[Maternal-Infant care in a refugee camp. Report from a refugee camp in southern Lebanon].
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Coupling transcription to signaling pathways: cAMP and nuclear factor cAMP-responsive element modulator.
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Animals↗
Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.
We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.
Adrenal Gland Neoplasms↗
Short-term feedback regulation of cAMP by accelerated degradation in rat tissues.
A recent study showed that cAMP analogs lowered cAMP levels in rat hepatocytes (Corbin, J.D., Beebe, S.J., and Blackmore, P.F. (1985) J. Biol. Chem. 260, 8731-8735). The present work demonstrates that cAMP analogs also lowered cAMP in a rapid, concentration-dependent manner in heart and fat cells. In order to determine if the cAMP-dependent protein kinase mediated this effect, techniques were developed to assay the protein kinase activity ratio in hepatocytes treated with cAMP analogs. The activation of protein kinase and phosphorylase in hepatocytes by 8-pCl phi S-cAMP (where 8-pCl phi S- indicates 8-parachlorothiophenyl-) was concentration-dependent and occurred in parallel to proportionate decreases in cAMP. More than 20% of the cAMP binding sites on the protein kinase were unoccupied at concentrations of 8-pCl phi S-cAMP that produced maximal cAMP lowering. Thus, the possibility that 8-pCl phi S-cAMP lowered cAMP by displacing it from protein kinase binding sites, making it available for hydrolysis, seemed unlikely. In adipocytes, the lowering of cAMP by 8-pCl phi S-cAMP occurred in parallel with increases in lipolysis and activation of low Km phosphodiesterase, suggesting that the phosphodiesterase was responsible for the cAMP lowering. Further evidence for this assertion was the finding that in hepatocytes preloaded with low concentrations of 8-pCl phi S-cAMP, glucagon lowered 8-pCl phi S-cAMP by about 50%, an amount similar to the cAMP lowering observed with 8-pCl phi S-cAMP treatment. The results were consistent with a cAMP-dependent protein kinase-catalyzed activation of a phosphodiesterase and suggested that 8-pCl phi S-cAMP-mediated hydrolysis of cAMP mimicked a physiologically significant response. The observation of this phenomenon in several tissues further suggested that it may be a general mechanism for dampening and terminating the hormonal signal through accelerated degradation of cAMP.
3',5'-Cyclic-AMP Phosphodiesterases↗
cAMP analogs inhibit gamma-aminobutyric acid-gated chloride flux and activate protein kinase A in brain synaptoneurosomes.
The effects of permeant cAMP analogs were studied on the function of the gamma-aminobutyric acidA (GABAA) receptor and on the activation of protein kinase A in brain synaptoneurosomes. Incubation of cerebral cortical synaptoneurosomes with permeant cAMP analogs decreased muscimol-induced 36Cl- uptake in a concentration-dependent manner. The order of potency was chlorophenylthio-cAMP (CPT-cAMP) greater than dibutyryl-cAMP greater than 8-bromo-cAMP. This order of potency was reflected by the ability of the analogs to gain access to the intravesicular compartment. cAMP, which failed to penetrate the membrane, had no effect. The half-maximal and maximal effects of the cAMP analogs were similar in the cerebral cortex, hippocampus, striatum, and cerebellum. To determine whether the cAMP analogs were acting through the activation of protein kinase A, protein kinase A activity was measured in lysed synaptoneurosomes, using kemptide as the substrate. In the lysed preparation, where the cAMP analogs have direct access to intracellular enzymes, the order of potencies of the cAMP analogs to activate protein kinase A (8-bromo-cAMP greater than CPT-cAMP greater than dibutyryl-cAMP) differed from the order of potencies to inhibit muscimol-induced 36Cl- uptake. In regional studies, the greatest effect of CPT-cAMP was observed in the cortex, whereas the smallest effect was observed in the hippocampus and cerebellum. To determine whether cAMP inhibition of GABA-gated ion flux was due to activation of protein kinase A, the time course for each response was measured. Inhibition of muscimol-induced 36Cl- uptake by cAMP analogs was nearly complete by 5 sec. Significant activation of protein kinase A by CPT-cAMP was also observed as early as 5 sec, but protein kinase A activation continued up to 10 min. The protein kinase inhibitor peptide inhibited protein kinase A activity in lysed synaptoneurosomes but had no effect on ion flux in intact synaptoneurosomes, as expected. However, a permeant kinase inhibitor, H-8, also failed to inhibit the effect of cAMP analogs on the muscimol response, yet it inhibited protein kinase A activity. The failure of H-8 to inhibit cAMP analog effects on GABAA receptor function was most likely due to the presence of ATP inside the synaptoneurosomes, because H-8 inhibition of protein kinase A was reduced in the presence of ATP. These results indicate that cAMP and cAMP analogs must penetrate the intravesicular compartment to inhibit GABAA receptor function. Although cAMP analogs decrease GABA-gated ion flux under conditions in which they activate protein kinase A, a causal relationship remains to be established.
