cGMP Phosphodiesterase Activity Research Articles

Activities of 3′:5′ cyclic nucleotide phosphodiesterases in the superior cervical ganglion of rat: Characterization, compartmentalization and observations in young and old animals

We investigated the presence and features of “low K m” 3′–5′ cyclic nucleotide phosphodiesterase activity in the homogenates and extracts of rat superior cervical ganglion. The DEAE chromatographic elution profile of a Triton X-100 extract showed two peaks of cAMP phosphodiesterase activity eluted at 280 and 600 mM sodium acetate and two peaks of cGMP phosphodiesterase activity eluted at 300 and at 500 mM sodium acetate. The activity was poorly stimulated by calcium-calmodulin and neither stimulated or inhibited by cGMP. Both cGMP PDE peaks were inhibited by zaprinast, with IC 50's of 1.4 μM and 0.28 μM; their K m values were 4.4 and 3.8 μM, respectively. These features, together with cGMP binding activity, indicate that both enzymes belong to the phosphodiesterase V family. The K m values of the first and second cAMP phosphodiesterase peaks were 1.7 and 3.8 μM. Although both peaks displayed a cAMP specific hydrolysis, only the second peak was inhibited by RO 20-1724, with an IC 50 of 8 μM. Preganglionic denervation indicated that the bulk of phosphodiesterase activity is localized in ganglion cells. In order to investigate possible effects of aging on the ganglionic function, phosphodiesterase activity was assayed in the ganglia of young (3 months) and old (25 months) male Fisher rats. The chromatographic profiles and kinetic features revealed no significant differences between young and old rats.

Phosphorylation of an inhibitory subunit of cGMP phosphodiesterase in Rana catesbeiana rod photoreceptors. II. A possible mechanism for the turnoff of cGMP phosphodiesterase without GTP hydrolysis.

In amphibian rod photoreceptor membranes, P gamma, an inhibitory subunit of cGMP phosphodiesterase, is phosphorylated by a protein kinase when P gamma is complexed with the guanosine 5'-O-(3-thiotriphosphate)-bound alpha subunit of transducin (GTP gamma S.T alpha). Five different experiments support the conclusion that the phosphorylated P gamma loses its interaction with GTP gamma S.T alpha. These observations include 1) detection of the inhibitory effect of the GTP gamma S.T alpha.P gamma complex on cGMP phosphodiesterase activity after P gamma in the complex is phosphorylated in a system reconstituted from isolated components, 2) no stimulating effect of GTP gamma S.T alpha on the phosphorylated P gamma-inhibited cGMP phosphodiesterase in the reconstituted system, 3) physical release of phosphorylated P gamma from GTP gamma S.T alpha in the reconstituted system, 4) no inhibitory effect of phosphorylated P gamma on both GTP hydrolysis by T alpha and GTP gamma S/GDP exchange on T alpha in the reconstituted system, and 5) no enhanced activity of cGMP phosphodiesterase by GTP gamma S.T alpha in rod outer segment membranes after incubation of the membranes with the kinase preparation in the presence of ATP. Together with our data that P gamma released with GTP.T alpha is not phosphorylated, and that phosphorylated P gamma inhibits more effectively cGMP phosphodiesterase activity than nonphosphorylated P gamma (Tsuboi, S., Matsumoto, H., Jackson, K. W., Tsujimoto, K., Williams, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023), these observations suggest that, after P gamma is released with GTP.T alpha from catalytic subunits of cGMP phosphodiesterase, P gamma complexed with GTP.T alpha is phosphorylated by a kinase. Then, the phosphorylated P gamma is released from GTP.T alpha and binds to active cGMP phosphodiesterase to inhibit the cGMP hydrolysis. It is suggested that in some G-protein-dependent signal transduction systems G-protein-activated effector may be phosphorylated with a specific kinase and that phosphorylation of the effector results in the turnoff of signal transduction without GTP hydrolysis.

A model for cAMP-mediated cGMP response in Dictyostelium discoideum.

