Absence Of Isoproterenol Research Articles

Beta-Adrenergic Activation of the Inward Rectifier K+ Current Is Mediated by the CaMKII Pathway in Canine Ventricular Cardiomyocytes.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K+ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (IK1) and the rapid delayed rectifier (IKr) K+ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. IK1 and IKr were dissected by 50 µM BaCl2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased IK1 under the plateau phase of the action potential (0-+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall IK1 integral. The ISO-induced enhancement of IK1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the IK1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the IKr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances IK1 in canine left ventricular cells through the CaMKII pathway, while IKr remains unaffected under the experimental conditions used.

Knockdown of SCN5A alters metabolic-associated genes and aggravates hypertrophy in the cardiomyoblast.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and βMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

Mathematical model of the ventricular action potential and effects of isoproterenol-induced cardiac hypertrophy in rats

Mathematical action potential (AP) modeling is a well-established but still-developing area of research to better understand physiological and pathological processes. In particular, changes in AP mechanisms in the isoproterenol (ISO) -induced hypertrophic heart model are incompletely understood. Here we present a mathematical model of the rat AP based on recordings from rat ventricular myocytes. In our model, for the first time, all channel kinetics are defined with a single type of function that is simple and easy to apply. The model AP and channels dynamics are consistent with the APs recorded from rats for both Control (absence of ISO) and ISO-treated cases. Our mathematical model helps us to understand the reason for the prolongation in AP duration after ISO application while ISO treatment helps us to validate our mathematical model. We reveal that the smaller density and the slower gating kinetics of the transient K+ current help explain the prolonged AP duration after ISO treatment and the increasing amplitude of the rapid and the slow inward rectifier currents also contribute to this prolongation alongside the flux in Ca2+ currents. ISO induced an increase in the density of the Na+ current that can explain the faster upstroke. We believe that AP dynamics from rat ventricular myocytes can be reproduced very well with this mathematical model and that it provides a powerful tool for improved insights into the underlying dynamics of clinically important AP properties such as ISO application.

Simvastatin nanoliposome induces myocardial and hepatic toxicities due to its absorption enhancement in mice

Nanoliposome is a useful dosage form to increase solubility and absorption of simvastatin (SMV), and consequently improves its therapeutic effects. However, in vivo toxicity of SMV could also be elevated accompanied by the absorption enhancement, which is a decisive factor for the clinical application of SMV nanoliposome (SMV-Lipo), but has not been studied systematically and reported so far. In this study, organ toxicity of SMV-Lipo was evaluated in mice in the presence and absence of isoproterenol and compared to those of free SMV. Results demonstrated that compared to free SMV, the SMV-Lipo administrated at an equal dose of 25 mg/kg/d led to severe myocardiotoxicity, hepatotoxicity at baseline and more pronounced liver injury with elevation of alanine aminotransferase. In addition, muscular adverse effect was also observed in SMV-Lipo treated group but not in SMV group. Pharmacokinetic studies revealed that compared to free SMV, the SMV-Lipo administration significantly improved the plasma SMV concentration, and the oral bioavailability was 6.5 times of free SMV. Notably, when the dosage of free SMV increased to 50 mg/kg/d, yielding the comparable plasma concentration as SMV-Lipo given at 25 mg/kg/d, the myocardiotoxicity was observed in free SMV treated mice as well, which further confirmed that the enhanced absorption of SMV by the nanoliposomal formulation resulted in more severe myocardiotoxicity than the equal dose of free SMV.

Cardiac-specific deficiency of the mitochondrial calcium uniporter augments fatty acid oxidation and functional reserve

The mitochondrial calcium uniporter (MCU) relays cytosolic Ca2+ transients to the mitochondria. We examined whether energy metabolism was compromised in hearts from mice with a cardiac-specific deficiency of MCU subjected to an isoproterenol (ISO) challenge. Surprisingly, isolated working hearts from cardiac MCU-deficient mice showed higher cardiac work, both in the presence or absence of ISO. These hearts were not energy-starved, with ISO inducing a similar increase in glucose oxidation rates compared to control hearts, but a greater increase in fatty acid oxidation rates. This correlated with lower levels of the fatty acid oxidation inhibitor malonyl CoA, and to an increased stimulatory acetylation of its degrading enzyme malonyl CoA decarboxylase and of the fatty acid β-oxidation enzyme β-hydroxyacyl CoA dehydrogenase. We conclude that impaired mitochondrial Ca2+ uptake does not compromise cardiac energetics due to a compensatory stimulation of fatty acid oxidation that provides a higher energy reserve during acute adrenergic stress.

