Differences in the association of calmodulin with cyclic nucleotide phosphodiesterase in relaxed and contracted arterial strips

Changes in the concentration of cytosolic Ca2+ are assumed to alter the activity of Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in intact cells. However, this assumption is based on indirect evidence and by analogy from studies of enzyme activities in broken cell systems. We have developed a procedure for estimating the fraction of Ca2+-calmodulin-sensitive phosphodiesterase that is in an activated, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) sensitive state in intact porcine coronary artery strips. The experimental approach involves homogenization of the strips and assay of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase activity under conditions that retard changes in the amount of the complex Ca2+-calmodulin-phosphodiesterase. Our findings indicate that cyclic GMP phosphodiesterase in intact coronary artery strips does associate with Ca2+-calmodulin and that interventions that change the concentration of Ca2+ in the cytosol of the intact strip change the extent of this functional association. Exposure to histamine (10 or 100 microM) or 50 mM KCl caused contraction and an increase in EGTA-sensitive cyclic GMP phosphodiesterase activity. Isoproterenol-induced relaxation of tissues that had been caused to contract with 10 microM histamine was accompanied by a reduction in EGTA-sensitive cyclic GMP phosphodiesterase activity to the same level as that present before contraction was initiated.     

Regulation by insulin of cyclic nucleotide phosphodiesterase (PDE) from rat luteal cells

The effect of insulin on cyclic nucleotide phosphodiesterase (PDE) in rat luteal cells was studied. Cells were obtained from PMSG/hCG primed rats and further incubated or not with insulin. The hormone produced an increase of enzyme activity after a 10 min incubation of intact cells. Maximal stimulation was achieved at 0.2 nM of insulin. Two peaks of cyclic nucleotide phosphodiesterase activity were resolved after chromatography of cell cytosolic extracts on DEAE-cellulose. These peaks (I and II) were active with cAMP as substrate but only peak I was active with cGMP. The enzyme activity of both peaks was increased in cells treated with insulin. Phosphodiesterase activity in the two peaks show two kinetic components for cAMP hydrolysis, one of high affinity (Km 2-4 microM) and the other of low affinity (47-56 microM). Treatment of the cells with insulin produced a 2 to 8 fold increase of the Vmax of these peaks. In addition after stimulation with insulin, the activation of peak I phosphodiesterase by calmodulin was less effective.     

Phosphorylation results in activation of a cAMP phosphodiesterase in human platelets

Agents such as prostaglandins E1 and I2 which elevate cAMP levels in platelets also increase cAMP phosphodiesterase activity. Since much of the cAMP phosphodiesterase activity in human platelets is due to the cGMP-inhibited isozyme (Macphee, C. H., Harrison, S. A., and Beavo, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6600-6663), we examined the regulation of this isozyme by prostaglandins E1 and I2 in intact platelets. Because this isozyme is a minor component of platelet protein, normally requiring several thousand-fold purification to achieve homogeneity, a specific monoclonal antibody (CGI-5) was utilized to identify and isolate the cGMP-inhibited phosphodiesterase activity. Treatment of intact platelets with the prostaglandins promoted an increase in the phosphorylation state of the cGMP-inhibited phosphodiesterase and a corresponding increase in phosphodiesterase activity. The effect on activity and phosphorylation of the cGMP-inhibited phosphodiesterase was observed within 2 min after intact platelets were exposed to the prostaglandins. The half-maximal effective dose for prostaglandin I2 (10 nM) was approximately 10-fold lower than that for prostaglandin E1. The phosphorylated, cGMP-inhibited isozyme migrated as a 110-kDa peptide following sodium dodecyl sulfate gel electrophoresis. Direct in vitro phosphorylation of the platelet cGMP-inhibited phosphodiesterase by the catalytic subunit of cAMP-dependent protein kinase caused a similar increase in phosphodiesterase activity. Treatment with PKI peptide, a specific inhibitor of cAMP-dependent protein kinase, blocked the phosphorylation and the effect on activity. Taken together, the data strongly suggest that the effects of prostaglandins E1 and I2 on platelet phosphodiesterase activity are mediated by a direct cAMP-dependent protein kinase-catalyzed phosphorylation of the cGMP-inhibited phosphodiesterase isozyme.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.     