8-Bromo Cyclic Adenosine Monophosphate↗
The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells.
Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP-dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.
Adenylyl Cyclase Inhibitors↗
Induction of tyrosine aminotransferase in H-35 hepatoma cells by cAMP captured in phospholipid vesicles.
The uptake, metabolism, and action of cAMP, captured within phospholipid vesicles, in H-35 hepatoma cells were studied. Sonication of lipids in buffer containing cAMP resulted in the formation of 300-A unilamellar lipid vesicles, capturing cAMP in the internal aqueous cavity. Incubation of H-35 hepatoma cells with vesicles containing cAMP (vesicle-cAMP) resulted in rapid incorporation of the vesicle content; apparent saturation of uptake was reached after approximately 30 min of incubation at 37 degrees C. Uptake of vesicle-cAMP was linear over a 10-fold vesicle concentration range. Pretreatment of cells with combined inhibitors of glycolysis and respiration inhibited vesicle uptake by 27%, suggesting vesicle fusion with the cell membrane as a predominant pathway of vesicle uptake. Studies on the metabolism of incorporated cAMP indicated that greater than 50% of the cell-associated radioactivity, derived from vesicle-[3H]cAMP, was preserved as cAMP at the end of a 20-min incubation at 37 degrees C. The incorporation of vesicle-cAMP by H-35 hepatoma cells resulted in increased tyrosine aminotransferase (TAT) activity. The concentration of vesicle-cAMP needed to produce a half-maximal increase in TAT activity was 10 microM, approximately two orders of magnitude lower than that of exogenously added dbcAMP. cAMP was ineffective when added extracellularly. The kinetic relationship of the cAMP-induced increase in TAT activity and the binding of cAMP to its receptor protein, in intact H-35 cells, was examined using vesicle-trapped 8-N3-cAMP, a photoaffinity labeling analogue of cAMP. Incubation of H-35 hepatoma cells with vesicle-8-N3-cAMP resulted in increased TAT activity, preceded by the binding of 8-N3-cAMP to the regulatory subunit of type II cAMP-dependent protein kinase. The use of lipid vesicles provides a means of modulating intracellular cAMP concentration without adding cyclic nucleotide in the millimolar concentration range to the extracellular medium. The increased efficiency of intracellular delivery of cyclic nucleotide with retention of biological activity, provides a useful technique in examining the relationship of occupancy of specific cAMP-receptor protein(s) and the occurrence of a cAMP-mediated biological response in intact cells.
Animals↗
Resolution of cAMP signals in three-dimensional microdomains using novel, real-time sensors.
