Export Of cAMP Research Articles

ABCC4 impacts megakaryopoiesis and protects megakaryocytes against 6-mercaptopurine induced cytotoxicity

The role of ABCC4, an ATP-binding cassette transporter, in the process of platelet formation, megakaryopoiesis, is unknown. Here, we show that ABCC4 is highly expressed in megakaryocytes (MKs). Mining of public genomic data (ATAC-seq and genome wide chromatin interactions, Hi-C) revealed that key megakaryopoiesis transcription factors (TFs) interacted with ABCC4 regulatory elements and likely accounted for high ABCC4 expression in MKs. Importantly these genomic interactions for ABCC4 ranked higher than for genes with known roles in megakaryopoiesis suggesting a role for ABCC4 in megakaryopoiesis. We then demonstrate that ABCC4 is required for optimal platelet formation as in vitro differentiation of fetal liver derived MKs from Abcc4-/- mice exhibited impaired proplatelet formation and polyploidization, features required for optimal megakaryopoiesis. Likewise, a human megakaryoblastic cell line, MEG-01 showed that acute ABCC4 inhibition markedly suppressed key processes in megakaryopoiesis and that these effects were related to reduced cAMP export and enhanced dissociation of a negative regulator of megakaryopoiesis, protein kinase A (PKA) from ABCC4. PKA activity concomitantly increased after ABCC4 inhibition which was coupled with significantly reduced GATA-1 expression, a TF needed for optimal megakaryopoiesis. Further, ABCC4 protected MKs from 6-mercaptopurine (6-MP) as Abcc4-/- mice show a profound reduction in MKs after 6-MP treatment. In total, our studies show that ABCC4 not only protects the MKs but is also required for maximal platelet production from MKs, suggesting modulation of ABCC4 function might be a potential therapeutic strategy to regulate platelet production.

Potentiation of long-acting \u03b22-agonist and glucocorticoid responses in human airway epithelial cells by modulation of intracellular cAMP

IntroductionOver 300 million people in the world live with asthma, resulting in 500,000 annual global deaths with future increases expected. It is estimated that around 50–80% of asthma exacerbations are due to viral infections. Currently, a combination of long-acting beta agonists (LABA) for bronchodilation and glucocorticoids (GCS) to control lung inflammation represent the dominant strategy for the management of asthma, however, it is still sub-optimal in 35–50% of moderate-severe asthmatics resulting in persistent lung inflammation, impairment of lung function, and risk of mortality. Mechanistically, LABA/GCS combination therapy results in synergistic efficacy mediated by intracellular cyclic adenosine monophosphate (cAMP).HypothesisIncreasing intracellular cAMP during LABA/GCS combination therapy via inhibiting phosphodiesterase 4 (PDE4) and/or blocking the export of cAMP by ATP Binding Cassette Transporter C4 (ABCC4), will potentiate anti-inflammatory responses of mainstay LABA/GCS therapy.MethodsExpression and localization experiments were performed using in situ hybridization and immunohistochemistry in human lung tissue from healthy subjects, while confirmatory transcript and protein expression analyses were performed in primary human airway epithelial cells and cell lines. Intervention experiments were performed on the human airway epithelial cell line, HBEC-6KT, by pre-treatment with combinations of LABA/GCS with PDE4 and/or ABCC4 inhibitors followed by Poly I:C or imiquimod challenge as a model for viral stimuli. Cytokine readouts for IL-6, IL-8, CXCL10/IP-10, and CCL5/RANTES were quantified by ELISA.ResultsUsing archived human lung and human airway epithelial cells, ABCC4 gene and protein expression were confirmed in vitro and in situ. LABA/GCS attenuation of Poly I:C or imiquimod-induced IL-6 and IL-8 were potentiated with ABCC4 and PDE4 inhibition, which was greater when ABCC4 and PDE4 inhibition was combined. Modulation of cAMP levels had no impact on LABA/GCS modulation of Poly I:C-induced CXCL10/IP-10 or CCL5/RANTES.ConclusionModulation of intracellular cAMP levels by PDE4 or ABCC4 inhibition potentiates LABA/GCS efficacy in human airway epithelial cells challenged with viral stimuli. The data suggest further exploration of the value of adding cAMP modulators to mainstay LABA/GCS therapy in asthma for potentiated anti-inflammatory efficacy.

