Effect of isoproterenol and taurine on heart calcium in normal and cardiomyopathic hamsters

Calcium accumulation has been implicated in the cardiac necrosis induced by isoproterenol and in the development of the cardiomyopathy in the BIO 14.6 hamster. Taurine, a natural constituent of the heart, has been shown to exert a modulating effect on calcium levels in the heart. Heart calcium and taurine levels were determined in BIO 14.6 and random bred (F₁B) hamsters treated with isoproterenol (80 mg/kg) following a 60 day drinking regimen of either taurine (100 mmol/l) or guanidinoethyl sulfonate (1%). Taurine supplementation provided some protection for the random bred hamster heart against isoproterenol induced calcium accumulation, but that protection was not demonstrable in the BIO 14.6 strain despite an elevated heart taurine content. The feeding of guanidinoethyl sulfonate decreased the taurine content of the heart in both strains, but guanidinoethyl sulfonate was unable to block taurine elevation following isoproterenol treatment in either strain. Since taurine feeding retards the usual calcium accumulation in the BIO 14.6, but is without statistically significant effect on the additional calcium accumulation induced by isoproterenol, the protecitive action of taurine seems insufficient to counteract the combined effect of isoproterenol and the myopathic process.     

Alterations in cyclic nucleotide phosphodiesterase activities in omental and subcutaneous adipose tissues in human obesity

Objective: To elucidate the activity and expression of cyclic nucleotide phosphodiesterase (PDE) families in omental (OM) and subcutaneous (SC) adipose tissue and adipocytes, and to study alterations in their activity in human obesity.Design: Cross-sectional, translational research study.Patients: In total, 25 obese and 9 non-obese subjects undergoing gastrointestinal surgery participated in the study.Results: Inverse correlations between PDE activities and body mass index (BMI) were seen in both SC and OM adipose tissue. Inverse correlations between total PDE and PDE3 activity and BMI were seen in OM adipocytes but not in SC adipocytes. In both SC and OM adipose tissue of obese patients, total PDE and PDE3 activities were decreased compared with the controls. In SC adipose tissue of Type 2 diabetes (T2D) patients, the PDE activity not inhibitable by PDE3 or PDE4 inhibitors (PDEn) was increased compared with obese non-diabetic patients. In addition to PDE3 and 4 isoforms, PDE7B, PDE9A and PDE10A proteins were also detected in adipose tissue or adipocytes.Conclusions: Multiple PDE families are present in human adipose tissue and their activities are differentially affected by obesity and T2D.     

高氟摄入后大鼠骨骼肌环核苷酸的变化及镁硒的影响

雄性Ｗｉｓｔａｒ大鼠腹腔ＮａＦ实验表明，亚急性氟中毒大鼠肌骼肌组织环磷酸腺苷（ＣＡＭＰ）水平明显市长中，环磷酸鸟苷（ＣＧＭＰ）水平下降，ＣＡＭＰ／ＣＧＭＰ升高。加镁后，由于ＣＧＭＰ水平升高，使ＣＡＭＰ／ＣＧＭＰ下降，这一作用可能与镁拮抗指一有关。而硒对这一生理过程无明显影响。     

Changes in the messenger RNA population from the heart of cardiomyopathic hamsters

Messenger RNAs were extracted from the heart of cardiomyopathic hamsters at different phases of the disease and from age-matched control hamsters. They were translated into proteins in a rabbit reticulocyte lysate in the presence of [35S]methionine, the translational products were fractionated by two-dimensional polyacrylamide gel electrophoresis and analyzed by fluorography. No difference between cardiomyopathic and control hamsters could be detected when comparing the spots corresponding to the major contractile proteins. However, we observed that three translational products of minor abundance were reproducibly decreased in cardiomyopathic hamsters at 60 days (necrotic phase) and 200 days (final phase) but not at 30 days (prenecrotic phase). At 120 days (hypertrophic phase), the decrease could also be detected but was much less pronounced.     

