Protein Kinase A Is a Master Regulator of Physiological and Pathological Cardiac Hypertrophy.

BACKGROUND: The sympathoadrenergic system and its major effector PKA (protein kinase A) are activated to maintain cardiac output coping with physiological or pathological stressors. If and how PKA plays a role in physiological cardiac hypertrophy (PhCH) and pathological CH (PaCH) are not clear. METHODS: Transgenic mouse models expressing the PKA inhibition domain (PKAi) of PKA inhibition peptide alpha (PKIalpha)-green fluorescence protein (GFP) fusion protein (PKAi-GFP) in a cardiac-specific and inducible manner (cPKAi) were used to determine the roles of PKA in physiological CH during postnatal growth or induced by swimming, and in PaCH induced by transaortic constriction (TAC) or augmented Ca2+ influx. Kinase profiling was used to determine cPKAi specificity. Echocardiography was used to determine cardiac morphology and function. Western blotting and immunostaining were used to measure protein abundance and phosphorylation. Protein synthesis was assessed by puromycin incorporation and protein degradation by measuring protein ubiquitination and proteasome activity. Neonatal rat cardiomyocytes (NRCMs) infected with AdGFP (GFP adenovirus) or AdPKAi-GFP (PKAi-GFP adenovirus) were used to determine the effects and mechanisms of cPKAi on myocyte hypertrophy. rAAV9.PKAi-GFP was used to treat TAC mice. RESULTS: (1) cPKAi delayed postnatal cardiac growth and blunted exercise-induced PhCH; (2) PKA was activated in hearts after TAC due to activated sympathoadrenergic system, the loss of endogenous PKIα (PKA inhibition peptide α), and the stimulation by noncanonical PKA activators; (3) cPKAi ameliorated PaCH induced by TAC and increased Ca2+ influxes and blunted neonatal rat cardiomyocyte hypertrophy by isoproterenol and phenylephrine; (4) cPKAi prevented TAC-induced protein synthesis by inhibiting mTOR (mammalian target of rapamycin) signaling through reducing Akt (protein kinase B) activity, but enhancing inhibitory GSK-3α (glycogen synthase kinase-3α) and GSK-3ß signals; (5) cPKAi reduced protein degradation by the ubiquitin-proteasome system via decreasing RPN6 phosphorylation; (6) cPKAi increased the expression of antihypertrophic atrial natriuretic peptide (ANP); (7) cPKAi ameliorated established PaCH and improved animal survival. CONCLUSIONS: Cardiomyocyte PKA is a master regulator of PhCH and PaCH through regulating protein synthesis and degradation. cPKAi can be a novel approach to treat PaCH.

Cardiomyocyte PKA Ablation Enhances Basal Contractility While Eliminates Cardiac ß-Adrenergic Response Without Adverse Effects on the Heart.