CAMP export by the fission yeast Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe produces a cAMP signal in response to glucose detection. Previous characterization of this signaling focused on intracellular levels of cAMP. Here, we find that the cAMP is secreted into the medium almost immediately. This is not due to PKA activation as might have been expected. In addition, a strain that is highly deficient in drug efflux shows only a modest reduction in the secretion of cAMP to the growth medium. These observations reveal a previously unappreciated aspect of cAMP metabolism in an important model organism, leading to new questions regarding the mechanism and benefit of cAMP export in S. pombe.

Determining the Effect of MRP4 Knockout on Pigment Granule Position in Mouse RPE

In the retinal pigment epithelium (RPE), pigment granules of wild type mice aggregate in darkness and disperse in light. In fish, pigment aggregation involves transport of cAMP into the RPE in vitro. We speculate that multidrug resistance protein 4 (MRP4) is responsible in vivo for the export of cAMP from the photoreceptors into the sub‐retinal space, making it available for uptake into the RPE in both fish and mice. We hypothesize if the MRP4 gene is knocked out in mice, we will not see a significant difference in pigment position between light and dark adapted mice. To test the hypothesis, we have quantified pigment position in RPE from wild type mice and knockout mice by estimating pigment granule density in the basal part of the cells examined by TEM. Bruch's membrane was used as a marker to determine the location of the base of the RPE. To measure pigment position, 2 μm × 2 μm boxes were aligned along Bruch's membrane, the pigment granules within each box were counted, and the average pigment density was calculated. The average pigment granule density for light adapted wildtype (LAWT; n = 3 mice) was 0.7 pigment granules/μm2 (± 0.2 pigment granules/μm2) and for dark adapted wildtype (DAWT; n = 3 mice) was 1.4 pigment granules/μm2 (± 0.3 pigment granules/μm2); pigment density was statistically significantly different (P < 0.02) between LAWT and DAWT. Preliminary data suggests the average pigment granule density for dark adapted MRP4 knockout is 0.57 pigment granules/μm2 (DAKO; n = 2). Our results with WT indicate basal pigment density increases in the dark, consistent with pigment aggregation and suggests dark adaptive pigment aggregation may be impaired in knockout mice.Support or Funding InformationThis project was supported by an Undergraduate Research Fellowship to AP and funds from the Office of the Provost awarded to DG in recognition of her service as Presidential Fellow. DEI received salary support from HSI STEM Program (84.031c) Award #P031C160035.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Comparative transcriptomic and proteomic analysis of Arthrobacter sp. CGMCC 3584 responding to dissolved oxygen for cAMP production

Arthrobacter sp. CGMCC 3584 is able to produce high yields of extracellular cyclic adenosine monophosphate (cAMP), which plays a vital role in the field of treatment of disease and animal food, during aerobic fermentation. However, the molecular basis of cAMP production in Arthrobacter species is rarely explored. Here, for the first time, we report the comparative transcriptomic and proteomic study of Arthrobacter cells to elucidate the higher productivity of cAMP under high oxygen supply. We finally obtained 14.1% and 19.3% of the Arthrobacter genome genes which were up-regulated and down-regulated notably, respectively, with high oxygen supply, and identified 54 differently expressed proteins. Our results revealed that high oxygen supply had two major effects on metabolism: inhibition of glycolysis, pyruvate metabolism, nitrogen metabolism, and amino acid metabolism (histidine, branched-chain amino acids and glutamate metabolism); enhancement of the tricarboxylic acid cycle and purine metabolism. We also found that regulation of adenylate cyclase and phosphodiesterase was not significant under high oxygen supply, suggesting efficient cAMP export might be important in cAMP production. These findings may contribute to further understanding of capacities of Arthrobacter species and would be highly useful in genetic regulation for desirable production.

Growth on glucose decreases cAMP-CRP activity while paradoxically increasing intracellular cAMP in the light-organ symbiont Vibrio fischeri.

