Changes in some properties of 3':5'-AMP-dependent protein kinase from rat skeletal muscles during physical exercise

During 6-week training of rats the activity of isoenzymes I and II of soluble 3':5'-AMP-dependent protein kinase increases by 22 and 33%, respectively. A long-term physical load does not cause any significant changes in the activity of both isoenzymes. The maximal activity of the isoenzymes from skeletal muscles of the control and experimental rats is observed at the same concentrations of 3':5'-AMP and pH of 6,0-6,5. During training and under physical load the apparent Km values for ATP of both isoenzymes of 3':5'-AMP-dependent protein kinase do not change significantly, whereas that of V shows an increase. The apparent Km and V values for the histone increase for isoenzyme I obtained from skeletal muscles of trained rats both at rest and under physical load. In case of isoenzyme II the Km value for the histone decreases, while that of V remains unchanged. The changes in the properties of isoenzymes I and II of 3':5'-AMP-dependent protein kinase from skeletal muscles suggest the participation of the enzyme in adaptation to systematic muscular activity.     

Studies on adenylate cyclase-cyclic AMP system of the myopathic hamster (UM-X7.1) skeletal and cardiac muscles.

Cyclic AMP content, adenylate cyclase (EC 4.6.1.1) activity and phosphodiesterase I (EC 3.1.4.1) activity of the hind leg skeletal muscle and cardiac muscle in 60- and 150-day-old normal and myopathic (UM-X7.1) hamsters were examined. In 60-day-old myopathic animals, cardiac cyclic AMP levels were higher and phosphodiesterase I activity was lower, without any changes in the basal adenylate cyclase activity, whereas in 150-day-old myopathic hamsters, cardiac cyclic AMP and basal adenylate cyclase activity were lower, without any changes in the homogenate phosphodiesterase I activity. On the other hand, basal adenylate cyclase and phosphodiesterase I activities in the skeletal muscle homogenate from 60- and 150-day-old myopathic animals were not different from the normal values but the skeletal muscle cyclic AMP levels were significantly less in 60-day-old myopathic hamsters only. The plasma cyclic AMP levels in 60-day-old myopathic hamsters, unlike 150-day-old myopathic animals, were higher than the normal. Although these results reveal differences in myopathic cardiac and skeletal muscles, it is concluded that changes in adenylate cyclase-cyclic AMP system in myopathy are dependent upon the degree of disease.     

Effect of immobilized adrenaline on the state of the adenylate cyclase system in the cardiac and skeletal muscles of rats in the recuperative period after physical exertion

It is shown that in the rehabilitation period after a single long physical load the adenylate cyclase activity and cAMP content in the skeletal muscle are low and normalize by the 48th and 36th h, respectively. These indices in the myocardium in the nearest rehabilitation period increase considerably, gradually normalizing by 24th and 48th h, respectively. Immobilized epinephrine accelerates normalization of the adenylate cyclase activity and cAMP content in the skeletal muscle and in myocardium in the rehabilitation period after physical load, the phosphodiesterase activity being unchanged.     

Two forms of cyclic nucleotide phosphodiesterase and Ca-dependent protein regulator from rabbit skeletal muscles]

In rabbit skeletal muscle extracts the activity of phosphodiesterase practically insensitive to the increase of Ca2+ concentration from 10(-8) M up to 10(-5) M. The Ca2+-dependent protein regulator is separated from phosphodiesterase at the stage of isolation and purification. The activity of phosphodiesterase devoid of the protein regulator is inhibited by Ca2+ (10(-5)--10(-3) M). An addition of Ca2+-dependent regulator protects the enzyme against inhibition by Ca2+. The Km values for 3',5'-AMP (5 mkM) and 3',5'-GMP (13 mkM) appear to be close; however, the maximal hydrolysis rates for these nucleotides differ considerably (14,0 and 0,25--0,50 nmoles/min/mg of protein). The hydrolysis of 3',5'-AMP is increased 1,6--3,2-fold under the effect of 3',5'-GMP and that of 3',5'-GMP is increased 1,8--2,7-fold under the effect of 3',5'-AMP. Using ion-exchange chromatography it was shown that only 1% of the total activity of skeletal muscle phosphodieterase belongs to the phosphodiesterase sensitive to the activating effect of Ca2+-dependent regulator the activity of this enzymic form is increased 4--5 fold. The Ca2+-dependent regulator of skeletal muscles is inactivated under the effects of trypsin and during gel-filtration is eluted together with the Ca2+-dependent regulator from the heart. The amount of Ca2+-dependent regulator in skeletal muscles is 30 times as low as that in brain and 3 times as low as that in the heart of the rabbit.     