RATIONALE: PKA (Protein Kinase A) is a major mediator of ß-AR (ß-adrenergic) regulation of cardiac function, but other mediators have also been suggested. Reduced PKA basal activity and activation are linked to cardiac diseases. However, how complete loss of PKA activity impacts on cardiac physiology and if it causes cardiac dysfunction have never been determined. OBJECTIVES: We set to determine how the heart adapts to the loss of cardiomyocyte PKA activity and if it elicits cardiac abnormalities. METHODS AND RESULTS: (1) Cardiac PKA activity was almost completely inhibited by expressing a PKA inhibitor peptide in cardiomyocytes (cPKAi) in mice; (2) cPKAi reduced basal phosphorylation of 2 myofilament proteins (TnI [troponin I] and cardiac myosin binding protein C), and one longitudinal SR (sarcoplasmic reticulum) protein (PLB [phospholamban]) but not of the sarcolemmal proteins (Cav1.2 α1c and PLM [phospholemman]), dyadic protein RyR2, and nuclear protein CREB (cAMP response element binding protein) at their PKA phosphorylation sites; (3) cPKAi increased the expression of CaMKII (Ca2+/calmodulin-dependent kinase II), the Cav1.2 ß subunits and current, but decreased CaMKII phosphorylation and CaMKII-mediated phosphorylation of PLB and RyR2; (4) These changes resulted in significantly enhanced myofilament Ca2+ sensitivity, prolonged contraction, slowed relaxation but increased myocyte Ca2+ transient and contraction amplitudes; (5) Isoproterenol-induced PKA and CaMKII activation and their phosphorylation of proteins were prevented by cPKAi; (6) cPKAi abolished the increases of heart rate, and cardiac and myocyte contractility by a ß-AR agonist (isoproterenol), showing an important role of PKA and a minimal role of PKA-independent ß-AR signaling in acute cardiac regulation; (7) cPKAi mice have partial exercise capability probably by enhancing vascular constriction and ventricular filling during ß-AR stimulation; and (8) cPKAi mice did not show any cardiac functional or structural abnormalities during the 1-year study period. CONCLUSIONS: PKA activity suppression induces a unique Ca2+ handling phenotype, eliminates ß-AR regulation of heart rates and cardiac contractility but does not cause cardiac abnormalities.

A novel human IL-2 mutein with minimal systemic toxicity exerts greater antitumor efficacy than wild-type IL-2.

IL-2 is critical to the activation, growth, and survival of T cells and NK cells, and maintains the delicate balance between auto-immunity and anti-neoplasm surveillance. High IL-2 doses have clear antitumor capabilities, but also have severe side effects that limit its clinical use. Side effects include the vascular leak syndrome (VLS), which results in lung edema and liver damage. Therefore, a new version of IL-2 that does not induce organ toxicity would improve IL-2-based immunotherapy. We conducted a systematic screening by changing one amino acid at a time at the interaction area of IL-2 with its receptor IL-2R to select one particular mutant IL-2, FSD13, in which the proline at position 65 was substituted by lysine (P65L). FSD13 had a greater ability than wild-type IL-2 in stimulating CD4+ T, CD8+ T, and NK cell proliferation, enhancing the expression of CD69, CD183, CD44, and CD54 in these cells, and triggering cancer cell apoptosis. FSD13 had three-time lower than wild-type IL-2 in inducing CD4+ T to Tregs. Compared with wild-type IL-2, FSD13 greatly limited the growth, invasion into adjacent tissues, and metastasis of melanoma metastatic into the lung. In contrast to wild-type IL-2, high dose of FSD3 did not alter structures and induce any pathogenic changes in the liver and lung. Thus, we generated a novel the IL-2 mutant, FSD13, by targeting a different area than previously reported. FSD13 surpasses the wild-type IL-2's ability in stimulating the antitumor immune cell functions, but exerts much less systemic toxicity.

Effect of enrofloxacin and roxarsone on CYP450s in pig.

Enrofloxacin (ENR) and roxarsone (ROX) have been widely used in animal breeding. In this study, the daily clinical dosage of ENR and daily additive amount of ROX were administrated to Bama pigs. After 5days, the activity and protein expression of three important enzymes in the cytochrome P450 family were measured in the porcine liver. CYP1A2 was induced by both ENR and ROX independently. CYP2E1 and CYP3A4 were inhibited by ENR, but not affected by ROX. The combined administration of ENR and ROX were antagonistic to CYP1A2 and CYP2E1, but not to CYP3A4. Drug-drug interactions should be considered during the administration of ENR, ROX and for their co-administration with other drugs to minimize adverse reactions.

Identification of a cytochrome P450 gene in the earthworm Eisenia fetida and its mRNA expression under enrofloxacin stress.