A large number of hormones, neurotransmitters, and odorants alter cellular behavior by triggering changes in intracellular levels of cAMP. Although the effector proteins that bind cAMP have been identified, it is not known how this one messenger can differentially regulate the activities of hundreds of cellular proteins. The spatial and temporal nature of cAMP signals and, thus, their information content remain largely unknown. We present here a high-resolution method for measuring cAMP signals near the plasma membrane in single cells. Cyclic nucleotide-gated (CNG) ion channels from olfactory receptor neurons have been genetically modified to improve their cAMP-sensing properties. We show how these channels can be used in electrophysiological experiments to accurately measure changes in cAMP concentration near the membrane, where most adenylyl cyclases reside. We have found in several cell types (both excitable and nonexcitable) that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is severely hindered. We also show that a uniform extracellular stimulus can initiate very distinct cAMP signals within different compartments of a simple, nonexcitable cell. Analysis of compartmental models indicates that diffusional restrictions between microdomains (near the membrane) and the cytosol, as well as differential regulation of phosphodiesterase activity, are necessary to explain such distinct signals. Using modified CNG channels as sensors has much greater spatial and temporal resolution than other methods for measuring cAMP, and should help to unravel the complexities of signaling by this ubiquitous messenger. Cyclic AMP (cAMP), the prototypical second messenger, regulates a wide variety of cellular processes. Changes in cAMP concentration transmit information to downstream effectors including protein kinase A (PKA), cyclic nucleotide-gated (CNG) channels, hyperpolarization activated (Ih) channels, and Epac. However, it is largely unclear how differential regulation of cellular targets occurs. The concept of compartmentation emerged over 20 years ago in studies of cardiac myocytes, to help explain how a variety of extracellular stimuli that primarily act through cAMP can have very different downstream effects on the cell. The basis for compartmentation, and indeed, the nature of cAMP signals themselves, have remained mysteries. To understand how these signals function within the cell it is important to answer the following questions: (i) How are cAMP signals localized? (ii) What are the kinetics of cAMP signals in localized domains? and (iii) What information is contained in the amplitude and frequency of cAMP signals? We describe here a high-resolution method for measuring cAMP signals near the plasma membrane, using modified cyclic nucleotide-gated ion channels. This approach was inspired by the field of retinal phototransduction, the best-studied second messenger signaling system, in which elegant biochemical studies have been complemented by real-time measurements of cGMP signals using endogenous cyclic nucleotide-gated (CNG) channels. CNG channels are directly opened by the binding of cyclic nucleotides. They were discovered in retinal photoreceptor cells and olfactory receptor neurons, where they generate the electrical response to light and odorants. The native retinal channel is cGMP specific, while the native olfactory channel is equally sensitive to cAMP and cGMP. Native CNG channels consist of A and B subunits, both of which bind cyclic nucleotides, although most A subunits form functional channels on their own. We have modified an olfactory channel A subunit (CNGA2) to improve its sensitivity and selectivity for cAMP. Two of the findings with this approach, summarized here, are: (i) cAMP in several cell types is produced in subcellular compartments under the plasma membrane with restricted diffusional access to the bulk cytosol; and (ii) the amplitude and kinetics of cAMP signals within these compartments are distinct from those in the remainder of the cell.
Biosensing Techniques↗
Kinetic analysis of cAMP-activated Na+ current in the molluscan neuron. A diffusion-reaction model.
