ONTOGENETIC DEVELOPMENT OF A PHOSPHODIESTERASE ACTIVATOR AND THE MULTIPLE FORMS OF CYCLIC AMP PHOSPHODIESTERASE OF RAT BRAIN

Abstract— The activity of cyclic AMP phosphodiesterase of rat cerebral homogenates increased several-fold between 1 and 60 days of age. Enzyme activity in the cerebellum, on the other hand, did not increase during this period. A kinetic analysis of the phosphodiesterase activity revealed evidence for multiple forms of the enzyme and indicated that the postnatal increase in phosphodiesterase activity of rat cerebrum was due almost exclusively to the high K_m enzyme. In cerebellum, the ratio of the high and low K_m enzyme remained fairly constant during ontogenetic development. Physical separation of the phosphodiesterases contained in 100,000 g soluble supernatant fractions of sonicated brain homogenates by polyacrylamide disc gel electrophoresis confirmed the presence of multiple enzyme forms. In adult rats we found six distinct peaks of phosphodiesterase activity (designated I to VI according to the order in which they were eluted from the column) in cerebellum and 4 forms of the enzyme (Peaks I through IV) in cerebrum. Brains of newborn rats had a different pattern and ratio of phosphodiesterase activities. For example, Peak I phosphodiesterase was undetectable in cerebrum or cerebellum of newborn rats. Moreover, in the cerebellum of newborn rats Peak II was the dominant peak whereas in the cerebellum of adult rats Peak III was the largest peak. A comparison of the multiple forms of phosphodiesterase from the cerebrum of newborn and adult animals suggested that the postnatal increase in phosphodiesterase activity previously seen in crude homogenates was due largely to an increase in a high K, Peak II phosphodiesterase. The ratios of activities of the other peaks and their sensitivities to an activator of phosphodiesterase were similar in newborn and adult rats. An endogenous heat-stable activator of phosphodiesterase was found in cerebrum, cerebellum and brain stem. In newborn rats, the cerebellum contained several-fold less activity of this activator than did cerebrum or brain stem. However, the activity of this activator increased with age in the cerebellum and would appear to have decreased postnatally in cerebrum and brain stem. These results suggest that some multiple forms of phosphodiesterase can develop independently and that changes in activities of these phosphodiesterases may occur by increases in the quantity of enzyme or by changes in the quantity of an endogenous activator of phosphodiesterase.     

Calcium-dependent 3':5'-cyclic nucleotide phosphodiesterase. Inhibition of basal activity at physiological levels of potassium ions.

A calcium-dependent cyclic nucleotide phosphodiesterase from rat cerebrum was, in the absence of activator protein, inhibited by various monovalent cations. The inhibition was rapid, readily reversible, and concentration-dependent, with 100 mM cesium, rubidium, or potassium ion inhibiting essentially all basal enzyme activity, while 100 mM sodium or lithium ions produced only moderate inhibition. The potency of the cations in inhibiting the enzyme was Cs greater than or equal to Rb greater than K greater than Na greater than or equal to Li. Potassium ions increased the apparent Km for cyclic GMP and cyclic AMP by 3- and 5-fold, respectively. At 100 mM, the monovalent cations inhibited enzyme activated by the calcium-dependent activator by only 15 to 30%, while at 55 mM no inhibition pertained. Potassium and sodium ions at 55 mM had no effect on the calcium-independent phosphodiesterase from rat cerebrum. The results indicate that at normal intracellular concentrations of potassium ions the activity of the calcium-dependent phosphodiesterase is virtually completely dependent on the presence of calcium plus activator protein.     

