Divergent cAMP signaling pathways regulate growth and pathogenesis in the rice blast fungus Magnaporthe grisea

cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA, do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea, we have identified strains in which a null mutation in the adenylate cyclase gene (MAC1) has an unstable phenotype such that the bypass suppressors of the Mac1(-) phenotype (sum) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation (sum1-99) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA (SUM1), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA, encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.     

Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits internalization and signal transduction but increases receptor affinity

A series of mutant porcine calcitonin receptors with progressively truncated carboxy termini have been expressed in COS and HEK 293 cells. All forms of the receptor, including those totally lacking the cytoplasmic tail, were able to bind 125I-labeled salmon calcitonin. However, removal of C-terminal domains resulted in multiple functional changes in the receptor. First, compared with the wild type receptor, affinity of binding of salmon calcitonin was increased for truncated receptors, whether determined in intact transfected cells or in cell membranes. Second, internalization of the ligand-receptor complex was greatly attenuated for mutants truncated by 44 or 83 amino acids but not for an intermediate form truncated by 63 amino acids. Third, truncation affected signal transduction, which for the porcine calcitonin receptor occurs by generation of intracellular cAMP and Ca2+. The magnitude of adenylate cyclase responses was much reduced for the same mutants defective in internalization. Under conditions where expression of each receptor form was approximately equal, the magnitude of intracellular Ca2+ responses was decreased by C-terminal truncation. These results draw attention to the functional significance of the cytoplasmic tail of the porcine calcitonin receptor and suggest intramolecular interactions between the carboxy terminus and other receptor domains and/or cellular regulatory elements.     

Sequence-specific protein interaction with a transcriptional enhancer involved in the autoregulated expression of cAMP receptor 1 in Dictyostelium

Major stages of Dictyostelium development are regulated by secreted, extracellular cAMP through activation of a serpentine receptor family. During early development, oscillations of extracellular cAMP mobilize cells for aggregation; later, continuous exposure to higher extracellular cAMP concentrations downregulates early gene expression and promotes cytodifferentiation and cell-specific gene expression. The cAMP receptor 1 gene CAR1 has two promoters that are differentially responsive to these extracellular cAMP stimuli. The early CAR1 promoter is induced by nM pulses of cAMP, which in turn are generated by CAR1-dependent activation of adenylyl cyclase (AC). Higher, non-fluctuating concentrations of cAMP will adapt this AC stimulus-response, repress the activated early promoter and induce the dormant late promoter. We now identify a critical element of the pulse-induced CAR1 promoter and a nuclear factor with sequence-specific interaction. Mutation of four nucleotides within the element prevents both in vitro protein binding and in vivo expression of an otherwise fully active early CAR1 promoter and multimerization of the wild-type, but not mutant, sequence will confer cAMP regulation to a quiescent heterologous promoter. These cis and trans elements, thus, constitute a part of the molecular response to the cAMP transmembrane signal cascade that regulates early development of Dictyostelium.     

Selective effects of ethanol on the generation of cAMP by particular members of the adenylyl cyclase family

A selective action of ethanol on major signal transduction proteins, such as adenylyl cyclase, has been considered to be important for certain actions of ethanol, and alcoholics have been demonstrated to differ from controls in measures of platelet adenylyl cyclase activity. Recent advances in identification and characterization of isoforms of adenylyl cyclase have demonstrated that there exists at least eight different forms of this enzyme. To examine whether the effect of ethanol on generation of cAMP is modified by the presence of particular isoforms of adenylyl cyclase within a cell, we transiently expressed each of six adenylyl cyclases in human embryonic kidney (HEK293) cells and measured cAMP accumulation in whole cells in the presence and absence of ethanol. The treatment of cells expressing the various adenylyl cyclases with ethanol alone did not enhance cAMP generation. In the presence of prostaglandin E1, cAMP generation by type I and type III adenylyl cyclases was insensitive to ethanol. cAMP accumulation generated by the other adenylyl cyclases was, however, increased by incubation of cells with ethanol in the presence of stimulatory agonists (e.g., prostaglandin E1). Stimulation by ethanol of cAMP generation by type VII adenylyl cyclase was 2- to 3-fold greater than that seen with the other tested adenylyl cyclases. The noted stimulation of cAMP generation by ethanol was dose-dependent and required concurrent activation of adenylyl cyclase through the stimulatory G protein. The effects of ethanol were reversible and mimicked by butanol but not by chloroform.     

