Effect of cold exposure on liver and muscle cAMP content and cAMP phosphodiesterase activity

Adenosine 3',5'-cyclic monophosphate (cAMP) concentration and 3',5'-cyclic-nucleotide phosphodiesterase (PDE) activity were measured in skeletal muscle, heart, and liver of rats exposed to 1, 3, 5, and 7 days of cold. Cyclic nucleotide concentration increased in fast-twitch red muscle at the same time that PDE activity was decreasing. Nucleotide concentration and enzyme activity of slow-twitch red muscle were not altered by the cold exposure. The PDE activity of fast-twitch white muscle was elevated approximately 50% above control after 1 and 3 days of cold exposure. By the 5th day in the cold, white muscle PDE activity had returned to control levels and remained there through the 7th day of experimentation. cAMP concentration in hearts of cold-exposed rats was significantly (P less than 0.01) elevated above control at all time points measured. Myocardial PDE activity was elevated above control (P less than 0.05) at 1 and 3 days of cold exposure but returned to control levels by the 5th day in the cold. Hepatic cAMP and PDE activity were elevated above control at all time points analyzed. These data suggest that changes in cyclic nucleotide metabolism play a role in attaining homeostasis during acute cold exposure.     

Relationship between myometrial cyclic nucleotide phosphodiesterase (PDE) activity and the RNA/DNA ratio at various stages of gestation in primates

Cyclic AMP and cGMP PDE activities were assayed in crude homogenates prepared from biopsies excised between day 39-162 of gestation (normal length of gestation, 165 days) in outer and inner layers of the macaque myometrium. In both layers, kinetic analysis of PDE indicated high (app Km approximately equal to 2 X 10(-6)M) and low (app Km approximately equal to 2 X 10(-5)M) affinity component for each substrate. Measured in high affinity conditions, specific activities were increased around day 40 and beyond day 130 of gestation. By contrast, low values were observed between days 50 and 100. No significant differences were observed between outer and inner layers. In both layers, the RNA/DNA ratio, which presumably reflects the rate of protein synthesis, culminated at the same time as the PDE activity. These variations were observed in the myometrium at specific stages of gestation. In late pregnancy, the human myometrium also displayed biphasic kinetics for cAMP and cGMP PDE activities. Non-human primates may be a partially representative model of what happens in the human myometrium.     

Expression of adenylate cyclase and phosphodiesterase in development of temperature-sensitive mutants with impaired metabolism of cAMP in drosophila melanogaster

The mode of the developmental expression of adenylate cyclase (AC) and phosphodiesterase (PDE) in D melanogaster indicates that PDE plays the major role in the maintenance of a certain level of cAMP in postembryonic development, while both enzymes function in concert in imago. The ts-mutants ts155 and ts622, characterized upon their isolation as having an increased cAMP content and normal PDE activity, manifest high levels of AC activity from the third day of imago life. The levels of PDE activity characteristic for adult mutants with altered enzyme activity (low in ts66 and ts980, high in ts398) are manifested in ts980 from larval instar II, and from the larval instar III in ts398 and ts66. Data on the dependence of PDE activity in adults upon temperature of incubation, being in agreement with the expectations for a ts-mutation in a gene coding for a form of PDE in case of ts66, suggest that ts398 affects not the enzyme-coding gene but rather one for an activator protein. The fact that in ts398 (the polyphasic ts-lethal mapping to 1-38.9) 1) AC activity is somewhat higher than normal at 22°C and is readily activated at 29°C, 2) activity of PDE-I assayed in heat-pretreated homogenates is higher than normal, 3) that boiled extracts of ts398 are potent activators of the wild type and of its own PDE-I indicates that it is a mutation affecting calmodulin, which is known to be stable at boiling and capable of activating both AC and PDE-I. Data on Ca²⁺ and EGTA effects suggest that the mutation presumably increases Ca²⁺-binding activity of calmodulin, ts980 and ts622, in which ts-lethality could be produced only by certain doses of haloperidol and triftazine, appear to be lethal in compounds with ts398, thus indicating that these mutations could affect the same calmodulin-controlling gene.     