In Dictyostelium discoideum extracellular cyclic AMP (cAMP), as shown by previous studies, induces a transient accumulation of intracellular cyclic guanosine-5'-monophosphate (cGMP), which peaks at 10 s and recovers basal levels at 30 s after stimulation, even with persistent cAMP stimulation. Additional investigations have shown that the cAMP-mediated cGMP response is built up from surface cAMP receptor-mediated activation of guanylyl cyclase and hydrolysis of cGMP by phosphodiesterase. The regulation of these activities was measured in detail on a seconds time-scale, demonstrating complex adaptation of the receptor, allosteric activation of cGMP-phosphodiesterase by cGMP, and potent inhibition of guanylyl cyclase by Ca2+. In this paper we present a computer model that combines all experimental data on the cGMP response. The model is used to investigate the contribution of each structural and regulatory component in the final cGMP response. Four models for the activation and adaptation of the receptor are compared with experimental observations. Only one model describes the magnitude and kinetics of the response accurately. The effect of Ca2+ on the cGMP response is simulated by changing the Ca2+ concentrations outside the cell (Ca2+ influx) and in stores (IP3-mediated release) and changing phospholipase C activity. The simulations show that Ca2+ mainly determines the magnitude of the cGMP accumulation; simulations are in good agreement with experiments on the effect of Ca2+ in electropermeabilized cells. Finally, when cGMP-phosphodiesterase activity is deleted from the model, the simulated cGMP response is elevated and prolonged, which is in close agreement with the experimental observations in mutant stmF that lacks this enzyme activity. We conclude that the computer model provides a good description of the observed response, suggesting that the main structural and regulatory components have been identified.

Functional effect of phosphorylation of the photoreceptor phosphodiesterase inhibitory subunit by protein kinase C

In rod outer segments the light activation of cGMP phosphodiesterase (PDE alpha beta gamma 2) is accomplished by removal of the gamma inhibitory subunit (PDE gamma) from the PDE alpha beta catalytic subunits. A light activation of the inositol signaling pathway also occurs, but there is little information linking these two signal transduction pathways. Here we report that protein kinase C (PKC) purified from bovine rod outer segment phosphorylates the bovine PDE gamma with incorporation of 0.9 +/- 0.1 mol of phosphate/mol of PDE gamma. Phosphorylation of PDE gamma increases its ability to inhibit PDE alpha beta catalytic activity (trypsin-activated PDE, tPDE) with an IC50 for phosphorylated PDE gamma of 26 +/- 4 pM and an IC50 of 60 +/- 5 pM for unphosphorylated PDE gamma. Inhibition of tPDE by PDE gamma is characterized by two values of Kd, Kd1 = 34 pM and Kd2 = 760 pM. Phosphorylation of PDE gamma by PKC eliminates the functional heterogeneity of the PDE gamma population resulting in a single value of Kd = 23 pM. Free PDE gamma (without PDE alpha beta catalytic subunits) is a better substrate for PKC than PDE gamma in a complex with PDE alpha beta. Phosphorylation of free PDE gamma by PKC is characterized by a value of Vmax = 1,550 +/- 148 units/mg (Km = 21.0 +/- 1.9 microM). In contrast, phosphorylation of PDE gamma in PDE alpha beta gamma 2 complex has two values of Vmax, Vmax1 = 0.3 +/- 0.1 units/mg of PDE gamma (Km1 = 0.4 +/- 0.2 microM) and Vmax2 = 0.7 +/- 0.2 units/mg of PDE gamma (Km2 = 4.6 +/- 0.9 microM). ROS PKC phosphorylates Thr35 in PDE gamma. We have previously reported (Morrison, D. F., Rider, M. A., and Takemoto, D. J. (1987) FEBS Lett. 222, 266-270; Lipkin, V. M., Udovichenko, I. P., Bodarenko, V. A., Yurovskaya, A. A., Telnykh, E. V., and Skiba, N. P. (1990) Biomed. Sci. (Lond.) 1, 305-308) that the central fragment of PDE gamma (24-45) is responsible for binding to PDE catalytic subunits. The new data suggests that this region of PDE gamma also includes the site for phosphorylation by PKC and that phosphorylation increases the ability of PDE gamma to inhibit PDE catalytic activity. This altered regulation of visual transduction may play a role in desensitization or light adaptation.

A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit.