Effect of phosphodiesterase inhibitors on heart contractile behaviour, protein kinase activity and cyclic nucleotide levels

Three cyclic nucleotide phosphodiesterase inhibitors were examined for their effects upon contractility, relaxation, cAMP-dependent protein kinase activity ratio and cyclic nucleotide levels in the perfused rat heart. Theophylline (2 × 10^?4m), papaverine (10^?5m) and pentoxifylline (10^?4m) were infused at constant heart rate and coronary flow. Developed tension (T), its maximal rate of rise (+?), its maximal rate of fall (??) and time to peak tension (TTP) were measured in fast records.No changes in contractility were observed with theophylline and a slightly positive inotropic effect at 1 min of perfusion with papaverine and pentoxifylline was detected. There was no prolongation in the duration of the systole and a small but significant decrease in TTP after 1 min of papaverine administration was found. After 1 min perfusion cAMP increased from control values of 0.503 ± 0.025 pmol/mg wet weight to 0.773 ± 0.062 (theophylline), 0.784 ± 0.077 (papaverine) and 0.604 ± 0.034 (pentoxifylline). After 15 min perfusion from 0.452 ± 0.031 to 0.762 ± 0.064 (papaverine) and 0.645 ± 0.047 (pentoxifylline). At this time of perfusion cAMP-dependent protein kinase activity ratio was also increased from 0.21 ± 0.01 to 0.30 ± 0.03 (theophylline), 0.31 ± 0.03 (papaverine) and 0.32 ± 0.03 (pentoxifylline). cGMP rose from 8.1 ± 1.2 fmol/mg wet weight to 15.0 ± 1.4 (theophylline) and 12.3 ± 1.4 (papaverine) after 1 min perfusion and after 15 min from 8.2 ± 1.2 to 13.2 ± 1.3 (theophylline) and 13.3 ± 1.9 (papaverine). Pentoxifylline did not produce significant changes.It is suggested that increased cGMP levels interfere with cAMP and cAMP-dependent protein kinase in the mechanical effects of cyclic nucleotide phosphodiesterase inhibitors.     

Adenosine depresses contractility and stimulates 3',5' cyclic nucleotide metabolism in the isolated frog ventricle

Adenosine depresses the contractile response of the isolated frog ventricle. An investigation has been made of its effects on the metabolism of endogenous 3′,5′ cyclic nucleotides. The levels of adenosine 3′,5′ cyclic monophosphate (3′,5′ cyclic AMP) and guanosine 3′,5′ cyclic monophosphate (3′,5′ cyclic GMP) were measured at different times during exposure of the ventricle to adenosine (10^?3m). The decline in isometric twitch tension is found to be accompanied by a progressive fall in intracellular 3′,5′ cyclic AMP and a concomitant rise in 3′,5′ cyclic GMP. These effects are dose related. It is suggested that the ability of adenosine to stimulate 3′,5′ cyclic nucleotide turnover is the result of a fall in intracellular Ca²⁺, acting via the Ca²⁺-binding protein modulator, calmodulin. Interestingly, the extent to which the contractile response is depressed is found to be paralleled closely by a quantitatively equivalent reduction in the ratio 3′,5′ cyclic AMP: 3′,5′ cyclic GMP. A possible biochemical basis for the antagonistic regulatory effects of 3′,5′ cyclic AMP and 3′,5′ cyclic GMP on ventricular contractility, implied by these (and other) results, is discussed briefly.     

Tyrosine hydroxylase activity in the heart of the cardiomyopathic syrian hamster.

The hearts of dystrophic hamsters have higher tyrosine hydroxylase activity (TH) than those of matched controls. The development of cardiac hypertrophy and failure is associated with parallel increases in cardiac TH activity and in the rate constant for norepinephrine turnover; but not with an increase in catecholamine synthesis. Reserpine pretreatment elevated TH activity in control hearts but did not further increase enzyme activity in failing ones; this supports our earlier observation that sympathetic input to the failing hamster heart at rest approaches the maximum achievable with stress.We conclude that TH activity may increase rather than decrease in some forms of heart failure, and that TH may not always be the limiting factor in determining the rate of norepinephrine production.     