The effect of diamide on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes

The effect of diamide (diazene dicarboxylic acid bis[N,N'-dimethylamide) on cyclic AMP levels and cyclic nucleotide phosphodiesterase in human peripheral blood lymphocytes was examined. In the absence of mitogenic lectins, 5 . 10(-3)-1 . 10(-4) M diamide markedly increased intracellular cyclic AMP with variable effects at higher levels. In the presence of phytohemagglutinin or concanavalin A, 5 . 10(-4) M or higher diamide concentrations consistently decreased cyclic AMP levels, usually to control levels or below, while 1 . 10(-4)-1 . 10(-5) M diamide augmented the lectin-induced rise in cyclic AMP. When intact lymphocytes were incubated with diamide, phosphodiesterase activity against both cyclic AMP and cyclic GMP, assayed in homogenates of these cells, was inhibited at concentrations as low as 1 . 10(-6) M. In contrast, when diamide was incubated with phosphodiesterase extracted from lymphocytes there was a dual effect. At low substrate concentrations and high diamide concentrations diamide was a non-competitive inhibitor of phosphodiesterase with a Ki of 1.3--2.5 mM for cyclic AMP and 3.3--10 mM for cyclic GMP. In contrast, at high substrate concentrations diamide was an 'uncompetitive' activator of phosphodiesterase activity for both cyclic AMP and cyclic GMP. The effects of diamide could be largely or completely blocked by glutathione or dithiothreitol, indicating that sulfhydryl reactivity was involved in diamide's action on lymphocyte phosphodiesterase activity and intracellular cyclic AMP levels. These data demonstrate that diamide is a phosphodiesterase inhibitor both on phosphodiesterase extracted from lymphocytes and when incubated with intact lymphocytes and that diamide may increase or decrease intracellular cyclic AMP levels depending on the concentration of diamide used.     

The phorbol ester TPA prevents the expression of both glucagon desensitisation and the glucagon-mediated block of insulin stimulation of the peripheral plasma membrane cyclic AMP phosphodiesterase in rat hepatocytes

The phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) causes a dose-dependent inhibition of the glucagon-stimulated adenylate cyclase activity expressed in plasma membranes isolated from TPA-treated hepatocytes. However, no observable inhibitory effect of TPA on adenylate cyclase activity was observed in cells which had been exposed to glucagon for 5 min, prior to isolation, to desensitise adenylate cyclase. The degree of inhibition of adenylate cyclase elicited by both glucagon desensitisation and TPA treatment of hepatocytes was identical. Pre-treatment of hepatocytes with TPA was also found to prevent glucagon from blocking insulin's activation of the peripheral plasma membrane cyclic AMP phosphodiesterase in intact hepatocytes. TPA treatment also inhibited the ability of cholera toxin to activate the peripheral cyclic AMP phosphodiesterase in intact hepatocytes. It is suggested that in these particular instances TPA and glucagon elicit mutually exclusive processes rather than TPA mimicking glucagon desensitisation per se.     

Evidence for regulatory control of canine platelet phosphodiesterase

Forskolin, epinephrine, and prostaglandin I2 were used to examine the adenylate cyclase-phosphodiesterase system of intact thrombopathic and normal canine platelets. The results provide indirect support for the hypothesis that the elevation of intraplatelet c-AMP in this unique hereditary defect is due to impaired phosphodiesterase activity. The inhibitory (Nj) and stimulatory (Ns) components of adenylate cyclase appeared functionally intact. Cytosolic fractions of normal and thrombopathic platelets had similar cAMP hydrolytic activities. The failure of intact forskolin-stimulated thrombopathic platelets to return elevated cAMP to non-stimulated levels after 15 min, despite significant phosphodiesterase activity in cytosolic fractions, implies that the platelet isoenzymes are under regulatory control.     

Solubilization and characterization of hormone- responsive phosphodiesterase activity of rat fat cells.

Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.     

