Termination and activation of store‐operated cyclic AMP production

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1‐AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca²⁺] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this ‘store‐operated’ pathway requires the ER Ca²⁺ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET‐based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store‐operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca²⁺ entry via the plasma membrane Ca²⁺ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca²⁺/cAMP crosstalk system.     

Beta-adrenergic induced potassium current in Xenopus oocyte: involvement of cyclic AMP

The effect of a beta-adrenergic agonist, on full-grown Xenopus oocytes, still surrounded by their ovarian envelopes, has been studied by electrophysiological methods. The oocytes were hyperpolarized by isoproterenol. Under voltage clamp, the elicited outward current reversed at a membrane potential of - 95 mV, a value close to the K+ equilibrium potential. The isoproterenol induced current varied linearly with the membrane potential in the range studied (- 120 mV, - 30 mV). Half-maximum current was obtained at 3.10(-8) M isoproterenol. Propranolol (10(-7) M) completely suppressed the response to isoproterenol (10(-9) to 10(-5) M). 8-Br-cAMP induced a current which also reversed at - 95 mV. Methyl-isobutyl-xanthine (MIX), a potent inhibitor of phosphodiesterases, potentiated the current induced by isoproterenol. These experiments strongly suggest that the increase in K+ permeability due to catecholamines is mediated by cAMP.     

Fluorescence study ofEscherichia coli cyclic AMP receptor protein

Time-resolved, steady-state fluorescence and fluorescence-detected circular dichroism (FDCD) have been used to resolve the fluorescence contributions of the two tryptophan residues, Trp-13 and Trp-85, in the cyclic AMP receptor protein (CRP). The iodide and acrylamide quenching data show that in CRP one tryptophan residue, Trp-85, is buried within the protein matrix and the other, Trp-13, is moderately exposed on the surface of the protein. Fluorescence-quenching-resolved spectra show that Trp-13 has emission at about 350 nm and contributes 76–83% to the total fluorescence emission. The Trp-85, unquenchable by iodide and acrylamide, has the fluorescence emission at about 337 nm. The time-resolved fluorescence measurements show that Trp-13 has a longer fluorescence decay time. The Trp-85 exhibits a shorter fluorescence decay time. In the CRP-cAMP complex the Trp-85, previously buried in the apoprotein becomes totally exposed to the iodide and acrylamide quenchers. The FDCD spectra indicate that in the CRP-cAMP complex Trp-85 remains in the same environment as in the protein alone. It has been proposed that the binding of cAMP to CRP is accompanied by a hinge reorientation of two protein domains. This allows for penetration of the quencher molecules into the Trp-85 residue previously buried in the protein matrix.Abbreviations CRP cyclic AMP receptor protein - NATA N-acetyltryptophanamide - FQRS fluorescence-quenching-resolved spectra - FDCD fluorescence-detected circular dichroism - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate - FPLC fast protein liquid chromatography     

Catabolite inactivation,cyclic AMP and protein phosphorylation in the methylotrophic yeastHansenula polymorpha

The inactivation of the peroxisomal enzyme alcohol oxidase and the cytoplasmic enzymes fructose-1,6-bisphosphatase, malate dehydrogenase and phosphoenolpyruvate carboxykinase was found to occur after addition of glucose to methanol-grown cells of the yeastHansenula polymorpha. The concentration of cyclic AMP increased nearly twofold within 3 min under the same conditions. In crude extracts ofH. polymorpha about 20 proteins are phosphorylated by cyclic AMP dependent protein kinases, among them also fructose-1,6-bisphosphatase. No phosphorylation of the alcohol oxidase protein could be detected. From this fact, it was concluded that the inactivation of the peroxisomal alcohol oxidase is independent of cyclic AMP-dependent protein phosphorylation.     

Dibutyryl cyclic AMP effects on calcium metabolism by mouse mastocytoma cells

The ability of mouse mastocytoma cells to take up 45Ca2+ was measured in normal growth medium. As previously observed in physiological buffers with succinate and Pi, cells grown for 18h with N6,O2'-dibutyryladenosine 3',5' cyclic monophosphate (DB cyclic AMP) to inhibit growth took up more 45Ca2+ than untreated cells. However 45Ca2+ uptake by cells in growth medium was less sensitive to respiratory inhibitors or uncouplers than 45Ca2+ uptake in physiological buffer. Increased 45Ca2+ uptake by 18h cyclic nucleotide-treated cells was not a result of tighter mitochondrial coupling since mitochondria prepared from cyclic nucleotide-treated cells were less coupled than those from untreated cells. Nevertheless studies with uncouplers suggested that the bulk of the intracellular Ca2+ was associated with mitochondria. DB cyclic AMP-treated cells contained less total Ca2+ than untreated cells indicating that net Ca2+ efflux occurred during the 18h period of drug treatment. These observations suggest that Ca2+ fluxes increase in DB cyclic AMP-treated PY815 cells and that a net efflux of Ca2+ occurs during growth inhibition by the cyclic nucleotide.     