Proteobacteria often co-ordinate responses to carbon sources using CRP and the second messenger cyclic 3', 5'-AMP (cAMP), which combine to control transcription of genes during growth on non-glucose substrates as part of the catabolite-repression response. Here we show that cAMP-CRP is active and important in Vibrio fischeri during colonization of its host squid Euprymna scolopes. Moreover, consistent with a classical role in catabolite repression, a cAMP-CRP-dependent reporter showed lower activity in cells grown in media amended with glucose rather than glycerol. Surprisingly though, intracellular cAMP levels were higher in glucose-grown cells. Mutant analyses were consistent with predictions that CyaA was responsible for cAMP generation, that the EIIA(Glc) component of glucose transport could enhance cAMP production and that the phophodiesterases CpdA and CpdP consumed intracellular and extracellular cAMP respectively. However, the observation of lower cAMP levels in glycerol-grown cells seemed best explained by changes in cAMP export, via an unknown mechanism. Our data also indicated that cAMP-CRP activity decreased during growth on glucose independently of crp's native transcriptional regulation or cAMP levels. We speculate that some unknown mechanism, perhaps carbon-source-dependent post-translational modulation of CRP, may help control cAMP-CRP activity in V.fischeri.

The ABC transporter, AbcB3, mediates cAMP export in D. discoideum development

Extracellular cAMP functions as a primary ligand for cell surface cAMP receptors throughout Dictyostelium discoideum development, controlling chemotaxis and morphogenesis. The developmental consequences of cAMP signaling and the metabolism of cAMP have been studied in great detail, but it has been unclear how cells export cAMP across the plasma membrane. Here we show pharmacologically and genetically that ABC transporters mediate cAMP export. Using an evolutionary-developmental biology approach, we identified several candidate abc genes and characterized one of them, abcB3, in more detail. Genetic and biochemical evidence suggest that AbcB3 is a component of the cAMP export mechanism in D. discoideum development.

Cardiac myocyte-secreted cAMP exerts paracrine action via adenosine receptor activation.

Acute stimulation of cardiac β-adrenoceptors is crucial to increasing cardiac function under stress; however, sustained β-adrenergic stimulation has been implicated in pathological myocardial remodeling and heart failure. Here, we have demonstrated that export of cAMP from cardiac myocytes is an intrinsic cardioprotective mechanism in response to cardiac stress. We report that infusion of cAMP into mice averted myocardial hypertrophy and fibrosis in a disease model of cardiac pressure overload. The protective effect of exogenous cAMP required adenosine receptor signaling. This observation led to the identification of a potent paracrine mechanism that is dependent on secreted cAMP. Specifically, FRET-based imaging of cAMP formation in primary cells and in myocardial tissue from murine hearts revealed that cardiomyocytes depend on the transporter ABCC4 to export cAMP as an extracellular signal. Extracellular cAMP, through its metabolite adenosine, reduced cardiomyocyte cAMP formation and hypertrophy by activating A1 adenosine receptors while delivering an antifibrotic signal to cardiac fibroblasts by A2 adenosine receptor activation. Together, our data reveal a paracrine role for secreted cAMP in intercellular signaling in the myocardium, and we postulate that secreted cAMP may also constitute an important signal in other tissues.

A cAMP-independent carbohydrate-driven mechanism inhibits tnaA expression and TnaA enzyme activity in Escherichia coli.

When Escherichia coli is grown in a medium lacking glucose or another preferred carbohydrate, the concentration of cAMP-cAMP receptor protein (cAMP-CRP) increases, and this latter complex regulates the expression of more than 180 genes. To respond rapidly to changes in carbohydrate availability, E. coli must maintain a suitable intracellular concentration of cAMP by either exporting or degrading excess cAMP. Currently, cAMP export via the TolC protein is thought to be more efficient at reducing these levels than is CpdA-mediated degradation of cAMP. Here, we compared the contributions of TolC and CpdA by measuring the expression of cAMP-regulated genes that encode tryptophanase (TnaA) and β-galactosidase. In the presence of exogenous cAMP, a tolC mutant produced intermediate levels of these enzymes, suggesting that cAMP levels were held in check by CpdA. Conversely, a cpdA mutant produced much higher amounts of these enzymes, indicating that CpdA was more efficient than TolC at reducing cAMP levels. Surprisingly, expression of the tnaA gene halted rapidly when glucose was added to cells lacking both TolC and CpdA, even though under these conditions cAMP could not be removed by either pathway and tnaA expression should have remained high. This result suggests the existence of an additional mechanism that eliminates intracellular cAMP or terminates expression of some cAMP-CRP-regulated genes. In addition, adding glucose and other carbohydrates rapidly inhibited the function of pre-formed TnaA, indicating that TnaA is regulated by a previously unknown carbohydrate-dependent post-translational mechanism.

