Dependent Cyclic Nucleotide Phosphodiesterase Research Articles

Microarray and Real-Time PCR Analyses of Gene Expression in the Honeybee Brain Following Caffeine Treatment

To test the idea that caffeine might induce changes in gene expression in the honeybee brain, we contrasted the transcriptional profiles of control and caffeine-treated brains using high-throughput cDNA microarrays. Additional quantitative real-time PCR was performed on a subset of eight transcripts to visualize the temporal changes induced by caffeine. Genes that were significantly upregulated in caffeine-treated brains included those involved in synaptic signaling (GABA:Na symporter, dopamine D2R-like receptor, and synapsin), cytoskeletal modifications (kinesin and microtubule motors), protein translation (ribosomal protein RpL4, elongation factors), and calcium-dependent processes (calcium transporter, calmodulin- dependent cyclic nucleotide phosphodiesterase). In addition, our study uncovered a number of novel, caffeine-inducible genes that appear to be unique to the honeybee. Time-dependent profiling of caffeine-sensitive gene expression shows significant upregulation 1 h after treatment followed by moderate downregulation after 4 h with no additional changes occuring after 24 h. Our results provide initial evidence that the dopaminergic system and calcium exchange are the main targets of caffeine in the honeybee brain and suggest that molecular responses to caffeine in an invertebrate brain are similar to those in vertebrate organisms.

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase (PDE1) Is a Pharmacological Target of Differentiation-Inducing Factor-1, an Antitumor Agent Isolated from Dictyostelium

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10-40 micro M dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5-20 micro M inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

Calmodulin-dependent cyclic nucleotide phosphodiesterase activity is altered by 20 microT magnetostatic fields.

Absorbance measurements at 660 nm of calmodulin (CaM) dependent cyclic nucleotide phosphodiesterase activity under cell free conditions indicate that 30-min exposures to weak magnetostatic field intensities alters this activity, compared to zero magnetic field exposures. This effect depends nonlinearly on the concentration of free calcium, with maximum magnetic interaction apparently occurring at an optimal Ca(2+) concentration corresponding to 50% activation (EC(50)). If one regards Ca(2+)/CaM activation as a switching process, then increasing the magnetic field at Ca(2+) levels in excess of optimal acts to bias this switch towards lower calcium concentrations. A magnetic dependence has been previously reported by others in an homologous system, CaM dependent myosin light chain phosphorylation, implying that there may be an underlying magnetic interaction that involves the initial Ca(2+)/CaM binding process common to both enzymatic pathways. The level of magnetostatic intensity at which this effect is observed ( approximately 20 microT) implies that CaM activation may be functionally sensitive to the geomagnetic field.

Effects of ruthenium red on activation of Ca 2+-dependent cyclic nucleotide phosphodiesterase

Ruthenium red inhibited Ca 2+-dependent phosphodiesterase (Ca 2+-PDE) selectively with an IC 50 value of 15 μM. Increasing calmodulin concentration in the presence of both 100 μM and 4000 μM Ca 2+ completely reversed the inhibition of Ca 2+-PDE activity by ruthenium red. Ruthenium red-induced inhibition of Ca 2+-PDE activity was also overcome by increasing the concentration of Ca 2+ in the presence of both 200 ng and 2000 ng calmodulin, in sharp contrast to fluphenazine-induced inhibition of Ca 2+-PDE. These results indicate that ruthenium red has distinct inhibitory mechanism which differs from that of calmodulin antagonists previously reported.

Effects of cilostazol, a selective cAMP phosphodiesterase inhibitor on the contraction of vascular smooth muscle.