cAMP-activated Na+ current (INa,cAMP) was studied in voltage-clamped neurons of the seaslug Pleurobranchaea californica. The current response to injected cAMP varied in both time course and amplitude as the tip of an intracellular injection electrode was moved from the periphery to the center of the neuron soma. The latency from injection to peak response was dependent on the amount of cAMP injected unless the electrode was centered within the cell. Decay of the INa,cAMP response was slowed by phosphodiesterase inhibition. These observations suggest that the kinetics of the INa,cAMP response are governed by cAMP diffusion and degradation. Phosphodiesterase inhibition induced a persistent inward current. At lower concentrations of inhibitor, INa,cAMP response amplitude increased as expected for decreased hydrolysis rate of injected cAMP. Higher inhibitor concentrations decreased INa,cAMP response amplitude, suggesting that inhibitor-induced increase in native cAMP increased basal INa,cAMP and thus caused partial saturation of the current. The Hill coefficient estimated from the plot of injected cAMP to INa,cAMP response amplitude was close to 1.0. An equation modeling INa,cAMP incorporated terms for diffusion and degradation. In it, the first-order rate constant of phosphodiesterase activity was taken as the rate constant of the exponential decay of the INa,cAMP response. The stoichiometry of INa,cAMP activation was inferred from the Hill coefficient as 1 cAMP/channel. The equation closely fitted the INa,cAMP response and simulated changes in the waveform of the response induced by phosphodiesterase inhibition. With modifications to accommodate asymmetric INa,cAMP activation, the equation also simulated effects of eccentric electrode position. The simple reaction-diffusion model of the kinetics of INa,cAMP may provide a useful conceptual framework within which to investigate the modulation of INa,cAMP by neuromodulators, intracellular regulatory factors, and pharmacological agents.
1-Methyl-3-isobutylxanthine↗
Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis.
cAMP has previously been shown to promote cell survival in a variety of cell types, but the downstream signaling pathway(s) of this antiapoptotic effect is unclear. Thus the role of cAMP signaling through PKA and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs) in cAMP's antiapoptotic action was investigated in the present study. cAMP's protective effect against bile acid-, Fas ligand-, and TNF-alpha-induced apoptosis in rat hepatocytes was largely unaffected by the selective PKA inhibitor, Rp-8-(4-chlorophenylthio)-cAMP (Rp-cAMP). In contrast, a novel cAMP analog, 8-(4-chlorophenylthio)-2'-O-methyl (CPT-2-Me)-cAMP, which activated cAMP-GEFs in hepatocytes without activating PKA, protected hepatocytes against apoptosis induced by bile acids, Fas ligand, and TNF-alpha. The role of cAMP-GEF and PKA on activation of Akt, a kinase implicated in cAMP survival signaling, was investigated. Inhibition of PKA with RP-cAMP had no effect on cAMP-mediated Akt phosphorylation, whereas CPT-2-Me-cAMP, which did not activate PKA, induced phosphatidylinositol 3-kinase (PI3-kinase)-dependent activation of Akt. Pretreatment of hepatocytes with the PI3-kinase inhibitor, Ly-294002, prevented CPT-2-Me-cAMP's protective effect against bile acid and Fas ligand, but not TNF-alpha-mediated apoptosis. Glucagon, CPT-cAMP, and CPT-2-Me-cAMP all activated Rap 1, a downstream effector of cAMP-GEF. These results suggest that a PKA-independent cAMP/cAMP-GEF/Rap pathway exists in hepatocytes and that activation of cAMP-GEFs promotes Akt phosphorylation and hepatocyte survival. Thus a cAMP/cAMP-GEF/Rap/PI3-kinase/Akt signaling pathway may confer protection against bile acid- and Fas-induced apoptosis in hepatocytes.
Animals↗
Differential effects of two structurally related N6-substituted cAMP analogues on C6 glioma cells.