Characteristics of the cyclic nucleotide phosphodiesterases of normal and leukemic lymphocytes

The specific activity of cylic AMP phosphodiesterase and cyclic GMP phosphodiesterase of leukemic lymphocytes was 5–10-fold greater than that of purified normal lymphocytes or homogenates of spleen, thymus or lymph nodes of normal mice. This rise was demonstrable over a wide range of substrate concentrations. Both normal and leukemic lymphocytes contained a heat-stable, calcium-dependent activator of phosphodiesterase. However, the increased activity of phosphodiesterase in leukemic lymphocytes was not due to this protein activator since (a) phophodiesterase activity from these cells was not stimulated by this activator and (b) phosphodiesterase activity of leukemic lymphocytes was not inhibited by the calcium chelator, ethyleneglycol-bis-(β-aminoethylether)-N′,N′-tetraacetic acid, suggesting that the enzyme was not already maximally activated. A comparison of several other properties of phosphodiesterase from normal and leukemic lymphocytes showed that the enzymes have similar pH optima, similar stabilitis to freezing and thawing and similar sensitivities to inhibition by the phosphodiesterase inhibitors, chlorpromazine, papaverine and isobutylmethylxanthine. However, the subcellular distribution of the phosphodiesterases was different, and the phosphodiesterase of leukemic lymphocytes was significantly more resistant to heat than that of normal lymphocytes.Although no differences were found between the phosphodiesterases of normal and leukemic lymphocytes in their sensitivities to drugs, there were marked differences in drug sensitivity between the phosphodiesterase of lymphocytes and that of other tissue. For example, concentrations of chlorpromazine which inhibited phosphodiesterase of cerebrum by 70% had no effect on phosphodiesterase activity of lymphocytes. On the other hand, the papaverine-induced inhibition of phosphodiesterase was similar in lymphocytes and cerebrum.Since an optimal concentration of cyclic nucleotides is essential to maintain normal cell growth, these results suggest that the abnormal growth characteristics of leukemic lymphocytes may be explained by their high activity of phosphodiesterase. Furthermore, the qualitative and quantitative differences between the phosphodiesterases of leukemic lymphocytes and other tissues raise the possibility of selectively inhibiting the phosphodiesterase of the leukemic lymphocytes, thereby reducing their rate of growth, without affecting other tissues.     

Phosphodiesterase activator from rat kidney cortex.

Incubation of homogenates of rat renal cortex at 4 degrees resulted in increased cAMP phosphodiesterase activity; the increase was much more rapid in hypotonic medium than in one of physiological tonicity. cAMP phosphodiesterase activity did not increase with incubation of supernatant fractions (48,000 x g, 20 min) prepared from isotonic homogenates. Extraction of the isotonic particulate fraction with hypotonic buffer released an activator which increased cAMP phosphodiesterase activity of the supernatant fraction. The kidney phosphodiesterase activator differed from a heat-stable, calcium-dependent protein activator of phosphodiesterase in that it was destroyed by heating (90 degrees for 10 min) and was not inhibited by EGTA. The phosphodiesterases of rat renal cortex were partially resolved by chromatography on DEAE-Bio-Gel, and a cAMP phosphodiesterase that is sensitive to the kidney activator was identified. This phosphodiesterase was separable from that affected by a calcium-dependent phosphodiesterase activator from bovine brain and from cGMP-stimulated cAMP phosphodiesterase. As determined by sucrose density gradient centrifugation, after incubation with the kidney activator, the activated form of phosphodiesterase had a lower sedimentation velocity than did the unactivated form.     

Calcium-dependent cyclic nucleotide phosphodiesterase inhibition of basal activity by heat-stable factors from rat cerebrum

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca²⁺-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca²⁺-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in K_m for cyclic GMP and a decrease in $\text{V}$. In the presence of calcium ions and purified activator protein, the Ca²⁺-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca²⁺-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was mimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca²⁺-independent activation of Ca²⁺-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca²⁺-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca²⁺-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phosphodiesterase activity.     

