Phosphodiesterase Activities for Cyclic Nucleotides in Nerve Endings from the Bovine Posterior Pituitary Gland

Abstract: The cyclic nucleotide phosphodiesterase (PDE) activities were studied in a nerve ending fraction from bovine neural lobes. Most of the activity was particulate and unaffected by calcium. Lineweaver-Burk plots for this fraction showed negative cooperativity with apparent K _m values for cyclic AMP of 11 μ M and for cyclic GMP of 4 μ M . The soluble activities for both cyclic nucleotides were activated by calcium and inhibited by calmodulin-binding drugs (trifluoperazine and calmidazolium). The apparent K _m values were 50 μ M for cyclic AMP and 20 μ M for cyclic GMP for the soluble activities. Sucrose density gradients resolved the soluble activities into two peaks. The activity with the higher sedimentation rate (MW 122,000 daltons) hydrolysed both cyclic nucleotides and was calcium-calmodulin-dependent. The other peak (MW 47,000 daltons) had a higher affinity for cyclic AMP than for cyclic GMP and was calcium-independent. Solubilized particulate activities gave two main peaks on the density gradient, both calcium-independent. One was mainly for cyclic AMP (MW 47,000 daltons) and the other mainly for cyclic GMP (MW 133,000 daltons). The function of PDEs in relation to secretion was discussed.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF HUMAN CEREBROSPINAL FLUID

Abstract— Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'–GMP (cGMP) phosphodiesterase activities were found in human cerebrospinal fluid (CSF) using low substrate concentration (0.4μM). More rapid hydrolysis of cGMP than that of cAMP was observed in human CSF. However, cGMP hydrolytic activity of CSF was very much lower (0.3 pmol/min/ml CSF) than that of human cerebral cortex (33.7 nmol/min/g wet cortex). The pH optimum was found to be 8.0 (cGMP phosphodiesterase) and 7.5 (cAMP phosphodiesterase). The maximum stimulation of both cAMP and cGMP phosphodiesterase was achieved at 4 mM-MgCl₂. Cyclic AMP had relatively little effect on the hydrolysis of cGMP in CSF and the cortex, while cGMP inhibited hydrolysis of cAMP in both tissues. Snake venom was found to stimulate cAMP and cGMP phosphodiesterase activity of CSF, by 60% and 110% respectively. This stimulation by snake venom was also observed in the cortex phosphodiesterase, but was not observed in human plasma or thyroid phosphodiesterase. When CSF was applied to Sepharose 6B column, cGMP phosphodiesterase was separated into three different molecular forms. A plot of activity against substrate concentration using peak I (largest molecular size) revealed a high affinity ( K _m= 2.6μM) and a low affinity ( K _m= 100μM) for cAMP suggesting the existence of at least two molecular forms of the enzyme. On the other hand, using a cGMP as substrate the only one K _m value (1.90 μm) was obtained. These K _m values of CSF enzymes described above were close to those obtained from human cerebral cortex preparations. The enzyme under peak I corresponded to the cortex enzyme when judged from its molecular size and stimulation by snake venom. It seems likely from our results that at least a part of CSF phosphodiesterase originates from the central nervous system.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF THE BOVINE RETINA: ACTIVITY, SUBCELLULAR DISTRIBUTION AND KINETIC PARAMETERS

Abstract— High phosphodiesterase activity for cyclic AMP and cyclic GMP was found in subcellular fractions of the bovine retina with more rapid hydrolysis of cyclic GMP than cyclic AMP in each fraction. Rod outer segments (ROS) and the supernatant fraction had highest activity. High enzyme activity remained associated with ROS membranes through several steps of purification by gradient centrifugation. A complex kinetic pattern was observed for cyclic AMP hydrolysis by the supernatant fraction yielding two values for K _m; a simple kinetic pattern was observed with cyclic GMP hydrolysis in supernatant and for both cyclic nucleotides in preparations of purified outer segments. Phosphodiesterase activity of outer segments was enhanced by Mg²⁺. Mn²⁺ and inhibited by EDTA. Cyclic AMP had relatively little effect on the hydrolysis of cyclic GMP in supernatant or ROS while cyclic GMP inhibited hydrolysis of cyclic AMP in both fractions.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