Low level cyclic adenosine 3',5'-monophosphate accumulation analysis of [des-His1, des- Phe6, Glu9] glucagon-NH2 identifies glucagon antagonists from weak partial agonists/antagonists

[des-His1, des-Phe6,Glu9]Glucagon-NH2 is a newly designed glucagon antagonist. This analog has a binding IC50 of 48 nM (compared to glucagon IC50 of 1.5 nM) and demonstrates pure antagonism in an adenylate cyclase assay. Although the number of glucagon antagonists has grown rapidly recently, closer examination suggested that many of these antagonists retained very low, almost imperceptible levels of cAMP accumulation that were sufficient to elicit an in vivo biological response. To investigate more carefully this secondary biological signal, we measured cAMP accumulation in a revised assay using isolated hepatocytes in the presence of the phosphodiesterase (PDE) inhibitor Rolipram. The PDE inhibitors Rolipram and isobutyl-1-methylxanthine (IBMX) increased the sensitivity of the cAMP accumulation assay from approximately 10-fold for the native hormone to 35-fold above basal levels. On the other hand, amrinone, another PDE inhibitor, did not affect the cAMP accumulation caused by glucagon. The use of PDE inhibitors indicated that three glucagon analogs that had previously been reported to have strong antagonist properties in classical adenylate cyclase assays were actually weak partial agonists in this new assay system. [N alpha-Trinitrophenyl-His1, homo-Arg12]glucagon, [des-amino-His1,D-Phe4,Tyr5, Arg12, Lys17,18,Glu21]glucagon, and [des-His1,Glu9]glucagon-NH2 demonstrated 233%, 21%, and 5.5% cAMP accumulation relative to the native hormone in the presence of 25 microM Rolipram. On the other hand, [des-His1,des-Phe6,Glu9]glucagon-NH2, a newly designed glucagon antagonist, did not activate adenylate cyclase in the presence of Rolipram up to a maximal physiological concentration of 1 microM, indicating that it was a pure antagonist of glucagon-induced adenylate cyclase activity and also the first one in this class. This compound and others were tested in a glycogen phosphorylase assay. As [des-His1,des- Phe6,Glu9]glucagon-NH2 did not activate phosphorylase activity, it was chosen as our candidate for in vivo testing in streptozotocin-induced diabetic rats. An initial dose of 0.75 mg/kg was found to cause the greatest lowering of blood glucose levels (to 63% of the initial levels in 15 min) when the bolus was followed by continuous infusion of 25 micrograms/kgxmin for 1 h.     

Regulation of adenylate cyclase from cultured human cell lines by adenosine

Broken cell particulate preparations of adenylate cyclase isolated from the human glioma cell line 132-1N1 were stimulated 2-to 3-fold by 30 muM adenosine. This concentration of adenosine produced a maximal stimulation of the cyclase while 3 to 5 muM adenosine produced half-maximal stimulation. Theophylline, at 40 muM, inhibited the adenosine stimulation of the adenylate cyclase by about 40% while 200 muM produced near complete inhibition. The inhibition by theophylline could be overcome by increasing adenosine to a concentration 10-fold that of theophylline, implying that the inhibition was competitive. Basal activity was not inhibited by even 1.0 mM theophylline, nor was the epinephrine stimulated activity. In contrast, 1.0 muM propranolol essentially completely inhibited the 8-fold stimulation of 1.0 muM epinephrine but had no effect on either basal or adenosine-stimulated activity. Adenosine and 2-chloroadenosine were equipotent in stimulating adenylate cyclase from the 132-1N1 line, whereas neither adenine nor guanosine had any detectable effect. GTP, 10 muM, produced a small variable stimulation of the adenylate cyclase while the GTP analogue, 5'-guanylylimidodiphosphate (Gpp(NH)p), produced a marked stimulation fo the cyclase. Preincubation of the adenylate cyclase preparation with the analogue greatly increased its potency and maximal effect. In contrast, both basal and adenosine-stimulated activity decreased markedly with preincubation. The effects of adenosine or epinephrine in combination with Gpp(NH)p were at least additive and often synergistic in comparison to the effects of the compounds alone. The effects of adenosine on intact and broken cell preparations of the human fibroblast lines WI-38 and VA13-2RA were also examined. In the intact VA13-2RA, adenosine produced rapid and large increases in intracellular and extracellular cyclic adenosine 3':5'-monophosphate (cAMP). In the parental fibroblast line, the WI-38, adenosine slightly elevated basal levels of cAMP, but only produced marked elevations in the presence of non-methylxanthine phosphodiesterase inhibitors. The effect of adenosine on the broken cell particulate preparations of adenylate cyclase from the fibroblasts was similar to its action on the cyclase from the 132-1N1; 30 muM adenosine produced a maximal stimulation of the adenylate cyclase, and the stimulation was inhibited by theophylline.     

Day/night differences in the stimulation of adenylate cyclase activity by calcium/calmodulin in chick pineal cell cultures: evidence for circadian regulation of cyclic AMP