Increase in the amount of adenylate cyclase in rat gastrocnemius muscle after denervation

After section of the sciatic nerve, the basal adenylate cyclase (AC) activity in rat gastrocnemius muscle increased 6-7 times per membrane protein and about 2 times per whole muscle in the following 30 or 40 days. The AC activity in the muscle 30 days after denervation was increased about 4 times by forskolin. Calcitonin gene-related peptide (CGRP) also increased the adenylate cyclase activity in the denervated muscle. The binding of [3H]-forskolin (10nM) to cells isolated from gastrocnemius muscle was examined to determine the amount of AC molecules. Inhibition of [3H]-forskolin binding by increasing amounts of unlabeled forskolin gave a sigmoid curve with a IC50 value of 3 x 10(-7) M. Results showed that the number of [3H]-forskolin binding sites per cell was higher on the denervated side than on the control side, like the basal AC activity. The IC50 values for inhibition by unlabeled forskolin of binding of [3H]-forskolin were similar to muscles on the control and denervated sides. These results suggest that an increase in the AC activity induced by denervation was due to an increase in the numbers of AC molecules in the muscle.     

Age-related alterations in cyclic nucleotide phosphodiesterase activity in dystrophic mouse leg muscle

Previous reports have described both increased and decreased cyclic nucleotide phosphodiesterase (PDE) activity in dystrophic muscle. Total PDE activity was measured in hind leg muscle from a mouse model of Duchenne muscular dystrophy (mdx) and a genetic control strain at 5, 8, 10, and 15 weeks of age. Total PDE activity declined in fractions isolated from mdx muscle over this time period, but was stable in fractions from control mice. Compared with age-matched controls, younger mdx muscle had higher cAMP and cGMP PDE activity. However, at 15 weeks, fractions from both strains had similar cGMP PDE activity and mdx fractions had lower cAMP PDE activity than controls. Particulate fractions from mdx muscle showed an age-related decline in sensitivity to the PDE4 inhibitor RO 20-1724. A similar loss of sensitivity to the PDE2 inhibitor erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA) was seen in a particulate fraction from mdx muscle and to a lesser degree in control muscle. These results suggest that the earlier disagreement regarding altered cyclic nucleotide metabolism in dystrophic muscle may be due to changes with age in PDE activity of dystrophic tissue. The age-related decline in particulate PDE activity seen in dystrophic muscle appears to be isozyme-specific and not due to a generalized decrease in total PDE activity.     

Expression of adenylyl cyclase mRNAs in the denervated and in the developing mouse skeletal muscle

Changes in theactivity and in the expression of adenylyl cyclase (AC) were examinedin mouse skeletal muscle after denervation and during development. Fourisoforms of AC (AC2, AC6, AC7, and AC9) were detected by Northern blotanalysis in gastrocnemius muscle, AC9 being the most abundant. Afterdenervation, the levels of AC2 and AC9 mRNA decreased, whereas those ofAC6 and AC7 increased. AC activity in response to severalneurotransmitters was increased after denervation. During development,AC activity was high in fetus and neonate and declined in the adult;the sensitivity of AC activity to various neurotransmitters was thehighest on the third postnatal day. The levels of AC6 and AC7 mRNAswere high on the third postnatal day and then decreased in adult,paralleling the decline in AC activity. All the characteristics of ACexpression and activity in fetus and neonate resembled those observedin denervated adult muscle. These results indicate that changes in ACactivity and AC mRNAs play an important role in the various physiopathological states of skeletal muscle, especially during muscleatrophy.

     

Cyclic nucleotide-mediated regulation of vascular smooth muscle cell cyclic nucleotide phosphodiesterase activity

Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents.     

Myosin heavy chain isoforms expression and cyclic AMP concentrations in hypoxia-induced hypertrophied right ventricle in rats

We have previously demonstrated that the relative expression of myosin heavy chain-beta (MHC-β) in both ventricles of rats exposed to long-term hypobaric hypoxia correlated significantly with the relative ventricular mass. In the present study, we investigated whether an increased expression of MHC-β was accompanied by a reduction in cyclic AMP (cAMP) activity in hypoxia-induced hypertrophied right ventricle (RV). We used male Wistar–Kyoto rats born and raised at simulated altitudes (2200 m: H2 group or 4000 m: H4 group) compared to age-matched sea level controls (SC group). There were no significant differences between the groups in basal and forskolin-stimulated adenylyl cyclase (AC) activities. The basal and IBMX-inhibited phosphodiesterase (PDE) activities were slightly higher in both hypoxic groups (p>0.05), except that the H2 group had a higher basal PDE activity than the SC group (p<0.05). The AC/PDE activity ratios were significantly decreased in both hypoxic groups (p<0.05), suggesting that low concentrations of cellular cAMP were maintained in the RV under hypoxic conditions. However, there were no correlations between MHC-β expression and either AC activity, PDE activity, or AC/PDE activity ratio. These results provided evidence against the causal role for cAMP concentration in the expression of MHC-β associated with hypoxia-induced ventricular hypertrophy.     