Activities and some properties of adenylate cyclase and phosphodiesterase in muscle, liver and nervous tissues from vertebrates and invertebrates in relation to the control of the concentration of adenosine 3'':5''-cyclic monophosphate.

1. The basal and fluoride-stimulated activities of adenylate cyclase, and the maximal activities of 3':5'-cyclic AMP phosphodiesterase and 3':5'-cyclic GMP phosphodiesterase, together with the Km values for their respective substrates, were measured in muscle, liver and nervous tissues from a large range of animals to provide information on the mechanism of control of cyclic AMP concentrations in these tissues. High activities of adenylate cyclase and cyclic AMP diesterase are found in nervous tissues and in the more aerobic muscles (e.g. insect flight muscles, cardiac muscle and some vertebrate skeletal muscles). The activities of these enzymes in liver are similar to those in the heart of the same animal. The Km values for the enzymes from different tissues and animals are remarkably similar. 2. The comparison of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities suggests that in vertebrate tissues only one enzyme (the high-Km enzyme), which possesses dual specificity, exists, whereas in invertebrate tissues there are at least two phosphodiesterases with separate specificities. 3. A simple quantitative model to explain the control of the steady-state concentrations of cyclic AMP is proposed. The maximum increase in cyclic AMP concentration predicted by comparison of basal with fluoride-stimulated activities of adenylate cyclase is compared with the maximum increases in concentration produced in the intact tissue by hormonal stimulation: reasonable agreement is obtained. The model is also used to predict the actual concentrations and the rates of turnover of cyclic AMP in different tissues and, where possible, these values are compared with reported values. Reasonable agreement is found between predicted and reported values. The possible physiological significances of different rates of turnover of cyclic AMP and the different ratios of high- and low-Km phosphodiesterases in different tissues are discussed.     

Effect of isobutylmethylxanthine on the adenylate cyclase system of the myocardium and skeletal muscles of the rat during the recovery period following physical loading

The in vivo experiments established that isobutyl methyl xanthine inhibits phosphodiesterase activity and activates adenylate cyclase in the skeletal muscles and myocardium. The preparation prevents phosphodiesterase activity from increasing due to physical exercises. Against a background of the latter it activates adenylate cyclase and prevents the cAMP level from declining in skeletal muscles. In the myocardium this effect of isobutyl methyl xanthine is less pronounced.     

Regulation of urocaninase activity in the liver: role of 3',5'-AMP]

A dependence of rat liver urocaninase activity on the agents affecting the adenylate cyclase system was studied in vitro and in vivo. Urocaninase is considerably activated after the injection of glucagone, NaF, theophylline and 3',5'-AMP. Under conditions optimal for the protein kinase activity of phosphorylase the urocaninase of liver extracts was activated 7-fold on the average. The nezyme retains its activity after gel-filtration through Sephadex G-25 and is capable of inactivation in the presence of Mg2+ and of reactivation after addition of ATP and 3',5'-AMP. These data suggest a possibility of regulation of mammalian liver urocaninase activity by 3',5'-AMP-dependent phosphorylation of the enzyme. Derivatives of hypoxanthine (theophylline and caffeine) in concentration 10(-4) M activate urocaninase in liver extracts 2--3 and 1.5-fold respectively. The activation is probably not due to the 3',5'-AMP phosphodiesterase inhibition, since another phosphodiesterase inhibitor--papaverine--has no activating effect on urocaninase.     

Myofibrillar and membrane-bound enzymes in skeletal muscle from myodystrophic mice.

1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. The proteolytic activity of myofibrils isolated from myd/myd was significantly higher than controls. 4. There was no significant difference in Ca2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. Mg2+ ATPase and Na(+)+K(+)-ATPase of myodystrophic SL showed significant increase compared to controls. 6. Isoproterenol stimulated adenylate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. GTP+isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X-100. cGMP and cAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control.     