\u03b2-Adrenergic signaling blocks murine CD8+ T-cell metabolic reprogramming during activation: a mechanism for immunosuppression by adrenergic stress

Primary and secondary lymphoid organs are heavily innervated by the autonomic nervous system. Norepinephrine, the primary neurotransmitter secreted by post-ganglionic sympathetic neurons, binds to and activates β-adrenergic receptors expressed on the surface of immune cells and regulates the functions of these cells. While it is known that both activated and memory CD8+ T-cells primarily express the β2-adrenergic receptor (β2-AR) and that signaling through this receptor can inhibit CD8+ T-cell effector function, the mechanism(s) underlying this suppression is not understood. Under normal activation conditions, T-cells increase glucose uptake and undergo metabolic reprogramming. In this study, we show that treatment of murine CD8+ T-cells with the pan β-AR agonist isoproterenol (ISO) was associated with a reduced expression of glucose transporter 1 following activation, as well as decreased glucose uptake and glycolysis compared to CD8+ T-cells activated in the absence of ISO. The effect of ISO was specifically dependent upon β2-AR, since it was not seen in adrb2−/− CD8+ T-cells and was blocked by the β-AR antagonist propranolol. In addition, we found that mitochondrial function in CD8+ T-cells was also impaired by β2-AR signaling. This study demonstrates that one mechanism by which β2-AR signaling can inhibit CD8+ T-cell activation is by suppressing the required metabolic reprogramming events which accompany activation of these immune cells and thus reveals a new mechanism by which adrenergic stress can suppress the effector activity of immune cells.

Early life stress reduces in vivo lipolysis efficiency in female mice fed a high fat diet

Previously, we have shown that female mice exposed to maternal separation and early weaning (MSEW), a model of postnatal neglect, increases fat mass and worsens the cardiometabolic function in response to high fat diet (HF) feeding. We also found that β3 adrenergic receptor mRNA expression, involved in local lipolysis stimulation, was reduced in gonadal white adipose tissue (gWAT). Thus, the aim of this study was to test the hypothesis that female MSEW mice display reduced lipolysis in‐vivo and in‐vitro. MSEW was performed by separating the pups from the mother for periods of 4–8 hr during postnatal days 2–16. Mice were weaned at P17. Control mice remained undisturbed in the home cage all times and weaned at P21. At weaning, mice were placed on a low fat (10% kcal from fat) or high fat (60% kcal from fat) diet for 16 weeks. At week 14, an in‐vivo lipolysis test was performed by injecting with 1mg/kg BW of isoproterenol, IP. Free fatty acids (FFA) were measured in plasma obtained after 15 minutes of the injection. At week 16, an in‐vitro lipolysis test was performed in gWAT. As such, gWAT was removed from female control and MSEW mice, weighed, and washed with ice‐cold PBS. In‐vitro lipolysis was performed in the presence or absence of isoproterenol (10uM, Sigma Aldrich). Free glycerol kit (Sigma Aldrich) was used to measure lipolytic products and results were normalized to tissue weight. In‐vivo, FFAs were reduced in female MSEW mice fed a HF (102±13 uM/ml) when compared with controls (144±24 uM/ml, p=0.066). In‐vitro, we found similar free glycerol levels in control and MSEW mice fed a LF (9.2±0.8 vs. 10.5±1.4 mg/dl/g tissue, respectively). Although HF reduced isoproterenol‐induced free glycerol levels, control and MSEW mice showed not statistically significant differences (4.4±0.6 vs. 3.5±0.6 mg/dl/g tissue, respectively). Taken together, these data indicate that HF reduced lipolysis capacity either in control and MSEW mice. Also, MSEW reduced lipolysis when determined in‐vivo conditions, suggesting that neuroendocrine and autonomic factors may be implicated in the mechanism by which MSEW mice are prone to increase fat mass.Support or Funding InformationThis study was supported by funds from the NIH National Heart, Lung, and Blood Institute to A.S.L (R00 HL111354), start‐up funds from the University of Kentucky to A.S.L, and the pilot project from the University of Kentucky Center of Research in Obesity and Cardiovascular Disease COBRE P20 GM103527‐06 to A.S.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Modulation of brown adipocyte activity by milk by-products: Stimulation of brown adipogenesis by buttermilk.