Membrane permeable derivatives of cAMP are widely used to investigate the role of cAMP in the regulation of cell growth and differentiation. To further investigate the molecular mechanisms, underlying the effects of cAMP analogues on growth control and differentiation, the concentration-dependent action of four structurally related cAMP analogues with substitutions at the N6-position in the adenine moiety, namely N6-benzyl-cAMP (Bn-cAMP), N6-benzoyl-cAMP (Bz-cAMP), N6-butyryl-cAMP (Bt-cAMP) and N6, O2'-cAMP (Bt2-cAMP), on C6 rat glioma cell proliferation was determined. The four analogues tested showed different specificities, and the order of growth inhibitory potency was: Bn-cAMP >> Bt-cAMP = Bt2-cAMP >> Bz-cAMP. Thus, although both derivatives have been described to equally bind and activate cAMP-dependent protein kinase (cAK) isozymes, Bn-cAMP most effectively inhibited C6 glioma cell proliferation with an IC50 of 25 microM, while Bz-cAMP was almost ineffective in C6 cells (IC50 >> 1000 microM). In vivo and in vitro studies using HPLC analysis, revealed that Bn-cAMP was subject to enzymatic degradation and that the metabolite Bn-adenosine (Bn-Ado) exerted growth inhibitory effects at a concentration even below 10 microM. Additionally, C6 glioma cells morphologically differentiated in the presence of Bn-cAMP (100 microM) and of Bn-Ado (10 microM), by extending long cellular processes. The growth inhibitory activity of Bn-Ado was not influenced, when dipyridamole, an inhibitor of adenosine uptake, was added to the incubation medium, indicating that adenosine action was mediated through a receptor-mediated mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Progesterone secretion by luteinizing human granulosa cells: a possible cAMP-dependent but PKA-independent mechanism involved in its regulation.
The corpus luteum formed after luteinization of follicular cells secretes progesterone under the control of luteinizing hormone (LH). Binding of LH to its G-protein-coupled receptor leads to the activation of the adenylate cyclase/ cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA) signalling pathway. The identification of a new class of cAMP-binding proteins termed 'guanine nucleotide exchange factors' (cAMP-GEFs) provides a means by which changes in cAMP could yield actions that are independent of PKA. Hence, in this study, we have explored the hypothesis that steroidogenesis in luteinizing cells is mediated in both a cAMP/PKA-dependent and cAMP-dependent, but PKA-independent, manner. Human granulosa cells were isolated from follicular aspirates of women undergoing assisted conception. Luteinizing human granulosa cells were cultured for up to 3 days in the presence of human (h)LH and the adenylate cyclase activator forskolin in the added presence or absence of increasing doses of the PKA inhibitors H89 (N-[2-(4-bromocinnamylamino)ethyl] 5-isoquinoline) and PKI (myristoylated protein kinase A inhibitor amide 14-22) or the cAMP antagonist, Rp-cAMP. Agonist-stimulated progesterone secretion was inhibited in a dose-dependent manner by the PKA inhibitors and the cAMP antagonist, with decreasing sensitivity as luteinization progressed. Pretreatment of granulosa cells for 4 h with human (h)LH reduced the effectiveness of H89 in inhibiting progesterone secretion. Under basal conditions, cAMP-GEFI expression increased progressively throughout culture, and this could be further enhanced when cells were incubated with increasing doses of LH and forskolin. Furthermore, incubation of cells in the presence of increasing concentrations of the novel cAMP-GEF-specific cAMP analogue, 8 CPT-2 ME-cAMP (8-(4-chloro-phenylthio)-2'-0-methyladenosine-3',5'-cyclic monophosphate), increased progesterone secretion in a dose-dependent manner. The results show that increases in cAMP generated by LH and forskolin, in addition to activating PKA, also induce increases in cAMP-GEFI protein expression in luteinizing human granulosa cells. In addition, activation of cAMP-GEFI results in increased progesterone secretion. Hence, increases in cAMP lead to the activation of PKA-dependent, as well as PKA-independent but cAMP-dependent (via cAMP-GEFI), signalling mechanisms. Since cAMP-GEFs have the capacity to activate the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB) signalling pathways, these may provide the potential mechanisms by which cAMP-dependent but PKA-independent progesterone synthesis is regulated.
Adenylyl Cyclases↗
cAMP cascade (PKA, Epac, adenylyl cyclase, Gi, and phosphodiesterases) regulates myelin phagocytosis mediated by complement receptor-3 and scavenger receptor-AI/II in microglia and macrophages.