Effects of streptozotocin-induced diabetes on calmodulin and cyclic AMP phosphodiesterase activity in rat lungs

Streptozotocin-induced diabetes caused a depression of rat lung tissue cyclic AMP-phosphodiesterase activity. This reduced activity was evident in both the 105,000×g particulate and supernatant fractions prepared from the tissue homogenates. Total calmodulin activity in the lungs from the diabetic rats was also reduced. Calmodulin and phosphodiesterase appeared to be translocated from the particulate to the supernatant fractions during the experimental diabetes. The presence of a heat-stable inhibitor of calmodulin-activated Ca²⁺-sensitive cyclic AMP-phosphodiesterase was evident in the rat lung and its activity was increased during diabetes. The data suggest that supression of the rat lung cyclic AMP-phosphodiesterase activity during experimental diabetes may result from changes in the subcellular distribution and concentration of the modulators of cyclic AMP-phosphodiesterase in rat lung.     

Calcium and calcium slow channel antagonists on cyclic nucleotide levels in the isolated rat heart

An investigation has been carried out into the effects of extracellular calcium on cyclic nucleotide levels in rat ventricle. Increasing extracellular calcium, over the range which directly affected contractility, produced a concentration related increase in the level of cyclic AMP. Positive correlations were found between extracellular calcium and cyclic AMP; and between cyclic AMP and isometrically developed tension. No reciprocal relationships were found for cyclic GMP over the same calcium concentration range. Increasing extracellular calcium above the maximal inotropic concentration did not produce a further increase in cyclic AMP, although increases in cyclic GMP were noted. Calcium slow channel antagonists, nifedipine and manganese both depressed developed tension at the concentrations used, but this effect was not associated with any significant change in cyclic nucleotide levels. Verapamil depressed developed tension and cyclic AMP levels but the effect on cyclic AMP was not concentration related. No calcium slow channel antagonist significantly affected cyclic GMP levels. In electrically paced hearts, increasing rate of stimulation depressed developed tension but had no significant effect on cyclic AMP.     

Cyclic AMP metabolism in pigeon arteries: comparison of atherosclerotic-resistant and -susceptible strains.

Cyclic AMP levels were higher in the aortas of pigeons susceptible to atherosclerosis (White Carneau) than in the aortas of resistant (Show Racer) pigeons. Basal levels, and fluoride-stimulated levels, of adenylate cyclase were similar in arteries of both kinds of pigeons. Cyclic AMP phosphodiesterase levels were similar in arteries from the two pigeon strains, but the cyclic GMP phosphodiesterase levels of the susceptible pigeon arteries were higher. Phosphodiesterase inhibitors produced similar effects in soluble extracts from both kinds of pigeon. Separation methods indicated the presence of the same phosphodiesterases in the arteries of White Carneau and Show Racer. DEAE-cellulose chromatography resolved two forms of phosphodiesterase and a heat-stable activator. Disc-gel electrophoresis resolved three peaks of phosphodiesterase activity; the heat-stable protein activator specifically affects one of these enzymes.     

染氟大鼠成骨细胞内钙调蛋白表达的变化

目的观察不同浓度的氟对体外培养大鼠成骨细胞内钙调蛋白(CaM)表达的影响。方法采用骨组织块法分离培养新生Wistar大鼠颅骨成骨细胞,然后将不同浓度的氟作用于大鼠成骨细胞,染氟培养48 h后,采用半定量RT-PCR方法分析CaM基因的表达,免疫细胞化学法检测CaM蛋白的表达,采用生物化学的方法检测超氧化物歧化酶(SOD)、超氧阴离子自由基、丙二醛(MDA)的含量。结果与对照组比较,各氟组CaM基因及蛋白的表达均显著增加;与对照组相比,各氟组成骨细胞内SOD含量明显降低,超氧阴离子自由基含量明显增高,差异均具有统计学意义,而MDA含量无明显变化。结论氟在一定浓度范围内可引起培养成骨细胞的SOD含量明显降低、超氧阴离子自由基含量明显增高,且对成骨细胞的CaM蛋白的表达具有促进作用。     

TSH, theophylline and cyclic AMP: in vitro thyroid activity in aging rats.