Brown adipocytes dissipate chemical energy in the form of heat through the expression of mitochondrial uncoupling protein 1 (Ucp1); Ucp1 expression is further upregulated by the stimulation of β-adrenergic receptors in brown adipocytes. An increase in energy expenditure by activated brown adipocytes potentially contributes to the prevention of or therapeutics for obesity. The present study examined the effects of milk by-products, buttermilk and butter oil, on brown adipogenesis and the function of brown adipocytes. The treatment with buttermilk modulated brown adipogenesis, depending on the product tested; during brown adipogenesis, buttermilk 1 inhibited the differentiation of HB2 brown preadipocytes. In contrast, buttermilk 3 and 5 increased the expression of Ucp1 in the absence of isoproterenol (Iso), a β-adrenergic receptor agonist, suggesting the stimulation of brown adipogenesis. In addition, the Iso-induced expression of Ucp1 was enhanced by buttermilk 2 and 3. The treatment with buttermilk did not affect the basal or induced expression of Ucp1 by Iso in HB2 brown adipocytes, except for buttermilk 5, which increased the basal expression of Ucp1. Conversely, butter oil did not significantly affect the expression of Ucp1, irrespective of the cell phase of HB2 cells, ie, treatment during brown adipogenesis or of brown adipocytes. The results of the present study indicate that buttermilk is a regulator of brown adipogenesis and suggest its usefulness as a potential food material for antiobesity.

Acetylated and propionated derivatives of swertiamarin have anti-adipogenic effects.

Objective:To investigate whether the acetylated and propionated derivatives (LMP-09-1 and -2) of swertiamarin have anti-adipogenic effects.Materials and Methods:3T3-L1 pre-adipocytes were grown in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% calf serum; fully confluent cells were differentiated with insulin, dexamethasone, and 3-isobutylmethylxanthine in the presence and absence of LMP-09-1 and -2 (100 μg/mL) for 10 days. Control cells received same amount of dimethylsulfoxide (DMSO). On day ten, cells were analyzed for triglycerides accumulation and the expression of genes involved in adipogenesis, lipogenesis, and lipolysis. In another set of experiment, effects of LMP-09-1 and 2 were studied for isoproterenol induced lipolysis using fully mature adipocytes.Results:LMP-09-1 and -2 caused a significant (P < 0.001) reduction in intracellular triglycerides accumulation. Both LMP-09-1 and -2 significantly (P < 0.001) decreased the mRNA expression of peroxisome proliferator activated receptor-γ and acetyl-CoA carboxylase-1, and increased isoproterenol induced lipolysis in adipocytes. LMP-09-1 induced lipolysis even in the absence of isoproterenol, and also showed a significant up-regulation of carnitine palmitoyl transferase-1α and hormone sensitive lipase (HSL) gene expression.Conclusions:These findings show that swertiamarin derivatives, LMP-09-1 and -2 have a potent anti-adipogenic effect.

Abstract 201: Beta-Adrenergic Stimulation Promotes Early Electrical Failure During Simulated VF-Induced Cardiac Arrest And Delays Electrical Recovery Upon Reperfusion

During cardiac arrest adrenergic stimulation is enhanced due to both endogenous increase in myocardial tissue catecholamine levels and exogenous epinephrine administration during resuscitation. Whereas alpha-adrenergic stimulation may be beneficial for hemodynamic recovery, there is growing evidence that beta-adrenergic stimulation is detrimental. Clinically, there is controversy as to whether epinephrine administration improves overall survival in cardiac arrest. Here we investigated the effects of beta-adrenergic stimulation on electrical activity during simulated ventricular fibrillation (VF) [[Unable to Display Character: –]] induced cardiac arrest and subsequent reperfusion. We performed optical mapping of the voltage-sensitive dye RH237 in the anterior right and left ventricles (RV and LV) of Langendorff-perfused rabbit hearts subjected to normothermic global no-flow ischemia and rapid pacing (cycle length 200 ms) in the presence or in the absence of isoproterenol (ISO, 30 nM). 2,3-butanedione monoxime (20 mM) was used to reduce motion artifact. Excitable area was measured in optical maps based on the criterion of the minimum time-derivative of the upstroke of the optical action potential. ISO markedly accelerated electrical depression in both LV and RV (more in LV) compared to control hearts (see Figure, * indicate comparisons with significant difference between groups at p&lt;0.05). In addition, ISO delayed recovery of electrical activity upon reperfusion (62±14% recovery in ISO vs.98±2% recovery in control at 5 min of reperfusion, p&lt;0.01). Furthermore, ISO tended to increase the incidence of post-reperfusion arrhythmias compared to control (83% vs. 25%, respectively; p=NS). We conclude that beta-adrenergic stimulation aggravates electrical dysfunction under conditions typically present during cardiac arrest/resuscitation, which might contribute to the overall detrimental outcomes of natural or induced catecholamine increase in this setting.