Effects of chymotrypsin and a calcium-dependent protein activator on guanosine 3′,5′-monophosphate phosphodiesterase from rat liver

Cyclic GMP phosphodiesterases from 100 00 × g rat liver supernatant were partially resolved by chromatography on DEAE-cellulose. Multiple forms of cyclic GMP phosphodiesterase(s) that were activated to different degrees by calcium plus a low molecular weight protein from rat liver and bovine brain supernantants, or by limited exposure to chymotrypsin, were identified. The cyclic GMP phosphodiesterase in some column fractions was activated over 10-fold by calcium plus activator or chymotrypsin. Activation by chymotrypsin was dependent both on the time of incubation with protease and its concentration. Prolonged exposure to chymotrypsin resulted in a decrease in s_20,w by sucrose density gradient centrifugation. The chymotrypsin-treated enzyme was no longer activated by exposure to calcium plus activator. The calcium- and protein activator-stimulated enzyme was inactivated by ethyleneglycol-bis-(β-aminoethylether)-N,N′-tetraacetic acid (EGTA). Exposure of this activated enzyme to chymotrypsin did not result in further activation, but the chymotrypsin-treated enzyme was no longer inhibited by EGTA. The apparently irreversible effects of chymotrypsin and the reversible effects of calcium plus activator on cyclic GMP hydrolysis by the phosphodiesterase over a wide range of cyclic GMP concentrations appeared to be identical.     

Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.     

Isolation of an activator of multiple forms of cyclic nucleotides phosphodiesterase of rat cerebrum by isoelectric focusing.

An isoelectric focusing technique was used to isolate multiple forms of cyclic nucleotide phosphodiesterase from a 105 000 times g soluble supernatant fraction of sonicated rat cerebrum. These separated peaks of activity had iso-electric points of 5.1, 5.6, 6.0, 6.6, 8.0, and 9.0. The activities were not stimulated by an endogenous activator of the enzyme but were inhibited by EGTA treatment. However, activator-sensitive forms of the enzyme could be separated from brain if the preparation of rat cerebrum was dialyzed against an EGTA containing buffer prior to electrofocusing. The procedure was also used to isolate a column fraction that stimulated maximum velocities of cyclic AMP and cyclic GMP hydrolysis. This fraction was itself devoid of phosphodiesterase activity and had an isoelectric point of 4.7.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

Ca++-dependent formation of brain adenylate cyclase-protein activator complex

In the presence of EGTA (ethyleneglycol-bis-(β-aminoethyl-ether) N,N′-tetraacetic acid), a Lubrol-PX solubilized rat brain adenylate cyclase (E.C. 4.4.1.1) and its protein activator were separated from each other in a Sephadex G-200 column. No activator was associated with the eluted enzyme, which required an exogenous activator for maximum activity. On the other hand, in the presence of Ca⁺⁺, some of the activator was eluted with the enzyme, which was independent of an exogenous activator for maximum activity. Because neither Ca⁺⁺ nor EGTA affected the elution profile of the activator in the filtration column, these results suggest that the formation of the enzyme-activator complex is dependent on Ca⁺⁺. Separate experiments indicated that the effect of Ca⁺⁺ on the formation of the enzyme-activator complex was immediate and reversible. Because the activator appears to be in excess of the enzyme, adenylate cyclase activityin vivo could be modulated by the cellular flux of Ca⁺⁺.     

Activity of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by bovine heart and rat liver cyclic nucleotide phosphodiesterases.

The effects of imidazole on the hydrolysis of cyclic AMP and cyclic GMP by crude and partially purified phosphodiesterases obtained from bovine heart and rat liver were studied in order to determine if imidazole has an activity on cyclic nucleotide hydrolysis under conditions which might explain its ability to antagonize the effects of several hormones. Imidazole-Cl (40 mm, pH 7.4) had no effect on the hydrolysis of cyclic AMP or cyclic GMP at substrate levels below 10 μm by the crude enzymes but increasing stimulation was observed with increasing substrate concentrations reaching a twofold stimulation at 1 mm cyclic nucleotide. Three phosphodiesterases with varying substrate specificities were partially purified from bovine heart by ammonium sulfate precipitation and diethyl aminoethyl cellulose chromatography. With these enzymes imidazole had less stimulatory activity and some inhibitory effect on the hydrolysis of 10^?4m cyclic AMP and cyclic GMP but was without significant effect on the hydrolysis of 10^?6m cyclic AMP or cyclic GMP. The stimulatory activity of imidazole on the hydrolysis of high levels of cyclic nucleotide was dependent on the presence of phosphodiesterase activator. The stimulatory effect of the activator and imidazole plus activator on the hydrolysis of 10^?4m cyclic GMP by the rather cyclic GMP-specific enzyme could be eliminated by the addition of ethylene glycol-bis-(β-aminoethyl ether)N,N′-tetraacetate (EGTA) and restored by Ca²⁺. Imidazole was without effect on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase from bovine heart. The lack of effect of imidazole on the hydrolysis of physiological levels of cyclic AMP or cyclic GMP suggests that the activity of imidazole to antagonize the effects of various hormones is probably not due to a direct action of imidazole on the hydrolysis of cyclic AMP or cyclic GMP.     

