricular myocyte (HVM) models. EAD formation in paced HVMs basically depended on bifurcation phenomena in non-paced HVMs, but was strongly affected by intracellular ion concentrations in stationary and dynamic states. EAD generation did not necessarily require IKs.

Leptin receptor signaling in the hypothalamus regulates hepatic autonomic nerve activity via phosphatidylinositol 3-kinase and AMP-activated protein kinase.

Leptin action in the brain has emerged as an important regulator of liver function independently from its effects on food intake and body weight. The autonomic nervous system plays a key role in the regulation of physiological processes by leptin. Here, we used direct recording of nerve activity from sympathetic or vagal nerves subserving the liver to investigate how brain action of leptin controls hepatic autonomic nerve activity. Intracerebroventricular (ICV) administration of leptin activated hepatic sympathetic traffic in rats and mice in dose- and receptor-dependent manners. The hepatic sympatho-excitatory effects of leptin were also observed when leptin was microinjected directly into the arcuate nucleus (ARC), but not into the ventromedial hypothalamus (VMH). Moreover, using pharmacological and genetic approaches, we show that leptin-induced increase in hepatic sympathetic outflow depends on PI3K but not AMP-activated protein kinase (AMPK), STAT3, or ERK1/2. Interestingly, ICV leptin also increased hepatic vagal nerve activity in rats. We show that this response is reproduced by intra-ARC, but not intra-VMH, leptin administration and requires PI3K and AMPK. We conclude that central leptin signaling conveys the information to the liver through the sympathetic and parasympathetic branches of the autonomic nervous system. Our data also provide important insight into the molecular events underlying leptin's control of hepatic autonomic nerve activity by implicating PI3K and AMPK pathways.

M3 Muscarinic Receptor Signaling Stabilizes a Novel Mutant Human Ether-a-Go-Go-Related Gene Channel Protein via Phosphorylation of Heat Shock Factor 1 in Transfected Cells.

BACKGROUND: Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS: M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

The Responses of Pulmonary and Systemic Circulation and Airway to Allergic Mediators in Anesthetized Rats.

Lung allergic diseases sometimes accompany pulmonary vaso- and broncho-constriction. Rats are currently used for the experimental study of lung allergies. However, their hemodynamic mechanisms are not fully understood. Therefore the effects of allergic mediators were determined systematically in vivo in rats in terms of pulmonary vascular resistance (PVR), airway pressure (AWP) and total peripheral resistance (TPR). We directly measured pulmonary arterial pressure, left atrial pressure, systemic arterial pressure, central venous pressure and aortic blood flow to determine PVR and TPR, as well as AWP, following injections of platelet-activating factor (PAF), histamine, serotonin, leukotriene (LT) C4, and prostaglandin (PG) D2 in anesthetized open-chest artificially ventilated Sprague-Dawley (SD) rats. PVR was dose-dependently increased by consecutive administration of PAF, LTC4, and PGD2, with the maximal responsiveness being PAF>LTC4>PGD2. However, neither histamine nor serotonin changed PVR. TPR was decreased by all agents except LTC4 which actually increased it. PAF and serotonin, but not the other agents, increased AWP. In conclusion, allergic mediators exert non-uniform actions on pulmonary and systemic circulation and airways in anesthetized SD rats: PAF, LTC4 and PGD2, but not histamine or serotonin, caused substantial pulmonary vasoconstriction; LTC4 yielded systemic vasoconstriction, while the others caused systemic vasodilatation; only two mediators, PAF and serotonin, induce airway constriction.

E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells.

Kv1.5 confers ultra-rapid delayed-rectifier potassium channel current (IKur) which contributes to repolarization of the atrial action potential. Kv1.5 proteins, degraded via the ubiquitin-proteasome pathway, decreased in some atrial fibrillation patients. Carboxyl-terminus heat shock cognate 70-interacting protein (CHIP), an E3 ubiquitin ligase, is known to ubiquitinate short-lived proteins. Here, we investigated the roles of CHIP in Kv1.5 degradation to provide insights into the mechanisms of Kv1.5 decreases and treatments targeting Kv1.5 for atrial fibrillation. Coexpression of CHIP with Kv1.5 in HEK293 cells increased Kv1.5 protein ubiquitination and decreased the protein level. Immunofluorescence revealed decreases of Kv1.5 proteins in the endoplasmic reticulum and on the cell membrane. A siRNA against CHIP suppressed Kv1.5 protein ubiquitination and increased its protein level. CHIP mutants, lacking either the N-terminal tetratricopeptide region domain or the C-terminal U-box domain, failed to exert these effects on Kv1.5 proteins. Immunoprecipitation showed that CHIP formed complexes with Kv1.5 proteins and heat shock cognate protein 70 (Hsc70). Effects of Hsc70 on Kv1.5 were similar to CHIP by altering interaction of CHIP with Kv1.5 protein. Coexpression of CHIP and Hsc70 with Kv1.5 additionally enhanced Kv1.5 ubiquitination. Kv1.5 currents were decreased by overexpression of CHIP or Hsc70 but were increased by knockdown of CHIP or Hsc70 in HEK 293 cells stably expressing Kv1.5. These effects of CHIP and Hsc70 were also observed on endogenous Kv1.5 in HL-1 mouse cardiomyocytes, decreasing IKur and prolonging action potential duration. These results indicate that CHIP decreases the Kv1.5 protein level and functional channel by facilitating its degradation in concert with chaperone Hsc70.

