Regulatory mechanisms of particulate cyclic nucleotide phosphodiesterases.
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2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Biomedical subjects
W J Thompson
Publications and source records attributed to W J Thompson.
Developmental and neural regulation of a subsarcolemmal component of the rat neuromuscular junction.
We have generated a monoclonal antibody, designated mAb 3G2, which reacts with a subsarcolemmal component of the neuromuscular junction in adult rats. mAb 3G2 immunoreactivity lies beneath and between the ACh receptor-rich synaptic gutters, around the sole plate nuclei, and at/near sarcomeric Z-disks in the vicinity of the synapse. Localization of mAb 3G2 immunoreactivity to neuromuscular junctions begins postnatally and gradually increases to adult levels. The establishment of this synaptic localization is neurally regulated, as neonatal denervation prevents its occurrence. In adults, denervation results in a loss of synaptic immunoreactivity that returns upon reinnervation. The antigen is also found at the myotendinous junction; its localization here is innervation independent. mAb 3G2 recognizes a 41 kDa protein on immunoblots of extracts of newborn muscle. Based on its distribution within muscle fibers, its developmental and neural regulation, and its molecular weight, the protein recognized by mAb 3G2 can be distinguished from other known postsynaptic proteins. Its neural dependence and developmental regulation suggest that it may participate in synaptic stabilization, perhaps as the intracellular component in a chain of proteins that serve to tether the nerve terminal to the perijunctional region of the muscle fiber.
Aging↗
HIV-1 protease inhibitory activity of L-694,746, a novel metabolite of L-689,502.
L-689,502 is a potent inhibitor of HIV-1 protease activity in vitro. Microbial biotransformations of L-689,502 by cultures belonging to the genus Streptomyces sp. were performed. Extracts of culture broths were examined for the production of metabolites of L-689,502 that could inhibit HIV-1 protease activity. One culture, MA 6804 (Streptomyces lavendulae, ATCC 55095), produced L-694,746 that, while being structurally related to L-689,502, is a novel metabolite and a potent inhibitor of HIV-1 protease.
Amino Acid Sequence↗
Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.
Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.
3',5'-Cyclic-AMP Phosphodiesterases↗
Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function.
This article is a review of cyclic nucleotide phosphodiesterase(s) (CN PDE) from the point of view of the relationships between the newer aspects of the complex enzymology of CN PDE and recent major advances in CN PDE pharmacology. A consolidation of isozyme nomenclature to the proposed family designations is recommended. Emphasis is placed on the importance of defining the subcellular localization of isozymes expressed in a given tissue and cyclic GMP substrate and regulatory roles in CN PDE isozyme functions. CN PDE inhibitors that may be useful for experimental and clinical purposes are discussed. Examples of these inhibitors include CGS 9343B, TCV-3B, KW-6, MIMAX, Dihydroisoquinolines, Trequinsin, bipyridine and dihydropyridazinone cardiotonics, Rolipram, SQ 65442, Zaprinast and Dipyridamole.
2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Benzocycloalkyl amines as novel C-termini for HIV protease inhibitors.
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Amides↗
Phosphodiesterase II, the cGMP-activatable cyclic nucleotide phosphodiesterase, regulates cyclic AMP metabolism in PC12 cells.