The removal by phagocytosis of degenerated myelin is central for repair in Wallerian degeneration that follows traumatic injury to axons and in autoimmune demyelinating diseases (e.g., multiple sclerosis). We tested for roles played by the cAMP cascade in the regulation of myelin phagocytosis mediated by complement receptor-3 (CR3/MAC-1) and scavenger receptor-AI/II (SRAI/II) separately and combined in mouse microglia and macrophages. Components of the cAMP cascade tested are cAMP, adenylyl cyclase (AC), Gi, protein kinase A (PKA), exchange protein directly activated by cAMP (Epac), and phosphodiesterases (PDE). PKA inhibitors H-89 and PKI(14-22) amide inhibited phagocytosis at normal operating cAMP levels (i.e., those occurring in the absence of reagents that alter cAMP levels), suggesting activation of phagocytosis through PKA at normal cAMP levels. Phagocytosis was inhibited by reagents that elevate endogenous cAMP levels to above normal: Gi-inhibitor Pertussis toxin (PTX), AC activator Forskolin, and PDE inhibitors IBMX and Rolipram. Phagocytosis was inhibited also by cAMP analogues whose addition mimics abnormal elevations in endogenous cAMP levels: nonselective 8-bromo-cAMP, PKA-specific 6-Benz-cAMP, and Epac-specific 8-CPT-2'-O-Me-cAMP, suggesting that abnormal high cAMP levels inhibit phagocytosis through PKA and Epac. Altogether, observations suggest a dual role for cAMP and PKA in phagocytosis: activation at normal cAMP levels and inhibition at higher. Furthermore, a balance between Gi-controlled cAMP production by AC and cAMP degradation by PDE maintains normal operating cAMP levels that enable efficient phagocytosis.
Acetylcysteine↗
Diastereomers of adenosine 3',5'-monothionophosphate (cAMP[S]) antagonize the activation of cGMP-dependent protein kinase.
cGMP-dependent protein kinase contains four cGMP-binding sites which are homologous to the four cAMP-binding sites of cAMP-dependent protein kinase. The interaction of the diastereomers of adenosine 3',5'-thionophosphate, (PS)-cAMP[S] and (PR)-cAMP[S], with cGMP-dependent protein kinase has been studied. Autophosphorylation of cGMP-dependent protein kinase is stimulated by cAMP and (PS)-cAMP[S] with apparent KA values of 7 microM and 94 microM, respectively. cAMP-stimulated autophosphorylation is inhibited competitively by (PR)-cAMP[S] with a Ki value of 15 microM. The phosphorylation of the peptide substrate (Leu-Arg-Arg-Ala-Ser-Leu-Gly) is stimulated by cGMP (approx. KA 1 microM) and cAMP (approx. KA 98 microM) but neither by the (PR) nor (PS) stereoisomer of cAMP[S]. (PR)-cAMP[S] and (PS)-cAMP[S] inhibit competitively cAMP-or cGMP-stimulated phosphorylation of the peptide substrate with Ki values of 52 microM and 73 microM, respectively. (PS)-cAMP[S] stimulates the phosphorylation of the peptide substrate by an autophosphorylated enzyme. Binding of [3H]cGMP to cGMP-dependent protein kinase is inhibited by (PS)-cAMP[S] and (PR)-cAMP[S] with IC50 values of 200 microM and 15 microM, respectively. These results show that both diastereomers of cAMP[S] bind to cGMP-dependent protein kinase. (PR)-cAMP[S] has properties of a pure antagonist whereas (PS)-cAMP[S] has properties of a partial agonist. The results provide further evidence that autophosphorylation of the enzyme affects the interaction between the cGMP-binding sites and the catalytic center of the enzyme by facilitating the activation of the phosphotransferase reaction.
Binding, Competitive↗
Co-regulation of cAMP-activated Na+ current by Ca2+ in neurones of the mollusc Pleurobranchaea.