Effect of in vitro cultivation andMycoplasma pneumoniae infection on intracellular cyclic AMP levels in hamster tracheal organ cultures

Summary Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains ofMycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels. This study was supported in part by Grant AI 12559 from the National Institutes of Health. The supply of cholera toxin from Dr. R. A. Finkelstein is most appreciated as are the assistance and advice of J. A. Engelhardt and Y. D. B. Stahl.     

Characterization of buccal motor programs elicited by a cholinergic agonist applied to the cerebral ganglion of Aplysia californica

Applying the non-hydrolyzable cholinergic agonist carbachol (CCh) to the cerebral ganglion of Aplysia elicits sustained, regular bursts of activity in the buccal ganglia resembling those seen during biting. The threshold for bursting is 10^2–4 M. Bursting begins after a 2 to 5 min delay. The burst frequency increases over the first 5 bursts, reaching a plateau value of 3 per minute. Bursting is maintained for over 10 min. Some of the effects of CCh may be attributed to its ability to depolarize and fire CBI-2, a command-like neuron in the cerebral ganglion that initiates biting. CBI-2 is also depolarized by ACh, and by stimulating peripheral sensory nerves. Excitation of CBI-2 caused by carbachol is partially blocked by the muscarinic antagonist atropine. We examined whether CCh-induced bursting is modified in ganglia taken from Aplysia that previously experienced treatments inhibiting feeding, such as satiation, head shock contingent or non-contingent with food, and training animals with an inedible food. No treatment consistently and repeatedly affected the latency, the peak burst period, the length of time that bursting was maintained, or the threshold CCh concentration for eliciting bursting. However, there was a decrease in the rate of the buildup of the buccal ganglion program in previously satiated animals.     

Activation of type I and type II cyclic AMP-dependent protein kinases by 2,8-disubstituted derivatives of cyclic AMP

Derivatives of cyclic AMP with substituents in both the 2-position (methyl or butyl) and the 8-position (bromo, benzylthio, p-chlorophenylthio or azido) and their singly modified parent compounds were examined for their abilities to activate type I isozymes of cyclic AMP-dependent protein kinases from rabbit and porcine muscle and type II isozymes of cyclic AMP-dependent protein kinases from bovine brain and heart. The specificity of 2-n-butyl-cyclic AMP for type II was substantially reduced or eliminated by the addition of 8-substituents. The lack of specificity of 2-methyl-cyclic AMP for either type I or II was not changed by the addition of 8-substituents.     

Parathyroid hormone promotes the disassembly of cytoskeletal actin and myosin in cultured osteoblastic cells: mediation by cyclic AMP.

Parathyroid hormone (PTH) alters the shape of osteoblastic cells both in vivo and in vitro. In this study, we examined the effect of PTH on cytoskeletal actin and myosin, estimated by polyacrylamide gel electrophoresis of Triton X-100 (1%) nonextractable proteins. After 2-5 minutes, PTH caused a rapid and transient decrease of 50-60% in polymerized actin and myosin associated with the Triton X-100 nonextractable cytoskeleton. Polymerized actin returned to control levels by 30 min. The PTH effect was dose-dependent with an IC50 of about 1 nM, and was partially inhibited by the (3-34) PTH antagonist. PTH caused a rapid transient rise in cyclic AMP (cAMP) in these cells that peaked at 4 min, while the nadir in cytoskeletal actin and myosin was recorded around 5 min. The intracellular calcium chelator Quin-2/AM (10 microM) also decreased cytoskeletal actin and myosin, to the same extent as did PTH (100 nM). To distinguish between cAMP elevation and Ca++ reduction as mediators of PTH action, we measured the phosphorylation of the 20 kD (PI 4.9) myosin light chain in cells preincubated with [32P]-orthophosphate. The phosphorylation of this protein decreased within 2-3 min after PTH addition and returned to control levels after 5 min. The calcium ionophore A-23187 did not antagonize this PTH effect. Visualization of microfilaments with rhodamine-conjugated phalloidin showed that PTH altered the cytoskeleton by decreasing the number of stress fibers. These changes in the cytoskeleton paralleled changes in the shape of the cells from a spread configuration to a stellate form with retracting processes. The above findings indicate that the alteration in osteoblast shape produced by PTH involve relatively rapid and transient changes in cytoskeletal organization that appear to be mediated by cAMP.     