Analysis of cyclic nucleotide phosphodiesterase (PDE) activity in cellular fractions from cultured rat pheochromocytoma (PC12) cells has shown that the predominant hydrolytic activity in both cytosolic and particulate compartments is characteristic of a PDE II, the cGMP-activatable family of PDE isozymes. Cytosolic PDE activity was purified to a high degree utilizing DE-52 anion exchange and cGMP-Sepharose affinity chromatographies. The physicochemical properties of PC12 PDE II were similar to those of PDE II isolated from particulate or soluble fractions of other tissues, including subunit molecular weight of approximately 102,000, activation of cAMP hydrolysis by cGMP, and positive cooperative kinetic behavior for cAMP and cGMP hydrolysis. The potential role of PDE II in regulating cAMP metabolism in intact PC12 cells was studied using an [3H]adenine prelabeling technique. Stimulation of PC12 cell adenosine receptors resulted in a 5-8-fold increase in cAMP accumulation. Removal of the adenosine stimulus by the addition of exogenous adenosine deaminase resulted in a rapid decay of cAMP to prestimulated basal levels within 2 min. Treatment of PC12 cells with atrial natriuretic factor or sodium nitroprusside caused 1) increased intracellular cGMP levels, 2) attenuation of adenosine-stimulated cAMP accumulation, and 3) increased rates of cAMP decay after removal of the adenosine stimulus. Treatment of PC12 cells with HL-725 (a potent inhibitor of isolated PDE II activity in vitro) caused 1) increased basal cAMP accumulation, 2) potentiation of adenosine-stimulated cAMP accumulation, and 3) retardation of the rate of cAMP decay after removal of the adenosine stimulus. HL-725 blocked both the attenuation of cAMP accumulation and the accelerated rate of cAMP decay observed with the cGMP-elevating agents. These results suggest that, in PC12 cells, drugs or hormones that inhibit PDE II or increase intracellular cGMP levels to activate PDE II can modulate cAMP metabolism by altering the catalytic status of the enzyme.
3',5'-Cyclic-GMP Phosphodiesterases↗
The origin and selective innervation of early muscle fiber types in the rat.
The diversity of muscle fiber types present in adult animals is present also in the fetus. Fibers generated early and late in fetal development undergo a stereotyped sequence of myosin expressions in giving rise to these fiber types. The differentiation of these fetal fiber types does not require innervation. However, evidence obtained from experiments identifying the types of fibers innervated by single motors suggests that the nervous system comes to recognize this diversity, at least during early postnatal life. Reinnervation experiments suggest that this recognition can occur in the absence of the timing cues normally present in the genesis of fiber types. Thus, a selective innervation of muscle fiber types occurs during development. The role of rearrangement of initial synaptic connections in generating this selectivity is discussed.
Animals↗
Development of muscle fiber types in the prenatal rat hindlimb.
Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447-459), all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H.M. Blau, and W.J. Thompson, 1990, Dev. Biol. 138, 275-295).
Acetylcholinesterase↗
Differentiation of fiber types in aneural musculature of the prenatal rat hindlimb.
The presynaptic neurotoxin, beta-bungarotoxin, was injected into rat fetuses in utero to destroy the innervation of their hindlimb muscles. These injections were made prior to the invasion of motor axons into the muscles and, in some cases, prior to the cleavage of individual muscles. Examination of the lateral motor column of the spinal cord showed a dramatic reduction (greater than 95%) in the number of motoneuron cell bodies. Staining of sections of the hindlimb with silver and with antibodies to neurofilament proteins and to a synaptic vesicle protein indicated that the muscles were aneural. Anti-myosin antibodies applied to sections of the hindlimb revealed that these aneural muscles by the 20th day of gestation had the same types of fibers as were present in normal muscles of the same age. Moreover, fiber types in most muscles showed their characteristic intramuscular distributions. These findings suggest that fiber types can differentiate in the absence of the nervous system. However, some fibers achieved their ultimate fiber type fate without passing through the normal sequence of myosin expressions. Moreover, some slow fibers lost their slow expression, suggesting that the maintenance of the slow differentiation may require innervation. Muscle growth was dramatically affected by the absence of motoneurons; some muscles were decreased in size and others disappeared completely. In muscles which had not degenerated by the time secondary myogenesis normally begins, secondary muscle fibers were generated indicating that the genesis of these fibers is not strictly nerve dependent. Because fiber types differentiate independently of the nervous system, this study suggests that motoneurons selectively innervate fiber types during normal development.
Animals↗
Synthesis and pharmacological evaluation of a series of dibenzo[a,d]cycloalkenimines as N-methyl-D-aspartate antagonists.
