Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.
Mol Cell Biol 1984 Oct
PMID:Mutants of PC12 cells with altered cyclic AMP responses. 609 39

Cyclic AMP (cAMP) and cyclic GMP (cGMP) have been implicated as intracellular signals in the transition from a resting to a growing state. This suggestion comes from observations showing that the addition of growth promoting factors to quiescent cell cultures causes a rapid and transient decrease in cAMP and an increase in cGMP contents [9, 11] and that exogenous cAMP or cGMP congeners reduce or stimulate cell growth respectively [6, 13]. In view of this antagonistic effect elicited by the two nucleotides, it has been suggested that a fall in cAMP/cGMP ratio might be the triggering event for the initiation of cell proliferation [6]. Since polyamines correlate positively with active cell division [7], a possible involvement of these biogenic polycations in the regulation of cellular cyclic nucleotide contents is worthwhile investigating. Our previous reports have shown indeed that in different cultured cell types, spermine, spermidine and putrescine, at relatively low doses, are able to reduce cAMP content [3] by increasing cAMP-dependent phosphodiesterase activity (cAMP-PDE) [4] and to counteract the action of different cAMP-mediated effectors [3]. Besides endogenous polyamines seem to be involved in the cAMP-mediated induction of cAMP-PDE, as observed in heart cell cultures [4]. This report shows that the addition of each individual polyamine to confluent and serum-restricted heart cell cultures, while lowering cAMP content, induces an early and rapid increase of cGMP content by reducing the rate of its degradation.
J Mol Cell Cardiol 1983 Feb
PMID:Increased cyclic GMP content in confluent and serum-restricted heart cell cultures exposed to polyamines. 630 28

The availability of the pure inhibitor of cAMP-dependent protein kinase prompted a re-examination of the inhibitor-induced meiotic maturation of Xenopus laevis oocytes. Injection of the inhibitor (1.5 microM) triggered 100% germinal vesicle breakdown faster than progesterone and slower than the maturation-promoting factor: at 0.15 microM, the inhibitor still triggered 100% meiosis, but with a much slower kinetics. In contrast, injection of 24 microM calmodulin resulted in less than 50% GVBD, and results were variable from female to female. Combined injection of inhibitor and calmodulin failed to show any synergism, which does not favour hypotheses according to which calmodulin acts by activation of cyclic nucleotide phosphodiesterase. The net effect of the inhibitor is to decrease the concentration of the free catalytic sub-unit of cAMP-dependent protein kinase, fully dissociated in the unstimulated oocyte, as shown by the absence of effect of pretreatment with cholera toxin on the inhibitor-induced maturation. After such decrease by about 1 microM, a maturation protein, Mp-P, is dephosphorylated by phosphoprotein phosphatases. Dephospho-Mp triggers the synthesis of MPF in cycloheximide-sensitive steps. Finally, MPF triggers GVBD in steps insensitive to cycloheximide. Evidence for such a 4-step scheme--fall in cAMP levels, then in C sub-unit levels, dephosphorylation of Mp leading to the synthesis of MPF and finally MPF-triggered GVBD--is presented and discussed.
Mol Cell Endocrinol 1981 May
PMID:The pure inhibitor of cAMP-dependent protein kinase initiates Xenopus laevis meiotic maturation. A 4-step scheme for meiotic maturation. 701 32

Calmodulin antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which bind to calmodulin (CaM) in the presence of Ca2+ and selectively inhibit CaM-induced enzyme activation, contain a hydrophobic moiety. In this study, the naphthalenesulfonamide derivatives that lacked the chlorine molecule were less hydrophobic than those with chlorine. The chlorine-deficient derivatives also were less able to suppress the fluorescence of the hydrophobic probe (2-p-toluidinylnaphthalene-6-sulfonate) in the presence of the Ca2+-CaM complex. The affinity of naphthalenesulfonamides for Ca2+-CaM correlated well with their hydrophobicity and their potency in inhibiting Ca2+-CaM-dependent enzymes such as Ca2+-dependent cyclic nucleotide phosphodiesterase. The correlation between their hydrophobicity and affinity for the Ca2+-CaM complex also was observed when derivatives with various lengths of alkyl chain were used and when bromine, fluorine, or cyanogen was substituted for chlorine. Our observations suggest that these CaM antagonists may bind to the Ca2+-CaM complex through a hydrophobic interaction.
Mol Pharmacol 1982 Sep
PMID:Hydrophobic interaction of the Ca2+-calmodulin complex with calmodulin antagonists. Naphthalenesulfonamide derivatives. 714 34

