Changes In Phosphodiesterase Activity Research Articles

Live-Cell Assays for Cell Stress Responses Reveal New Patterns of Cell Signaling Caused by Mutations in Rhodopsin, α-Synuclein and TDP-43.

Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling, we created live-cell assays for endoplasmic reticulum (ER)-mediated cell stress and second messenger signaling. We first examined neurodegenerative mutations associated with direct ER stress by exploring the effect of rhodopsin mutations on ER stress and Ca2+ signaling. The rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa (RP), produced increased ER stress levels compared to wild type (WT) rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress-dependent manner. Analysis of single-cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing WT or P23H rhodopsin, consistent with the idea that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in neurodegenerative diseases that may not have a direct effect on ER-mediated cell stress, we examined how various mutants of α-synuclein and TDP-43 affected ER stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson’s disease (PD) and Amyotrophic lateral sclerosis (ALS) demonstrated increased ER stress compared to WT proteins. To examine the effect of α-synuclein and TDP-43 mutants on cellular signaling, we created a second live-cell assay to monitor changes in cAMP signaling during expression of various forms of α-synuclein and TDP-43. The increased cell stress caused by expression of the mutant proteins was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors for cellular stress and signaling can provide nuanced, new views of neurodegenerative disease processes.

Molt-inhibiting hormone-mediated regulation of ecdysteroid synthesis in Y-organs of the crayfish ( Procambarus clarkii): Involvement of cyclic GMP and cyclic nucleotide phosphodiesterase

Crustacean molt-inhibiting hormone (MIH), a polypeptide secreted by the X-organ/sinus gland complex of the eyestalks, regulates molting by inhibiting the synthesis of ecdysteroids by Y-organs. Previous results indicate the biosynthetic activity of Y-organs is likely controlled not only by the level of hemolymphatic MIH, but also by the responsiveness of Y-organs to MIH. The present studies were conducted to (a) identify the second messenger that mediates MIH-induced suppression of ecdysteroidogenesis, and (b) assess the possible involvement of cyclic nucleotide phosphodiesterase (PDE) in determining the responsiveness of Y-organs to MIH. Adding 8-bromo cAMP or 8-bromo cGMP to incubation medium significantly suppressed ecdysteroid production by Y-organs of the crayfish ( Procambarus clarkii). Incubating Y-organs with MIH produced a significant increase in glandular cGMP, but MIH had no effect on glandular cAMP. The composite data indicate that MIH-induced suppression of ecdysteroidogenesis in Y-organs of P. clarkii is mediated by cGMP . Subsequently, Y-organs from various stages of the molt cycle were incubated with MIH, 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of PDE), or both. Y-Organs from middle and late premolt stages were poorly responsive to MIH alone. Including IBMX in the incubation medium enhanced the responsiveness of the Y-organs to MIH at these stages. Moreover, glandular PDE activity in the Y-organs at these stages was significantly higher than other stages. The combined results suggest that molt cycle-associated changes in PDE activity affect the ability of MIH to stimulate cGMP accumulation and suppress ecdysteroidogenesis in Y-organs of P. clarkii.

Alterations of the expression and activity of midbrain nitric oxide synthase and soluble guanylyl cyclase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

The study was aimed at investigating the expression and the activity of neuronal nitric oxide synthase, and of soluble guanylyl cyclase and phosphodiesterase activities that regulate guanosine 3′,5′-cyclic monophosphate level in the midbrain, in a mouse model of PD using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine injections. Adult male mice of the C57/BL strain were given three i.p. injections of physiological saline or three i.p. injections of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine solution in physiological saline at 2 h intervals (summary 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine dose: 40 mg/kg), and were killed 3, 7, or 14 days later. mRNA, protein level, and/or activities of neuronal nitric oxide synthase, soluble guanylyl cyclase, phosphodiesterase and guanosine 3′,5′-cyclic monophosphate were determined. Immunohistochemistry showed about 75% decrease in the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta. Mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed increased midbrain guanylyl cyclase and total nitric oxide synthase activities at 3, 7, and 14 days post-treatment. The specific neuronal nitric oxide synthase inhibitor 7-nitroindazole (10 μM) and the specific inducible nitric oxide synthase inhibitor 1400W (10 μM) inhibited the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced excess in nitric oxide synthase activity by 63–70 and 13–25%, respectively. The increases in total midbrain nitric oxide synthase activity were accompanied by elevated guanosine 3′,5′-cyclic monophosphate, enhanced expression of neuronal nitric oxide synthase and of the β1 subunit of guanylyl cyclase at both mRNA and protein levels that persisted up to the end of the observation period, and by enhanced neuronal nitric oxide synthase and guanylyl cyclase β1 immunoreactivities in substantia nigra pars compacta 7 and 14 days after the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The increases in guanylyl cyclase activity were found to occur exclusively due to increased maximal enzyme activity. No 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced change in phosphodiesterase activity has been detected in any brain region studied. 7-Nitroindazole prevented a significant increase in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced midbrain guanosine 3′,5′-cyclic monophosphate level and neurodegeneration of dopaminergic neurons. These results raise the possibility that the nitric oxide/guanylyl cyclase/guanosine 3′,5′-cyclic monophosphate signaling pathway may play a role in maintaining dopaminergic neurons function in substantia nigra pars compacta.