A series of 73 dibenzo[a,d]cycloalkenimines were synthesized and evaluated for their ability to displace (+)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]-(+)-10) from its specific binding site on rat cortical membranes. A number of the more active compounds (Ki ranging from 0.006 to 0.21 microM) were evaluated for N-methyl-D-aspartate (NMDA) antagonist activity in the rat cortical slice (Kb ranging from 0.08 to 0.9 microM) and anticonvulsant activity in the mouse against NMDA induced convulsions. The ED50 values ranged from 0.22 to 7.76 mg/kg and correlated reasonably well with the Kb determination. In the dibenzo[a,d]cyclohepten-5,10-imine series, the (+)-5S,10R enantiomer displayed consistently higher levels of biological activity. While substitution at the 3-position of (+)-10 with electronegative atoms generally increased in vitro activity, a loss of potency relative to (+)-10 (MK-801) was observed in vivo for all of the compounds tested.
Animals↗
Structure and activity of hydrogenated derivatives of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801).
Several hydrogenated derivatives of the potent NMDA antagonist 1 have been prepared and evaluated as competitive inhibitors of [3H]-1 binding. These compounds were also tested for their ability to act as noncompetitive antagonists of NMDA in vitro. These studies indicate that two aromatic rings are not strictly required for high-affinity binding or NMDA antagonism.
Animals↗
Idiopathic meralgia paresthetica.
Idiopathic meralgia paresthetica was treated surgically in 14 men and 11 women, the latter group were a decade older on average. Treatment was unsatisfactory if symptoms had been present for longer than 18 months. Reexploration of the nerve was not therapeutic in those cases in which the initial decompression provided no relief. The usual course of the lateral femoral cutaneous nerve is deep to the lateral end of the inguinal ligament but superficial to the sartorius muscle. However, the nerve may overlie the anterior iliac wing or pass between two slips of the inguinal ligament and may also be compressed if it passes deep to or through the sartorius muscle.
Adult↗
Fura 2 analysis of cytosolic calcium regulation in elutriated rat gastric parietal cells.
The calcium probe, Fura 2, is used to establish and partially characterize histamine-, carbachol-, and forskolin-induced calcium transients in enriched parietal cell populations prepared by centrifugal elutriation of dispersed rat fundic mucosa cell isolates. The magnitude of the maximal carbachol response, which is blocked by atropine but not cimetidine, is nearly five times that of histamine or forskolin. Time to peak responses for carbachol, forskolin, and histamine are approximately 7, 17, and 28 sec, respectively. Carbachol-, histamine-, and forskolin-induced increases in Fura 2 fluorescence appear dependent upon extracellular calcium, since these responses are attenuated in low calcium media and blocked by EGTA in low-calcium media or by lanthanum in high- or low-calcium medium. Trifluoperazine and fenoctimine, at concentrations that inhibit secretion, have no effect on either carbachol- or histamine-induced increases in cytosolic calcium. Seven major calcium/EGTA-sensitive phosphoproteins are identified by SDS-PAGE electrophoresis of ATP 32P-labeled cell sonicates. We conclude that cytosolic calcium in enriched rat gastric parietal cell populations is regulated by secretagogue receptor-controlled calcium channels. We postulate that these channels may be controlled by cyclic AMP-dependent phosphorylation, since neither changes in cyclic AMP nor calcium alone mediate the effects of secretagogues entirely, but the interplay between these two second-messenger systems potentiates the actions of these agents. The role of cytosolic calcium as a second messenger in secretagogue action appears similar to that of cyclic AMP in that a specific cellular concentration must be reached to initiate acid secretion.
1-Methyl-3-isobutylxanthine↗
Molecular properties of cyclic nucleotide phosphodiesterase isozymes.