Induction of Beige-Like Adipocytes in 3T3-L1 Cells

ABSTRACTThere are two types of brown adipocytes: classical brown adipocytes that form the brown fat depots and beige adipocytes that emerge in the white fat depots. Beige adipocytes have a low level of uncoupling protein 1 (Ucp1) expression in the basal state, but Ucp1 expression is increased in response to β adrenergic receptor activation. The present study explored the factors responsible for the differentiation of 3T3-L1 white preadipocytes to beige adipocytes. Significant expression of Ucp1 was not detected under any tested conditions in the absence of isoproterenol (Iso), an agonist of β adrenergic receptor. Iso-induced Ucp1 expression was significantly higher in the cells treated with a mixture of triiodothyronine (T3) and 3-isobutyl-1-methylxanthine (IBMX) for days 0–8 than in the control cells. Chronic IBMX treatment was indispensable for the enhanced Iso-induced Ucp1 expression, and treatment with additional rosiglitazone (Rosi) for days 0–8 further increased the Ucp1 expression. Recently, genes were identified that are predominantly expressed in beige adipocytes, which were induced from stromal vascular cells in white fat depots. However, the expression levels of the beige adipocyte-selective genes in the adipocytes induced by the mixture of T3, IBMX and Rosi did not differ from those in the control adipocytes. The present study indicates that 3T3-L1 cells can differentiate to beige-like adipocytes by prolonged treatment with the mixture of T3, IBMX and Rosi and that the gene expression profile of the adipocytes is distinct from those previously induced from white fat depots.

Enhanced Cardiac Function in Gravin Mutant Mice Involves Alterations in the β-Adrenergic Receptor Signaling Cascade

Gravin, an A-kinase anchoring protein, targets protein kinase A (PKA), protein kinase C (PKC), calcineurin and other signaling molecules to the beta2-adrenergic receptor (β2-AR). Gravin mediates desensitization/resensitization of the receptor by facilitating its phosphorylation by PKA and PKC. The role of gravin in β-AR mediated regulation of cardiac function is unclear. The purpose of this study was to determine the effect of acute β-AR stimulation on cardiac contractility in mice lacking functional gravin. Using echocardiographic analysis, we observed that contractility parameters such as left ventricular fractional shortening and ejection fraction were increased in gravin mutant (gravin-t/t) animals lacking functional protein compared to wild-type (WT) animals both at baseline and following acute isoproterenol (ISO) administration. In isolated gravin-t/t cardiomyocytes, we observed increased cell shortening fraction and decreased intracellular Ca2+ in response to 1 µmol/L ISO stimulation. These physiological responses occurred in the presence of decreased β2-AR phosphorylation in gravin-t/t hearts, where PKA-dependent β2-AR phosphorylation has been shown to lead to receptor desensitization. cAMP production, PKA activity and phosphorylation of phospholamban and troponin I was comparable in WT and gravin-t/t hearts both with and without ISO stimulation. However, cardiac myosin binding protein C (cMyBPC) phosphorylation site at position 273 was significantly increased in gravin-t/t versus WT hearts, in the absence of ISO. Additionally, the cardioprotective heat shock protein 20 (Hsp20) was significantly more phosphorylated in gravin-t/t versus WT hearts, in response to ISO. Our results suggest that disruption of gravin’s scaffold mediated signaling is able to increase baseline cardiac function as well as to augment contractility in response to acute β-AR stimulation by decreasing β2-AR phosphorylation and thus attenuating receptor desensitization and perhaps by altering PKA localization to increase the phosphorylation of cMyBPC and the nonclassical PKA substrate Hsp20.