Some features of cyclic adenosine monophosphate metabolism in mouse liver and hepatoma 22]

The levels of cyclic adenosine monophosphate (cAMP) and two forms of cAMP phosphodiesterase with low (PDE1) and high (PDE2) affinity for the substrate were determined in homogenates from mouse liver and transplanted hepatoma 22. The level of cAMP in the tumour is 3 times lower than that in liver. By te kinetic parameters (Vmax, Km, pH optimum) adenylate cyclase from tumour does not show any significant differences as compared to the liver enzyme; the enzyme from hepatoma is, however, more sensitive to activation by F- ions. The activities of adenylate cyclase in liver and tumour cells are the same. Phosphodiesterases of cAMP from tumour and liver cells are similar in their Km values (3,3-10(-4) M for PDE1 and 2-10(-6) M for PDE2); however, the maximal and real rates of cAMP hydrolysis in hepatoma are much higher than in liver. The fact that both cAMP phosphodiesterase activities have similar dependence on Mg2+ and Ca2+ concentrations, suggests that PDE1 is a latent form of PDE2. In tumour cells the equilibrium between these two forms is probably shifted towards the enzyme with high affinity for the substrate. The results suggest that a decreased cAMP level in hepatoma cells (as compared to the liver) is due to the activation of PDE2.     

Hepatic adenosine-3',5'-monophosphate metabolism in newborn rats irradiated during the period of organogenesis]

The adenylate cylcase (AC) and phosphodiesterase (PDE) activities, as well as the 3'5'-AMP (cAMP) fund were studied in the liver of newborn rats, intact, and after irradiation on the 9th day of the embryonic development. A decrease of AC and PDE activities was noted with a dose of 50 r. The stationary level of cAMP in the tissue remained unchanged. The adrenaline-stimulated AC activity only tended to decrease in case of irradiation. As suggested, during critical periods of development, in the presence of the hormone inductor in the liver of irradiated rats, conditions can be created for an increase of the cAMP pool.     

Inhibitor of adenosine 3',5'-monophosphate binding and protein kinase activity in leucocyte lysosomes

In contrast to other tissues (e.g. brain, heart), no cAMP dependent protein kinase activity and little cAMP-binding activity could be detected in crude homogenates of purified human PMN leucocytes. This was due to the presence of an inhibitor of cAMP binding and protein kinase activity in PMN leucocytes. Since the inhibitor was entirely segregated in PMN lysosomes (rich in β-glucuronidase and acid phosphatase), lysosomefree supernatants yielded cAMP-dependent protein kinase (> 5-fold stimulation with 5 μM cAMP) and considerable cAMP binding activity. The inhibitor was not dialyzable, and unlike the usual protein kinase modulators, was heat-labile. Preparations of beef-heart protein kinase, treated with the PMN inhibitor, lost cAMP-binding and protein kinase activities simultaneously. The presence of this lysosomal inhibitor may invalidate studies of cAMP binding and protein kinase activities in crude homogenates prepared from lysosome-rich tissues.     

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres.

Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.     

Antioxidant Defense Systems in the Brains of Type II Diabetic Mice

Abstract: The specific activities of superoxide dismutase, catalase, and glutathione S -transferase (μ subtype) were significantly lower in the brains of mice with type II diabetes than in the brains of control mice. On the other hand, the specific activity of glutathione peroxidase was unaltered. The concentration of vitamin E, but not that of total glutathione and ascorbate, was increased in the brains of the type II diabetic mice. The relative amount of polyunsaturated fatty acids (as determined with soybean lipoxygenase) was increased in whole brains and crude synaptosomal membranes of the type II diabetic mice. Endogenous levels of thiobarbituric acid-positive material were decreased in both whole brain homogenates and crude synaptosomal membranes of the db/db mice. Susceptibility of lipids within whole brain homogenates and crude synaptosomal membranes of mice with type II diabetes to peroxidation with iron/ascorbate was also markedly decreased compared with that of controls. Vitamin E is known to quench lipid peroxidation. Therefore, decreased lipid peroxidation in the type II mouse brain may be due to increased vitamin E content.     

Increased endocytotic and lysosomal activities in denervated type I and type II muscle fibres

Summary Previous work has shown that increased endocytotic and lysosomal activities occur in the endplate region of denervated skeletal muscle fibres. This, however, does not engage all fibres of a muscle at a given time after denervation. The present study was carried out in order to determine if both type I (slow) and type II (fast) muscle fibres can react to denervation by increased endocytotic and lysosomal activities. Uptake of horseradish peroxidase as a marker for endocytosis was studied in conjunction with acid phosphatase staining for lysosomal activity in type I and type II fibres of the denervated mouse hemidiaphragm. Fibre typing was performed using a monoclonal antibody against fast skeletal myosin and by adenosine triphosphatase staining. The results show that increased endocytosis and lysosomal activation occur in both type I and type II fibres after denervation.     