Catalytic and regulatory properties of two forms of bovine heart cyclic nucleotide phosphodiesterase.

The soluble supernatant fraction of bovine heart homogenates may be fractionated on a DEAE cellulose column into two cyclic nucleotide phosphodiesterases (EC 3.1.4.-):PI and PII phosphodiesterases, in the order of emergence from the column. In the presence of free Ca2+, the PI enzyme may be activated several fold by the protein activator which was discovered by Cheung((1971) J. Biol. Chem. 246, 2859-2869). The PII enzyme is refractory to this activator, and is not inhibited by the Ca2+ chelating agent, ethylene glycol bis (beta-aminoethyl ether)-N, N'-tetraacetate (EGTA). The activated activity of PI phosphodiesterase may be further stimulated by imidazole or NH+4, and inhibited by high concentrations of Mg2+. These reagents have no significant effect on either the PII enzyme or the basal activity of PI phosphodiesterase. Although both forms of phosphodiesterase can hydrolyze either cyclic AMP or cyclic GMP, they exhibit different relative affinities towards these two cyclic nucleotides. The PI enzyme appears to have much higher affinities toward cyclic GMP than cyclic AMP. Km values for cyclic AMP and cyclic GMP are respectively 1.7 and 0.33 mM for the non-activated PI phosphodiesterase; and 0.2 and 0.007 mM for the activated enzyme. Each cyclic nucleotide acts as a competitive inhibitor for the other with Ki values similar to the respective Km values. In contrast with PI phosphodiesterase, PII phosphodiesterase exhibits similar affinity toward cyclic AMP and cyclic GMP. The apparent Km values of cyclic AMP and cyclic GMP for the PII enzyme are approx. 0.05 and 0.03 mM, respectively. The kinetic plot with respect to cyclic GMP shows positive cooperativity. Each cyclic nucleotide acts as a non-competitive inhibitor for the other nucleotide. These kinetic properties of PI and PII phosphodiesterase of bovine heart are very similar to those of rat liver cyclic GMP and high Km cyclic AMP phosphodiesterases, respectively (Russel, Terasaki and Appleman, (1973) J. Biol. Chem. 248, 1334).     

Protein activator of cyclic 3':5'-nucleotide phosphodiesterase of bovine or rat brain also activates its adenylate cyclase.

Bovine or rat brain adenylate cyclase (EC 4.6.1.1) solubilized by Lubrol PX contained an activator which was separated from the enzyme by an anionic exchange resin column. Dissociation of the activator from adenylate cyclase rendered the enzyme less active, and reconstituting with an exogenous activator restored full enzyme activity. A pure protein activator of cyclic 3′:5′-nucleotide phosphodiesterase (EC 3.1.4.17) isolated from bovine brain also stimulated this adenylate cyclase. Stimulation of adenylate cyclase by the activator required Ca⁺⁺, the effect being immediate and reversible. Although the activator was specific, it lacked tissue specificity; an activator isolated from bovine brain cross-activated effectively adenylate cyclase from rat, and vice versa. These findings indicate that brain adenylate cyclase required an activator for activity and that this activator is functionally identical to the protein activator of phosphodiesterase (J.B.C. 249: 4943–4954, 1974).     