Change in some properties of soluble cAMP-dependent protein kinase from rat myocardium during physical exercise

During 1.5- and 3-months physical exercise the activity of soluble 3':5'-AMP-dependent protein kinase of the rat increases 2.4- and 4.6-fold, respectively. The maximal activity of the enzymes from heart muscles of control and experimental animals is observed at the same concentration of 3':5'-AMP (10(-6) M) and pH (6.8-7.0). The degree of changes in V and apparent Km for ATP and histone H2b depend on the duration of physical exercise. The changes in the properties of soluble 3':5'-AMP-dependent protein kinase suggest the participation of the enzyme in adaptation to systematic muscular activity.     

Effect of growth conditions on the activity of the enzymes of cyclic 3':5'-AMP synthesis and decay in phototrophic bacteria]

Activity, ratio and summary content of cyclic AMP enzymes, adenylate cyclase and phosphodiesterase varied depending on growth conditions of phototrophic bacteria (Rhodospirillum rubrum and Rhodopseudomonas palustris). It suggests, that membrane-bound and soluble enzymes carry different functions. The increase of adenylate cyclase under chaning growth conditions was usually accompanied by the increase of phosphodiesterase. Sharp increase of both enzymes activity was observed when bacteria were growth in aerobic conditions. The activity of both enzymes in chromatophores was 2.8-fold higher when bacteria were grown in the light in anaerobic conditions, than in chromatophores of bacteria grown under stationary aerobic conditions in the light. It is suggested that 3':5' AMP can participate in autotrophic carbon assimilation or in the synthesis of pigments and other components of bacterial photosynthetizing apparatus. Substitution of NH4+ into NO3- and glutamate under the growing of R. rubrum in anaerobic conditions in the light resulted in the increase of the enzymes activities, which is the evidence of possible role of 3':5' AMP in mineral nitrogen uptake and nitrogen fixation. Glutamate concentration of 4 g/l stimulated the enzymes both in vivo and in vitro. The data obtained suggest that 3':5' AMP can carry multiple functions, participating in regulation of a number of metabolic processes in photorophic bacteria.     

Differential regulation of beta-adrenergic receptor-coupled adenylate cyclase by thyroid hormones in rat liver and heart: possible role of corticosteroids

Thyroid hormone regulation of beta-adrenergic receptor-coupled adenylate cyclase activity was studied in rat liver and heart particulate fractions. Thyroidectomy (Tx) increased isoproterenol-stimulated cAMP accumulation in the liver and decreased it in the heart. Administration of L-thyroxine (L-T4) or L-3,3',5-triiodothyronine (L-T3) reversed these changes in both liver and heart. The changes observed in liver beta-receptor-coupled adenylate cyclase activity after Tx were similar to those reported after adrenalectomy (ADX). Thus the hypothesis was considered that these changes with altered thyroid status are produced indirectly through alteration in adrenal corticosteroids. Hydrocortisone in Tx rats decreased liver isoproterenol-stimulated adenylate cyclase activity but had no significant effect on the heart. Serum corticosterone levels were decreased significantly (by 34%) in Tx rats, as compared to euthyroid rats. Administration of L-T4 to Tx rats doubled the serum corticosterone levels. In Tx-ADX rats, L-T4 had no significant effect on liver beta-receptor-coupled adenylate cyclase. However, L-T4 significantly increased heart beta-receptor-coupled adenylate cyclase in these animals. Dexamethasone, but not deoxycorticosterone, decreased liver isoproterenol-stimulated cAMP accumulation in Tx animals to the same extent as was observed with L-T4 and hydrocortisone. Thus overall the results indicate that in the liver, as opposed to the heart, thyroid hormones regulate beta-adrenergic receptor-coupled adenylate cyclase indirectly through corticosteroids. Glucocorticoid rather than mineralocorticoid activity seems to be responsible for this regulation.     

Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

A MODULATING ROLE OF PITUITARY-ADRENAL AXIS IN CEREBRAL METABOLISM OF ADENOSINE 3'',5''-MONOPHOSPHATE

Abstract— The effect of adrenalectomy or hypophysectomy on the metabolism of adenosine 3',5'-monophosphate (cyclic AMP) in the cerebral cortex of male Wistar rats was investigated.
The bilateral removal of adrenal glands reduced significantly the activity of cerebral adenylate cyclase [EC 4.6.1.1]. whereas that of cyclic 3'.5'-nucleotide phosphodiesterase [EC 3.1.4.17] remained unchanged. The formation of cyclic AMP measured in cerebral cortical slices from adrenalectomized or hypophysectomized rats was also diminished. Decreases in the activity of adenylate cyclase and formation of cyclic AMP following adrenalectomy were antagonized by in vivo administration of dexamethasone or aldosterone, while those observed in hypophysectomized rats were restored by ACTH or dexamethasone. It is suggested that the pituitary adrenal axis has a modulating role in the metabolism of cerebral cyclic AMP, possibly by changing adenylate cyclase activity.     