1. The cAMP-gated Na+ current (INa, cAMP) was studied in axotomized neurons of the pedal ganglion of the sea slug Pleurobranchaea. INa, cAMP responses were elicited by iontophoretic injection of cAMP and recorded in voltage clamp. 2. The current-voltage relation for INa, cAMP was flat between -90 and -50 mV, but declined steeply with depolarization from -50 to -30 mV. Depolarizing pulses also suppressed the INa, cAMP response, which recovered slowly over tens of seconds. 3. The inactivating effects of depolarization on the current were abolished both by blockade of Ca2+ current and intracellular injection of Ca2+ chelator. Thus, Ca2+ influx through voltage-dependent Ca2+ channels probably mediates inactivation of INa, cAMP within its normal physiological range of action. 4. Increasing intracellular cAMP levels antagonized the effects of Ca2+ influx on INa, cAMP. The mutual antagonism of the ions suggests that cAMP and Ca2+ act competitively in regulation of the INa, cAMP channel. 5. Measures of fractional inactivation of INa, cAMP provided evidence for the existence of an appreciable basal level of current, and hence cAMP, in the unstimulated neuron. Since INa, cAMP is a direct function of cAMP activity, measures of fractional inactivation permit quantification of cAMP levels in the living neuron. 6. Calcium inactivation of INa, cAMP completes a negative feedback loop that can contribute to endogenous burst activity. Over the burst cycle, depolarization and action potential activity driven by INa, cAMP would lead to Ca2+ influx, consequent inactivation of the inward current, and hyperpolarization. This mechanism of endogenous bursting resembles other in which the burst cycle has been found to be regulated by kinetics of Ca2+ influx and removal. However, INa, cAMP may vary in its Ca2+ sensitivity in different neurons and these variations may affect the functional expression of endogenous oscillatory activity.
Animals↗
Differential effects of cAMP and serotonin on membrane current, action-potential duration, and excitability in somata of pleural sensory neurons of Aplysia.
1. In somata of sensory neurons in the pleural ganglia of Aplysia californica, serotonin (5-HT) modulates at least three K+ currents: the S K+ current (IK,S), a slow component of the Ca2(+)-activated K+ current (IK,Ca), and the delayed or voltage-dependent K+ current (IK,V). The modulation of IK,S and the slow component of IK,Ca by 5-HT has been shown previously to be mediated via adenosine 3',5'-cyclic monophosphate (cAMP). To determine whether the modulation of IK,V by 5-HT also is mediated via cAMP, we used two-electrode voltage-clamp techniques to compare the modulation of membrane current by cAMP and 5-HT. 2. Current responses were elicited by brief (200 ms) voltage-clamp pulses before and after the bath application of analogues of cAMP. At all voltage-clamp potentials examined (-40-30 mV), analogues of cAMP reduced the amplitude of the current response. The properties of the cAMP-sensitive component of membrane current were revealed by computer subtraction of current responses elicited in the presence of the analogue of cAMP from current responses elicited before application of the analogue. The characteristics of the resulting cAMP difference current (IcAMP) suggested that cAMP modulated a component of membrane current that was relatively voltage independent, did not inactivate, and was active over a wide range of membrane potentials. In addition, the current-voltage (I-V) relationship of the cAMP difference current had a positive slope. These properties of the cAMP difference current were consistent with those of IK,S but did not indicate that IK,V was modulated by cAMP. 3. The cAMP-independent modulation of membrane current by 5-HT was examined by eliciting current responses first in the presence of an analogue of cAMP and again after the addition of 5-HT to the bath, which still contained the analogue. The presence of the analogue of cAMP occluded further modulation of IK,S by 5-HT. However, the analogue of cAMP did not occlude the modulation of IK,V by 5-HT. This cAMP-independent effect of 5-HT on membrane current was revealed by computer subtraction of current responses elicited in the presence of 5-HT from current responses elicited before the application of 5-HT (the analogue of cAMP was present throughout). The resulting cAMP-independent 5-HT difference current (I5-HT) was highly voltage dependent, had complex kinetics, and its I-V relationship had a negative slope at membrane potentials above 0 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
Action Potentials↗
Contact alters cAMP metabolism in aggregation-competent Dictyostelium amoebae.