Cytochrome P450 (CYP450) enzymes are a family of hemoproteins primarily responsible for detoxification functions. Earthworms have been used as a bioindicator of soil pollution in numerous studies, but no CYP450 gene has so far been cloned. RT-PCR and RACE-PCR were employed to construct and sequence the CYP450 gene DNA from the extracted mRNA in the earthworm Eisenia fetida. The cloned gene (EW1) has an open reading frame of 477bp. The 3'-terminal region contained both the consensus and the signature sequences characteristic of CYP450. It was closely related to the CYP450 gene from the flatworm genus Opisthorchis felineus with 87% homology. The predicted structure of the putative protein was 97% homologous to human CYP450 family 27. This gene has been deposited in GenBank (accession no. KM881474). Earthworms (E. fetida) were then exposed to 1, 10, 100, and 500mgkg-1 enrofloxacin in soils to explore the mRNA expression by real time qPCR. The effect of enrofloxacin on mRNA expression levels of EW1 exhibited a marked hormesis pattern across the enrofloxacin dose range tested. This is believed to be the first reported CYP450 gene in earthworms, with reference value for molecular studies on detoxification processes in earthworms.

Effect of enrofloxacin on the proteome of earthworms.

The environmental and human health risks of veterinary drugs are becoming public health issues. Enrofloxacin (EF) is an extensively used animal-specific antibacterial agent that leaves drug residues in the environment. This study investigated the proteomic response of the earthworm Eisenia fetida to EF exposure. Earthworms were exposed to EF in soil at 1-500mg·kg-1, and samples were collected at intervals during a 28 day period. The extracted proteins were separated by two dimensional electrophoresis to detect differentially expressed proteins (DEPs) in EF-exposed earthworms. In total, 35 unique DEPs were found. These proteins were subjected to MALDI-TOF/TOF-MS analysis and identified through comparison of their mass spectra with those in protein databases. The DEPs were grouped on the basis of their function, into metabolism, stress-related, transport, transcription, and predicted/hypothetical protein categories. Knowledge of proteins that are induced or repressed by EF in earthworms could provide insight into mechanisms of sub-clinical physiological effects of xenobiotic residues in the environment, and may also help understand synergy between pollutants. As several DEPs in E. fetida showed similarity to human protein sequences, E. fetida has potential as an indicator species to assess the environmental and biological risks of drug residues.

Reduced Myocardial Reserve in Young X-Linked Muscular Dystrophy Mice Diagnosed by Two-Dimensional Strain Analysis Combined with Stress Echocardiography.

BACKGROUND: Early, sensitive, and reproducible evaluation of left ventricular function is imperative for the diagnosis of cardiac dysfunction in patients with Duchene muscular dystrophy. The aim of this study was to test the hypothesis that combining two-dimensional strain analysis with catecholamine stress could be a sensitive method for detecting early cardiac dysfunction. METHODS: Mdx (C57BL/10ScSn-Dmdmdx/J, a mouse model of DMD) and control (C57BL/10ScSn) mice were studied with conventional M-mode and high-frequency ultrasound-based two-dimensional speckle-tracking echocardiography using long- and short-axis images of the left ventricle at baseline and after intraperitoneal isoprenaline (ISO) administration (2 µg/g body weight). RESULTS: Conventional M-mode analysis showed no differences in left ventricular fractional shortening, wall thickness, or internal diameter at diastole between mdx and control mice before the age of 6 months. ISO increased left ventricular ejection fraction and fractional shortening to the same extent in mdx and control mice at young ages (3, 4, and 5 months). No differences in basal peak systolic strain (PSS) but increased SDs of times to PSS between young mdx and control mice were found. After ISO, PSS and percentile changes of PSS were significantly diminished in mdx mice compared with control mice at young ages. ISO increased the normalized maximum difference of times to PSS in young mdx mice but not in young control mice, suggesting that ISO reduces cardiac contractile synchrony in young mdx mice. CONCLUSIONS: This study suggests that catecholamine stress coupled with two-dimensional strain analysis is a feasible and sensitive approach for detecting early onset of cardiac dysfunction, which is instrumental for early diagnosis of cardiac dysfunction and early treatment.