MODULATION OF CYCLIC AMP LEVELS IN THE BOVINE SUPERIOR CERVICAL GANGLION BY PROSTAGLANDIN E, AND DOPAMINE

Abstract— Dopamine, norepinephrine, carbamylcholine and PGE₁ (prostaglandin E₁). increased cyclic AMP concentrations in slices of bovine superior cervical ganglia. PGF_1α was less effective and neither PGE₂ nor PGF_2α had any effect. Dopamine and PGE, alone or in combination, did not modify low K _m cyclic AMP phosphodiesterase activity. Combinations of dopamine and PGE, showed a marked synergistic effect, increasing ganglionic cyclic AMP to a much greater extent than that observed when the two compounds were tested alone. Norepinephrine (10 μ M) , which increased cyclic AMP as much as 10 μ m -dopamine, showed no synergistic effect when tested in the presence of PGE₁ or other PGs. Phentolamine, fluphenazine and triflupromazine blocked the dopamine effect without suppressing its synergism with PGE₁ Adenylate cyclase of synaptosomes isolated from the ganglia under a variety of experimental conditions appeared to be as responsive to PGE₁ as the slices, but it was poorly stimulated by dopamine and was not synergistically modulated by dopamine in the presence of PGE₁
These and other data are interpreted as indicating the presence of both a PGE₁-sensitive and a PGE₁-modulated dopamine-sensitive adenylate cyclase in the cervical ganglion. These adenylate cyclases are tentatively assigned to pre- and post-synaptic structures respectively.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Regional, cellular and subcellular distribution of calcium-activated cyclic nucleotide phosphodiesterase and calcium-dependent regulator in porcine brain.

Abstract— Cyclic nucleotide phosphodiesterase activity (calcium-dependent and calcium-independent) and CDR (calcium-dependent regulator protein of phosphodiesterase) are present in all ten brain regions examined, with the specific activity of both being highest in areas predominating in grey matter and lowest in areas consisting largely of white matter. Fractionation of cerebral cortex into neuronal and glial perikarya shows that cyclic nucleotide phosphodiesterase and CDR are present at approximately equal levels in both cell types. Subcellular fractionation reveals that calcium-sensitive enzyme specific activity, as well as CDR are highly localized in the 100,000 g supernatant fraction. Regional, cellular and subcellular distribution studies indicate that the distribution of CDR closely parallels the distribution of enzyme specific activity, suggesting that the levels of cyclic nucleotide phosphodiesterase and CDR may be synchronized.     

Purification, characterization and production of rabbit antibodies to rat liver particulate, high-affinity, cyclic AMP phosphodiesterase

The cyclic nucleotide phosphodiesterase (EC 3.4.16) activities of a rat liver particulate fraction were analyzed after solubilization by detergent or by freeze-thawing. Analysis of the two extracts by DEAE-cellulose chromatography revealed that they contain different complements of phosphodiesterase activities. The detergent-solubilized extract contained a cyclic GMP phosphodiesterase, a low affinity cyclic nucleotide phosphodiesterase whose hydrolysis of cyclic AMP was activated by cyclic GMP and a high affinity cyclic AMP phosphodiesterase. The freeze-thaw extract contained a cyclic GMP phosphodiesterase and two high affinity cyclic AMP phosphodiesterase, but no low affinity cyclic nucleotide phosphodiesterase. The cyclic AMP phosphodiesterase activities from the freeze-thaw extract and from the detergent extract all had negatively cooperative kinetics. One of the cyclic AMP phosphodiesterases from the freeze-thaw extract (form A) was insensitive to inhibition by cyclic GMP; the other freeze-thaw solubilized cyclic AMP phosphodiesterase (form B) and the detergent-solubilized cyclic AMP phosphodiesterase were strongly inhibited by cyclic GMP. The B enzyme appeared to be converted into the A enzyme when the particulate fraction was stored for prolonged periods at -20 degrees C. The B form was purified extensively, using DEAE-cellulose, a guanine-Sepharose column and gel filtration. The enzyme retained its negatively cooperative kinetics and high affinity for both cyclic AMP and cyclic GMP throughout the purification, although catalytic activity was always much greater for cyclic AMP. Rabbit antiserum was raised against the purified B enzyme and tested via a precipitin reaction against other forms of phosphodiesterase. The antiserum cross-reacted with the A enzyme and the detergent-solubilized cyclic AMP phosphodiesterase from rat liver. It did not react with the calmodulin-activated cyclic GMP phosphodiesterase of rat brain, the soluble low affinity cyclic nucleotide phosphodiesterase of rat liver or a commercial phosphodiesterase preparation from bovine heart. These results suggest a possible interrelationship between the high affinity cyclic nucleotide phosphodiesterase of rat liver.     