Upregulation of guanylyl cyclase expression and activity in striatum of MPTP-induced parkinsonism in mice

The aim of our study was to investigate the expression and the activity of soluble guanylyl cyclase (GC) and phosphodiesterase (PDE) activities that regulate cGMP level in the striatum, hippocampus, and brain cortex in an animal model of PD, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). We observed the increase of total activity and protein level of GC in striatum after MPTP injection. It was accompanied by an enhancement of both mRNA expression and protein level of GCβ1 subunit. MPTP induces mRNA expression and elevates protein concentration of GCβ1 in striatum up to 14 days after its injection, which in turn causes a marked enhancement of cGMP formation. Furthermore, the activation of GC occurs through change of maximal enzyme activity ( V max). Simultaneously, no change in PDE activity has been detected in all investigated regions of the brain after MPTP. MPTP injection caused the elevation of GCβ1 protein level in both the membrane and cytosol fractions being significantly higher in cytosol. Western blot analysis demonstrated about 45–67% decrease of tyrosine hydroxylase protein content in striatum. These data suggest that NO/cGMP signaling pathway may at least partially contribute to dopaminergic fiber degeneration in the striatum, the damage attributed to PD.

Changes in phosphodiesterase activity in the developing rat submandibular gland

Developmental changes (from 2 to 26 weeks) in phosphodiesterase (PDE) activity in the rat submandibular gland were investigated. Major activities for both cAMP- and cGMP-PDE were present in the 100,000× g supernatant fractions (70–90% of total activities), but not in the pellet fractions, during development. The effects of stimulators (Ca 2+/calmodulin and cGMP) and inhibitors (cGMP, cilostamide, rolipram and zaprinast) were investigated in the supernatant fractions. During development, PDE4 (cAMP-specific PDE) was a major PDE, indicating that the majority of cAMP is hydrolysed by PDE4. In the young rat, PDE1 hydrolysed cGMP three-fold more than the control, and PDE2 (cGMP-stimulated PDE) was present, indicating that the concentration of intracellular cGMP may be enhanced, and cGMP may function in the growth pathway in the submandibular gland. Chromatograms eluted on a Mono Q HR5/5 ion-exchange column supported the results of the inhibition studies: PDE1, PDE2, PDE3, PDE4 and PDE5 were present in the young submandibular gland, and PDE1, PDE3, PDE4 and PDE5 in the adult gland. Expression of PDE5 was detected by inhibition studies, reverse transcriptase-polymerase chain reaction and Western blotting in the submandibular gland.

Sub-family selective actions in the ability of Erk2 MAP kinase to phosphorylate and regulate the activity of PDE4 cyclic AMP-specific phosphodiesterases.

Expressed in intact cells and in vitro, PDE4B and PDE4C isoenzymes of cyclic nucleotide phosphodiesterase (PDE), in common with PDE4D isoenzymes, are shown to provide substrates for C-terminal catalytic unit phosphorylation by the extracellular signal-regulated kinase Erk2 (p42(MAPK)). In contrast, PDE4A isoenzymes do not provide substrates for C-terminal catalytic unit phosphorylation by Erk2. Mutant PDE4 enzymes were generated to show that Erk2 phosphorylation occurs at a single, cognate serine residue located within the C-terminal portion of the PDE4 catalytic unit. PDE4 long-form isoenzymes were markedly inhibited by Erk2 phosphorylation. The short-form PDE4B2 isoenzyme was activated by Erk2 phosphorylation. These functional changes in PDE activity were mimicked by mutation of the target serine for Erk2 phosphorylation to the negatively charged amino acid, aspartic acid. Epidermal growth factor (EGF) challenge caused diametrically opposed changes in cyclic AMP levels in COS1 cells transfected to express the long PDE4B1 isoenzyme compared to cells expressing the short PDE4B2 isoenzyme. We suggest that PDE4 enzymes may provide a pivotal point for integrating cyclic AMP and Erk signal transduction in cells with 4 genes encoding enzymes that are either insensitive to Erk2 action or may either be activated or inhibited. This indicates that PDE4 isoenzymes have distinct functional roles, giving credence to the notion that distinct therapeutic benefits may accrue using either PDE4 subfamily or isoenzyme-selective inhibitors.