Activation of Glibenclamide-Sensitive ATP-Sensitive K + Channels During β-Adrenergically Induced Metabolic Stress Produces a Substrate for Atrial Tachyarrhythmia

Cardiac ATP-sensitive K(+) channels have been suggested to contribute to the adaptive physiological response to metabolic challenge after β-adrenoceptor stimulation. However, an increased atrial K(+)-conductance might be expected to be proarrhythmic. We investigated the effect of ATP-sensitive K(+) channel blockade on the electrophysiological responses to β-adrenoceptor-induced metabolic challenge in intact atria. Atrial electrograms were recorded from the left atrial epicardial surface of Langendorff-perfused rat hearts using a 5×5 electrode array. Atrial effective refractory period and conduction velocity were measured using an S(1)-S(2) protocol. The proportion of hearts in which atrial tachyarrhythmia was produced by burst-pacing was used as an index of atrial tachyarrhythmia-inducibility. Atrial nucleotide concentrations were measured by high performance liquid chromatography. Perfusion with ≥10(-9) mol/L of the β-adrenoceptor agonist, isoproterenol (ISO), resulted in a concentration-dependent reduction of atrial effective refractory period and conduction velocity. The ISO-induced changes produced a proarrhythmic substrate such that atrial tachyarrhythmia could be induced by burst-pacing. Atrial [ATP] was significantly reduced by ISO (10(-6) mol/L). Perfusion with either of the ATP-sensitive K(+) channel blockers, glibenclamide (10(-5) mol/L) or tolbutamide (10(-3) mol/L), in the absence of ISO had no effect on basal atrial electrophysiology. On the other hand, the proarrhythmic substrate induced by 10(-6) mol/L ISO was abolished by either of the sulfonylureas, which prevented induction of atrial tachyarrhythmia. Atrial ATP-sensitive K(+) channels activate in response to β-adrenergic metabolic stress in Langendorff-perfused rat hearts, resulting in a proarrhythmic substrate.

Proinsulin C-Peptide Inhibits Lipolysis in Diabetic Rat Adipose Tissue Through Phosphodiestrase-3B Enzyme

We have previously reported that C-peptide modulates insulin-mediated inhibition of lipolysis and glucose consumption but has no significant effects per se on adipose tissue of normal rats. It has been repeatedly observed that certain actions of C-peptide are restricted to the diabetic states. In the present study, therefore, we examined whether C-peptide alters lipolysis in adipose tissue of diabetic rats. Rats were rendered diabetic by streptozotocin and divided into 2 groups; insulin treated and untreated. Retroperitoneal adipose tissue was excised aseptically, subjected to organ culture and incubated with rat C-peptide, insulin, or a combination of both peptides in the presence or absence of isoproterenol. Tissue lipolysis was assessed by the rate of glycerol release into the culture media. The cultures were pretreated with cilostamide, a phosphodiesterase-3B enzyme inhibitor, when the role of this enzyme was to be examined. C-Peptide on its own, like insulin, significantly inhibited isoproterenol-stimulated lipolysis in the adipose tissue of untreated diabetic rats. The effect was enhanced by a combination of C-peptide and insulin. Notably, the C-peptide's effect was totally blocked in the presence of cilostamide. In the adipose tissue of insulin treated rats, however, C-peptide failed to show any significant antilipolytic effects. These data show that C-peptide has the potential to act, conditionally, as an antilipolytic hormone by activating phosphodiesterase-3B and suggest that the action may contribute to the C-peptide's beneficial effects on diabetes-induced complications.

Interaction between Ca2+ channel blockers and isoproterenol on L‐type Ca2+ current in canine ventricular cardiomyocytes

The aim of this work was to study antagonistic interactions between the effects of various types of Ca(2+) channel blockers and isoproterenol on the amplitude of L-type Ca(2+) current in canine ventricular cells. Whole-cell version of the patch clamp technique was used to study the effect of isoproterenol on Ca(2+) current in the absence and presence of Ca(2+) channel-blocking agents, including nifedipine, nisoldipine, diltiazem, verapamil, CoCl(2) and MnCl(2) . Five micromolar Nifedipine, 1μM nisoldipine, 10μM diltiazem, 5μM verapamil, 3mM CoCl(2) and 5mM MnCl(2) evoked uniformly a 90-95% blockade of Ca(2+) current in the absence of isoproterenol. Isoproterenol (100nM) alone increased the amplitude of Ca(2+) current from 6.8±1.3 to 23.7±2.2 pA/pF in a reversible manner. Isoproterenol caused a marked enhancement of Ca(2+) current even in the presence of nifedipine, nisoldipine, diltiazem and verapamil, but not in the presence of CoCl(2) or MnCl(2) . The results indicate that the action of isoproterenol is different in the presence of organic and inorganic Ca(2+) channel blockers. CoCl(2) and MnCl(2) were able to fully prevent the effect of isoproterenol on Ca(2+) current, while the organic Ca(2+) channel blockers failed to do so. This has to be born in mind when the effects of organic Ca(2+) channel blockers are evaluated either experimentally or clinically under conditions of increased sympathetic tone.