Escherichia coli Exports Cyclic AMP via TolC

In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

Efflux of cAMP in the exocrine pancreas: Role of secretin and atrial natriuretic factor (ANF)

We previously reported that ANF dose‐dependently reduces secretin secretory response and intracellular signaling in the exocrine pancreas through NPR‐C receptors coupled to the PLC/PKC pathway. In this study we evaluated if cAMP efflux contributed to ANF response in isolated pancreatic acini. Results showed that ANF favored the efflux of cAMP formed by secretin. The export of cAMP was also observed with secretin alone but not with isoproterenol or amthamine. Evoked‐cAMP efflux was blocked by probenecid, suggesting that multidrug resistant proteins (MRPs) were involved. RT‐PCR analysis revealed that MRP‐4 but not MRP‐5 (transport cyclic nucleotides) was expressed in the pancreas. Time course studies in the presence of phosphodiesterase (PDE) activity showed that tissue cAMP increased peaking at 3 min onset secretin stimulation and declining thereafter. With ANF, secretin‐induced cAMP augmented reaching a peak at 1 min after which it rapidly decreased. Extracellular cAMP accumulation occurred earlier and persisted throughout the experiment whereas with secretin alone it was delayed. Similar findings were observed in studies with PDE inhibition, except that cAMP content was higher and its appearance delayed in tissues and the media. Present findings suggest that cAMP export is likely mediated by MRP‐4 and further it may contribute to ANF‐induced desensitization of secretin response in the exocrine pancreas.(CONICET PIP5929/ UBACYT B079)

Receptor (MT(1)) mediated influence of melatonin on cAMP concentration and insulin secretion of rat insulinoma cells INS-1.

Recent functional, autoradiographic, and molecular investigations have shown that the pineal secretory product melatonin reduces the forskolin-stimulated insulin secretion from isolated pancreatic islets of neonate rats. Autoradiographic and binding studies as well as reverse transcriptase-polymerase chain reaction (RT-PCR) experiments proved that these effects are mediated through specific, high-affinity pertussis-toxin-sensitive Gi-protein-coupled MT(1) receptors and subsequent inhibition of the adenylyl cyclase/cyclic adenosine monophosphate (cAMP) system. This hypothesis was proved by blocking the intracellular signal transduction pathway using the non-hydrolyzable guanosine triphosphate analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) or the competitive melatonin receptor antagonist luzindole. Both GTPgammaS and luzindole diminished the melatonin effect. We have published these prior results elsewhere. So far, however, no information is available on both whether the MT1 receptors are located on the beta-cells and whether the consecutive functional reactions are based on a direct influence of melatonin on the insulin producing beta-cells. In order to examine this question, we used a glucose responsive insulin producing insulinoma cell line INS-1 isolated from rats. Comparable with the results of islets the competitive receptor antagonist luzindole diminished the insulin-decreasing effect of melatonin. In addition, our RT-PCR experiments, using specific primers for the rat melatonin receptor MT(1) showed that this melatonin receptor mRNA is also expressed in the INS-1 cells. Furthermore we radioimmunologically analyzed the forskolin-stimulated cAMP concentration in the superfusate. Similar to insulin secretion, the cAMP concentration was significantly reduced by melatonin. Following the hypothesis that cAMP is actively secreted from INS-1 cells by an energy-dependent mechanism based on either a OAT1/ROAT1 like anion exchanger or MDR-like transport systems, we used probenecid (p-[dipropylsulfamoyl] benzoic acid), a known inhibitor of cAMP extrusion. Probenecid blocks the export of cAMP by acting on transport mechanisms which are as yet not completely understood. Consistently, insulin secretion was increased and cAMP concentration diminished. The application of the phosphodiesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) caused a marked rise of insulin secretion as well as cAMP concentration in the perifusate. From these data we conclude that the MT1 receptor is located on the INS-1 cell and therefore in general on pancreatic beta-cells.

Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues.

Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the MRP subfamily, which in turn is a member of the superfamily of ATP-binding-cassette (ABC) transporters. Within the MRP subfamily, ABCC4,ABCC5 (MRP5), ABCC11 (MRP8) and ABCC12 (MRP9) have similar predicted membrane topologies. All lack the additional transmembrane domain, TMD(0), which is present in the other MRPs. Using cells stably overexpressing ABCC4, this study shows that ABCC4 exports GSH. ABCC4 also facilitates the efflux of cAMP. Depletion of intracellular GSH with DL-buthionine-(S,R)-sulphoximine led to decreased export of cAMP and a corresponding increase in intracellular cAMP was observed. ABCC4 also mediates resistance to purine analogues 9-(2-phosphonylmethoxyethyl)-adenine and 6-thioguanine. This resistance can be reversed by the presence of DL-buthionine-(S,R)-sulphoximine. We conclude that as well as nucleotide and nucleoside analogues, ABCC4 can mediate the export of GSH. In addition, GSH plays an important role in the function of ABCC4. Depletion of intracellular GSH adversely affects the export of cAMP by ABCC4. Resistance to nucleoside analogues is also adversely affected by depletion of cellular GSH.