Kinetics of a cellular nitric oxide/cGMP/phosphodiesterase-5 pathway

Rat platelets served as a model to evaluate quantitatively how guanylate cyclase (GC)-coupled nitric oxide (NO) receptors and phosphodiesterases (here phosphodiesterase-5) interact to transduce NO signals in cells. The platelets expressed mRNA only for the alpha(1) and beta(1) GC-coupled receptor subunits. In intact platelets, the potency of NO for elevating cGMP (EC(50) = 10 nm) was lower than in lysed platelets (EC(50) = 1.7 nm). The limiting activities of GC and phosphodiesterase in intact platelets were both very high, being equivalent to about 100 microm/s. With low phosphodiesterase activity (imposed by 100 microm sildenafil), the cGMP response over time was hyperbolic in shape for a range of NO concentrations or GC activities due to GC desensitization. Without a phosphodiesterase inhibitor, NO generated only brief cGMP transients, peaking after 2-5 s but amounting maximally to about 150 microm cGMP. The transients were caused partly by GC desensitization, which varied in rate (half-time up to 3 s) and extent (up to 80%) depending on the NO concentration, and partly by an enhancement of the phosphodiesterase catalytic activity with time, which was deduced to be up to 30-fold and to occur with a half-time of up to 5 s. The results were simulated by a quantitative model, which also explains the varied shapes of cGMP responses to NO found in other cells. Downstream phosphorylation in platelets was detectable within 2 s, and, with continuous exposure (1 min), this pathway could be engaged by subnanomolar NO concentrations (EC(50) = 0.5 nm).     

Glycerophosphocholine metabolism in higher plant cells. Evidence of a new glyceryl-phosphodiester phosphodiesterase

Glycerophosphocholine (GroPCho) is a diester that accumulates in different physiological processes leading to phospholipid remodeling. However, very little is known about its metabolism in higher plant cells. (31)P-Nuclear magnetic resonance spectroscopy and biochemical analyses performed on carrot (Daucus carota) cells fed with GroPCho revealed the existence of an extracellular GroPCho phosphodiesterase. This enzymatic activity splits GroPCho into sn-glycerol-3-phosphate and free choline. In vivo, sn-glycerol-3-phosphate is further hydrolyzed into glycerol and inorganic phosphate by acid phosphatase. We visualized the incorporation and the compartmentation of choline and observed that the major choline pool was phosphorylated and accumulated in the cytosol, whereas a minor fraction was incorporated in the vacuole as free choline. Isolation of plasma membranes, culture medium, and cell wall proteins enabled us to localize this phosphodiesterase activity on the cell wall. We also report the existence of an intracellular glycerophosphodiesterase. This second activity is localized in the vacuole and hydrolyzes GroPCho in a similar fashion to the cell wall phosphodiesterase. Both extra- and intracellular phosphodiesterases are widespread among different plant species and are often enhanced during phosphate deprivation. Finally, competition experiments on the extracellular phosphodiesterase suggested a specificity for glycerophosphodiesters (apparent K(m) of 50 microM), which distinguishes it from other phosphodiesterases previously described in the literature.     

A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cells, neither total cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.     

Adenylate cyclase activity in cultured epithelial cells.

The cyclic AMP metabolism of cultured epithelial cells was investigated. Epinephrine or 1-methyl,3-isobutylxanthine (MIX) alone had no effect on cyclic AMP levels in intact cells, whereas the combination of the two agents yielded a 6- to 10-fold increase in cyclic AMP levels. Both basal and stimulated cyclic AMP levels decreased with increasing cell density. Cell-free adenylate cyclase preparations were stimulated markedly by epinephrine or isoproterenol in the absence of MIX. Since the epithelial cells were found to have a relatively small amount of cyclic nucleotide phosphodiesterase (PDE) activity, the requirement for MIX to visualize intact cell responsiveness to epinephrine could be explained only partially by its PDE inhibitory properties.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: effect of growth.

Adenosine 3',5'-cyclic monophosphate (cyclic AMP) phsophodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3'5'-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E1-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine.     