PKA-dependent activation of PDE3A and PDE4 and inhibition of adenylyl cyclase V/VI in smooth muscle

Regulation of adenylyl cyclase type V/VI and cAMP-specific, cGMP-inhibited phosphodiesterase (PDE) 3 and cAMP-specific PDE4 by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) was examined in gastric smooth muscle cells. Expression of PDE3A but not PDE3B was demonstrated by RT-PCR and Western blot. Basal PDE3 and PDE4 activities were present in a ratio of 2:1. Forskolin, isoproterenol, and the PKA activator 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole 3',5'-cyclic monophosphate, SP-isomer, stimulated PDE3A phosphorylation and both PDE3A and PDE4 activities. Phosphorylation of PDE3A and activation of PDE3A and PDE4 were blocked by the PKA inhibitors [protein kinase inhibitor (PKI) and H-89] but not by the PKG inhibitor (KT-5823). Sodium nitroprusside inhibited PDE3 activity and augmented forskolin- and isoproterenol-stimulated cAMP levels; PDE3 inhibition was reversed by blockade of cGMP synthesis. Forskolin stimulated adenylyl cyclase phosphorylation and activity; PKI blocked phosphorylation and enhanced activity. Stimulation of cAMP and inhibition of inositol 1,4,5-trisphosphate-induced Ca(2+) release and muscle contraction by isoproterenol were augmented additively by PDE3 and PDE4 inhibitors. The results indicate that PKA regulates cAMP levels in smooth muscle via stimulatory phosphorylation of PDE3A and PDE4 and inhibitory phosphorylation of adenylyl cyclase type V/VI. Concurrent generation of cGMP inhibits PDE3 activity and augments cAMP levels.     

Increased muscle calcium. A possible cause of mitochondrial dysfunction and cellular necrosis in denervated rat skeletal muscle.

Mitochondrial preparations derived from denervated rat skeletal muscle and paired controls were characterized with respect to their ability to take up externally added Ca2+. The denervated and control muscle homogenates and mitochondrial [Ca2+] were also determined. Our data indicate that the denervated mitochondria are able to take up less Ca2+ than the controls before uncoupling occurs. This defect is associated with elevated [Ca2+] in homogenate and mitochondrial fractions in the denervated state. The causal relationship between Ca2+ overload, mitochondrial functional damage and cell necrosis is discussed.     

Expression and activity of cAMP phosphodiesterase isoforms in pulmonary artery smooth muscle cells from patients with pulmonary hypertension: role for PDE1

Pulmonary hypertension (PHT) is associated with increased vascular resistance due to sustained contraction and enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC); the abnormal tone and remodeling in the pulmonary vasculature may relate, at least in part, to decreased cyclic nucleotide levels. Cyclic nucleotide phosphodiesterases (PDEs), of which 11 families have been identified, catalyze the hydrolysis of cAMP and cGMP. We tested the hypothesis that PASMC isolated from patients with PHT, either idiopathic pulmonary arterial hypertension (IPAH) or secondary pulmonary hypertension (SPH), have increased expression and activity of PDE isoforms that reduce the responsiveness of agents that raise cellular cAMP. Real-time PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMC from both IPAH and SPH patients compared with control PASMC. Consistent with this enhanced expression of PDEs, agonist-stimulated cAMP levels were significantly reduced in IPAH and SPH PASMC unless a PDE inhibitor was present. The use of specific PDE inhibitors revealed that an increase in PDE1 and PDE3 activity largely accounted for reduced agonist-induced cAMP levels and increased proliferation in IPAH and SPH PASMC. Treatment with PDE1C-targeted small interference RNA enhanced cAMP accumulation and inhibited cellular proliferation to a greater extent in PHT PASMC than controls. The results imply that an increase in PDE isoforms, in particular PDE1C, contributes to decreased cAMP and increased proliferation of PASMC in patients with PHT. PDE1 isoforms may provide novel targets for the treatment of both primary and secondary forms of the disease.     