The effects of cilostazol (OPC-13013, 6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quin olinone) on cyclic nucleotide metabolism and Ca2+-induced contraction of intact and skinned rabbit arterial smooth muscles were investigated. The concentrations of cilostazol producing 50% inhibition of cyclic adenosine monophosphate phosphodiesterase and Ca2+-dependent cyclic nucleotide phosphodiesterase were 0.4 microM and above 100 microM, respectively. This compound has no significant effect on adenylate cyclase in concentrations of up to 100 microM. Addition of cilostazol increased significantly the cAMP content without significant effect on cyclic guanosine monophosphate level of rabbit thoracic aorta in the presence of forskolin. Moreover, the ED50 value of cilostazol in relaxation of rabbit mesenteric arterial strips was decreased selectively by addition of 0.01 microM forskolin, which alone at this concentration has no effect on vascular contraction. Cilostazol of up to 30 microM did not suppress the Ca2+-induced contraction of the chemically skinned rabbit mesenteric artery. Therefore, cilostazol may produce the relaxation of intact vascular smooth muscle by its inhibition of cyclic adenosine monophosphate hydrolysis.

Different sensitivities of Ca2+, calmodulin-dependent cyclic nucleotide phosphodiesterases from rabbit aorta and brain to dihydropyridine calcium channel blockers

The concentrations of the dihydropyridines, CD-349, nicardipine, and nimodipine, producing 50% inhibition of Ca2+, calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (CaPDE) from rabbit aorta in the absence of Ca2+-CaM complex were approximately 7 to 13-fold higher than these of aorta CaPDE in the presence of Ca2+-CaM complex and of the trypsin treated enzyme form. On the other hand, these dihydropyridine derivatives inhibited CaPDE from rabbit brain at much the same IC50 values seen in the absence and presence of the Ca2+-CaM complex and the trypsin-treated enzyme. Kinetic analysis revealed that these dihydropyridines inhibited the activities of CaPDEs from both the aorta and brain, competitively with cyclic GMP as substrate, and the Ki values of CD-349 for CaPDE from aorta or brain in the absence or presence of Ca2+-CaM complex and trypsin-treated enzyme were 9.6, 0.75, 0.75 or 0.69, 0.70, 0.66 microM, respectively. These results suggest that CaPDE from the rabbit aorta differs from this enzyme in the brain, with regard to the relationship between the dihydropyridine binding sites on CaPDE molecules and the domains regulated by the Ca2+-CaM complex or limited proteolysis.

Purification of calmodulin from bean rust uredospores

Calmodulin has been isolated from uredospores of the bean rust fungus and purified to apparent homogeneity as judged using sodium dodecyl sulfatepolyacrylamide gels. The protein substituted for bovine calmodulin as an activator of Ca 2+-dependent cyclic nucleotide phosphodiesterase. Its molecular mass was 15.6 kDa in the presence of Ca 2+ and 17.0 kDa in its absence. Thep I was 4.4. The amino acid composition was generally similar to those of calmodulin from other fungi.

The Ca 2+-activated protease (calpain) modulates Ca 2+/calmodulin dependent activity of smooth muscle myosin light chain kinase

The Ca 2+-activated neutral protease can proteolyze both Ca 2+-dependent cyclic nucleotide phosphodiesterase and smooth muscle myosin light chain kinase. Ca 2+-dependent cyclic nucleotide phosphodiesterase from rat brain was converted to the Ca 2+-independent active form by Ca 2+-activated protease. The proteolytic effects on myosin light chain kinase of Ca 2+-activated protease differed in the presence and absence of the Ca 2+-calmodulin (CaM) complex. In the presence of bound CaM, myosin light chain kinase (130k dalton) was degradated to a major fragment of 62 kDa, which had Ca 2+ CaM -dependent enzyme and CaM-binding activity. When digestion occurred in the absence of bound CaM, myosin light chain kinase cleaved to a fragment of 60 kDa. This peptide had no enzymatic activity in the presence or absence of the Ca 2+-CaM complex. Available evidence suggests that the Ca 2+-activated proteases may recognize the conformational change of smooth muscle myosin light chain kinase induced by Ca 2+-CaM complex.

Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons.

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.

Protective effects of the calmodulin antagonist bepridil on ischaemia induced in the rat myocardium.