A Biochemical and Functional Assessment of Monocyte Phosphodiesterase Activity in Healthy and Asthmatic Subjects

The aim of this study was to investigate whether cyclic adenosine 3′5-monophosphate (cAMP) phosphodiesterase (PDE) activity is altered in monocytes from mild asthmatic subjects. Total cAMP PDE activity (pmol/min per mg protein) was significantly greater in homogenates prepared from monocytes from asthmatic subjects (68.3 ± 7.0, n=9) compared to healthy individuals (46.3 ± 3.3, n=14, P<0.05). The PDE inhibitors siguazodan (PDE3-selective), rolipram (PDE4-selective) and theophylline (non-selective) produced a concentration-dependent inhibition of cAMP PDE activity in homogenates from monocytes from normal and asthmatic subjects. However, siguazodan produced significantly greater (P<0.05), and rolipram significantly less (P<0.05), inhibition of total cAMP PDE activity in monocytes from asthmatics (n=4) than from healthy individuals (n=5). cAMP PDE activity was inhibited with equal potency by theophylline in monocytes from healthy and asthmatic subjects. We also investigated the functional consequences of the changes in PDE activity in mononuclear cells obtained from asthmatic subjects. There was no significant difference in the ability of PDE4 inhibitors to attenuate TNF α release from monocytes obtained from asthmatic compared with healthy subjects (P>0.05). Despite a significant increase in the biochemical activity of PDE3 in monocytes from asthmatic subjects, the PDE3 inhibitor siguazodan, failed to significantly reduce TNF α release from human monocytes. Thus, total cAMP PDE activity is increased in monocytes taken from mild asymptomatic asthmatics compared to healthy subjects and is reflected by an increase in the proportion of PDE3 and a decrease in the proportion of PDE4. This augmented enzyme activity was not associated with an alteration in the ability of PDE4 inhibitors to attenuate mononuclear cell function from asthmatics compared to healthy individuals.

Reduced alpha 1- and beta 2-adrenoceptor-mediated positive inotropic effects in human end-stage heart failure.

1. alpha 1-Adrenoceptor (phenylephrine in the presence of propranolol) and beta 2-adrenoceptor (fenoterol)-mediated positive inotropic effects were investigated in human ventricular preparations isolated from five non-failing (prospective organ donors) and from eight explanted failing hearts with end-stage idiopathic dilative cardiomyopathy (NYHA IV). 2. For comparison, the nonselective beta-adrenoceptor agonist isoprenaline, the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), the cardiac glycoside dihydroouabain, and calcium were studied. 3. Furthermore, the influence of IBMX on adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDE activity as well as total beta-adrenoceptor density, beta 1- and beta 2-adrenoceptor subtype distribution, and alpha 1-adrenoceptor density were compared in nonfailing and failing human heart preparations. The radioligands (-)-[125I]-iodocyanopindolol for beta-adrenoceptor binding and [3H]-prazosin for alpha 1-adrenoceptor binding were used. 4. The inotropic responses to calcium and dihydroouabain in failing human hearts were unchanged, whereas the maximal alpha 1- and beta 2-adrenoceptor-mediated positive inotropic effects were greatly reduced. The inotropic effects of the other cyclic AMP increasing compounds, i.e. isoprenaline and IBMX, were also reduced to about 60% of the effects observed in nonfailing controls. The potency of these compounds was decreased by factors 4-10. 5. The basal PDE activity and the PDE inhibition by IBMX were similar in nonfailing and failing preparations. 6. The total beta-adrenoceptor density in nonfailing hearts was about 70 fmol mg-1 protein. In failing hearts the total number of beta-adrenoceptors was markedly reduced by about 60%. The betal/beta2-adrenoceptor ratio was shifted from about 80/20% in nonfailing to approximately 60/40% in failing hearts which was due to a selective reduction of beta1-adrenoceptors. The beta2-adrenoceptor population remaining unchanged. alpha-Adrenoceptor density was increased from about 4 fmol mg-' protein in nonfailing to 10 fmol mgprotein in failing hearts.7. Changes in PDE activity and adrenoceptor downregulation cannot completely explain the reduced positive inotropic effects of alpha 1- and beta 2-adrenoceptor agonists in failing human hearts. This supports the hypothesis that impairment of other processes such as the coupling between receptor and effector system, i.e. the respective G-proteins, are equally important in end-stage heart failure.