In vitro thyroid accumulation of cyclic 3',5'-adenosine monophosphate (cyclic AMP) and release of triiodothyronine (T₃) and thyroxine (T₄) in response to TSH, theophylline or cyclic AMP treatment was assessed in 60- and 340-day-old male rats. Plasma levels of T₃ and T₄ at the time of sacrifice were determined. Mature animals exhibited significantly lower plasma T₃ and T₄ levels but slightly elevated in vitro secretory rates of T₃ and T₄. TSH stimulation elicited little effect in the mature gland in terms of cyclic AMP accumulation or thyroid hormone release. Conversely, cyclic AMP enhanced in vitro thyroid hormone release in both age groups. The data suggest an age-related alteration in thyroid responsiveness to TSH which in turn may be a function of changes in the thyroidal adenylate cyclase-cyclic AMP-phosphodiesterase system. Evidence is also presented which suggests either cyclic AMP-mediated T₄ to T₃ conversion or a differential action of the nucleotide upon T₃ as compared to T₄ synthesis.     

Characterization of cyclic 3', 5'-nucleotide phosphodiesterase activity in an islet cell tumor of the syrian hamster

During the purification of cyclic nucleotide phosphodiesterase (diesterase) activity from an insulin-secreting islet cell tumor of the syrian hamster, two soluble diesterases (diesterase I and diesterase II) and a particulate diesterase were resolved. Based on gel filtration, the estimated molecular weights of diesterases I and II were approximately 180,000. All of the diesterases had ‘low’ (approximately 1 μM) and ‘high’ (10–20 μM) K_M's for both cAMP and cGMP. A heat-stable protein activator was partially separated from the soluble diesterases upon DEAE-cellulose chromatography. The activator enhanced the activity of the soluble diesterase 2$\text{1}\text{2}$-fold at cyclic nucleotide concentrations of 5 μM. No stimulation of diesterase activity was observed at higher (50–500 uM) substrate concentrations.The following compounds (in order of decreasing potency) inhibited both the soluble and particulate diesterases: papaverine, SQ 20009, xanthine derivatives, diphenylhydantoin, diazoxide, sulfonylureas, nicotinamide and catecholamines. Insulin, proinsulin, prostaglandins E₁ and F_1α, secretin, glucagon, colchicine, glucose, streptozotocin and imidazole were all without effect. Sulfhydryl-containing compounds and a number of amino acids enhanced the activity of soluble and paniculate diesterases. These investigations coupled with studies of adenylate cyclase and protein kinase activities, intracellular cyclic nucleotide levels and insulin secretion will provide a basis for understanding the role of cAMP in insulin secretion.     

Altered expression of the adenine nucleotide translocase isoforms and decreased ATP synthase activity in skeletal muscle mitochondria in heart failure

Exercise intolerance is a component of heart failure (HF) syndrome. We aimed to identify the defects in skeletal muscle mitochondria which may contribute to the development of peripheral myopathy. Subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria were isolated from gastrocnemius muscle of control dogs (N = 5) and dogs with pacing-induced HF (N = 5). The measurement of integrated mitochondrial function (oxidative phosphorylation) and of individual activities of mitochondrial electron transport chain (ETC) complexes was complemented with the assessment of the amount and activity of the components of the phosphorylation apparatus. Both populations of skeletal muscle mitochondria isolated from HF have significantly decreased ADP-stimulated (state 3) respiratory rates with complex I, II and III substrates. The decrease in respiratory rates of skeletal muscle SSM are neither relieved upon collapsing the mitochondrial potential with an uncoupler nor increased in the presence of maximal ADP concentrations showing a defect in the ETC, which needs further investigation. In contrast, respiratory rates of skeletal muscle IFM from HF were relieved with the uncoupler and partially improved in the presence of maximal ADP concentrations. In these IFM, alterations in the phosphorylation apparatus were detected with a decreased amount of ANT isoform 2 and increased amount of isoform 1. The IFM dysfunction may be explained by this shift in ANT isoforms. In conclusion, pacing-induced HF causes a decrease in the oxidative phosphorylation of skeletal muscle mitochondria due to defects in the ETC and phosphorylation apparatus.     