Pulmonary vascular and airway responses to systemic vasoconstrictors in anesthetized BALB/c mice.

There is no systematic study in which the effects of vasoactive substances were investigated on pulmonary vascular resistance (PVR) in in vivo mouse by directly measuring cardiac output and the inflow and outflow pressures in the pulmonary circulation. We determined the responses of PVR, total peripheral resistance (TPR), and airway pressure (AWP) to angiotensin II, endothelin-1, vasopressin, phenylephrine, and thromboxane A2 analog U46619 in anesthetized BALB/c mice. Pulmonary arterial pressure, left atrial pressure (LAP), and aortic blood flow were measured. TPR increased dose-dependently in response to consecutive administration of all vasoconstrictors except vasopressin which reduced TPR at the highest dose of 100 nmol/kg. At high doses of vasoconstrictors, pulmonary arterial pressure and AWP increased due to increased LAP, as demonstrated by the separate LAP elevation experiments. When LAP transiently increased at high doses, PVR did not increase but decreased. Nonetheless, enodothelin-1, angiotensin II, and U46619 increased PVR. Vasopressin at 100 nmol/kg increased AWP without LAP elevation. In conclusion, the high doses of the vasoconstrictors studied here exert indirectly a transient pulmonary vasodilatory and AWP increasing actions due to pulmonary congestion evoked by strong systemic vasoconstriction. Nevertheless, enodothelin-1, angiotensin II, and U46619 cause pulmonary vasoconstriction, and vasopressin constricts airway in anesthetized BALB/c mice.

Molecular Mechanisms Underlying Urate-Induced Enhancement of Kv1.5 Channel Expression in HL-1 Atrial Myocytes.

BACKGROUND: Hyperuricemia induces endothelial dysfunction, oxidative stress and inflammation, increasing cardiovascular morbidities. It also raises the incidence of atrial fibrillation; however, underlying mechanisms are unknown. METHODS AND RESULTS: The effects of urate on expression of Kv1.5 in cultured mouse atrial myocytes (HL-1 cells) using reverse transcriptase-PCR, immunoblots, flow cytometry and patch-clamp experiments were studied. Treatment with urate at 7 mg/dl for 24 h increased the Kv1.5 protein level, enhanced ultra-rapid delayed-rectifier K(+)channel currents and shortened action potential duration in HL-1 cells. HL-1 cells expressed the influx uric acid transporter (UAT), URATv1, and the efflux UATs, ABCG2 and MRP4. An inhibitor against URATv1, benzbromarone, abolished the urate effects, whereas an inhibitor against ABCG2, KO143, augmented them. Flow cytometry showed that urate induced an increase in reactive oxygen species, which was abolished by the antioxidant, N-acetylcysteine (NAC), and the NADPH-oxidase inhibitor, apocynin. Both NAC and apocynin abolished the enhancing effects of urate on Kv1.5 expression. A urate-induced increase in the Kv1.5 proteins was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and was abolished by an ERK inhibitor, PD98059. NAC abolished phosphorylation of ERK by urate. CONCLUSIONS: Intracellular urate taken up by UATs enhanced Kv1.5 protein expression and function in HL-1 atrial myocytes, which could be attributable to ERK phosphorylation and oxidative stress derived from nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.

Systemic vasoconstriction modulates the responses of pulmonary vasculature and airway to vasoconstrictors in anesthetized rats.

PURPOSE: The physiological responses of the pulmonary vasculature and airway to various vasoconstrictors were studied using isolated perfused lungs and pulmonary arteries, but these responses were not systematically studied in in vivo rats. We determined these responses and modulating effects of systemic circulation in anesthetized rats. METHODS: We measured directly pulmonary arterial pressure (PAP), left atrial pressure (LAP), aortic blood flow, and airway pressure (AWP) to determine pulmonary vascular resistance (PVR), following injections of angiotensin II (ANG II), endothelin-1 (ET-1), vasopressin, phenylephrine and thromboxane A2 mimetic U46619 in anesthetized SD rats. RESULTS: ANG II, phenylephrine and vasopressin at high doses caused strong systemic vasoconstriction and left heart overload, resulting in a transient increase in LAP and pulmonary congestion, which consequently decreased PVR. Nonetheless, prior to LAP elevation, PVR was slightly but significantly increased by ANG II and phenylephrine. In contrast, ET-1 and U46619 substantially increased PVR in the absence of LAP elevation, while vasopressin did not increase PVR. In separate experiments, PAP and AWP increased when LAP was forcedly elevated. AWP was increased by U46619 through bronchoconstriction and by the other agents through increased LAP-induced pulmonary congestion. CONCLUSION: Airway constriction is induced by U46619, and pulmonary vasoconstriction is induced strongly by U46619 and ET-1, and weakly by ANG II and phenylephrine, but not by vasopressin in anesthetized rats. ANG II, vasopressin and phenylephrine exert indirectly a transient pulmonary vasodilatory action due to pulmonary congestion evoked by strong systemic vasoconstriction, which may account for weak pulmonary pressor responses to these agents.