Cyclic changes in cAMP concentration and phosphodiesterase activity in a mammalian circadian clock studied in vitro

The mammalian suprachiasmatic nuclei (SCN) contain a circadian pacemaker that continues to keep 24-h time when isolated in vitro. We are investigating the role of cAMP in the cellular mechanisms underlying SCN function. We have previously shown that increasing intracellular cAMP during the subjective day resets the SCN pacemaker in the in vitro rat brain slice preparation 8, 20. We now report that the level of cAMP fluctuates within the rat SCN under constant conditions in vitro. The level of endogenous cAMP is high during late day and late night, and low during early night. These changes in cAMP concentration are accompanied by opposite changes in phosphodiesterase activity; we detected no significant change in adenylate cyclase activity. These results provide further support for the hypothesis that cAMP is involved in circadian function in the SCN.

Developmental changes in phosphodiesterase activity and hormonal response in the prothoracic glands of Manduca sexta

Prothoracicotropic hormone (PTTH) stimulates ecdysteroid secretion by the prothoracic glands of Manduca sexta in a cAMP-dependent manner. However, larval and pupal glands differ markedly in the degree to which PTTH stimulates cAMP accumulation, suggesting a stage-specific difference in phoshodiesterase activity. The present study was designed to determine if and when such a difference arose during development, and its effect on PTTH-stimulated ecdysteroid secretion. The results reveal that soluble phosphodiesterase activity in the prothoracic glands changes significantly during the course of the fifth (last) larval instar, with a marked increase in activity occurring at the onset of prepupal development. Phosphodiesterase activity, particularly in the soluble cell fraction, is inversely correlated with PTTH-stimulated cAMP accumulation. Hormone-stimulated ecdysteroid secretion does not require cAMP accumulation, but does appear to require detectable cAMP synthesis as measured in the presence of phosphodiesterase inhibitors. The amount of ecdysteroid secreted, however, is not proportional to the amount of cAMP synthesized but rather is more closely correlated with developmental changes in glandular protein content.

Cellular Tolerance to Adenosine Receptor-Mediated Inhibition of Lipolysis: Altered Adenosine 3',5'- Monophosphate Metabolism and Protein Kinase Activation*

Prolonged exposure of many types of cells to drugs or hormones that inhibit the activity of the enzyme adenylate cyclase, such as narcotics and alpha 2-adrenergic agonists, leads to enhanced accumulation of cAMP upon removal of the inhibitory drug. We have found previously that chronic infusion of the adenosine A1 receptor agonist phenylisopropyladenosine (PIA), an inhibitor of adenylate cyclase, into rats leads to enhanced isoproterenol-stimulated cAMP accumulation in adipocytes isolated from these animals. The enhanced cAMP accumulation was associated with an impaired ability of PIA to inhibit lipolysis in these cells. In the present study we have investigated the mechanism of the enhanced cAMP accumulation in adipocytes from PIA-infused rats and the relationship of these changes to the impaired antilipolytic action of the drug. The enhanced isoproterenol-stimulated cAMP accumulation in adipocytes prepared from PIA-infused rats was due to both an increased rate of cAMP synthesis and a decreased rate of cAMP metabolism at high concentrations of cAMP without a change in phosphodiesterase activity. There was heterologous desensitization of the ability of PIA, prostaglandin E1, and nicotinic acid to inhibit cAMP accumulation in the adipocytes from PIA-infused rats; there was an increase in the EC50 of each of these agonists, although maximal inhibition of cAMP accumulation was similar. The relationship between the activation of cAMP-dependent kinase and extent of lipolysis was similar in the two groups of cells. We demonstrated that the explanation for the impaired ability of PIA to decrease the rate of isoproterenol (10(-7) M)-stimulated lipolysis in the cells from the PIA-infused rats was due to the markedly increased concentrations of cAMP in these cells, which led to sufficient activation of the kinase to maintain a high rate of lipolysis even in the presence of PIA. In addition, we found that the changes induced by the PIA infusion were largely reversible over a 2-day period after discontinuing the PIA infusion. These results demonstrate that adipocytes from PIA-infused rats provide an interesting model to investigate the mechanisms of tolerance to inhibitory drugs.