Heterogeneity of β-Adrenoceptors in Guinea-Pig Brain: Radioligand Binding and Cyclic Nucleotide Generation

Abstract: In this report, we have examined the radioligand binding and second messenger signalling characteristics of β-adrenoceptors in the guinea-pig brain. [¹²⁵I]lodocyanopindolol ([¹²⁵I]ICYP)-labelled sites in the cerebellum and cerebral cortex were of similar densities ( B _max 34 and 24 fmol·mg⁻¹) and affinities ( K _D 20 and 55 p M ), respectively. Analysis of competition for [¹²⁵I]ICYP binding in the cerebellum was compatible with the presence of a β₂-adrenoceptor. In this tissue, isoprenaline evoked a cyclic AMP stimulation, and also potentiated cyclic GMP accumulations evoked in the presence of a nitric oxide donor, consistent with mediation via a β₂-adrenoceptor. The [¹²⁵I]ICYP binding profile in the cerebral cortex did not comply with those previously described for β-adrenoceptor subtypes, and isoprenaline failed to alter significantly cyclic AMP accumulation in the cerebral cortex, hippocampus, or neostriatum, even in the presence of forskolin or a phosphodiesterase inhibitor. Isoprenaline was also without effect on cyclic GMP accumulation or phosphoinositide turnover in the cerebral cortex. These results suggest that the guinea-pig cerebellum expresses a functional β₂-adrenoceptor coupled to cyclic AMP generation, and potentiation of cyclic GMP accumulation. However, the guinea-pig cerebral cortex displays binding sites that exhibit β-adrenoceptor-like pharmacology but fail to show functional coupling to cyclic AMP, cyclic GMP, or phosphoinositide signalling systems.     

COMPARISON OF THE EFFECTS OF DEPOLARIZING AGENTS AND NEUROTRANSMITTERS ON REGIONAL CNS CYCLIC GMP LEVELS IN VARIOUS''ANIMALS

Abstract— The effects of 121 m m -K⁺, 10 m m -glutamate, 5 m m -GABA, 1 m m -glycine, 0.1 m m -NE, and 1–10 μ m ACh on cyclic GMP levels in tissue slices prepared from cerebral cortex and cerebellum of mouse, rabbit, guinea-pig, cat, and rat were studied. Basal levels of cyclic GMP in the cerebella of mice, guinea-pigs and cats were 4–15 and 70 pmol/mg prot in rat, whereas in the cerebral cortex of the same animals, levels were only 0.6–2 pmol/mg prot. In contrast, basal levels of the cyclic nucleotide were 1–2 pmol/mg prot in both of these regions in rabbit brain. Only 121 m m -K⁺ was capable of increasing cyclic GMP levels in all the tissues studied. Elevations ranged from 30% in rat cerebral cortex to 2800% in mouse cerebellum. Glutamate produced a 30–1000% rise of cyclic GMP levels in all tissues except rabbit cerebellum. NE elevated levels of cyclic nucleotide 2- to 3-fold in slices of cerebellum from all species studied but had no effect in cerebral cortex. GABA and glycine had no effect in any tissue except mouse cerebellum. ACh had no consistent effect on levels of cyclic GMP in any brain region investigated. These results suggest that mechanisms regulating cyclic GMP levels in mammalian CNS vary among brain regions and among animal species.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

Levels of cyclic nucleotides in mouse regional brain following 300 ms microwave inactivation.

