Cyclic AMP phosphodiesterase in Leydig cell preparations of the rat testis

Enzymatic digestion of the interstitial tissue of early juvenile and adult rat testes resulted in an enrichment of the Leydig cell population. The cells of the intertubular preparation from adult testes were separated by centrifugal elutriation, according to differences in sedimentation velocity, a counter-flow centrifugation technique leading to 70% Leydig cell purity. Using this approach, it was possible to demonstrate that Leydig cells from adult testes contain only low affinity isoenzymes of cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17), an intracellular regulator of cAMP. Starch gel electrophoresis showed that the isozyme of cAMP PDe of Leydig cells is masked in crude testis homogenates due to the relatively low level of these cells in the total population. In Leydig cells, there are two different electrophoretic forms expressed which resemble two of eleven different molecular forms of cAMP PDE demonstrated for comparison in 21 different organs of the adult rat. An interstitial cell preparation from early juvenile testes, with a Leydig cell content of up to 20%, was also investigated electrophoretically with regard to molecular forms of cAMP PDE, the properties of which were characterized by kinetics analysis of cAMP hydrolysis. The results presented are discussed in relation to the onset of testosterone synthesis in Leydig cells of prepubertal rats leading to the initiation of male puberty.     

Protein kinase activation of heparin-releasable lipoprotein lipase in rat heart

An attempt was made to activate the capillary-bound fraction of lipoprotein lipase (LPL) with cAMP-dependent protein kinase catalytic subunit (PKC). Following a 30s washout period, hearts were perfused for 1 min with buffer containing heparin. Medium was collected during the second 30s of heparin perfusion. Addition of PKC+Mg-ATP to this capillary bed perfusate increased LPL activity from 6.84 +/- 0.72 nmol/ml/min to 13.76 +/- 1.12 nmol/ml/min (P less than 0.001). A similar 2-fold increase in activity was observed when results were expressed on a mg protein basis. Removal of serum from, or addition of 1.0M NaCl to, the assay system inhibited PKC-stimulated LPL activity approximately 85%. These results indicate that capillary alkaline LPL can be activated by PKC assayed under experimental conditions free of other TG lipases. Moreover, these findings suggest that the intracellular fraction of LPL can be activated by cAMP and that this activation is mediated through protein phosphorylation by cAMP-dependent protein kinase.     

Effects of cold exposure on heart clearing factor lipase and triglyceride utilization in the rat

The clearing factor lipase activity of the rat heart was measured in animals kept at 4 degrees C for several hours and was compared with that in control animals kept at 25 degrees C. The total activity of the enzyme in the heart increased markedly on exposure to the low temperature, whether the animals were in a fed or a fasted state. The activities of both the heparin-releasable and the heparin-nonreleasable enzyme fractions were usually raised. However, only increases in the former could be correlated satisfactorily with corresponding increases in the capacity of the heart to utilize chylomicron triglyceride fatty acids perfused through it. Cold exposure also raised the plasma clearing factor lipase activity and reduced the plasma triglyceride concentration. These changes may have been due, at least in part, to the alterations in the activity of the tissue enzyme.     

Enhanced cell aggregation in Dictyostelium discoideum by ATP activation of Cyclic AMP receptors.

Cell aggregation in the cellular slime mold Dictyostelium discoideum is mediated by cyclic AMP. In the presence of ATP the onset of cell aggregation is enhanced and cyclic AMP receptors are activated. The number of phosphorylation sites is species dependent, and two main phosphorylated proteins with MW of, respectively, 20,000 and 15,000 are localized.     

Effects of hormones, fasting and diabetes on triglyceride lipase activities in rat heart and liver

Male rats were fasted for 3 days, subjected to streptozotocin-diabetes or injected with L-thyroxine, Kenacort-A40 (corticosteroid) and Synacthen (ACTH). Cardiac heparin-releasable lipoprotein lipase (LPL) activity was increased after fasting, experimental diabetes and all hormone treatments. Cardiac neutral lipase activity was decreased during diabetes and enhanced in the fasted state and by L-thyroxine, corticosteroid and ACTH administration. The close correlation between vascular LPL and tissue neutral lipase with cardiac triglyceride content is in agreement with the contention that tissue neutral lipase is similar to LPL (Hülsmann, Stam and Breeman 1982). Myocardial acid lipase activity was reduced during diabetes and L-thyroxine treatment, increased during fasting and corticosteroid administration and not affected by short-term ACTH treatment. Hepatic acid lipase activity was increased during fasting, diabetes and by L-thyroxine and reduced after corticosteroid and ACTH treatment. The alkaline liver lipase activity was depressed by fasting, experimental diabetes, corticosteroid and ACTH treatment, whereas L-thyroxine induced a slight increase in enzyme activity. The possible mechanism underlying the observed changes in acid, neutral, alkaline, and LPL activities in heart and liver are discussed.     