Effect of cyclic 3':5'-AMP derivatives prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture.

The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N6, O2'-dibutyryl cyclic 3':5'-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process, malignant trophoblast cultures were incubated with 3':5'-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmunoassayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretion of hCG, the N6--and O2'-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3':5'-GMP (cGMP), dbcGMP, 5'-AMP, adenosine, L-epinephrine and prostaglandins E1, E2, F1a and F2a were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast.     

Theoretical analysis of the consequences of cyclic nucleotide phosphodiesterase negative co-operativity. Amplification and positive co-operativity of cyclic AMP accumulation.

Most tissues contain multiple forms of cyclic nucleotide phosphodiesterases (3':5'-cyclic-nucleotide 5' nucleotidohydrolase, EC 3.1.4.17). Consequently, in most, if not in all, tissues, substrate-velocity curves deviate from Michaelian kinetics and exhibit an apparent negative co-operativity. We have studied the possible theoretical consequences of this property on the quantitative features of cyclic AMP accumulation in response to activation of adenylate cyclase. Negative co-operativity of phosphodiesterases tends to generate a "positively co-operative" cyclic AMP accumulation curve. It amplifies the stimulation of cyclic AMP accumulation as compared with the stimulation of cyclic AMP synthesis. It enhances the sensitivity of cyclic AMP accumulation to slight variation of phosphodiesterase maximal velocity. It tends to shift the cyclic AMP accumulation curve to higher concentrations of stimulator as compared with the adenylate cyclase activation curve. This accounts for much of the data in the literature of hormonal effects on phosphodiesterase activity. It shows that the characteristics of cyclic nucleotide phosphodiesterases are as important as those of adenylate cyclase in determining the response of the system.     

Gossypol modulation of nucleotide metabolizing enzymes in the reproductive tract of male rats

The activities of several pivotal nucleotide metabolizing enzymes from the testis and vasal sperm of rats treated for 7 wk with 0, 20 or 30 mg X kg X day gossypol acetic acid were examined. Total testicular lactate dehydrogenase (LDH) activity increased 40% above control in the highest treatment group examined. However, the specific activity of the testis-specific isozyme of LDH, LDH-C4, decreased to 50 and 20% of control in the 20 and 30 mg X kg X day treatment groups, respectively. Basal soluble adenylate cyclase from a 100,000 X g supernatant of testis homogenate exhibited a 25% decrease in activity only in the 30-mg treatment group. Basal adenylate cyclase activity in the testicular membrane fraction increased 20 to 30% above control in response to gossypol administration. Testis membranes from the 20- and 30-mg treatment group exhibited a 2- and 4-fold greater activation of adenylate cyclase by guanine nucleotides. In vitro dose-response curves showed a half-maximal inhibitory concentration (IC50) for inhibition of soluble testicular adenylate cyclase by gossypol of 400 microM in each treatment group. Caudal epididymal sperm adenylate cyclase activity decreased to 25% of control levels in gossypol-treated animals, and the in vitro sensitivity of the enzyme to the inhibitory effects of gossypol increased 4-fold. IC50 values for gossypol inhibition of sperm adenylate cyclase decreased from 200 microM in control animals to 75 and 50 microM in the 20 and 30 mg X kg X day treatment groups, respectively. Cyclic adenosine 3':5' monophosphate phosphodiesterase activity in caudal sperm increased 6-fold in the 20- and 30-mg treatment groups. These results demonstrate that nucleotide metabolizing enzymes in sperm are major targets for the actions of gossypol and provide a possible mechanism for the inhibition of normal sperm function by this compound.     

Triglyceride lipase activity and fatty acid transport in physical endurance in rats adapted to the muscular activity

Aerobic training led to enhancement of lipase activity in type IIA type muscles. Still more obvious changes were found in rats trained to aerobic swimming with maximal intensity. In latter activity, a rise of the fatty acid-binding protein (FABP) was revealed in types I and IIA skeletal muscles. These adaptive changes led to enhancement of lipid metabolism. It was also shown that the FABP content decreased after physical exercise more obviously in the trained animals due, probably, to their substance turnover enhancement.     

Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.