Enrofloxacin at environmentally relevant concentrations enhances uptake and toxicity of cadmium in the earthworm Eisenia fetida in farm soils.

Individual and combined effects of enrofloxacin (EF) and cadmium (Cd) on the earthworm Eisenia fetida at environmentally relevant concentrations were investigated. EF is a veterinary antibiotic; Cd is an impurity in phosphatic fertiliser. For both, residues may accumulate in farm soils. In laboratory tests, over 98% of spiked EF was adsorbed by farm soils, with a half-life >8 weeks. However, earthworms absorbed less than 20% of spiked EF. Earthworms in soil with EF concentration 10 mg kg(-1) soil experienced transient oxidative stress and exhibited reduced burrowing activity and respiration after an 8-week exposure; EF at 0.1 and 1.0 mg kg(-1) soil did not elicit toxicity symptoms. When both were added, Cd did not affect EF uptake, but each increment of spiked EF increased Cd bioaccumulation and associated oxidative stress of earthworms, and also caused decreased burrow length and CO2 production. However, metallothionein induction was not affected. The enhanced toxicity of Cd to earthworms in the presence of EF at low environmental concentrations may have implications for the health and reproductive success of earthworm populations and highlights the importance of understanding effects of antibiotic contamination of farm soils, and of awareness of environmental effects from interaction between multiple contaminants.

Persistent increases in Ca(2+) influx through Cav1.2 shortens action potential and causes Ca(2+) overload-induced afterdepolarizations and arrhythmias.

Persistent elevation of Ca(2+) influx due to prolongation of the action potential (AP), chronic activation of the ß-adrenergic system and molecular remodeling occurs in stressed and diseased hearts. Increases in Ca(2+) influx are usually linked to prolonged myocyte action potentials and arrhythmias. However, the contribution of chronic enhancement of Cav1.2 activity on cardiac electrical remodeling and arrhythmogenicity has not been completely defined and is the subject of this study. Chronically increased Cav1.2 activity was produced with a cardiac specific, inducible double transgenic (DTG) mouse system overexpressing the ß2a subunit of Cav (Cavß2a). DTG myocytes had increased L-type Ca(2+) current (ICa-L), myocyte shortening, and Ca(2+) transients. DTG mice had enhanced cardiac performance, but died suddenly and prematurely. Telemetric electrocardiograms revealed shortened QT intervals in DTG mice. The action potential duration (APD) was shortened in DTG myocytes due to significant increases of potassium currents and channel abundance. However, shortened AP in DTG myocytes did not fully limit excess Ca(2+) influx and increased the peak and tail ICa-L. Enhanced ICa promoted sarcoplasmic reticulum (SR) Ca(2+) overload, diastolic Ca(2+) sparks and waves, and increased NCX activity, causing increased occurrence of early and delayed afterdepolarizations (EADs and DADs) that may contribute to premature ventricular beats and ventricular tachycardia. AV blocks that could be related to fibrosis of the AV node were also observed. Our study suggests that increasing ICa-L does not necessarily result in AP prolongation but causes SR Ca(2+) overload and fibrosis of AV node and myocardium to induce cellular arrhythmogenicity, arrhythmias, and conduction abnormalities.

Blunted cardiac beta-adrenergic response as an early indication of cardiac dysfunction in Duchenne muscular dystrophy.