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha t*) and the PDE inhibitory gamma-subunit (P gamma) is a major component of PDE activation. The central polycationic region of P gamma, P gamma-24-45, has been implicated as one of the sites involved in alpha t*.P gamma interaction. Here we determine the site on alpha t* that interacts with P gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH2 termini, respectively, and then cross-linked to alpha t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha t GTP gamma S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha t GDP. The site of cross-linking between Cys(ACM)Tyr-P gamma-24-45-Cys and alpha t GTP gamma S was localized to within alpha t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

Regulation of G protein function by an effector in GTP-dependent signal transduction. An inhibitory subunit of cGMP phosphodiesterase inhibits GTP hydrolysis by transducin in vertebrate rod photoreceptors.

The regulation of cGMP phosphodiesterase in vertebrate rod photoreceptors is a typical G protein-dependent signal transduction mechanism. The interaction of P gamma, an inhibitory subunit of cGMP phosphodiesterase, with transducin alpha subunit (T alpha) is essential for the activation of cGMP phosphodiesterase. It has been shown that, in a homogenized preparation of frog (Rana catesbeiana) rods, P gamma interacts with GTP.T alpha and remains tightly bound to GDP.T alpha after GTP hydrolysis on T alpha. Association of this complex with beta gamma subunits of transducin (T beta gamma) triggers the release of P gamma from the complex and the subsequent inactivation of cGMP phosphodiesterase. In a system reconstituted with purified components, both GTP- and GDP-bound forms of T alpha were found to interact with P gamma. Under these conditions, P gamma inhibited GTP hydrolysis by transducin in a noncompetitive manner with a Ki of 92 nM. Binding of an hydrolysis-resistant GTP analog to T alpha was also inhibited by P gamma. These inhibitions of transducin function were resulted from the inhibition of both hydrolysis of GTP bound to T alpha and interaction of GDP.T alpha with membrane-bound T beta gamma. However, after GDP.T alpha reassociated with membrane-bound T beta gamma, the inhibitory effect of P gamma on the binding of an hydrolysis-resistant GTP analog to T alpha was greatly diminished, suggesting that the GTP/GDP exchange on T alpha was not inhibited by P gamma. These data indicate that the T alpha function is altered during complexing with P gamma. G protein functions may be modified by interacting with an effector in the G protein-dependent signal transduction.

Developmental appearance of pineal adrenergic-->guanosine 3',5'-monophosphate response is determined by a process down-stream from elevation of intracellular Ca2+: possible involvement of a diffusible factor.

Adrenergic stimulation of the adult pineal gland increases cAMP and cGMP production by over 100-fold. beta-Adrenergic stimulation results in Gs alpha-mediated cyclase activation; alpha 1-adrenergic activation potentiates the beta-adrenergic effects through mechanisms mediated by the intracellular Ca2+ concentration ([Ca2+]i) and Ca(2+)-phospholipid-dependent protein kinase. Development analysis of these responses has indicated that the adrenergic stimulation of cAMP is present several days after birth, but the cGMP response develops only after the second week of life. In the study presented here, the adrenergic-->cGMP response was analyzed in pineal glands from 10- and 25-day-old rats, with the intention of determining the basis of the developmental appearance of this response. Organ culture and tissue homogenate studies indicated that guanylate cyclase activity, cGMP phosphodiesterase activity, and adrenergic elevation of phospholipase-A2 were similar in pineal glands from 10- and 25-day-old rats. Norepinephrine stimulated an increase in [Ca2+]i in dispersed pinealocytes from 10-day-old rats, as has been previously demonstrated in adult pinealocytes. In contrast, several treatments that elevate [Ca2+]i had no effect on cGMP accumulation in forskolin-treated or beta-adrenergically activated glands from 10-day-old rats, but were fully effective in similarly treated glands from 25-day-old rats. However, glands from 10-day-old animals showed a 33-fold accumulation of cGMP when they were cultured together with glands from 25-day-old rats. These studies indicate that whereas many elements in the system that mediate adrenergic regulation of pineal cGMP are fully developed at 10 days of age, the developmental appearance of the cGMP response is triggered by the development of a process down-stream of the alpha 1-adrenergic stimulation of [Ca2+]i, and this process may involve a diffusible factor.