Inhibition of cyclic AMP phosphodiesterase activity of human blood platelet membrane by ADP

Purified human blood platelet membrane showed the presence of one low Km (1.1 microM) and one high Km (5.0 microM) cyclic AMP phosphodiesterase(s). Incubation of platelet-rich plasma or gel-filtered platelets with ADP (4.0 microM), a well-known platelet aggregating agent, resulted in the inhibition of phosphodiesterase activity of the isolated membrane by 25% in 5 min at 23 degrees C. A Lineweaver-Burk plot of the enzymic activity of the membrane preparation showed that ADP specifically inhibited the low Km (1.1 microM) phosphodiesterase by reducing the Vmax from 241 to 176 pmol/mg per min with concomitant lowering of Km to 0.5 microM. In contrast, neither the high Km (5.0 microM) enzymic activity of the membrane preparation nor the phosphodiesterase activities of the cytosolic fraction of the ADP-treated platelets was affected. This effect of ADP, which was independent of platelet aggregation, reached maximal level within 5 min of incubation. When platelet-rich plasma was incubated longer in the presence of nucleotide, the inhibition of phosphodiesterase activity began to decrease, and after 20 min of incubation approx. 90% of the original enzymic activity was regained. The incubation of platelet-rich plasma with 4.0 microM ADP also increased the cyclic AMP level to twice the basal level. The effect of ADP on the phosphodiesterase activity could be demonstrated only by incubating the intact platelets with the nucleotide. The treatment of isolated membrane from platelets, previously unexposed to ADP, with the nucleotide did not inhibit the enzymic activity. The inhibition of phosphodiesterase by the nucleotide in the absence of stirring, as expected, resulted in the inhibition of platelet aggregation when these cells were subsequently stirred with 1-epinephrine or an increased concentration of ADP.     

A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cell whole sonicate or cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.     

Effect of imidazole on renal gluconeogenesis

The metabolic effects of imidazole were tested in rat renal cortex. Imidazole enhanced the activity of renal cortical phosphodiesterase in vitro. Imidazole inhibited glucose production in a dose-dependent fashion from a variety of substrates in the gluconeogenic pathway proximal to the triose phosphates. The stimulation in renal gluconeogenesis resulting from isoproterenol and parathyroid hormone was inhibited by imidazole. These changes correlated with an inhibition of the augmented levels of renal cortical cyclic AMP levels produced by these hormones. These studies indicate that imidazole is an effective activator of phosphodiesterase in intact renal cells and lend further support to the suggestion that the stimulation of renal gluconeogenesis produced by isoproterenol and parathyroid hormone is mediated by a release of cyclic AMP.     

In vivo regulation of cyclic AMP phosphodiesterase in Xenopus oocytes. Stimulation by insulin and insulin-like growth factor 1

Cyclic AMP phosphodiesterase activity was measured in vivo after microinjection of [3H]cAMP into intact Xenopus oocytes. This activity was inhibited by extracellular application of methylxanthines, and the dose-dependent inhibition of phosphodiesterase activity correlated with the abilities of isobutylmethylxanthine and theophylline to inhibit oocyte maturation induced by progesterone, with IC50 values of approximately 0.3 and 1.5 mM, respectively. Insulin stimulated in vivo phosphodiesterase activity measured after microinjection of 200 microM [3H]cAMP in a time- and dose-dependent fashion without affecting phosphodiesterase activity measured after microinjection of 2 microM [3H]cAMP. Although progesterone alone had no effect on in vivo phosphodiesterase activity, low concentrations of progesterone (0.01 microM) accelerated the time course of insulin stimulation of both phosphodiesterase activity and oocyte maturation. The EC50 for stimulation of in vivo phosphodiesterase activity by insulin correlated with the IC50 for inhibition of oocyte membrane adenylate cyclase activity measured in vitro (2 and 4 nM, respectively). Twenty-fold higher concentrations of insulin were required to stimulate oocyte maturation. In contrast, insulin-like growth factor 1 stimulated in vivo phosphodiesterase, inhibited in vitro adenylate cyclase, and induced oocyte maturation at concentrations of 0.3-1.0 nM. These results demonstrate a dual regulation of oocyte phosphodiesterase and adenylate cyclase by insulin and insulin-like growth factor 1.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.