In chick pineal cell culture, stimulation of adenylate cyclase with the diterpene forskolin was greater during the subjective night than during the subjective day. This rhythm of cyclic AMP (cAMP) stimulation mimicked the rhythm of unstimulated cAMP measured previously during LD cycles from flow-through culture. Direct measurement of adenylate cyclase activity in permeabilized cells revealed an adenylate cyclase activity activated by Ca2+/calmodulin during the night but not during the day. However, this difference in adenylate cyclase activity at two times of the circadian cycle is apparent only when permeabilized cells were prewashed with buffer containing GTE When cAMP was measured from flow-through cultures maintained in continuous darkness to determine whether a circadian clock may regulate cAMP, a low-amplitude rhythm was measured. The circadian rhythm of cAMP was similar to the cAMP rhythm previously measured on LD cycles except that the rhythm in darkness had a lower amplitude. Similar to the suppression of melatonin, cAMP was suppressed by light presented during the middle of the night. LD differences in nocturnal cAMP levels were abolished with dipyridamole, an inhibitor of cyclic GMP (cGMP) phosphodiesterase. These results suggest that the rhythm of cAMP in chick pineal cells involves the stimulation of adenylate cyclase by Ca2+/calmodulin during the night and a GTP-dependent suppression of adenylate cyclase activity during the day. The photic suppression of cAMP at night involves the activation of a dipyridamole-sensitive, cGMP phosphodiesterase.     

Restoration of the cAMP second messenger pathway enhances cardiac preservation for transplantation in a heterotopic rat model

Current organ preservation strategies subject graft vasculature to severe hypoxia (PO2 approximately 20 Torr), potentially compromising vascular function and limiting successful transplantation. Previous work has shown that cAMP modulates endothelial cell (EC) antithrombogenicity, barrier function, and leukocyte/EC interactions, and that hypoxia suppresses EC cAMP levels. To explore the possible benefits of cAMP analogs/agonists in organ preservation, we used a rat heterotopic cardiac transplant model; dibutyryl cAMP added to preservation solutions was associated with a time- and dose-dependent increase in the duration of cold storage associated with successful graft function. Preservation was also enhanced by 8-bromo-cAMP, the Sp isomer of adenosine 3',5'monophosphorothioate, and types III (indolidan) and IV (rolipram) phosphodiesterase inhibitors. Neither butyrate alone nor 8-bromoadenosine were effective, and the cAMP-dependent protein kinase antagonist Rp isomer of adenosine 3',5'monophosphorothioate prevented preservation enhancement induced by 8-bromo-cAMP. Grafts stored with dibutyryl cAMP demonstrated a 5.5-fold increase in blood flow and a 3.2-fold decreased neutrophil infiltration after transplantation. To explore the role of cAMP in another cell type critical for vascular homeostasis, vascular smooth muscle cells were subjected to hypoxia, causing a time-dependent decline in cAMP levels. Although adenylate cyclase activity was unchanged, diminished oxygen tensions were associated with enhanced phosphodiesterase activity (59 and 30% increase in soluble types III and IV activity, respectively). These data suggest that hypoxia or graft ischemia disrupt vascular homeostasis, at least in part, by perturbing the cAMP second messenger pathway. Supplementation of this pathway provides a new approach for enhancing cardiac preservation, promoting myocardial function, and maintaining vascular homeostatic properties.     

Morphine dependence in human neuroblastoma SH-SY5Y cells is associated with adaptive changes in both the quantity and functional interaction of PGE1 receptors and stimulatory G proteins

Chronic exposure of all-trans-retinoic acid-differentiated SH-SY5Y cells to morphine (10 mu M; 2 days) results in sensitization of adenylate cyclase as characterized by a significant increase in both PGE1 receptor-mediated as well as receptor-independent (NaF, 10 mM; forskolin, 100 mu M) stimulation of effector activity. To investigate the underlying biochemical alterations, chronic opioid regulation of each of the components comprising the stimulatory PGE1 receptor system was examined. On receptor level, chronic morphine treatment was found to reduce PGE1 receptor number (Bmax) by approximately 40%, whereas their affinity slightly increased. Binding experiments performed in the presence of GTPgammaS (100 mu M) further indicate that the decrease in PGE1 receptor density is associated with a loss of functionally G protein-coupled receptors. On post-receptor level, chronic morphine treatment substantially increased the abundance and functional activity of stimulatory G proteins, as assessed by cholera toxin-catalyzed ADP-ribosylation of GSalpha and S49 cyc- reconstitution assays. No changes were found on the level of adenylate cyclase. Evaluation of the functional interaction between PGE1 receptors and GS in situ by application of a C-terminal anti-GSalpha antibody revealed a more intense coupling efficiency between these two entities, since a significant higher amount of antibody (2.3-fold) was required in morphine dependent cell membranes to half-maximally attenuate PGE1 receptor-stimulated adenylate cyclase activity. In addition, limitation of the amount of functionally available GSalpha within the PGE1 receptor/adenylate cyclase signal transduction cascade abolished the generation of a supersensitive adenylate cyclase response during the state of naloxone (100 mu M)-precipitated withdrawal. These data demonstrate that in human neuroblastoma SH-SY5Y cells chronic morphine-induced sensitization of adenylate cyclase is associated with distinct quantitative and qualitative adaptations within the stimulatory adenylate cyclase-coupled PGE1 receptor system. Thus, alterations in the functional activity of stimulatory receptor systems are suggested to contribute to the cellular mechanisms underlying opioid dependence.     

Autoxidation of pyridoxalated hemoglobin polyoxyethylene conjugate

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10(-5) M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.