AIMS: To determine whether altered beta-adrenergic responses contribute to early cardiac dysfunction in mdx (X-linked muscular dystrophy) mice, an animal model for human Duchenne muscular dystrophy. METHODS AND RESULTS: Replacement fibrosis in mdx hearts gradually increased with age, suggesting a gradual loss of cardiomyocytes. Echocardiography and intra-left ventricular haemodynamic measurements detected baseline cardiac dysfunction in mdx mice at ≥8 months. However, a reduction of cardiac beta-adrenergic response to isoproterenol (ISO) was already present in mdx mice at 4 months. Ventricular myocytes (VMs) isolated from 4- and 8-month-old mdx mice had greater baseline contractile function {fractional shortening, [Ca(2+)]i, and sarcoplasmic reticulum (SR) Ca(2+) content} and ICa-L than age-matched control VMs and than myocytes isolated from 2-month-old mdx mice. ISO increased myocyte function in the VMs of 4- and 8-month-old mdx mice to the same level as in age-matched control VMs. In the VMs of 12-month-old mdx mice, ISO failed to increase myocyte function to the level in VMs of 12-month-old control mice and could not further increaseICa-L. No differences were observed in the expression of Cav1.2α1c, Cav1.2ß1, Cav1.2ß2, sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA), and the Na(+)/Ca(2+) exchanger. In contrast, total ryanodine receptor 2 (RyR2) and basal phosphorylation of RyR2, phospholamban, and Cav1.2α1c were found to be increased in hearts of 4-month-old mdx mice; baseline protein kinase A activity was also increased. After ISO treatment, phosphorylation levels were the same in mdx and control hearts. VMs of 4-month-old mdx mice had reduced beta1-adrenergic receptor (ß1-AR) density and beta-adrenergic sensitivity. CONCLUSION: In young mdx mice, the myocyte increases its contractile function to compensate for myocyte loss. However, these myocytes with enhanced baseline function have reduced potential for stimulation, decreased ß1-AR density/sensitivity, leading to blunted cardiac beta-adrenergic response.

Cardiotoxic and cardioprotective features of chronic ß-adrenergic signaling.

RATIONALE: In the failing heart, persistent ß-adrenergic receptor activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II. ß-adrenergic signaling pathways also are capable of activating cardioprotective mechanisms. OBJECTIVE: This study used a novel PKA inhibitor peptide to inhibit PKA activity to test the hypothesis that ß-adrenergic receptor signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways. METHODS AND RESULTS: In PKA inhibitor peptide transgenic mice, chronic isoproterenol failed to induce cardiac hypertrophy, fibrosis, and myocyte apoptosis, and decreased cardiac function. In cultured adult feline ventricular myocytes, PKA inhibition protected myocytes from death induced by ß1-adrenergic receptor agonists by preventing cytosolic and sarcoplasmic reticulum Ca(2+) overload and calcium/calmodulin-dependent kinase II activation. PKA inhibition revealed a cardioprotective role of ß-adrenergic signaling via cAMP/exchange protein directly activated by cAMP/Rap1/Rac/extracellular signal-regulated kinase pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction that was superior to ß-blocker therapy. CONCLUSIONS: These results suggest that selective block of PKA could be a novel heart failure therapy.

Calcium influx through Cav1.2 is a proximal signal for pathological cardiomyocyte hypertrophy.

Pathological cardiac hypertrophy (PCH) is associated with the development of arrhythmia and congestive heart failure. While calcium (Ca(2+)) is implicated in hypertrophic signaling pathways, the specific role of Ca(2+) influx through the L-type Ca(2+) channel (I(Ca-L)) has been controversial and is the topic of this study. To determine if and how sustained increases in I(Ca-L) induce PCH, transgenic mouse models with low (LE) and high (HE) expression levels of the ß2a subunit of Ca(2+) channels (ß2a) and in cultured adult feline (AF) and neonatal rat (NR) ventricular myocytes (VMs) infected with an adenovirus containing a ß2a-GFP were used. In vivo, ß2a LE and HE mice had increased heart weight to body weight ratio, posterior wall and interventricular septal thickness, tissue fibrosis, myocyte volume, and cross-sectional area and the expression of PCH markers in a time- and dose-dependent manner. PCH was associated with a hypercontractile phenotype including enhanced I(Ca-L), fractional shortening, peak Ca(2+) transient, at the myocyte level, greater ejection fraction, and fractional shortening at the organ level. In addition, LE mice had an exaggerated hypertrophic response to transverse aortic constriction. In vitro overexpression of ß2a in cultured AFVMs increased I(Ca-L), cell volume, protein synthesis, NFAT, and HDAC translocations and in NRVMs increased surface area. These effects were abolished by the blockade of I(Ca-L), intracellular Ca(2+), calcineurin, CaMKII, and SERCA. In conclusion, increasing I(Ca-L) is sufficient to induce PCH through the calcineurin/NFAT and CaMKII/HDAC pathways. Both cytosolic and SR/ER-nuclear envelop Ca(2+) pools were shown to be involved.