Mammalian cells contain multiple molecular forms of cyclic nucleotide phosphodiesterase that differ in substrate specificity and kinetic and regulatory properties. Calcium/calmodulin and cyclic GMP are important regulators of the hydrolysis of cyclic AMP by either stimulating or inhibiting the activity of distinct forms of phosphodiesterase. Several isozymes of cyclic nucleotide phosphodiesterase have been purified to apparent homogeneity. Although some sequence homology is observed the isozymes appear genetically distinct by immunological criteria. Cyclic AMP- and calmodulin-dependent protein kinases can phosphorylate these enzymes and alter their kinetic and regulatory properties. Both tissue specificity and pharmacological selectivity of isozymes have been demonstrated for several drugs. In certain cases, e.g. cardiac muscle, the selective inhibition of a high affinity cAMP phosphodiesterase activity in a specific subcellular fraction correlates with pharmacologic responses. The results from molecular and pharmacologic studies of cyclic nucleotide phosphodiesterases have indeed expanded the role this system of isoenzymes exerts in the regulation of cellular function.
2',3'-Cyclic-Nucleotide Phosphodiesterases↗
Subcellular distribution of high-affinity type IV cyclic AMP phosphodiesterase activities in rabbit ventricular myocardium: relations to post-natal maturation.
Cytosolic and particulate Type IV (high-affinity) cAMP phosphodiesterase (PDE) activities were isolated from the ventricular myocardium of newborn (NB; 24 to 48 h), immature (IM; 14 to 16 days) and adult (AD; 6 to 8 months) rabbits. Cytosolic activity from each age group was resolved into three distinct peaks of activity by DEAE cellulose anion exchange chromatography. Type IV PDE activity was identified as a predominant activity in the cytosolic peak III activity in all three age groups when measured with 0.25 microM cAMP as substrate. A particulate Type IV PDE activity was associated with the sarcoplasmic reticulum (SR) fractions in each age group. No significant age-related changes in the affinity of the particulate enzyme for cAMP (apparent Km = 0.3 to 0.5 microM) were evident, but the Vmax for this SR-associated activity increased from 553 +/- 7 pmol/min/mg in the NB to 725 +/- 9 pmol/min/mg in the IM and 2450 +/- 33 pmol/min/mg in the AD. In each age group, milrinone, imazodan, piroximone and indolidan were more potent inhibitors of the SR-associated activity as compared with the cytosolic peak III activity. In contrast, RO 20-1724 and rolipram were relatively more selective inhibitors of the cytosolic peak III activity. Age-related differences in the sensitivity of type IV PDE to inhibition was dependent upon the selectivity of the inhibitor and the subcellular enzymic distribution. Cytosolic peak III PDE activity was further resolved by gel filtration chromatography into two peaks. Hydrolysis of cAMP by the higher molecular weight peak was inhibitable by cGMP (IC50 = 0.25 +/- 0.07 microM in NB and 0.07 +/- 0.01 microM in AD) whereas the lower molecular weight peak activity was relatively insensitive to inhibition by cGMP (IC50 greater than 100 microM). The lower molecular weight peak constituted a relatively greater proportion of the total peak III activity in the NB as compared to the AD. Analysis of the kinetics of cGMP inhibition of high-affinity cAMP hydrolysis was consistent with the presence of a greater number of high-affinity (presumably drug-sensitive) binding sites in the SR-associated activity as compared to the cytosolic peak III activity in both NB and AD. These results support the hypothesis that the cGMP-inhibitable Type IV PDE activity may be the primary site of action for certain newer cardiotonic drugs. Differences in drug action in young versus adult myocardium may be related to the selectivity of the cardiotonic drugs for this specific isozyme and its lower specific activity during the early stages of maturation.
3',5'-Cyclic-AMP Phosphodiesterases↗
Inhibition of gastric acid secretion in vivo and in vitro by a new calmodulin antagonist, CGS 9343B.