The disruption of the intracellular Ca2+ homeostasis is associated with heart disfunction in chronically malnourished Wistar rats

It is known the association between malnutrition and hypertension in the adult life. The aim of this study was to determine the consequences of the brazilian regional basic diet (RBD) submitted chronically to Wistar rats in the function and in the intracellular Ca2+ homeostasis of the heart. After weaning, the rats were fed with RBD and conventional diet for 10 weeks (n=10 each). The rats were subjected to echocardiography. A cohort of 5 hearts was isolated, perfused with Krebs buffer in the presence or absence of isoproterenol (ISP, from 0.3 to 1000 nM) by the Langendorff method. The microssomal fractions of the ventricles were obtained to measure the Ca2+‐ATPase activities. Ecocardiographic analysis showed a decrease in the cardiac output (53%), ejection fraction (71%), stroke volume (48%), and left diastolic ventricular diameter (22%) in the RBD. The left ventricular developed pressure was decreased in the RBD (34%). Furthermore, the contractility (+LVdP/dt) and relaxation (−LVdP/dt) in the presence of ISP are disrupted. The lung weight/body weight ratio were increased in 75%. We observed that the total Ca2+‐ATPase activity was two times higher in the RBD; due to a decrease in SERCA and an increase in the PMCA activities. Our data indicate that chronic malnutrition unbalances the SERCA and PMCA activities that could affect the heart hemodynamic.

Regional increase in extracellular potassium can be arrhythmogenic due to nonuniform muscle contraction in rat ventricular muscle

In the ischemic myocardium, extracellular potassium ([K(+)](o)) increases to ≥20 mmol/l. To determine how lethal arrhythmias occur during ischemia, we investigated whether the increased spatial pattern of [K(+)](o), i.e., a regional or a global increase, affects the incidence of arrhythmias. Force, sarcomere length, membrane potential, and nonuniform intracellular Ca(2+) ([Ca(2+)](i)) were measured in rat ventricular trabeculae. A "regional" or "global" increase in [K(+)](o) was produced by exposing a restricted region of muscle to a jet of 30 mmol/l KCl or by superfusing trabeculae with a solution containing 30 mmol/l KCl, respectively. The increase in [Ca(2+)](i) (Ca(CW)) during Ca(2+) waves was measured (24°C, 3.0 mmol/l [Ca(2+)](o)). A regional increase in [K(+)](o) caused nonuniform [Ca(2+)](i) and contraction. In the presence of isoproterenol, the regional increase in [K(+)](o) induced sustained arrhythmias in 10 of 14 trabeculae, whereas the global increase did not induce such arrhythmias. During sustained arrhythmias, Ca(2+) surged within the jet-exposed region. In the absence of isoproterenol, the regional increase in [K(+)](o) increased Ca(CW), whereas the global increase decreased it. This increase in Ca(CW) with the regional increase in [K(+)](o) was not suppressed by 100 μmol/l streptomycin, whereas it was suppressed by 1) a combination of 10 μmol/l cilnidipine and 3 μmol/l SEA0400; 2) 20 mmol/l 2,3-butanedione monoxime; and 3) 10 μmol/l blebbistatin. A regional but not a global increase in [K(+)](o) induces sustained arrhythmias, probably due to nonuniform excitation-contraction coupling. The same mechanism may underlie arrhythmias during ischemia.

Inhibition of Protein Phosphatase 2A Activity by PI3Kγ Regulates β-Adrenergic Receptor Function

Phosphoinositide 3-kinase γ (PI3Kγ) is activated by Gprotein-coupled receptors (GPCRs). We show here that PI3Kγ inhibits protein phosphatase 2A (PP2A) at the β-adrenergic receptor (βAR, a GPCR) complex altering G protein coupling. PI3Kγ inhibition results in significant increase of βAR-associated phosphatase activity leading to receptor dephosphorylation and resensitization preserving cardiac function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A) on serine residues 9 and 93, resulting in enhanced binding to PP2A. Indeed, enhanced phosphorylation of β2ARs is observed with a phosphomimetic I2PP2A mutant that was completely reversed with a mutant mimicking dephosphorylated state. siRNA depletion of endogenous I2PP2A augments PP2A activity despite active PI3K resulting in β2AR dephosphorylation and sustained signaling. Our study provides the underpinnings of a PI3Kγ-mediated regulation of PP2A activity that has significant consequences on receptor function with broad implications in cellular signaling.