Cyclic AMP distinguishes between two functions of acidic FGF in a rat bladder carcinoma cell line

The rat bladder carcinoma cell line NBT-II exhibits two completely different responses to acidic FGF (aFGF): at high cell density, aFGF is a potent mitogen whereas at low cell density, aFGF acts as a scattering agent that can convert the epithelial NBT-II cells into fibroblastic- like, motile cells. The basis of the dual action of aFGF has been approached by using substances interfering with the transducing pathways known to be activated by growth factors. Genistein and tyrphostin, two inhibitors of tyrosine kinases, inhibit both cell scattering and mitogenesis induced by aFGF. Conversely, sodium orthovanadate, a potent inhibitor of tyrosine phosphatases can reproduce the two effects of aFGF, indicating that protein tyrosine phosphorylations are determinant in the two pathways. In contrast, transforming growth factor (TGF)-beta 1 is a strong inhibitor of DNA synthesis induced by aFGF but has no effect on cell scattering, providing evidence that the two pathways are divergent. In an attempt to determine the specificity of the pathways of aFGF we found that the level of cAMP, which can be externally elevated, is of pivotal importance in distinguishing between the two transducing pathways leading to either DNA replication or cell dispersion. Forskolin, 8- bromo cAMP, dibutyryl-cAMP, and cholera toxin are all capable of potentiating the mitogenic effect of aFGF while strongly inhibiting its scattering action. Moreover, addition of any of these substances to NBT- II cells converted into fibroblasts immediately induces their reversion towards an epithelial phenotype. These findings support a role for cAMP as a modulator of the effects of aFGF. Moreover, basal cAMP synthesis, which is not affected by aFGF, is higher in sparse than in dense cultures indicating that the level of cAMP depends on the status of the cell. Altogether, these results suggest that establishment and maintenance of the epithelial state require a precise regulation of cAMP level.     

Enzyme-linked immunosorbent assay for rat hepatic triglyceride lipase

A noncompetitive enzyme-linked immunosorbent assay to measure rat hepatic triglyceride lipase (H-TGL) was developed. Antibodies to rat H-TGL were purified from goat antisera by immunoadsorption on an H-TGL-Sepharose 4B column. Routinely, Immulon 2 Removawell strips were coated with the purified antibody overnight at 4 degrees C. After blocking the wells with bovine serum albumin (BSA) for 2 hr at room temperature, standards (0.85 ng/ml-13.1 ng/ml) or samples were added to the wells and were incubated with the bound anti-rat H-TGL overnight at 4 degrees C. The standards and samples had been pretreated with 5-20 mM SDS for 30 min at room temperature and were then diluted so that the final SDS concentration in the assay was 1 mM or less. The pretreatment with SDS was necessary to achieve maximal immunoreactivity. The sample incubation was followed by an overnight incubation at 4 degrees C with an anti-rat H-TGL-horseradish peroxidase conjugate. Rat H-TGL was detected by the color development after the addition of 0.4 mg/ml of o-phenylenediamine in 0.01% H2O2, 0.1 M citrate phosphate, pH 5.0. A linear relationship was obtained between absorbance at 490 nm and the amount of highly purified rat H-TGL used as a standard. Inclusion of 1 M NaCl in the assay buffer (1% BSA, 0.05% Tween 20, 10 mM phosphate, pH 7.4) during the sample and conjugate incubations minimized non-specific interactions. Recoveries of purified rat H-TGL added to a rat liver perfusate sample ranged from 98.6% to 103%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP and steroidogenesis in superovulated rat ovaries

During the gonadotropin stimulated differentiation of ovarian follicles into corpora lutea, the concentrations of total cyclic AMP and protein bound cyclic AMP increased only marginally. In contrast, there was a 10 fold increase in progesterone production. After acute stimulation of luteinized ovaries with choriogonadotropin total cyclic AMP levels increase more than 6 times while bound cyclic AMP and plasma progesterone rose by 80 and 60% respectively. Our results question the role of cyclic AMP in the basal production of progesterone by the ovary, and suggest a relationship between bound cyclic AMP and progesterone synthesis only exists after acute gonadotropin stimulation.     

Effect of colchicine on alkaline triglyceride lipase activity and triglyceride content in rat skeletal muscle

One purpose of this study was to determine if colchicine increased intracellular alkaline triglyceride (TG) lipase activity above control levels in rat skeletal muscle. The second aim was to determine the effects of colchicine treatment on the concentration of TG in skeletal muscle. The results show that colchicine was a potent inducer of alkaline TG lipase activity, increasing enzyme activity approximately twofold in slow-twitch red, fast-twitch red, and fast-twitch white muscle types. It was found that in slow-twitch red soleus and fast-twitch red vastus, the two muscle groups with the highest levels of enzyme activity, 76% or more of enzyme activity resides in the intracellular compartment. These results provide evidence that colchicine blocks the export of alkaline TG lipase from skeletal muscle cells similar to that seen in the heart. The finding that TG were reduced at a time when enzyme activity was elevated suggests that intracellular alkaline TG lipase may be playing a role in the hydrolysis of the intramuscular TG droplet.     