Role of glutathione in the multidrug resistance protein 4 (MRP4/ABCC4)-mediated efflux of cAMP and resistance to purine analogues

Multidrug resistance protein 4 (MRP4/ABCC4) is a member of the MRP subfamily, which in turn is a member of the superfamily of ATP-binding-cassette (ABC) transporters. Within the MRP subfamily, ABCC4, ABCC5 (MRP5), ABCC11 (MRP8) and ABCC12 (MRP9) have similar predicted membrane topologies. All lack the additional transmembrane domain, TMD0, which is present in the other MRPs. Using cells stably overexpressing ABCC4, this study shows that ABCC4 exports GSH. ABCC4 also facilitates the efflux of cAMP. Depletion of intracellular GSH with dl-buthionine-(S,R)-sulphoximine led to decreased export of cAMP and a corresponding increase in intracellular cAMP was observed. ABCC4 also mediates resistance to purine analogues 9-(2-phosphonylmethoxyethyl)-adenine and 6-thioguanine. This resistance can be reversed by the presence of dl-buthionine-(S,R)-sulphoximine. We conclude that as well as nucleotide and nucleoside analogues, ABCC4 can mediate the export of GSH. In addition, GSH plays an important role in the function of ABCC4. Depletion of intracellular GSH adversely affects the export of cAMP by ABCC4. Resistance to nucleoside analogues is also adversely affected by depletion of cellular GSH.

Cyclic Adenosine 3‘:5‘-Monophosphate-Dependent Protein Kinase on the External Surface of LS-174T Human Colon Carcinoma Cells

The analysis of purified plasma membranes and the surface of intact cells revealed the presence of cyclic adenosine 3':5'-monophosphate-(cAMP) dependent protein kinase (PKA) on the external surface of LS-174T human colon carcinoma cells. Photoaffinity labeling of intact cells at confluence with 8-azido-[32P]cAMP identified the cAMP-binding proteins on the surface. Immunoprecipitation identified the photoaffinity-labeled cAMP-binding proteins as the RIIalpha regulatory subunit of PKA. During the logarithmic stage of growth, both the RIalpha and RIIalpha subunits of PKA were localized on the cell surface. Intact LS-174T cells catalyzed the phosphorylation of Kemptide in a cAMP-dependent manner; upon substitution of cAMP in the medium with 8-chloroadenosine, which did not compete with cAMP for the binding on intact cells, the ecto-PKA was no longer activated. The specific inhibitory protein for PKA, PKI, abolished the stimulation of phosphorylation by cAMP. Forskolin, which elevates intracellular levels of cAMP, activated ecto-PKA. Moreover, probenecid, which blocks the export of cAMP, inhibited the forskolin-mediated activation of ecto-PKA. These results demonstrate that LS-174T colon carcinoma cells possess an ecto-PKA on the external surface. This ecto-PKA is similar, if not identical, to the soluble intracellular PKA.

Epidermal growth factor potentiates cyclic AMP accumulation in A-431 cells.

Epidermal growth factor (EGF) treatment of A-431 cells potentiates up to 5-fold the intracellular cyclic AMP (cAMP) accumulation induced by isoproterenol, cholera toxin, forskolin, or 3-isobutyl-1-methylxanthine (IBMX). EGF potentiates cAMP accumulation in several epithelial cell lines which overexpress the EGF receptor including A-431 cells, HSC-1 cells, and MDA-468 cells, and in the A-431-29S clone which expresses a normal complement of EGF receptors. Although EGF potentiates cAMP accumulation, EGF by itself does not measurably alter the basal level of cAMP. EGF rapidly enhances cAMP accumulation (within 1 to 3 min) in A-431 cells treated with these cAMP-elevating agents. EGF potentiation of cAMP accumulation does not reflect enhancement of beta-adrenergic receptor activation and is not a consequence of intracellular cAMP elevation or the concomitant activation of cAMP-dependent protein kinase. Since EGF potentiates accumulation of both intracellular and extracellular cAMP in isoproterenol-treated A-431 cells, EGF does not potentiate intracellular cAMP accumulation by inhibition of cAMP export. EGF potentiation of cAMP accumulation is pertussis toxin-insensitive and does not result from EGF inhibition of cAMP degradation in A-431 cells. These results demonstrate that EGF transmembrane signaling includes an interaction with a component of the adenylate cyclase system and that this interaction stimulates cAMP synthesis resulting in enhancement of cAMP accumulation.