Effects of trans-10, cis-12 conjugated linoleic acid on gene expression and lipid metabolism of adipose tissue of growing pigs

The objective of the present study was to determine the effects of trans-10, cis-12 conjugated linoleic acid (CLA) in adipose tissue explant cultures of growing pigs on the following responses: lipogenesis (measured as rate of (14)C-labeled glucose incorporation over a subsequent 2-h incubation in the presence or absence of insulin), lipolysis (release of non-esterified fatty acid over a 2-h incubation in the presence or absence of isoproterenol), activities of lipogenic enzymes, and mRNA abundance of fatty acid synthase (FAS). Adipose tissue explants from nine growing pigs (78 +/- 3 kg) were cultured in 199 medium with insulin, dexamethasone and antibiotics for 4, 12, 24, and 48 h. The treatments were 1) control: 100 microM polyvinyl alcohol (PVA); 2) pGH: 100 ng/mL porcine growth hormone (pGH) plus 100 microM PVA; 3) CLA200: 200 microM trans-10, cis-12 CLA; 4) CLA50: 50 microM trans-10, cis-12 CLA, and 5) LA: 200 microM linoleic acid. Fatty acids were added along with PVA (2:1), respectively, for 24 h. Explants were collected after each culture period and assayed for lipogenesis. Transcripts of FAS mRNA were quantified by real-time RT-PCR after 24 and 48 h. Lipolysis and activities of FAS, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and NADP-malate dehydrogenase were determined after 48 h. As expected, glucose incorporation was decreased (P < 0.05) in response to pGH treatment (positive control). LA had no effect on any parameter evaluated. Treatment with trans-10, cis-12 CLA decreased FAS activity (P < 0.05), but NADPH-generating enzymes were unaffected by treatments. Consistent with reduction in FAS activity, both lipid synthesis and FAS mRNA abundance were reduced with chronic CLA treatment, pGH increased baseline and stimulated lipolysis (P < 0.05) after 48 h of culture, while CLA treatment had no effect on non-esterified fatty acid release. Results of this study showed that trans-10, cis-12 CLA alters lipogenesis but has no effect on lipolysis in cultures of pig adipose tissue.

Mutation of Important Amino Acid Residue of Asp104Lys in Human .BETA.1-Adrenergic Receptor Triggers Functional and Constitutive Inactivation

Based on our previous molecular modeling and radioligand binding study, we have demonstrated that aspartic acid of 104 in transmembrane helix (TMH) II of beta(1)-adrenergic receptor (beta(1)-AR) is important for functional characteristics of these receptors. We have also showed that mutation of negatively charged aspartic acid to neutral charged alanine exhibited constitutive activity of beta(1)-AR. However, the mutation of negatively charged aspartic acid to positively charged lysine is still remained to be examined, which is very important to know for fully understanding the characteristics of beta(1)-AR. At the present study, we mutated aspartic acid to lysine (Asp104Lys) residue in human beta(1)-AR. This resultant mutant (Asp104Lys) markedly reduced the binding affinity of isoproterenol and (-)-epinephrine. On the other hand, antagonist binding with this mutant was similar to the wild type receptor. Isoproterenol at its saturation concentrations produced lower amount of intracellular cyclic adenosine-3',5' cyclic monophosphate (cAMP) in HEK-293 cells expressing Asp104Lys mutant receptor as compared to cells expressing wild type receptor. Moreover, cAMP accumulation of Asp104Lys mutant was unchanged in the presence or absence of isoproterenol. Therefore, it has been demonstrated that Asp104Lys mutation in the human beta(1)-AR differentially affects the binding of antagonist and exhibits a functional uncoupling of G-protein-coupled receptors. Thus, we may suggest that mutation of negatively charged aspartic acid to positively charged lysine as well as neutral charged alanine may help to understand the mechanism of the activation or inactivation of beta(1)-AR by its conformational changes and this finding would be helpful for clarifying the functional responses mediated by beta(1)-AR.