cAMP and cell-cell contact are involved in the coordination of differentiation and morphogenesis in Dictyostelium discoideum. The experiments described in this paper establish a relationship between cAMP and cell-cell contact. Contact between Enterobacter aerogenes and aggregation-competent Dictyostelium amoebae and contact between Dictyostelium amoebae themselves results in the transient secretion of cAMP and an alteration in the amount of cAMP secreted in response to subsequent stimulation by cAMP, i.e., an alteration in magnitude of a cAMP relay response. The subsequent cAMP relay response can be enhanced or diminished depending upon the number of contacts formed and the concentration of cAMP present at the time of contact. Latex beads are capable of evoking cAMP secretion. However, the bead/amoebal contact is unable to alter the magnitude of a subsequent response to cAMP. This suggests that a nonspecific interaction via cell-cell contact elicits transient cAMP secretion in aggregation-competent Dictyostelium amoebae. The two responses to cell-cell contact are distinct from each other and distinct from the cAMP relay response. 1) The dose-response curves for the responses to Enterobacter contact are clearly different. 2) Contact with latex beads can elicit cAMP secretion but not alter the magnitude of a subsequent cAMP relay response. 3) The temperature dependences of the contact-induced responses and the cAMP relay response show that only the contact-induced cAMP secretion is inhibited at 12 and 15 degrees C, while only the cAMP relay response is inhibited at 28 degrees C. A 4-second application of cAMP at the time that contact is initiated enhances both contact-induced responses. Whether the relationship between these two developmental regulators is important for the regulation of Dictyostelium development has yet to be established.
Cell Communication↗
Cyclic AMP modulation of human B cell proliferative responses: role of cAMP-dependent protein kinases in enhancing B cell responses to phorbol diesters and ionomycin.
The ability of cyclic AMP (cAMP) to modulate human B cell proliferative responses and the possible role of cAMP-dependent kinases (PKA) in cAMP modulation of proliferative responses were investigated. The addition of dibutyl cAMP (Bt2 cAMP) or the cAMP-elevating agent forskolin to B cells stimulated by crosslinking surface immunoglobulins (sIg) resulted in a concentration-dependent inhibition of proliferative responses. By contrast, Bt2 cAMP or forskolin enhanced the proliferative responses of B cells after direct stimulation by phorbol myristate acetate (PMA) and the calcium ionophore ionomycin. The inhibition and enhancement of B cell proliferative responses by Bt2 cAMP were observed at different incubation intervals and were not due to temporal shifts of optimal responses. Also, Bt2 cAMP caused only small changes in B cell RNA synthesis compared to modulation of proliferative responses. Exposure of B cells to Bt2 cAMP rapidly activated PKA. Blocking Bt2 cAMP activation of PKA with the kinase inhibitor HA1004 prevented Bt2 cAMP enhancement of B cell responses after direct stimulation by PMA and ionomycin. In reciprocal experiments, the kinase inhibitor H7 resulted in some inhibition of PKC activation but did not inhibit Bt2 cAMP activation of PKA or Bt2 cAMP enhancement of proliferative responses. Other experiments demonstrated that B cells treated with Bt2 cAMP had selective increases in the de novo phosphorylations of two endogenous substrates which reflected PKA activation. Furthermore, concentrations of HA1004 or H8 which inhibited Bt2 cAMP enhancement of proliferative responses also inhibited PKA phosphorylations of these substrates whereas H7 did not. Thus, elevations of cAMP can enhance or inhibit human B cell proliferative responses to different stimuli and the activation of PKA is important for cAMP enhancement of certain responses.
Amino Acid Sequence↗