The effects of a block of the slow calcium channel by bepridil and its antagonising effects on calmodulin were determined in the ischaemic rat myocardium. When isolated rat hearts were treated with a cardioplegic solution containing bepridil or verapamil there was a significant diminution in ischaemic damage in the myocardium. Bepridil was more effective than verapamil in protecting against this ischaemic damage. Bepridil effectively inhibited the activation of calmodulin dependent enzymes, such as calcium calmodulin dependent cyclic nucleotide phosphodiesterase and myosin light chain kinase. The inhibitory effect of verapamil on these enzymes was weaker than that of bepridil. Since bepridil protects the myocardium and minimises ischaemia induced damage these events might be related to a calmodulin antagonistic action as well as a calcium channel blocking action.

Ca2+-dependent cyclic nucleotide phosphodiesterase is activated by poly(L-aspartic acid).

Ca2+-dependent cyclic nucleotide phosphodiesterase (Ca2+-PDE) activity was stimulated by poly(L-aspartic acid) but not by poly(L-glutamic acid), poly(L-arginine), poly(L-lysine), and poly(L-proline). This activation was Ca2+ independent and did not further enhance the activation of Ca2+-PDE by Ca2+-calmodulin (CaM). Poly(L-aspartic acid) produced an increase in the Vmax of the phosphodiesterase, associated with a decrease in the apparent Km for the substrate, such being similar to results obtained with Ca2+-CaM. Poly(L-aspartic acid) did not significantly stimulate myosin light chain kinase and other types of cyclic nucleotide phosphodiesterase. CaM antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine selectively antagonized activation of the enzyme by poly(L-aspartic acid). Kinetic analysis of W-7-induced inhibition of activation of phosphodiesterase by poly(L-aspartic acid) was in a competitive fashion, and the Ki value was 0.19 mM. On the other hand, prenylamine, another type of calmodulin antagonist that binds to CaM at sites different from the W-7 binding sites, did not inhibit the poly(L-aspartic acid)-induced activation of Ca2+-dependent cyclic nucleotide phosphodiesterase. These results imply that poly(L-aspartic acid) is a calcium-independent activator of Ca2+-dependent phosphodiesterase and that aspartic acids in the CaM molecule may play an important role in the activation of Ca2+-PDE.

The differential stimulation of brain and heart cyclic-AMP phosphodiesterase by oncomodulin

Ca 2+ / calmodulin dependent cyclic nucleotide phosphodiesterase, from the bovine heart and brain, purified by monoclonal antibody chromatography were tested with respect to activation by oncomodulin. The heart and brain enzymes which have previously been shown to have slightly different electrophoretic mobilites (1), were found to also differ in the oncomodulin dose-dependent activation of cAMP hydrolysis. Oncomodulin was shown to activate the heart enzyme to the same extent as calmodulin. However, this study indicates that the heart phosphodiesterase has approximately 25-fold higher affinity for oncomodulin than the brain enzyme. The oncomodulin concentration required for the half-maximal activation of the heart phosphodiesterase was estimated to be 2 × 10 −7M. In addition, the possibility of the observed activation by oncomodulin being due to calmodulin contamination can be ruled out as the oncomodulin activation profiles were unaltered subsequent to chromatography on organomercurial agarose and the activation by oncomodulin could not be reversed by anti-calmodulin IgG.

Calcium-dependent interactions of an ionophore A23187 with calmodulin

We found that ionophore A23187 interacted reversibly with calmodulin (CaM), in a calcium-dependent fashion. It was found that A23187 interacts selectively with CaM, among calcium binding proteins (such as troponin C and S-100 protein) and other proteins. However, apparently differing from W-7, A23187 did not suppress CaM-dependent enzyme activity such as myosin light chain kinase and Ca 2+-dependent cyclic nucleotide phosphodiesterase. Our observations suggest that there are novel calcium-dependent regions of CaM which can be monitored using ionophore A23187 and may not be related to enzyme activation.