Enhanced basal contractility but reduced excitation-contraction coupling efficiency and beta-adrenergic reserve of hearts with increased Cav1.2 activity.

Cardiac remodeling during heart failure development induces a significant increase in the activity of the L-type Ca(2+) channel (Cav1.2). However, the effects of enhanced Cav1.2 activity on myocyte excitation-contraction (E-C) coupling, cardiac contractility, and its regulation by the beta-adrenergic system are not clear. To recapitulate the increased Cav1.2 activity, a double transgenic (DTG) mouse model overexpressing the Cavbeta2a subunit in a cardiac-specific and inducible manner was established. We studied cardiac (in vivo) and myocyte (in vitro) contractility at baseline and upon beta-adrenergic stimulation. E-C coupling efficiency was evaluated in isolated myocytes as well. The following results were found: 1) in DTG myocytes, L-type Ca(2+) current (I(Ca,L)) density, myocyte fractional shortening (FS), peak Ca(2+) transients, and sarcoplasmic reticulum (SR) Ca(2+) content (caffeine-induced Ca(2+) transient peak) were significantly increased (by 100.8%, 48.8%, 49.8%, and 46.8%, respectively); and 2) cardiac contractility evaluated with echocardiography [ejection fraction (EF) and (FS)] and invasive intra-left ventricular pressure (maximum dP/dt and -dP/dt) measurements were significantly greater in DTG mice than in control mice. However, 1) the cardiac contractility (EF, FS, dP/dt, and -dP/dt)-enhancing effect of the beta-adrenergic agonist isoproterenol (2 microg/g body wt ip) was significantly reduced in DTG mice, which could be attributed to the loss of beta-adrenergic stimulation on contraction, Ca(2+) transients, I(Ca,L), and SR Ca(2+) content in DTG myocytes; and 2) E-C couplng efficiency was significantly lower in DTG myocytes. In conclusion, increasing Cav1.2 activity by promoting its high-activity mode enhances cardiac contractility but decreases E-C coupling efficiency and the adrenergic reserve of the heart.

Nicotine could augment adhesion molecule expression in human endothelial cells through macrophages secreting TNF-alpha, IL-1beta.

Nicotine, the major immunomodulatory components of cigarette smoking, is among the leading risk factors in atherosclerosis and various other diseases. The subject of this study is to observe how nicotine affects the function of macrophages and vascular endothelial cells. The changes of nicotine on releasing of cytokines from Ana-1 were detected by radio-immunoassay (RIA) or enzyme-link immunosorbent assay (ELISA). The adhesion of monocytes to human umbilical vein endothelial cells (HUVECs) with Ana-1 supernatant-activated was evaluated through adhesion experiments. ELISA and RT-PCR methods examined expression of soluble adhesion molecular protein and their mRNA. Which cytokines in Ana-1 supernatant affecting HUVECs ability to express adhesion molecular were tested by adhesion blockade analysis and ELISA. The results showed TNF-alpha, IL-1beta could reach the peak with 0.06mM nicotine treated for 24 and 12 h on Ana-1, respectively, but IL-8 and IFN-gamma had no significant alter. Adhesion experiments proved treatment of HUVECs with supernatant of Ana-1 for 24 h obviously augmented the adhesion of monocytes to HUVECs. ELISA and PCR demonstrated expression of soluble intracellular adhesion molecule-1 protein (sICAM-1) increased sharply at 24 h, while soluble vascular cell adhesion molecule-1 protein (sVCAM-1) and soluble endothelial selectin protein (sE-selectin) rose at 9 h; ICAM-1, VCAM-1 and E-selectin mRNA had a similar tendency. Treatment of HUVECs with anti-TNF-alpha, anti-IL-1beta antibodies pre-neutralized supernatant of Ana-1 could block monocytes adhesion. In conclusion, our findings suggest that nicotine could augment macrophages releasing TNF-alpha and IL-1beta, furthermore TNF-alpha and IL-1beta could up-regulate the expression of adhesion molecule and increase adhesion of monocytes to HUVECs. These might be one of the reasons that leaded to endothelial dysfunction.