Patterns of cyclic AMP phosphodiesterases during rat testis development

Summary Cyclic AMP phosphodiesterase (PDE; E.C.: 3.1.4.17) plays a crucial role in the regulation of intracellular cAMP levels arising from the hormonal activation of membrane-bound adenylcyclase in the target cell. In the present study, we revealed a complex sequence of appearance and disappearance of individual molecular electrophoretic forms of PDE during the development of the rat testis. Kinetic analysis of cAMP hydrolysis in crude testis homogenates of developmental stages where multiple PDE isozymes are expressed revealed complex kinetic behavior of PDE. After separation of individual isozymes by elution from starch gel blocks, several enzymatic forms still act with complex kinetics, indicating negatively cooperative behavior. The stage specificity of the kinetic properties of PDE appears to be related to the hormonally regulated events leading to the initiation of male puberty.     

Effects of Surgical Sympathetic Denervation on myo-Inositol Trisphosphate Production and Contraction in the Dilator and Sphincter Smooth Muscles of the Rabbit Iris: Evidence for Interaction Between the Cyclic AMP and Calcium Signaling Systems

The effects of norepinephrine (NE), carbachol (CCh), NaF, 3-isobutyl-1-methylxanthine (IBMX), and high K+ concentration (80 mM) depolarization on inositol trisphosphate (IP3) accumulation, cyclic AMP (cAMP) formation, and contraction were investigated in the dilator and sphincter smooth muscles of the sympathetically denervated as well as the normal rabbit eye. (a) In the denervated dilator muscle, NE-stimulated IP3 production and contraction are enhanced. (b) In the sphincter muscle of rabbits that have undergone sympathetic denervation. CCh-stimulated IP3 production and contraction are attenuated. (c) The increase in tension by a maximal effective dose of NaF (209 mM) in the dilator was 12.5 and 18 mg of tension/mg wet weight in normal and denervated tissue, respectively, and in the sphincter was 33.8 and 15.2 mg of tension/mg wet weight in normal and denervated tissue, respectively. NaF had no effect on cAMP formation. (d) Addition of NE had no effect on cAMP formation in both the normal and denervated dilator, whereas basal and IBMX-induced cAMP formation increased. in the denervated sphincter over that of the normal tissue by 15 and 60%, respectively. (e) Isoproterenol (5 microM) increased cAMP formation in the normal and denervated sphincter by 47 and 91%, respectively. (f) Whereas CCh inhibits cAMP formation in the normal sphincter, it lost its inhibitory effect in the sphincter with denervation. (g) IBMX (0.1 mM) attenuated the CCh-stimulated IP3 production and contraction of the sphincter by approximately 30% of their respective controls. (h) High K+ concentration depolarization attenuated contraction in both dilator and sphincter muscles with denervation. These observations suggest that an increase in the level of cAMP in the iris sphincter due to sympathetic denervation could lead to inhibition of phospholipase C (or other target sites, such as phosphorylation of the muscarinic receptor, Gp protein itself, myosin light chain kinase, or the IP3 receptor), IP3 production, and contraction. In conclusion, we suggest that the supersensitivity and subsensitivity observed after surgical sympathetic denervation of the iris dilator and sphincter muscles, respectively, are caused by alterations in the efficiency of coupling, probably through the Gp proteins, between their respective receptors and the breakdown of polyphosphoinositides by phospholipase C. In addition, we propose that the sympathetic nervous system can regulate, through alterations in cAMP levels, the muscarinic stimulation of IP3 accumulation and contraction in the iris sphincter. These findings add further support to the hypothesis that there are reciprocal interactions between the cAMP and IP3-Ca2+ signaling systems and the contractile response in the iris smooth muscle.     

Expression of Glut-4 and Glut-1 transporters in rat diaphragm muscle

Glucose transporters (Gluts) are a family of membrane proteins responsible for the transport of glucose across cellular membranes. Generally, alterations of Gluts expression in limb skeletal muscle have been reported. However, the changes of Glut isoforms in respiratory muscle which contracts with a duty cycle have rarely been studied. This study was performed to evaluate at the light microscopy level the expression of Glut-4 and Glut-1 transporters in normal and denervated diaphragm by immunohistochemistry method with specific Gluts antibodies. The results showed Glut-4 immunoreactivity in both the cell periphery and the interior of myocytes. Glut-1 was also present in the cell border and in the interior of myocytes in control diaphragm. However, Glut-4 staining was stronger than Glut-1 staining in control diaphragm. In denervated hemidiaphragm, the Glut-4 immunolabelling decreased and Glut-1 increased. These data indicated that (1) Glut-4 and Glut-1 transporters were observed in diaphragm; and (2) there were alterations in the expression of both glucose transporters after denervation. These alterations in Glut isoforms after denervation may be associated with the removal of innervation itself, and/or may partly result from passive stretch imposed by inspiratory activation of the contralateral side.