Control of light‐sensitive current in salamander rods.

1. The exponential decline of light-sensitive current seen after switch from Na+ to Li+ in the presence of Ca2+ probably depends on the activity of the phosphodiesterase (PDE) which hydrolyses cyclic GMP. 2. This probability is supported by experiments with suction electrodes which show that in toad and salamander rods the rate constant, b, of the exponential decline of current was increased at least 10-fold by moderate light intensities and decreased about 10-fold by 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of PDE. 3. The rate constant b is about 3 times more sensitive to weak lights or to IBMX than the membrane current. This may be explained by a feed-back involving calcium ions which tends to hold current constant, perhaps by calcium inhibition of guanylate cyclase. 4. The time course of b, which probably represents the changes in PDE activity, was measured by switching from Na+ to Li+ at various times after a flash. The results suggest that a moderate flash (140 Rh) increased b about 7 times in 0.5 s and that b then declined with a time constant of 1.5-2 s. 5. Extrapolated values of the parameter b suggest that strong flashes (5000-10,000 Rh) increased b from 1 s-1 in the dark to perhaps 60 s-1 and that b continued to increase with flash strength for several log units after the current had reached saturation. 6. The observations in 4 and 5 fit well with the idea that b is related to PDE activity and that changes in the latter are sufficient to account for the rising phase of the flash response. 7. After a flash the light-sensitive current recovers much more rapidly than the time constant b-1, a discrepancy which is explained if a light flash causes a delayed increase in guanylate cyclase activity. 8. The apparent delayed increase in cyclase activation is consistent with an inhibitory effect of [Ca2+]i which is reduced when calcium is pumped out during the plateau of the response. 9. Experiments in which pulses of IBMX were applied at different times during a flash response support the idea that a flash causes a delayed increase in the rate of supply of cyclic GMP. Quantitative analysis of these and other tests with IBMX gave rate constants similar to those obtained by the Na+----Li+ method.

Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro.

Cyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation-related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5'-nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.

Biochemistry of spore germination inPhycomyces

The constitutionally dormant spores of Phycomyces blakesleeanus can be activated by heat shock or treatment with several monocarboxylic acids. Activation is followed first by a general stimulation of metabolism, e.g. respiration, protein-, RNA- and cell-wall synthesis, and subsequently by nuclear division and germ-tube emergence. Initial germination is not dependent on RNA synthesis and can even start without protein synthesis. The first common effect of different activating treatments is a transient rise in cyclic AMP (cAMP) content, caused by a change in phosphodiesterase activity after heat activation, and by unknown factors during activation by acids. cAMP transiently activates trehalase and glycerol-3-phosphatase in the spores. The activation of these enzymes causes a quick turnover of trehalose into glycerol. During the same period, the water status of the cells is altered so dramatically that perhaps this may explain at least part of the stimulation of metabolism in the germinating spore.

Hormonal regulation of a plasma membrane phosphodiesterase in differentiating granulosa cells. Reciprocal actions of follicle-stimulating hormone and a gonadotropin-releasing hormone agonist on cAMP degradation.