[Effects of cysteamine on the plasma levels of SS and some metabolic hormones in adult geese].

AIM: To know the effect of cysteamine (CS) on the plasma levels of somatostatin (SS) and some metabolic hormones in adult geese. METHODS: Fourteen adult crossbred geese (Chuan white x Tai lake) fitted with chronic wing vein cannulas were used in this study to evaluate the effect of CS on SS, TSH, T3 and T4 levels. The experiment was consisted of control and treated phase. The diet was added CS at dosage of 100 mg/kg bw on the first day of the treated phase. The blood samples were collected from the cannulas and analyzed by radioimmunoassay. RESULTS: The plasma SS concentration was (1.87 +/- 0.10) microg/L in control phase. Whereas SS concentrations on day 1, 3, 5, 7 of treated phase were decreased markedly (P < 0.05 or P < 0.01). Thereafter it was rose on the seventh day, however it was still lower than that of control. The thyroid stimulating hormone (TSH) content (2.45 +/- 0.31 mIU/L) was significantly decreased by 21.63% (P < 0.01) on day 1, and 18.37% (P > 0.05) on day 3 and day 5. Comparing with control phase (5.41 +/- 0.98 microg/L), T4 contents were elevated by 60.26% (P < 0.01), 43.25% (P < 0.01), 37.15% (P < 0.01) and 16. 82% (P < 0.01) respectively on day 1, 3, 5, 7. T3 level was (1.05 +/- 0.06) microg/L in control phase, whereas the levels was significantly increased by 36.19% (P < 0.01) on day 3. Also, the insulin concentration was higher than that of control (4.43 +/- 0.41 mU/ L) by 18.28% (P < 0.05) on the day 5. CONCLUSION: These results indicate that CS can decrease the plasma SS and TSH levels, whereas increase the levels of T4, T3 and insulin, therefore change metabolism, improve the nutrition transform and accelerate the growth in geese.

[Effects of clenbuterol on nitrogen metabolism and G6PDH activity of rat hepatocyte].

AIM: To study the effects of beta 2-adrenergic receptor-selective agonist clenbuterol on nitrogen metabolism and glucose-6-phosphate dehydrogenase activity of rat hepatocyte and its pharmacological mechanism. METHODS: Biochemical methods were used to study the influence of clenbuterol on urea-nitrogen concentration of hepatocyte culture medium, 3H-leucine incorporation into hepatocyte, insulin-like growth factor I (IGF-I) production and glucose-6-phosphate dehydrogenase (G6PDH) activity of rat hepatocyte. RESULTS: The results showed that urea-nitrogen production by cultured rat hepatocytes was markedly affected with clenbuterol treatment (1 x 10(-6) mol.L-1), urea-nitrogen concentration of culture medium was decreased by 25.51% (P < 0.05) compared with control. The inhibitory effect of hepatocyte urea-nitrogen production of clenbuterol was blocked by propranolol, a beta-adrenoreceptor antagonist (1 x 10(-6) mol.L-1), but hepatocyte urea-nitrogen level was not affected with propranolol treatment only (P > 0.05). The content of 3H-leucine incorporation in rat hepatocyte was significantly increased by 23.35% (P < 0.05) with clenbuterol-treatment (1 x 10(-6) mol.L-1), and the enhanced effect of 3H-leucine incorporation into hepatocyte was antagonized by propranolol (1 x 10(-6) mol.L-1. The level of 3H-leucine incorporation of rat hepatocyte was not influenced by propranolol alone. IGF-I production of rat hepatocyte might be affected by clenbuterol. IGF-I concentration of culture medium was increased by 39.46% with clenbuterol (1 x 10(-6) mol.L-1), but no significant difference was found compared with the control (P > 0.05). Moreover, G6PDH activity of rat hepatocyte was significantly decreased by 43.36% (P < 0.05) with clenbuterol treatment (1 x 10(-6) mol.L-1), and the declined effect of clenbuterol was antagonized by propranolol. G6PDH activity of rat hepatocyte was not affected on condition that propranolol was administered alone (P > 0.05). CONCLUSION: It is suggested that clenbuterol may regulate nitrogen and fat metabolism by means of increasing nitrogen retention and protein synthesis, and decreasing G6PDH activity of rat hepatocyte for pharmacological effects.