Alterations of receptor-G-protein-regulated adenylyl cyclase activity have been suggested to represent an important alteration leading to contractile dysfunction in the failing human heart. Recent experiments suggest that the beta 1-adrenoceptor (beta 1 AR) density and mRNA levels are reduced, while beta 2-adrenoceptors and stimulatory G-proteins are unchanged (mRNA and protein level). Functional assays demonstrated that the catalyst of the adenylyl cyclase is not different between failing and nonfailing myocardium. Inhibitory G-proteins are increased (pertussis toxin substrates, protein and mRNA) and correlate to the reduced inotropic effects of beta-adrenoceptor agonists and of cAMP-PDE inhibitors. Gi alpha-coupled m-cholinoceptors and A1-adrenergic receptors are unchanged in density and affinity. Stimulation of these receptors resulted in an unchanged antiadrenergic effect on force of contraction. In conclusion, a downregulation of beta 1 AR and an increase of Gi alpha have been observed as signal transduction alteration in failing human myocardium. These alterations are due to alterations of gene expression in the failing heart and are related to a defective regulation of force of contraction in heart failure.
Mol Cell Biochem
PMID:Alterations of beta-adrenoceptor-G-protein-regulated adenylyl cyclase in heart failure. 749 44

Throughout vegetative growth, Dictyostelium amoebae secrete an autocrine factor, prestarvation factor, PSF, which accumulates in proportion to cell density. During late exponential growth, PSF induces the expression of several genes whose products are needed for cAMP signaling and cell aggregation. Among these genes are discoidin-I and the 2.4-kb transcript of cyclic nucleotide phosphodiesterase (PDE). We have identified several parameters that modulate expression of one or both of these prestarvation response genes; all effects were monitored in cells growing exponentially on bacteria. Under these conditions, axenic mutants produce higher levels of PSF activity than wild-type cells. Consistent with the high PSF levels, the 2.4-kb PDE transcript is more abundant in axenic strains than wild-type cells at the same cell density. In contrast, the density-dependent induction of discoidin-I is greatly delayed in axenic strains, occurring only at the very end of exponential growth. Analysis of axenic strains of independent origin suggested that this negative effect on discoidin-I expression is attributable to the axenic mutations themselves. The effects of two environmental factors that inhibit the prestarvation response (the bacteria upon which the cells feed and a bacterial product, folic acid) were also analyzed. We found that folate does not account for the inhibitory effect of bacteria. Cells deficient in the G-protein beta subunit, which is thought to be common to all heterotrimeric G-proteins in Dictyostelium, respond to PSF in the same manner as G beta+ cells, and this response is inhibited by bacteria. However, folate has no inhibitory effect on g beta- cells, indicating that folate inhibition is mediated by a heterotrimeric G-protein. In cells lacking the catalytic subunit of protein kinase A, the prestarvation response is severely impaired, but about 3% of the pka- cells manifest an apparently normal density-dependent induction of discoidin-I. This behavior and the heterogeneity of the prestarvation response in wild-type cells lead us to speculate that protein kinase A may not be required for PSF signal transduction per se, but rather may render the cells responsive to PSF. Based on analysis of adenylyl cyclase mutants (aca-), the effect of protein kinase A is not cAMP-dependent.
Mol Biol Cell 1995 Mar
PMID:Genetic and physiologic modulation of the prestarvation response in Dictyostelium discoideum. 761 66