The activity of a plasma membrane cAMP-phosphodiesterase in cultured ovarian granulosa cells was regulated by follicle-stimulating hormone (FSH) and the gonadotropin-releasing hormone (GnRH) agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). Degradation of cAMP was similar in cultures treated with FSH alone or FSH plus GnRHa when the labeled cyclic nucleotide was added from 24 to 42 h of culture. However, at 48 h and subsequent times of incubation, cAMP phosphodiesterase activity was significantly higher in cells incubated with FSH plus GnRHa. Phosphodiesterase activity was progressively increased by GnRHa concentrations between 10(-13) and 10(-10) M, and was maximally stimulated by 10(-9) M GnRHa. In comparison with control cells, FSH lowered the Vmax of cAMP catabolism by the high (1 microM cAMP substrate) and the low (50 microM) affinity phosphodiesterase, while GnRHa raised enzyme activity toward control levels. These actions of FSH and GnRHa were specific for a plasma membrane phosphodiesterase that was accessible to extracellular cAMP, since extracellular substrate was hydrolyzed, no intracellular uptake of [3H]cAMP was observed, and only a small fraction (10%) of cAMP was catabolized in the incubation medium in the absence of cells. Further, the actions of FSH and GnRHa on the membrane enzyme were the opposite of those observed when total phosphodiesterase activity was measured in cellular sonicates. Hormonal changes in phosphodiesterase activity were not due to leakage of the enzyme from damaged cells since a constant percentage of cAMP hydrolysis in the medium was observed during culture. Analysis of cAMP catabolites in granulosa cells indicated that the phosphodiesterase reaction product, 5'-AMP, was rapidly converted to adenosine by a plasma membrane 5'-nucleotidase, independent of the cellular hormonal status. These results indicate that the opposing actions of FSH and GnRHa upon granulosa cell differentiation include modulation of cAMP degradation at the plasma membrane level.

Hormone-sensitive particulate cAMP phosphodiesterase activity in 3T3-L1 adipocytes. Regulation of responsiveness by dexamethasone.

Confluent 3T3-L1 fibroblasts incubated for 72 h with methylisobutylxanthine, dexamethasone, and insulin differentiate and acquire phenotypic characteristics of mature adipocytes, including hormone-sensitive cAMP phosphodiesterase activity located in a particulate fraction of homogenates. About 10 days after initiating differentiation, a maximally effective concentration of insulin (100 pM) increased particulate cAMP phosphodiesterase activity 40 to 60% in 8 min; activation persisted for at least 30 min in the presence of insulin. Incubation of adipocytes for 6-8 min with agents that increased cAMP, e.g. 1 microM epinephrine, 0.1 microM isoproterenol, corticotropin (2 mu units/ml), or thyroid-stimulating hormone (15 ng/ml), also increased particulate phosphodiesterase activity 40-60%. Changes in phosphodiesterase activity produced by epinephrine tended to lag behind changes in cAMP. Insulin, epinephrine, and corticotropin increased Vmax, not Km (0.5 microM), for cAMP. Particulate phosphodiesterase activity, solubilized with detergent, eluted in a single peak from DEAE-Bio-Gel. Insulin and epinephrine increased the activity eluted in this peak. Neither insulin nor lipolytic hormones increased activity in soluble fractions from differentiated cells or particulate or soluble fractions from undifferentiated cells. Incubation of adipocytes for 48 h with 1 microM dexamethasone prevented insulin-induced activation of the particulate phosphodiesterase and did not alter basal activity. After incubation for 72 h with 0.1 microM dexamethasone, insulin and epinephrine activation were abolished. These effects of dexamethasone on hormonal regulation of particulate phosphodiesterase activity could account for some of the so-called permissive effects of glucocorticoids on cAMP-mediated processes as well as the "anti-insulin" effects of glucocorticoids.

Different effects of selenium on cyclic AMP metabolism in hepatoma cells and normal liver cells

Sodium selenite administered to normal and hepatoma (HA)-bearing mice sc (2 mg/kg) or ip (1 mg/kg) led to a significant increase in cyclic AMP (cAMP) level and a decrease in cAMP phosphodiesterase (PDE) activity in HA cells. In contrast, in tumor host liver and normal liver the cAMP level was reduced, and the PDE activity was slightly elevated, whereas the cAMP adenylate cyclase (AC) was little affected by Na2SeO3, if at all. These results imply that selenite-induced changes in PDE activity play a decisive role in regulating intracellular cAMP level. Kinetic studies revealed that there were different forms of PDE. TheK m value of PDE isozymes in normal liver and host liver were identical, but differed from those of HA. It seems likely that the selective responsiveness of PDE to selenite may be related to the difference in PDE isozyme patterns. Addition of DBcAMP to the culture medium resulted in an inhibition of(3)H-thymidine incorporation into hepatoma cells, indicating that the inhibition of HA cell proliferation was cAMP-mediated. Thus, selenium has been shown to exert a selective effect on cAMP metabolism of hepatoma cells, which may account for its inhibitory effects on cancer cells.