Partial purification of cyclic guanosine monophosphate phosphodiesterase from sea urchin ( Strongylocentrotus purpuratus) sperm

1. 1. A cGMP-stimulated cyclic nucleotide phosphodiesterase has been extracted from intact sea urchin sperm by means of detergents. 2. 2. CHAPS and octyl glucoside inhibited enzyme activity less than did Triton X-100, Lubrol PX, Nonidet P-40 and sodium deoxycholate. 3. 3. Homogenization in 1% CHAPS with a sperm concentration of 10 mg/ml resulted in maximal solubilization (55%) of the cGMP phosphodiesterase from whole sperm. 4. 4. Partial purification of cGMP phosphodiesterase from intact sperm was achieved by 1% CHAPS extraction and chromatography on DEAE-Sephacel. 5. 5. Chromatography separated cAMP and cGMP phosphodiesterases from each other, with an increase in specific activity of the cGMP enzyme up to 9-fold. 6. 6. The partially purified cGMP phosphodiesterase activity was markedly unstable. 7. 7. It displayed Michaelis-Menten kinetic for cGMP dependence with a K m of 66 μM. With cGMP as substrate, both Mg 2+ and Mn 2+ but not Ca 2+ were effective activators of the partially purified cGMP phosphodiesterase. 8. 8. The K 0.5 of Mg 2+ and of Mn 2+ were 0.50 and 0.32 mM, respectively.

Impaired G-proteins and cyclic nucleotide phosphodiesterase activity in T-lymphocytes from patients with sarcoidosis.

Among the various immune abnormalities which characterize active sarcoidosis, a low proliferative response of peripheral blood lymphocytes to mitogenic lectins has long been observed. Since membrane-associated G-proteins are very likely to be crucial elements in lectin signal transduction, we investigated the binding of 5'-guanylylimidodiphosphate (GppNHp), a non hydrolyzable GTP analogue, to blood total lymphocyte membranes and to blood T-lymphocyte membranes from patients with active sarcoidosis, and from healthy control subjects. GppNHp binding was markedly decreased in peripheral cells from patients with sarcoidosis as compared to controls, suggesting the occurrence of a defect at the level of G-protein(s). A further characterization of G-proteins in these cells by means of ADP-ribose-labelling in the presence of bacterial toxins brought forward a significant decrease in the labelling of a 40 kDa protein, the major pertussis toxin substrate, in membranes from sarcoid patients, while the labelling of the major 44 kDa cholera toxin substrate proved to be unchanged with respect to control membranes. It is hypothesized that, in sarcoid lymphocytes, a defect in the negative control of adenylate cyclase mediated by the inhibitory G-protein Gi, prevents the lowering of cAMP necessary to normal mitogenic response of blood lymphocytes to stimulation. cAMP degradation by the specialized enzyme phosphodiesterase constitutes another critical step in the control of cAMP levels. Both cAMP and cGMP phosphodiesterase activities were found decreased in blood total lymphocyte preparations from sarcoid patients. With purified T-cells, although the mean cAMP and cGMP phosphodiesterase activities from sarcoid patients were found more markedly decreased with respect to healthy donors, only the decrease in cGMP phosphodiesterase was found statistically significant. The role these defects in cyclic nucleotide degradation potentially play in the disturbance of blood lymphocytes response associated with sarcoidosis is discussed.

Light-sensitivity modulating protein in frog rods.

Cyclic GMP is the second messenger in the phototransduction mechanism in rod photoreceptors. Light-induced activation of cGMP phosphodiesterase (PDE), the hydrolyzing enzyme of cGMP, reduces cytoplasmic cGMP concentration to close the cGMP-activated channel and thereby causes a hyperpolarizing light response. Ca2+ concentration decreases during light-adaptation and this decrease is thought to be at least one of the underlying mechanisms of light-adaptation. Our previous electrophysiological work suggested that PDE in frog rod photoreceptors is regulated by this Ca2+ concentration decrease. In the present work, we isolated a protein that binds to disk membranes at high Ca2+ concentrations. In the presence of this protein (a 26 kDa protein), PDE light sensitivity becomes high at high Ca2+ concentrations. The effect was observed at physiological ranges of Ca2+ concentrations. Thus we could explain high light-sensitivity of photoreceptors under the dark-adapted condition. According to its function, we termed the 26 kDa protein 'sensitivity-modulating protein' or 'S-modulin'. During the purification we noticed that there are additional mechanisms present that may contribute to light-adaptation in frog rod photoreceptors.