Mutations induced by activated benzo[a]pyrene ((+)-anti-B[a]PDE) in Escherichia coli are being investigated, by using both random and adduct-site-specific mutagenesis approaches. A working hypothesis was proposed that the major adduct of (+)-anti-B[a]PDE (formed at N2-Gua) is able to induce different base-substitution mutations (e.g., GC-->TA vs. GC-->AT) depending upon its conformation in DNA, which can be influenced by various factors, notably DNA sequence context. Frameshift mutations are also common with (+)-anti-B[a]PDE, and other work suggested that the frameshift and base-substitution mutagenesis pathways are coupled. The simplest hypothesis to rationalize this interrelationship is that a single (+)-anti-B[a]PDE adduct in a single conformation can be bypassed via either a frameshift or a base-substitution pathway. This counterintuitive notion can be reconciled if there are two different kinds of conformations on the pathway to mutagenesis: a class I conformation, which is the initial conformation of a DNA adduct in double-stranded DNA before its encounter with a DNA polymerase, and a class II conformation, which is the conformation that forms at a single-strand/double-strand DNA junction during replication by a DNA polymerase. Thus, GC-->TA and GC-->AT mutations may be induced by different class I conformations, whereas base substitution and frameshift mutations may be induced by the same class I conformation but by different class II conformations. The pathway of mutagenesis would be dictated by the relevant class I and II conformations, which in turn would be controlled by various factors, notably DNA sequence context.
Mol Carcinog 1995 Aug
PMID:How are potent bulky carcinogens able to induce such a diverse array of mutations? 764 60

Greater knowledge over the past decade on the biochemical properties, as well as the identification of specific pharmacological tools has led to a marked improvement in the methods employed for the analysis and assay of PDE isoenzymes. A major message has been the marked species and tissue-dependent variation in the distribution of the various isoenzymes and their subtypes. This has great implications not only in terms of extrapolating animal data to the human situation, but also from one tissue to another. Thus, it is critical, in particular for drug discovery efforts, to characterize human PDEs in the relevant tissue. Molecular cloning is probably the best and most direct route to achieve this objective since access to disease-free human tissue is heavily limited. Alternatively, well-characterized animal tissues showing PDE and pharmacological profiles similar to human tissues may be utilized. The methods described in this chapter have been successfully applied to study to the biochemistry and pharmacology of PDEs in both animal and human tissues, and in the discovery of novel selective inhibitors for these proteins.
Methods Mol Biol 1995
PMID:The analysis and assay of cyclic nucleotide phosphodiesterase isoenzyme activity. 765 51

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.
Mol Cell Biochem 1993 Feb 17
PMID:Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine. 768 Nov 41

Calmodulin, the ubiquitous and multifunctional Ca(2+)-binding protein, mediates many of the regulatory effects of Ca2+, including the contractile state of smooth muscle. The principal function of calmodulin in smooth muscle is to activate crossbridge cycling and the development of force in response to a [Ca2+]i transient via the activation of myosin light-chain kinase and phosphorylation of myosin. A distinct calmodulin-dependent kinase, Ca2+/calmodulin-dependent protein kinase II, has been implicated in modulation of smooth-muscle contraction. This kinase phosphorylates myosin light-chain kinase, resulting in an increase in the calmodulin concentration required for half-maximal activation of myosin light-chain kinase, and may account for desensitization of the contractile response to Ca2+. In addition, the thin filament-associated proteins, caldesmon and calponin, which inhibit the actin-activated MgATPase activity of smooth-muscle myosin (the cross-bridge cycling rate), appear to be regulated by calmodulin, either by the direct binding of Ca2+/calmodulin or indirectly by phosphorylation catalysed by Ca2+/calmodulin-dependent protein kinase II. Another level at which calmodulin can regulate smooth-muscle contraction involves proteins which control the movement of Ca2+ across the sarcolemmal and sarcoplasmic reticulum membranes and which are regulated by Ca2+/calmodulin, e.g. the sarcolemmal Ca2+ pump and the ryanodine receptor/Ca2+ release channel, and other proteins which indirectly regulate [Ca2+]i via cyclic nucleotide synthesis and breakdown, e.g. NO synthase and cyclic nucleotide phosphodiesterase. The interplay of such regulatory mechanisms provides the flexibility and adaptability required for the normal functioning of smooth-muscle tissues.
Mol Cell Biochem 1994 Jun 15
PMID:Calmodulin and the regulation of smooth muscle contraction. 781 54


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