Effect of proteases and protease inhibitors on adenylate cyclase activity in rat cerebral cortical membranes

Mild proteolysis of membrane preparations from rat cerebral cortex with low concentrations of endopeptidases such as trypsin or chymotrypsin caused a 50–400% increase in the basal adenylate cyclase activity. Maximal activation of adenylate cyclase was obtained by including the protease in the adenylate cyclase assay, although an activated preparation could be obtained by pretreatment of the membranes with proteolytic enzymes. The proteolytically activated enzyme showed an increased V, with very little change in the K_m for the substrate, ATP. The proteolytically activated enzyme retained responsiveness to activation by sodium fluoride and 5′-guanylylimidodiphosphate (GppNHp), but was no longer activated by gangliosides or calcium-dependent activator protein. Activation by alcohols and detergent was lost or reduced in magnitude. The activity of adenylate cyclase after protease treatment showed a very marked temperature dependence, with maximal activity expressed in the 30–40 °C range and no activation due to the prior protease treatment expressed at either 10 or 50 °C. Basal adenylate cyclase activity was usually slightly inhibited in the presence of various protease inhibitors. Activation by fluoride, gangliosides, or GppNHp was little affected by protease inhibitors although one inhibitor, N-α-tosyl-l-lysine chloromethyl ketone, caused an inhibition of the ganglioside and GppNHp responses, slightly inhibited the fluoride response, and blocked the norepinephrine response normally seen in the presence of gangliosides or GppNHp. This inhibitor caused a loss of β-adrenergic binding sites for dihydroalprenolol in rat cortical membranes which paralleled the loss of the responsiveness of adenylate cyclase to a GppNHp-norepinephrine combination.     

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.     

Measurement of protein activator levels in experimental diabetic rat adipose tissue.

Experimental diabetes induced by streptozotocin has been shown to decrease the level of cyclic AMP phosphodiesterase activity in rat adipose tissue. This reduced activity was restored with insulin. Protein activator, a small molecular weight substance, is essential for full activity of some component phosphodiesterases. Herein we demonstrate a significant decrease in protein activator level in the 13,000 X g boiled supernatant from streptozotocin-diabetic rat adipose tissue. However, although a decrease in protein activator level is consistent with diabetic inactivation of phosphodiesterase activity, additional studies presented here suggest that a defect in the diabetic phosphodiesterase enzyme itself also contributed to the decrease of total phosphodiesterase activity.     

Cyclic nucleotide phosphodiesterase and protein activator in fetal rabbit tissues.

Cyclic nucleotide phosphodiesterase activity (EC 3.1.4.17) was studied in fetal and newborn rabbit brain, heart, liver, kidney, and lung. Kinetic analysis of phosphodiesterase activity from homogenates of organs from the 25-day embryo suggested the presence of a high K_m and a low K_m activity for both cyclic AMP and cyclic GMP hydrolysis. The addition of 1 μm cyclic GMP to the assay stimulated the hydrolysis of cyclic AMP by whole homogenates of liver, brain, lung, and kidney, but not heart, at all of the ages studied. The addition of micromolar levels of calcium ion stimulated cyclic GMP hydrolysis by homogenates of fetal brain, heart, and kidney, with or without added protein activator. Cyclic GMP phosphodiesterase activity was not stimulated by the addition of calcium ion in homogenates of early fetal rabbit liver and lung, but stimulation was detected in the late embryo and newborn. The presence of the heat-stable protein activator was demonstrated in brain, heart, kidney, liver, and lung tissue at all of the fetal ages studied, and in the newborn rabbit. DEAE-cellulose chromatography demonstrated the presence of three separable enzymes in brain and liver at 15 days, heart at 19 days, and lung and kidney at 25 days of gestation, with no changes in the kinetic properties of the isolated enzymes during development. These experiments suggest that all of the organs studied have the mature array of phosphodiesterases early in development, but an enzyme from liver and lung becomes sensitive to regulatory control by calcium only late in gestation.     