P-13) - Activation of Ca2+-dependent cyclic nucleotide phospho diesterase by poly-L-aspartic acid and inhibition by calmodu lin antagonsits

P-13) - Activation of Ca2+-dependent cyclic nucleotide phospho diesterase by poly-L-aspartic acid and inhibition by calmodu lin antagonsits

Function of calmodulin in mammalian sperm: Presence of a calmodulin-dependent cyclic nucleotide phosphodiesterase associated with demembranated rat caudal epididymal sperm

A calmodulin dependent cyclic nucleotide phosphodiesterase is associated with the head and tailpieces of demembranated rat caudal epididymal sperm. The phosphodiesterase was stimulated two-fold in the presence of Ca 2+, while the simultaneous addition of Ca 2+ and calmodulin resulted in a four-fold increase in activity. Ca 2+ stimulation was abolished if demembranated sperm were extracted with EGTA and was recovered upon the addition of exogenous calmodulin. Micromolar levels of Ca 2+ were required for full stimulation. Trifluoperazine inhibited the Ca 2+ stimulated enzyme in a dose dependent manner (ID 50 = 50 μM) but had no effect on the basal phosphodiesterase activity.

Calcium-independent activation of calcium ion dependent cyclic nucleotide phosphodiesterase by synthetic compounds: quinazolinesulfonamide derivatives.

Quinazolinesulfonamides are synthetic compounds which calcium-independently stimulate Ca2+-dependent cyclic nucleotide phosphodiesterase. As this activation was observed with 2,4-dipiperidino-6-quinazolinesulfonamides but not with 4-piperidino-6-quinazolinesulfonamides, the activation seems to be dependent on the piperidine residue at the 2 and 4 position of the quinazoline ring, and the extent of hydrophobicity of each compound was thus enhanced. 2,4-Dipiperidino-6-quinazolinesulfonamide activates Ca2+-dependent phosphodiesterase in the absence of Ca2+-calmodulin (CaM). These quinazolinesulfonamides did not further enhance the activity of Ca2+-dependent phosphodiesterase activated by the Ca2+-CaM complex. These compounds are also potent inhibitors of cyclic AMP and GMP phosphodiesterases. CaM antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), its derivatives, and chlorpromazine and prenylamine inhibited selectively the quinazolinesulfonamide-induced activations of the phosphodiesterase. These quinazolinesulfonamides, in a high concentration, had only a slight stimulatory effect on myosin light chain kinase activity. All these findings suggest that the quinazolinesulfonamides are calcium-independent activators of Ca2+-dependent phosphodiesterase and they are proving to be useful tools for the study of CaM and phosphodiesterase, in vitro.

P-83 - A new activator of Ca2+-dependent cyclic nucleotide phosphodiesterase; PDase

P-83 - A new activator of Ca2+-dependent cyclic nucleotide phosphodiesterase; PDase

Proteolytic activation of Ca2+-dependent cyclic nucleotide phosphodiesterase from Escherichia coli.

Ca2+ -dependent cyclic nucleotide phosphodiesterase from Escherichia coli was activated by limited proteolysis. The proteolytically activated enzyme lost the sensitivity to Ca2+ but its kinetic properties were not altered.

A calcium-dependent cyclic nucleotide phosphodiesterase from Escherichia coli

Ca2+ is considered to have an important role in cellular response to hormonal stimulation; the Ca2+dependent cyclic nucleotide phosphodiesterase (EC 3.1.4.17) present in many eukaryotic cells [I] plays an important role. In prokaryotes, phosphodiesterases have been reported but are Ca’+-independent [2,3]. The enzyme of Escherichia coli requires Fe2+ or an activator protein (M, 90 000) for its activity [2]. Here, we report the occurrence of an additional species of phosphodiesterase in the soluble fraction of E. coli. The enzyme is Ca’+-dependent and hydrolyzes both cyclic AMP and cyclic GMP. The existence of Ca’+-dependent phosphodiesterase suggests that, in prokaryotes as well as in eukaryotes, one of the roles of Ca2+ in hormonal regulation is acceleration of cyclic nucleotide degradation through the activation of the enzyme.

Ca 2+-Dependent cyclic nucleotide phosphodiesterase from rabbit lung

Studies are presented which demonstrate that rabbit lung contains both Ca 2+-activated cyclic nucleotide phosphodiesterase and calmodulin activity. The Ca 2+-activatable cyclic nucleotide phosphodiesterase is different from the common type in that it contains tightly bound calmodulin. The bound calmodulin is not dissociated from the enzyme even in very low concentrations of Ca 2+ after DEAE-cellulose and Sephadex G-200 column chromatography.