A small population of rods in the human retina has the IPM labeled with peanut agglutinin

Production of high titer of antibodies against nuclear components is a hallmark of systemic lupus erythematosus, an autoimmune disease characterized by the progressive chronic inflammation of multiple joints and organs. Organ damage and dysfunction such as renal failure are typical clinical features in lupus. Cell hypermetabolism and hypertrophy can accelerate organ dysfunction. In this study we focus on a specific murine model of lupus, the MRL/lpr strain, and investigated the role of cyclic guanosine monophosphate (cGMP) catabolism in organ remodeling of main target tissues (kidney, spleen and liver) in comparison with age-matched control mice. In MRL/lpr-prone mice, the cGMP-phosphodiesterase (PDE) activities were significantly increased in the kidney (3-fold, P < 0.001), spleen (2-fold, P < 0.001) and liver (1.6-fold, P < 0.05). These raised activity levels were paralleled by both an increased activity of PDE1 in the kidney (associated with nephromegaly) and in the liver, and PDE2 in the spleen of lupus-prone mice. The up-regulation of PDE1 and PDE2 activities were associated with a decrease in intracellular cGMP levels. This underlines an alteration of cGMP-PDE signaling in the kidney, spleen and liver targeting different PDEs according to organs. In good agreement with these findings, a single intravenous administration to MRL/lpr mice of nimodipine (PDE1 inhibitor) but not of EHNA (PDE2 inhibitor) was able to significantly lower peripheral hypercellularity (P = 0.0401), a characteristic feature of this strain of lupus-prone mice. Collectively, our findings are important for generating personalized strategies to prevent certain forms of the lupus disease as well as for understanding the role of PDEs and cGMP in the pathophysiology of lupus.

Time course of cGMP-phosphodiesterase activity elicited by light flashes in retinal rods

Time course of cGMP-phosphodiesterase activity elicited by light flashes in retinal rods

CGMP-phosphodiesterase immunoreactivity parallels the morphological differentiation of photoreceptors in the retina of the rat

Second messenger cAMP and cGMP represent a key step in the action of dopamine that modulates directly or indirectly their synthesis. We aimed to verify whether levodopa-induced dyskinesias are associated with changes of the time course of levodopa/dopamine stimulated cAMP and cGMP levels, and/or with changes of their catabolism by phosphodiesterase activity in rats with experimental hemiparkinsonism. Microdialysis and tissue homogenates of the striatal tissues demonstrated that extracellular and intracellular cAMP/cGMP levels were lower in dyskinetic animals during the increasing phase of dyskinesias compared to eukinetic animals, but cAMP/cGMP levels increased in dyskinetic animals during the phase of decreasing and extinction of dyskinesias. Dyskinesias and the abnormal lowering of striatal cGMP and cAMP after levodopa were prevented by pretreatment with the multipotent drug amantadine, outlining the inverse relationship of cAMP/cGMP to dyskinesias. Moreover, dyskinetic animals showed higher striatal hydrolyzing cGMP-phosphodiesterase but not hydrolyzing cAMP-phosphodiesterase activity, suggesting that low cGMP but not cAMP levels could be due to increased catabolism. However, expressions of isozyme phosphodiesterase-1B and -10A highly and specifically located in the basal ganglia were not changed after levodopa in dyskinetic and eukinetic animals: accordingly, selective inhibitors of phosphodiesterase-1B and -10A were ineffective on levodopa dyskinesias. Therefore, the isozyme(s) expressing higher cGMP-phosphodiesterase activity in the striatum of dyskinetic animal should be determined. These observations suggest that dopamine-mediated processes of synthesis and/or degradation of cAMP/cGMP could be acutely impaired in levodopa dyskinesias, opening new ways to understanding physiopathology and treatment.

The β subunit of cyclic GMP phosphodiesterase mRNA is deficient in canine rod-cone dysplasia 1

Irish setter dogs affected with rod-cone dysplasia 1 have elevated levels of retinal cGMP resulting from deficient rod-specific cGMP phosphodiesterase (cGMP PDE) activity. We investigated the mRNAs coding for the three subunits of cGMP PDE and for the proteins involved in the activation/deactivation of this enzyme in the retinas of developing affected and control dogs. While the photoreceptor cells are viable in the diseased retinas, opsin, transducin α 1 and β 1, 48 and 33 kd proteins, and cGMP PDEα and γ mRNAs have normal transcript sizes and levels. In contrast, a different pattern of cGMP PDEβ mRNAs with lower than normal concentrations is present in the developing affected retinas prior to degeneration. Our observations suggest that an abnormality involving cGMP PDEβ expression is implicated in rod-cone dysplasia 1.