Crystallization of alpha 1-acid glycoprotein

A possible link between cellular cyclic AMP content and Na+K+ATPase activity was investigated in homogenates of rat kidney. Enzyme kinetics of Mg2+ and Na+K+ATPase were run in the presence of cyclic AMP, dibutyryl cAMP and compounds expected to elevate cyclic AMP levels such as forskolin, a potent adenylate cyclase activator, IBMX, an inhibitor of phosphodiesterases, and the beta-agonist isoproterenol. Medullary Na+K+ATPase is strongly inhibited by cyclic AMP whereas cortical Na+K+ATPase was stimulated in the same conditions. The correlation between ATPase activity and cellular cyclic AMP content supports the concept of a possible regulation of the enzyme by cyclic AMP.     

Ca2+-independent cyclic GMP phosphodiesterases from rat liver and HTC hepatoma cells.

We have separated and characterized a Ca2+- and calmodulin-insensitive cyclic nucleotide phosphodiesterase from rat liver supernatant as well as an analogous enzyme from HTC hepatoma cells. Chromatography of rat liver supernatant on DEAE-cellulose in the presence and subsequently in the absence of 0.1 mM-CaCl2 resulted in the separation of two distinct phosphodiesterase activities, both of which preferentially hydrolysed cyclic GMP rather than cyclic AMP. One enzyme, E-Ib, was activated in the presence of Ca2+ and calmodulin, and the other, E-Ia, was not. The E-Ia enzyme, which did not bind to calmodulin-Sepharose, had Mr 325 000 and displayed anomalous kinetic behaviour [Km (cyclic GMP) 1.2 microM; Km (cyclic AMP) 15.4 microM]. The E-Ib enzyme, which bound to calmodulin-Sepharose in the presence of Ca2+, had Mr 150 000 and exhibited Michaelis-Menten kinetics for hydrolysis of cyclic GMP [Km (basal) 6.5 microM; Km (activated) 12.0 microM]. E-Ia activity was diminished by incubation with alpha-chymotrypsin and was unaffected by the action of a rat kidney lysosomal proteinase. Partial hydrolysis of E-Ib enzyme by alpha-chymotrypsin or the kidney proteinase resulted in irreversible activation of the enzyme. The E-I enzyme isolated from HTC hepatoma cells was similar to the rat liver E-Ia enzyme in many respects. Its apparent Mr was 325 000. Its activity was unaffected by calmodulin in the presence of Ca2+ or by incubation with the kidney proteinase, and was decreased by digestion with alpha-chymotrypsin. Unlike the liver E-Ia enzyme, however, the hepatoma enzyme exhibited normal kinetic behaviour, with Km (cyclic GMP) 3.2 microM. Although HTC cells contain two other phosphodiesterases analogous to those in rat liver and a calmodulin-like activator of phosphodiesterase, no calmodulin-sensitive phosphodiesterase was detected.     

Specificity of the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase and to a series of other calcium-binding proteins

Trifluoperazine inhibits the activation of phosphodiesterase by binding to the calcium-dependent activator. To determine further the specificity by which trifluoperazine binds to activator, we compared the binding of trifluoperazine to activator prepared from several species and tissues and to a number of other calcium-binding proteins devoid of activator activity.Trifluoperazine binds to activator prepared from human, bovine, rat and rabbit brain and from chick embryo fibroblasts. In each case, the binding of trifluoperazine to activator was qualitatively similar and related quantitatively to the ability of the preparation to activate phosphodiesterase.Of the other calcium-binding proteins examined, namely, troponin-C, S-100 protein, phospholipase A, phospholipase B and myosin light chain, only troponin-C displayed any significant calcium-specific binding of trifluoperazine. The binding to troponin-C, however, appeared to be different from the binding to activator; whereas the binding of trifluoperazine to actovator showed no cooperativity, the binding to troponin-C showed positive cooperatively.These results and earlier data showing that trifluoperazine fails to bind to a variety of other proteins, indicate that the binding of trifluoperazine to the calcium-dependent activator of phosphodiesterase is selective and suggest that this binding may explain some of the biochemical and pharmacological actions of this antipsychotic agent.