The new calmodulin antagonist, CGS-9343B, was found to inhibit both histamine plus 3-isobutyl-1-methylxanthine and carbachol-induced [14C]aminopyrine accumulation in dispersed, fundic mucosal cells of rats. The IC50 value for CGS-9343B inhibition of histamine plus 3-isobutyl-1-methylxanthine-induced [14C]aminopyrine accumulation was 306 nM. The drug was more potent than the H2-histamine receptor antagonist, cimetidine (1128 nM), less potent than the nonspecific calmodulin antagonists, trifluoperazine and fenoctimine (IC50 = 40 and 224 nM, respectively), and equipotent with the H+, K+-adenosine triphosphatase inhibitor, omeprazole (365 nM). CGS-9343B showed an IC50 of 369 nM for carbachol-induced [14C]aminopyrine accumulation in dispersed mucosal cells. CGS-9343B must be added to cells before or simultaneously with acid secretagogues in order to be effective. The drug did not reverse previously stimulated secretion. Unlike trifluoperazine and fenoctimine, CGS-9343B had anticamodulin activity for inhibition of calmodulin-activated (Type I) phosphodiesterase in the same range of potency as observed for the inhibition of aminopyrine accumulation. In anesthetized rats and dogs the i.v. infusion of CGS-9343B did not block histamine plus pentagastrin-stimulated acid secretion. However, i.a. administration of CGS-9343B to anesthetized rats produced a significant inhibition of acid secretion. In vivo the order of potency was omeprazole greater than cimetidine much greater than CGS-9343B. These data provide evidence for involvement of calmodulin in the acid secretory process and suggest that the pursuit of selective calmodulin antagonists such as CGS-9343B may prove useful for understanding the regulation of the hydrogen ion secretory process.
1-Methyl-3-isobutylxanthine↗
Analysis of the binding sites for the cardiotonic phosphodiesterase inhibitor [3H]LY186126 in ventricular myocardium.
The positive inotropic action of the newer cardiotonic phosphodiesterase inhibitors such as indolidan, milrinone, and imazodan has been previously attributed to selective inhibition of cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity. However, the subcellular binding site(s) for this class of compounds has not been defined. We have characterized the binding of [3H]LY186126, an analogue of indolidan, in subcellular fractions prepared from rabbit and sheep ventricular myocardium. Binding required magnesium ion and exhibited rapid association and dissociation kinetics. Specific binding (defined by ligand displacement with 5 microM indolidan) to enriched rabbit sarcoplasmic reticulum (SR) membrane vesicles was saturable (Bmax = 714 +/- 77 fmol/mg of protein) and of high affinity (Kd = 6.2 +/- 1.4 nM). Linear and nonlinear analyses of the binding isotherms fit a single-site model. Mixed SR preparations from sheep myocardium exhibited binding characteristics (Bmax = 944 +/- 115 fmol/mg; Kd = 8.5 +/- 2.3 nM) comparable to those of rabbit cardiac SR. Further subfractionation of sheep SR indicated that the binding sites were equally distributed between free (Bmax = 630 fmol/mg; Kd = 4.4 nM) and junctional SR (Bmax = 569 fmol/mg; Kd = 10.9 nM). Specific binding of [3H]LY186126 was also demonstrated in the cytosolic subfraction of rabbit myocardium that contained Type IV phosphodiesterase activity (Peak III from anion exchange chromatography). Competition for [3H] LY186126 binding studied in rabbit SR showed that, of the compounds tested, lixazinone (RS 82856) competed most effectively (IC50 = 0.030 +/- 0.008 nM), followed by indolidan (0.14 +/- 0.05 nM), cGMP (17.8 +/- 2.6 nM), milrinone (39.3 +/- 13.2 nM), and imazodan (192 +/- 73 nM). In contrast, rolipram, which does not inhibit SR-associated Type IV phosphodiesterase activity, was not effective at competing for [3H]LY186126 binding (IC50 greater than 30 microM). These results indicate that [3H]LY186126 has specific binding sites in myocardial subcellular fractions that contain cGMP-inhibitable Type IV (high affinity) cAMP phosphodiesterase activity.
Animals↗