Retinal degeneration is rescued in transgenic rd mice by expression of the cGMP phosphodiesterase beta subunit.

The beta subunit of the cGMP phosphodiesterase (PDE) gene has been identified as the candidate gene for retinal degeneration in the rd mouse. To study the molecular mechanisms underlying degeneration and the potential for gene repair, we have expressed a functional bovine cGMP PDE beta subunit in transgenic rd mice. One transgenic mouse line showed complete photoreceptor rescue across the entire span of the retina. A second independently derived line showed partial rescue in which photoreceptors in the superior but not the inferior hemisphere of the retina were rescued. In the latter animals, intermediate stages of degeneration were observed in the transition zone between rescued and diseased photoreceptors. Pathologic changes in the retina ranged from vesiculation of the basalmost outer segment discs in otherwise structurally intact rod cells to photoreceptors with highly disorganized outer segments and intact inner segments. Totally or partially rescued retinas showed a corresponding restoration of cGMP PDE activity, whereas nonrescued retinas had minimal enzyme activity, characteristic of the rd phenotype. These transgenic animals provide models for studying the molecular basis of retinal degenerative disease and conclusively demonstrate that the phenotype of rd mice is produced by a defect in the beta subunit of cGMP PDE.

Myofibrillar and membrane-bound enzymes in skeletal muscle from myodystrophic mice

1. 1. Experiments were carried out to examine the biochemical changes, such as contractile protein biochemistry and membrane bound enzyme alterations associated with skeletal muscles of myd/myd. 2. 2. Our studies demonstrate that there was a progressive decline in myofibrillar ATPase activity, and this decrease is greatest in 30 weeks old animals of myd/myd as compared to controls. 3. 3. The proteolytic activity of myofibrils isolated from myd/myd was significantly higher than controls. 4. 4. There was no significant difference in Ca 2+ ATPase activity of myosin and actin-activated myosin ATPase activity of myd/myd and their controls. 5. 5. Mg 2+ ATPase and Na + + K +-ATPase of myodystrophic SL showed significant increase compared to controls. 6. 6. Isoproterenol stimulated adenylate cyclase activity was significantly lower in the SL of dystrophic mice compared to controls. 7. 7. GTP + isoproterenol stimulate adenylate cyclase was significantly higher in control SL and SR when compared to SL and SR isolated from myd/myd. 8. 8. Guanylate cyclase activity was greater in myodystrophic mice both in the absence and presence of Triton X-100. cGMP and cAMP phosphodiesterase activities were greater in dystrophic mice as compared to controls. 9. 9. These observations suggest that there are significant changes in myofibrillar ATPase, myofibrillar protease and membrane bound enzymes of myd/myd compared to control.

Nucleotide exchange and cGMP phosphodiesterase activation by pertussis toxin inactivated transducin.

Transducin, the signal coupling protein of retinal rod photoreceptor cells, is one of a family of G proteins that can be inactivated by pertussis toxin. We have investigated the nature of this inactivation in order to determine (1) whether it requires the toxin-catalyzed transfer of ADP-ribose from NAD+ to cysteine-347 of the alpha subunit and (2) whether it involves locking the alpha subunit in the inactive conformation characteristic of its GDP-bound state, or is limited to disruption of binding to photoexcited rhodopsin (R*). Our results indicate that all observed effects of pertussis toxin treatment, including a shift in the electrophoretic mobility of transducin's alpha subunit and functional inactivation, require NAD+ and that the appearance of the shift parallels incorporation of ADP-ribose. We have also found that, apart from interactions with photoexcited rhodopsin, the functional properties of ADP-ribosylated transducin are essentially the same as those of unmodified transducin. Normal spontaneous nucleotide exchange kinetics and the ability to activate cGMP phosphodiesterase are preserved following quantitative ADP-ribosylation, as are the abilities to hydrolyze GTP, to bind to a dye affinity column, and to display enhanced fluorescence upon addition of Al3+ and F-. Thus, ADP-ribosylation merely blocks catalysis of transducin nucleotide exchange by R* and does not lock transducin in an inactive state.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a nonsense mutation in the rod photoreceptor cGMP phosphodiesterase beta-subunit gene of the rd mouse.