Mechanisms of cyclic AMP phosphodiesterase regulation

The mechanisms of regulation of cyclic AMP phosphodieterases were studied using the cytoplasmic fraction of PC-12 cells sensitive to the action of nerve growth factor. The cells contain phosphodiesterases of two types. One of them possesses a high affinity for cyclic AMP (K m = 2.46 m m), whereas the other has the affinity by an order worse (K m = 37.1 m m). PC-12 cell differentiation under the action of nerve growth factor is connected with the cyclic nucleotide elevation; however, activities of both phosphodiesterases remain unchanged. This indicates that the regulation of activity of these enzymes in PC-12 cells is mediated by second messenger effects. The main object of cell regulation is phosphodiesterase with low affinity for the substrate. Its activity is modulated by the calmodulin-Ca 2+ complex, cyclic GMP and NAD + at micromolar concentrations. The effect of the phosphodiesterase system of both a “quick” messenger, Ca 2+ and “slow” messengers, cyclic GMP and NAD +, has the same consequences: the turnover number of the enzymic reaction increases that is accompanied by a proportional decrease in the enzyme affinity for cyclic AMP so that the ratio V max/ K m remains constant. A possible explanation of functional significance of such an activity modulation may be the necessity to maintain the conditions for phosphodiesterase functioning when K m ⪢ [cyclic AMP] and the reaction rate are directly proportional to the substrate concentration: v = V max /K m [cyclic AMP]. Then the cells are transferred into such a mode when autoregulation of the cyclic nucleotide level takes place. Besides the transient effects causing changes in phosphodiesterase activity, studies of PC-12 cells revealed a chronic effect of phosphodiesterase activity change under the action of staphylococcal enterotoxin A. This protein which induces differentiation of PC-12 cells and possesses a NAD +-glycohydrolase activity is translocated into cytoplasm of cells in the presence of NAD + and accomplishes ADP-ribosylation of phosphodiestarase. As a result, the enzyme activity falls, cyclic AMP level increases and cell differentiation starts. The activity of soluble phosphodiesterase of PC-12 cells also decreases under the effect of two neurotoxins from bee venon, melittin and tertiapin. Both the toxins at concentration of 10 μ m completely block calcium regulation of the enzyme. The mechanism of tertiapin action was investigated on a model system of calmodulin-bovine brain phosphodiesterase. It appeared that inhibition of Ca 2+ action is achieved as the result of binding of two toxin molecules with K d = 2 m m to the activated calmodulin molecule. The triple complex formed completely loses the modulating properties. A kinetic model is proposed which can be used in investigations on the mechanism of action ob biologically active substrances blocking calcium regulation.

Changes in phosphodiesterase activity of human subcutaneous adipose tissue during starvation

The phosphodiesterase (PDE) activity, the basal rate of lipolysis and the basal cyclic AMP level in adipose tissue were determined in hypogastric and gluteal specimens obtained from 14 obese healthy subjects before and after one week of starvation. During starvation there was a significant increase in both the tissue level of cyclic AMP and the rate of lipolysis, whereas the apparent values of V max of the low and high K m forms of PDE decreased significantly—i.e., by about 50 and 30 percent, respectively. The substrate concentration of cyclic AMP at V max of the low K m PDE corresponded to the tissue level of the nucleotide. During, but not before, starvation there was a positive correlation between V max of the low K m PDE and the cyclic AMP level ( r = 0.7–0.8). The metabolism in the tissues from the two fat depots exhibited similar variations during starvation. The findings suggest that the low K m form of PDE is inhibited during starvation. This may be one factor responsible for the starvation-mediated increase in the cyclic AMP level and the rate of lipolysis in adipose tissue.

The periodic change in adenosine 3′,5′-monophosphate concentration in sea-urchin eggs

The level of adenosine 3′,5′-monophosphate (cyclic AMP) in the eggs of the sea urchin, Anthocidaris crassispina, was found to change periodically after fertilization. The minimum and maximum levels of cyclic AMP were 1.0·10 −7 M and 1.5·10 −6 M, respectively. The activity of adenylate cyclase in a 105 000 × g precipitate reached a plateau at 20 min after fertilization and stayed constant for at least 2 h. It was also found that 1.0 mM CaCl 2 increased the activity of adenylate cyclase in the same precipitate from unfertilized eggs. In contrast, phosphodiesterase activity changed periodically and correlated with cyclic AMP levels in the eggs. Up to a concentration of 1.5·10 −6 M cyclic AMP, phosphodiesterase activity was low, but it became activated when the level of cyclic AMP rose beyond this level. These results indicate that the change in the intracellular level of cyclic AMP is regulated mainly by the change in phosphodiesterase activity.