Metabolism of cyclic AMP in non-pathogenic Mycobacterium smegmatis

The intracellular concentration of cyclic AMP reached a maximum in 3.5-day old cultures of Mycobacterium smegmatis grown in the presence of glycerol as the main source of carbon. Glucose-grown cells exhibited decreased cyclic AMP levels at all stages of growth. When M. smegmatis cells were incubated with various metabolites, pyruvate increased whereas glucose, citric acid, succinic acid and lactic acid decreased intracellular cyclic AMP levels. No cyclic AMP was detected in the incubation medium. The presence of a cyclic AMP-binding protein was demonstrated in cellfree extracts of M. smegmatis.     

Response of cyclic AMP by DMSO differentiated HL-60 cells exposed to electric interferential current after prestimulation

The action of interferential current (IFC) upon intracellular content of cyclic adenosine monophosphate (cAMP) after prestimulation with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) has been investigated. Human promyelocytes (HL-60) differentiated to granulocytes by dimethylsulphoxide (DMSO) have been treated with different concentrations of fMLP. This enhances their cAMP content. The half maximal effective concentration (EC50) was about 4 nM. Exposure to IFC with modulation frequencies of 35 and 125 Hz (5 min, 250 microA/cm2) after prestimulation with various concentrations of fMLP had no enhancing effect at low or high concentrations of fMLP. In contrast, at medium concentrations in the range of about 100 pM fMLP, a significant enhancement of cAMP could be observed. This synergistic effect of fMLP and IFC has been examined in detail by varying the modulation frequency, current density, and exposure time. Enhancement of cAMP content could be observed at certain modulation frequencies and exposure times suggesting a window effect, whereas for the current density in the range measured (8.5 microA/cm2-2.5 mA/cm2) a constant enhancement could be observed. The synergistic effect of fMLP and IFC could only be observed in the treatment sequence of fMLP followed by IFC; an inverse sequence had no effect on the cAMP content. .     

Studies of the cyclic adenosine monophosphate chemoreceptor ofParamecium

Summary A doublet of proteins (48,000M _r) from theParamecium cell body membrane fits several criteria for the external cAMP chemoreceptor. These criteria include: (i) selective elution from a cAMP affinity column, matching a specificity that could be predicted from the behavioral response and whole-cell binding; (ii) binding to wheat germ agglutinin indicating the presence of carbohydrate moieties indicating surface exposure; and (iii) selective inhibition of the intact cells' chemoresponse to cAMP by antibodies against the doublet. Additional evidence for the existence of a receptor, in general, comes from selective elimination of the cAMP chemoresponse by photoaffinity labeling of whole cells with 8-N₃-cAMP. The doublet proteins are not identical to the regulatory subunit of a cAMP-dependent protein kinase fromParamecium, theDictyostelium cAMP chemoreceptor, or the 42–45 kDa range proteins related to the large surface glycoprotein inParamecium. The doublet proteins are not readily separable and, as inDictyostelium, may represent two different covalent modification states of the same protein. Amino acid analysis indicates that the proteins are similar, but does not distinguish between the possibilities of proteolysis and covalent modification. Once cloned, this doublet may prove to be only the fifth external, eukaryotic chemoreceptor to be identified.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Inhibitory effect of regucalcin on Ca2+/calmodulin-dependent cyclic AMP phosphodiesterase activity in rat kidney cytosol

The effect of regucalcin, a novel Ca²⁺-binding protein, on Ca²⁺/ calmodulin-dependent cyclic adenosine monophosphate (AMP) phosphodiesterase activity in the cytosol of rat renal cortex was investigated. Regucalcin with physiologic concentration (10^-7 M) in rat kidney had no effect on cyclic AMP phosphodiesterase activity in the absence of CaCl₂ and calmodulin. However, the activatory effect of both CaCl₂ (10 µM) and calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was markedly inhibited by the addition of regucalcin (10^-8 to 10^-6 M) in the enzyme reaction mixture. The inhibitory effect of regucalcin on the enzyme activity was also seen in the presence of CaCl₂ (5-50 µM) or calmodulin (5-50 U/ml) with increasing concentrations. The presence of trifluoperazine (10 µM), an antagonist of calmodulin, caused a partial inhibition of Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activity. This inhibition was further enhanced by the addition of regucalcin (10^-7 M). The inhibitory effect of regucalcin (10^-7 M) was not seen in the presence of 20 µM trifluoperazine. Moreover, the activatory effect of calmodulin (20 U/ml) on cyclic AMP phosphodiesterase was not entirely seen, when calmodulin was added 10 min after incubation in the presence of CaCl₂ (10 µM) and regucalcin (10^-7 M). The present results demonstrates that regucalcin has an inhibitory effect on Ca²⁺ /calmodulin-dependent cyclic AMP phosphodiesterase activation in the cytosol of rat renal cortex.     