Cyclic AMP decreases the phosphorylation state of myelin basic proteins in rat brain cell cultures

Previous work has suggested that myelin basic proteins are phosphorylated prior to their appearance in the myelin sheath (Ulmer, J. B. and Braun, P. E. (1984) Dev. Neurosci. 6, 345-355). In order to corroborate this finding we have examined the phosphorylation of myelin basic proteins in rat brain cell cultures containing 14-17% oligodendrocytes. Incorporation of 32P into the 14-, 17-, 18.5-, and 21.5-kDa myelin basic proteins was observed in cells incubated with 32P at 7, 14, and 21 days in culture. Myelin basic proteins in 14-day cells incorporated 32P linearly until at least 120 min after the addition of isotope. The apparent half-life of myelin basic protein phosphate groups was determined to be approximately 80 min in pulse-chase experiments. However, this value may be an overestimation due to the presence of significant levels of acid-soluble radioactivity in the cells throughout the chase period. The presence of dibutyryl cAMP or 8-bromo-cAMP in the incubation medium substantially inhibited the incorporation of 32P into the myelin basic proteins at all time points studied. The presence of dibutyryl cAMP in the chase medium in pulse-chase experiments resulted in an increase in the turnover rate of [32P] phosphate in the myelin basic proteins. These results indicate that cAMP decreases the phosphorylation state of myelin basic proteins in oligodendrocytes by inhibiting the phosphorylation and/or stimulating the dephosphorylation of myelin basic proteins.     

Investigating cADPR and NAADP in intact and broken cell preparations

The body of literature characterizing cyclic adenosine diphosphoribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) as Ca2+-mobilizing second messengers is growing apace. However, their unique properties may, for the uninitiated, make them difficult to work with. This article reviews many of the available techniques (and associated pitfalls) for investigating these nucleotide messengers, predominantly focusing upon optical techniques using fluorescent reporters to measure Ca2+ in the cytosol as well as Ca2+ or pH within the lumen of intracellular organelles.     

Effects of diet and age on lipoprotein lipase and hepatic triglyceride lipase activities in the rat

Plasma clearance of triglyceride-rich lipoproteins appears decreased in aged humans and rats and may be due to lowered activities of the lipases responsible for lipid degradation. This study was designed to examine differential effects of age and diet on lipoprotein lipase (LPL) activity of adipose and heart tissue and hepatic triglyceride lipase (HTGL) activity. LPL and HTGL activities were examined in 3- and 13-month-old Sprague-Dawley rats after they had consumed either a high-carbohydrate or a high-fat diet for 14 days. The data were analyzed for age and diet differences by two-way analysis of variance. Although animals in the two age groups consumed diets of equal caloric content, the older rats gained less weight. Rats on the high-carbohydrate diet consumed less calories and gained less weight than the fat fed rats in both age groups. Neither heart nor adipose tissue LPL activity differed when examined for age or diet. HTGL activity levels, while not affected by age, were higher in the carbohydrate fed rats (P = 0.014). Regardless of age group, fasting plasma cholesterol levels were significantly higher in the carbohydrate-fed rats than fat-fed rats (P = 0.002). Thus, the diet effect was much stronger than the age effect for HTGL and plasma cholesterol levels.     

Cyclic AMP and cell division in Escherichia coli.

We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex.     

Cyclic AMP and cell differentiation in amphibian embryonic explants

Conflicting results have been published concerning the effects of cyclic nucleotides on amphibian cell differentiation. Here we report the effects of cyclic adenosine monophosphate (cAMP) and dibutyryl-cyclic adenosine monophosphate (db-cAMP) on isolated explants from late blastulae of Ambystoma mexicanum and Xenopus laevis. Both cAMP and db-cAMP (10(-4)-10(-9) M) promote 'neuralizing' differentiation in Ambystoma explants. Xenopus explants treated with the nucleotides (10(-4), 10(-6), 10(-8) M) LiCl or heparan sulphate only give rise to ciliated aggregates or dissociation. The results confirm observations that different amphibian species react in different ways to activating chemicals.     

Tunicamycin-treated rat heart cell cultures synthesize an inactive nonreleasable lipoprotein lipase

Cells isolated from newborn rat hearts were cultured in the presence of 100 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Lipoprotein lipase activity was present in an intracellular and heparin-releasable pool and was also secreted into the culture medium. Treatment of the cultures with 5 micrograms/ml tunicamycin caused almost complete loss of lipoprotein lipase activity in all three compartments. In control cultures, immunoblotting of lipoprotein lipase derived from all three pools revealed a single band of lipoprotein lipase with an apparent Mr of 56,000. In the tunicamycin-treated cultures, the enzyme appeared only intracellularly and had an apparent Mr of 49,000. No immunoreactive enzyme was found in the medium. Thus, glycosylation of lipoprotein lipase in heart cell cultures is mandatory for enzyme activity and translocation from an intracellular to the heparin-releasable pool and for secretion into the medium.     

Reprogramming a broken heart

Fibrosis resulting from cardiac injury presents a major challenge to restoring heart function after myocardial infarction. Two recent papers in Nature report successful in vivo reprogramming of fibroblasts to cardiomyocytes in injured mouse hearts (Qian et al., 2012; Song et al., 2012), resulting in improved cardiac function and reduced scar formation.