Abstract— The uniformity and speed of inactivation of mouse brain adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterase were measured after 6 kW microwave irradiation (MWR). Inactivation of enzymes was uniform throughout the brain during heating and 100% loss of activity was evident after 300 ms. MWR. For comparison of effects of inactivation times on levels of cyclic nucleotides measured in regional brain areas, cyclic AMP and cyclic GMP were estimated after 1.5 kW MWR requiring 4 s of heating and 6 kW MWR requiring 300 ms. Except for corpus striatum, uniformly lower levels of cyclic AMP were measured following 300 ms vs. 4s MWR . There was no change in cyclic GMP levels in regional brain areas after 4s vs. 300 ms MWR . Cyclic AMP and cyclic GMP were measured from the same regional brain tissue samples after 300 ms and ratios calculated. The finding of much lower cyclic AMP:cyclic GMP ratios than had previously been reported suggests that slow inactivation times provide for the measurement of regional brain cyclic nucleotide values which are not consistent with the in-vivo state.     

REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES OF CEREBRAL CORTEX BY Ca2+ AND CYCLIC GMP

Cyclic nucleotide phosphodiesterase activity of porcine cerebral cortical extracts was measured with 0.1–100 μM-cyclic AMP and cyclic GMP and found to be dependent on both Ca²⁺ and added cyclic nucleotides. With decreasing substrate concentration activity with cyclic GMP became more dependent on Ca²⁺ whereas hydrolysis of cyclic AMP became less dependent. Cyclic GMP at 3 μM stimulated the hydrolysis of 0.1–10μM-cyclic AMP in the absence of Ca²⁺ (< 10^-10M) but inhibited activity with 200 μM-Ca²⁺ present. This differential, substrate- and Ca²⁺-dependent regulation was attributed to the presence of at least two types of phosphodiesterase distinguishable by DEAE-column chromatography. In the absence of Ca²⁺, activity with 1 μM-cyclic GMP eluted in one minor peak followed by two major peaks, D-I and D-II. Activity with 1 μM-cyclic AMP eluted almost entirely in D-II. Hydrolysis of cyclic AMP in D-II was activated by cyclic GMP. With added Ca²⁺ plus a Ca²⁺-dependent regulator (CDR), activity with 1 μM-cyclic GMP was markedly increased and eluted entirely at D-I. Total activity with 1 μM-cyclic AMP was only moderately increased and eluted as D-I with a shoulder at D-II. Elution profiles with 100 μM-substrate were relatively independent of substrate, with D-I predominant with Ca²⁺·CDR present and D-II predominant in its absence. Kinetic analysis of rechromatographed D-I showed a 20- to 40-fold activation by Ca²⁺·CDR that was largely due to an increase in V_max, with only 50% decreases in K_m Both substrates competitively inhibited hydrolysis of the other with K_i values equal to their respective K_m values (1.7 μM for cyclic GMP and 48 μM for cyclic AMP with Ca²⁺-CDR present). Studies with theophylline and trifluoperazine indicate differential, substrate-dependent inhibitions of both enzymes. These findings demonstrate that phosphodiesterase activity in neural tissue is subject to regulation by Ca²⁺, cyclic GMP, and inhibitors in a complex, substrate-specific and concentration-dependent manner.     

Differential Effects of Two Protein Kinase C Inhibitors, Calphostin C and Gö6976, on Pineal Cyclic Nucleotide Accumulation

Abstract: In rat pinealocytes, protein kinase C (PKC) is involved in the α₁-adrenergic-mediated potentiation of β-adrenergic-stimulated cyclic nucleotide responses; however, the specific PKC isozyme(s) involved in the potentiation mechanism remain unknown. In the present study, we compared the effects of two PKC inhibitors, calphostin C, a specific inhibitor of PKC, and Gö6976, a selective inhibitor of PKCα and PKCβ₁, on the adrenergic-stimulated cyclic nucleotide accumulation in rat pinealocytes. Surprisingly, Gö6976 was found to have an enhancing effect on basal cyclic GMP and isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, an effect not shared by calphostin C. Gö6976 also increased the norepinephrine- and ionomycin-induced potentiation of isoproterenol-stimulated cyclic AMP and cyclic GMP accumulation, whereas the effect of calphostin C was inhibitory. The enhancing effect of Gö6976 was abolished in the presence of isobutylmethylxanthine or zaprinast, but not rolipram, suggesting that this effect of Gö6976 may be mediated through type V or the retinal type of phosphodiesterase. Based on these observations, we propose that some of the PKC isozyme(s) inhibited by calphostin C are involved in the potentiation of β-adrenergic-stimulated cyclic nucleotide responses and that they act by enhancing synthesis. However, PKC isozymes inhibited by Gö6976 appear to be basally active and tonically inhibit cyclic nucleotide accumulation through their stimulatory action on phosphodiesterase.     