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis.Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function,such as adhesion,aggregation,and secretion.Elevation of intracellular cAMP,which induces the activation of PKA,results in the inhibition of platelet function.Thus,tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy.In this review,we summarize the PKA substrates and their contributions to platelet function,especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology.In addition,we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.     

Cyclic AMP and intracellular ionic activities innecturus gallbladder

Summary Open-tip and liquid ion-exchanger microelectrodes were used to study the effects of cAMP (6mm, added to the serosal medium) on apical membrane potential (E _m ) and intracellular sodium, potassium, and chloride activities (a _Na ⁱ ,a _K ⁱ ,a _Cl ⁱ ) inNecturus gallbladder under open-circuit conditions. Transepithelial potential difference (E _Tr ) was also measured. In the presence of cAMP,a _Cl ⁱ fell from about 1.5 times its equilibrium value to a level that corresponded to electrochemical equilibrium across the apical and basolateral cell membranes. Under these conditionsa _Na ⁱ decreased anda _K ⁱ increased,E _m was unchanged andE _Tr increased from virtually zero to a small but significant serosal positive value. The cAMP-induced increase ina _K ⁱ was abolished when Cl^–-free incubation media were used. Addition of the Ca⁺⁺-ionophore A23187 (0.5 g/ml) to the serosal medium had no effect onE _m ,E _Tr , ora _Cl ⁱ . When A23187 was added to the mucosal medium,E _m and the basolateral membrane potential hyperpolarized by about 20 mV and an increase in the outwardly directed electrochemical driving force for Cl^– was observed. These results indicate that cAMP inhibits coupled transapical Na–Cl entry into epithelial cells ofNecturus gallbladder and suggest that this inhibition may not be mediated by an increase in intracellular Ca⁺⁺ concentration.     

The critical roles of cyclic AMP/cyclic AMP-dependent protein kinase in platelet physiology

Platelets are the primary players in both thrombosis and hemostasis. Cyclic AMP (cAMP) and cAMP-dependent protein kinase (PKA) are important signaling molecules in the regulation of platelet function, such as adhesion, aggregation, and secretion. Elevation of intracellular cAMP, which induces the activation of PKA, results in the inhibition of platelet function. Thus, tight control of the intracellular cAMP/PKA signaling pathway has great implications for platelet-dependent hemostasis and effective cardiovascular therapy. In this review, we summarize the PKA substrates and their contributions to platelet function, especially the advancing understanding of the cAMP/PKA-dependent signaling pathway in platelet physiology. In addition, we suggest the possibility that cAMP/PKA is involved in the platelet procoagulant process and receptor ectodomain shedding.     

Relationship between cyclic AMP level and accumulation of carotenoid pigments in Neurospora crassa

Dark grown mycelial cells of Neurospora crassa bearing mutant genes crisp-I or frost and having a decreased level of cyclic adenosine 3,5-monophosphate contained more carotenoid pigments than the cells with wild alleles of these genes. A transient decrease of the cyclic AMP occurred following photoinduction of carotenoid synthesis during its lag-period. Its intensity correlated with the increase of carotenoid pigment level due to photoinduction. No correlation in the content of cyclic guanosine 5-phosphate with both constitutive level of carotenoids and its photoinduced increase was observed.     

A voltage-dependent and pH-sensitive proton current in Rana esculenta oocytes

Voltage clamp technique was used to study macroscopic ionic currents in Rana esculenta oocytes. Depolarization steps led to the activation of a single type of outward current (I _out) when contaminant potassium and calcium-dependent chloride currents were pharmacologically inhibited. The voltage threshold of I _out activation was 10 mV and this current, which did not inactivate, presented a deactivation the time constant of 73±21 msec (n=26) corresponding to a membrane voltage of –60 mV. Its reversal potential (E _rev) was dependent on the magnitude of the depolarization and also on pulse duration. These changes in E _rev were thought to reflect intracellular ion depletion occurring during activation of the remaining outward current. Furthermore, the activation threshold of I _out was clearly affected by modifications in extracellular and intracellular H⁺ concentrations. Indeed, intracellular alkalinization (evoked by external application of ammonium chloride) or extracellular acidification induced a rightward shift in the activation threshold while intracellular acidification (evoked by external application of sodium acetate) or extracellular alkalinization shifted this threshold toward a more negative value. Lastly, I _out was dramatically reduced by divalent cations such as Cd²⁺, Ni²⁺ or Zn²⁺ and was strongly decreased by 4 Aminopyridine (4-AP), wellknown H⁺ current antagonists already described in many cell types. Therefore, it was suggested that the outward current was prominently carried by H⁺ ions, which may play a key role in the regulation of intracellular pH and subsequent pH dependent processes in Rana oocyte.