Expression of GRK2 is increased in the left ventricles of cardiomyopathic hamsters

Reduced β-adrenergic responsiveness in the heart is a characteristic feature of heart failure. G protein-coupled receptor kinase 2 (GRK2) phosphorylates β-adrenoceptors in an agonist-dependent manner, causing receptor uncoupling and desensitisation. Elevated levels of both GRK2 mRNA and activity have been shown to occur in the failing human heart (Ungerer et al. (1992) Circulation 87: 454–463). We have analysed levels of GRK2 protein in heart tissue from the cardiomyopathic Syrian hamster CHF 147 and compared these to GRK2 levels in age-matched, non-cardiomyopathic control hamsters (CHF 148). GRK2 protein levels were found to be significantly increased in the left ventricles of the cardiomyopathic hamsters compared to the controls. The relative amounts of GRK2 in the cardiomyopathic hamsters, as compared to normal controls, increased with age from 2-fold at 100 days to 5-fold at 350 days. These animals should provide a useful model for testing the effect of GRK2 inhibitors on the development of heart failure. Received: 6 November 2000 / Returned for revision: 2 January 2001 / Revision received: 11 January 2001 / Accepted: 15 January 2001     

Neurotransmitter and cyclic nucleotide modulation of frog cardiac contractility.

The experiments performed in this study were designed to test the hypothesis that cyclic nucleotides mediate the effects of adrenergic and cholinergic neurotransmitters on frog ventricle. A physiological approach was to measure the twitch tension and contracture tension of ventricular strips in the presence of exogenous neurotransmitter agonists. These observations were correlated to biochemical measurements of cyclic AMP and cyclic GMP levels in ventricular strips exposed to the same physiological conditions. Epinephrine augmented twitch tension, depressed contracture tension, and augmented cyclic AMP levels. Phosphodiesterase inhibitors potentiated the effects of epinephrine. Exogenous cyclic AMP usually mimicked the epinephrine effect on both twitch and contracture tension. Carbachol and acetylcholine depressed twitch tension without affecting contracture, and carbachol transiently augmented cyclic GMP while depressing cyclic AMP levels. These muscarinic agonists inhibited the effects of epinephrine in depressing contracture tension and increasing cyclic AMP levels. Cyclic GMP did not mimic the twitch depressant effect of carbachol but did tend to mimic the cholinergic antagonism of epinephrine. Our observations generally support the idea that cyclic AMP mediates the effects of epinephrine but do not clearly relate cyclic GMP to the action of cholinergic agonists.     

Thyrotrophin and prostaglandin E2 increase calmodulin levels and cyclic AMP phosphodiesterase activity in cultured porcine thyroid cells

Thyrotrophin (TSH) and prostaglandin E2 (PGE2) increased cellular cyclic AMP (cAMP), calmodulin levels and cAMP phosphodiesterase activity in cultured porcine thyroid cells. Dibutyryl cAMP (dbcAMP), a stable analogue of cAMP, increased calmodulin levels and cAMP phosphodiesterase activity. These results indicate that TSH- and PGE2-stimulated increases in calmodulin are mediated by cAMP. This increased concentration of calmodulin in turn stimulates cAMP phosphodiesterase. Double reciprocal plots of cAMP hydrolysis yielded two apparent Michaelis constants (Km); the lower in the 1 mumol/l and the higher in the 10 mumol/l range. Thyrotrophin, PGE2 and dbcAMP increased the values of maximal velocity without changing the Km values.