Retinal degeneration in the mouse mutant, rd, was previously shown to be a disorder of cyclic nucleotide metabolism involving a deficiency in the activity of the rod photoreceptor cGMP phosphodiesterase (PDE). We have characterized the normal and rd PDE beta-subunit gene, and their respective transcripts, by PCR and direct sequence analysis. We show that the gene consists of at least 22 exons ranging in size from 48 base pairs to several hundred base pairs, covering greater than 25 kilobases. Within a 67-base-pair exon of the rd PDE beta-subunit gene, we identified a nonsense ochre mutation (a C----A transversion in codon 347) that truncates the normal gene product, eliminating more than one-half of the peptide chain, including the putative catalytic domain. The consequences of the truncation are consistent with the observed phenotypes in rd mice heterozygous and homozygous for the disorder. The nonsense mutation was also found in another related and in six unrelated strains displaying the rd phenotype, indicating that the rd allele arose from a single genetic event. The results strongly argue for the nonsense mutation being responsible for retinal degeneration in the rd mouse.

Characterization of guinea-pig eosinophil phosphodiesterase activity: Assessment of its involvement in regulating superoxide generation

Experiments have been performed to characterize guinea-pig peritoneal eosinophil cyclic nucleotide phosphodiesterase (PDE) activity and establish whether it is involved in regulating superoxide (.O − 2) generation. Eosinophils were found to contain a predominantly membrane-bound cAMP PDE(s) (92.5 ± 2.4% of total activity) which was resistant to solubilization with Triton X-100 (1%). This particulate PDE exhibited complex kinetics ( K m = 1.3 and 31.4 μM) and was unaffected by cGMP ( ic 50 > 100 μ M) or CaCl 2 (2 mM) + calmodulin (10 units/mL). Little cGMP PDE activity was detected in either the soluble or particulate fractions. Inhibitors of the Ro-20-1724-inhibited (Type IV) cAMP PDE, namely Ro-20-1724 ( ic 50 = 0.92 ± 0.43 μ M) , rolipram ( ic 50 = 0.20 ± 0.04 μ M) and denbufylline ( ic 50 = 0.20 ± 0.01 μ M) , potently inhibited the particulate cAMP PDE, as did the non-selective inhibitors trequinsin ( ic 50 = 0.11 ± 0.02 μ M) and AH-21-132 ( ic 50 = 2.57 ± 0.02 μ M) . Eosinophil cAMP PDE was resistant to SK&F 94120 ( ic 50 > 1000 μM), the cGMP-inhibited (Type III) cAMP PDE inhibitor, and the cGMP PDE (Type I) inhibitor, zaprinast, was only weakly active ( ic 50 = 35.33 ± 10.74 μ M) . O − 2 release from resting cells was potently inhibited by rolipram ( ic 50 = 0.05 ± 0.03 μ M) and denbufylline ( ic 50 = 0.06 ± 0.04 μ M) but surprisingly, in view of its potent cAMP PDE inhibitory activity, was only weakly decreased by trequinsin ( ic 50 = 8.0 ± 2.7 μ M) . AH-21-132 ( ic 50 > 10 μ M) , SK&F 94120 ( ic 50 > 10 μ M) and zaprinast ( ic 50 > 10 μ M) were without effect. Rolipram and denbufylline alone exerted little effect on cAMP in intact cells but, in the presence of 10 μM isoprenaline, potently increased intracellular accumulation ( ec 50 = 0.45 ± 0.16 and 0.28 ± 0.08 μ M , respectively). Trequinsin and AH21–132 only weakly enhanced isoprenaline-stimulated cAMP accumulation. Although it induced a marked rise in cAMP only in the presence of isoprenaline, rolipram (50 μM) alone was able to increase the activity ratio of cAMP-dependent protein kinase from 0.24 to 0.84. The results suggest that Ro-201724-inhibited cAMP PDE plays a role in regulating eosinophil. O − 2 generation. The poor correlation between the PDE inhibitory actions of certain compounds and their effectiveness in elevating cAMP and inhibiting. O − 2 suggests the existence of a barrier impeding access to the enzyme.