[Effects of clenbuterol on the hepatic flux of nitrogen, VFA and glucose in sheep].

AIM: To examine the liver mechanism with which clenbuterol (CL) is explained how to affect growth metabolism. METHODS: The technique of chronic poly catheter was used to study the effects of CL (0.8 mg/kg b w) on the hepatic flux of nitrogen, VFA and glucose in 4 sheep. RESULTS: The urea-nitrogen flux in CL-treated period always was lower than that in control during 24 h. The average flux of urea-nitrogen in hepatic and portal vein were decreased by 16.86% (P < 0.01) and 15.51% (P < 0.05), respectively, compared with that of control. The peptide level in hepatic vein was decreased with the treatment of CL, average flux of peptide was decreased by 38.71% (P < 0.01). But the peptide level of portal vein in CL treatment period was similar to control. Moreover, VFA level in the portal vein was enhanced by CL, the average flux of acetate in portal vein was increased by 19.49% (P < 0.01). No difference of VFA level in hepatic vein was noted between CL-treated period and control. In addition, the glucose flux in hepatic vein was obviously increased with CL treatment, the average flux of glucose was increased by 25.96% (P < 0.01). And glucose flux in portal vein was also elevated during CL-treated period. CONCLUSION: CL can affect growth metabolism of animal with increasing nitrogen deposition, improving absorption and utilization of VFA and enhancing glucose synthesis in sheep liver.

[Effect of cysteamine on the pancreatic secretion and enzymatic activity in geese].

AIM: To know the effect of cysteamine on the pancreatic secretion and enzyme activity in geese. METHODS: Eight adult geese fitted chronic pancreatic and duodenal cannulas were used to evaluate the effect of cysteamine (CS) on the pancreatic secretion and enzyme activity. The experiment was consist of control and treated phase. CS was added in the diet at the dosage of 100 mg/kg bw on the first day of treated phase. The birds were free fed at daytime (8:00-20:00) and fasted at nighttime (20:00-8:00). The pancreatic juice samples were collected continuously for three days in each phase. RESULTS: CS increased the average rate of pancreatic secretion by 240.16% (P < 0.01), in which that of daytime was elevated by 234.45% (P < 0.01), while that of nighttime elevated by 253.70% (P < 0.01). The secretion volume at daytime was more than that of night. CS increased trypsin activity by 49.05% (P < 0.01), whereas lipase and amylase activity was reduced by 25.44% (P < 0.01) and 21.95% (P < 0.01) separately. The one hour total activity of trypsin, lipase and amylase were elevated by 406.88% (P < 0.01), 153.58% (P < 0.01) and 166.59% (P < 0.01) respectively. Ratios of pancreatic secretion were different between day and night. CONCLUSION: These results indicate that CS can affect the pancreatic juice secretion and pancreatic enzyme activity by depleting the somatostatin, so that benefits to improve the digestive foundation and supply more nutrition for quickly growing in geese.