Assessment of selective inhibition of rat cerebral cortical calcium-independent and calcium-dependent phosphodiesterases in crude extracts using deoxycyclic AMP and potassium ions

The effects of various inhibitors on the activity of calcium-independent and calcium-dependent phosphodiesterases from rat cerebral cortex were examined. While the agents varied greatly in their relative potency, each was found to be approximately equipotent in inhibiting the calcium-dependent hydrolysis of either cyclic AMP or cyclic GMP. In contrast, the inhibitors displayed a marked substrate specificity for the calcium-independent enzyme with ratios of IC₅₀ values for inhibition of cyclic GMP hydrolysis when compared to cyclic AMP hydrolysis in decreasing order being: ZK 62711 (? 100) > Ro 20–1724 (?>25) papaverine (13) > 7-benzyl IBMX (4) > quercetin and kaempferol (2). The differential selectivity of the inhibitors for the two enzymes was most pronounced for ZK 62711 and Ro 20–1724 which were at least 25–100-times more potent in inhibiting the calcium-independent hydrolysis of cyclic AMP when compared to the calcium-dependent hydrolysis of cyclic AMP. In contrast, 7-benzyl IBMX, kaempferol and quercetin were 8–100-times more effective as inhibitors of cycluc GMP hydrolysis by the calcium-dependent phosphodiesterase while 7-benzyl IBMX and trimazosin displayed a similar enzyme selectivity using cyclic AMP as substrate. With the exception of papaverine, all agents were competitive inhibitors of the calcium-dependent phosphodiesterase. The type of inhibition observed with the calcium-independent enzyme was dependent on the substrate employed. The specificity of potassium ions in inhibiting the activity of the calcium-dependent phosphodiesterase and deoxycyclic AMP in inhibiting the calcium-independent enzyme was found to provide a convenient means to assess the effects of agents on these activities in crude extracts of cerebral cortex.     

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.     

Evidence for convertible forms of soluble uterine cyclic nucleotide phosphodiesterase

The cyclic nucleotide phosphodiesterase (3':5'-cyclic nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. Km of approx. 3 and 20 microM) and linear kinetics for cyclic GMP (app. Km of approx. 3 microM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (Ki approx. 32 microM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (Ki approx 16 microM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (Ki approx 34 microM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 microgram/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc- and displays anomalous kinetics for cyclic AMP hydrolysis (app. Km of 2 and 20 microM) and linear kinetics for cyclic GMP (app. Km approx. 5 microM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc- and displays a higher apparent affinity for cyclic AMP (app. Km approx. 1.5 mu) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissues may be conversions from a single enzyme species.     

Cyclic nucleotide phosphodiesterases in the cricket, Acheta domesticus.

Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second peak specific for cyclic AMP. There were no appreciable changes in the specific activity or kinetic properties of accessory gland cyclic GMP phosphodiesterase during a developmental period over which cyclic GMP levels rise more than 500-fold. Thus, the accumulation of cyclic GMP in the accessory gland is probably not associated with concomitant developmental modulation of phosphodiesterase activity.     

Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein.

The calcium-dependent regulatory protein (CDR) purified from bovine brain was iodinated with Na[125I]I using the lactoperoxidase-glucose oxidase system. The iodinated protein retained its ability to stimulate the Ca2+-sensitive CDR-depleted cyclic nucleotide phosphodiesterase from bovine heart. Stimulation of the phosphodiesterase by 125I-CDR was Ca2+-dependent and the labeled protein had a Ka for activation of cyclic nucleotide phosphodiesterase that was 4 times greater than unmodified CDR. 125I-CDR formed a Ca2+-dependent complex with the partially purified cyclic nucleotide phosphodiesterase which was detectable by autorradiography following electrophoresis of the complex on nondenaturing gels. This technique was used to detect CDR binding components in crude homogenates prepared from bovine heart and brain.     

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.