Evidence for Ecto-Protein Kinase Activity That Phosphorylates Kemptide in a Cyclic AMP-dependent Mode

The heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) is a synthetic construct of a substrate for cAMP-dependent protein kinase (PK). In this work we show that Kemptide has all the properties of a cytophilic substrate, i.e. it is a molecule preserving cell membrane intactness when added to cultured cells. Kemptide thus satisfies the prerequisites for employment in assays for cell surface-located ecto-PK activity. Different types of intact cells catalyze the phosphorylation of Kemptide in the presence of extracellular ATP and cAMP with Km values of 3-4 microM for Kemptide. Kemptide phosphorylation was influenced by PKI, the inhibitory protein specific for cAMP-PK. The results of comparative experiments with intact cells and with cell extracts demonstrate the ectoenzyme nature of this cAMP-PK. Further, the possibility was ruled out of a transfer of enzyme activity from damaged cells to the surface of intact cells. The anchorage of the surface cAMP-PK activity to the plasma membrane appears to be relatively stable since (i) cell supernatants, obtained after preincubation of intact cells with cAMP or Kemptide, did not show Kemptide phosphorylation, and (ii) the cAMP-dependent PK activity remained with cells even after five consecutive washes with cAMP or Kemptide. This is in contrast to the ecto-cAMP-independent phosvitin/casein type PK (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025) which is released from intact cells through the addition of substrate. Data are presented which show that both ectokinase activities are exhibited independently. In conjunction with published evidence for an active export of cAMP from cells as well as for the appearance of extracellular ATP the demonstration of an ecto-cAMP-PK further supports the potential of PK for intercellular regulation. The potential of ecto-cAMP-PK is demonstrated by its ability to phosphorylate biologically active forms of atrial natriuretic peptide, the atrial natriuretic peptide, which possesses the specific sequence for a cAMP-PK-catalyzed phosphorylation.

8] cAMP export and its regulation by prostaglandin A1

This chapter examines cAMP export and its regulation by prostaglandin A1. The main requirement for studying export is a method for the rapid separation of the intracellular and extracellular spaces, with collection of both, if possible. Export of cAMP is remarkably sensitive to temperature, occurring only slightly, if at all, below 22° in avian red cells and greatly inhibited by decreased temperatures in S49 lymphoma cells and mammalian reticulocytes. Thus, one may “freeze” the level of exported cAMP by the simple expedient of cooling or by dilution with cold buffer, a handy maneuver in many experimental protocols. Despite the simplicity of these comments and procedures and the relative ease of demonstrating efflux of cellular cAMP, readers should realize that the study of solute export is not subject to rigorous analysis because the exact intracellular activity or concentration of the solute being transported is not known or readily controlled. The export of cAMP seems limited by the cell's ability to produce and accumulate the solute and properties such as saturability are not directly demonstrable.

Phosphoenolpyruvate:sugar phosphotransferase system-mediated regulation of carbohydrate metabolism in Salmonella typhimurium.

The crr mutation was shown to affect the phosphoenolpyruvate:sugar phosphotransferase system-mediated transient repression of the lac operon, intracellular cAMP levels, and sensitivity to inducer exclusion. Our results indicate that the presumed crr gene product, factor IIIGlc, plays a direct role in the regulation of inducer exclusion. We propose a mechanism in which inducer exclusion depends on both the level and state of phosphorylation of factor IIIGlc and the level of an inducer exclusion-sensitive transport system. The results of studies on the sensitivity to inducer exclusion of glycerol and maltose in cultures induced for short periods of time on these substrates (resulting in varying degrees of activity of the respective transport systems) support this model of inducer exclusion. Previously, the crp*-771 mutation has been shown to result in an altered cAMP receptor protein, which has a changed affinity for cAMP, and to affect the sensitivity for inducer exclusion of glycerol. Changes in other functions of the altered cAMP receptor protein were indicated by our results; these changes were in the roles of this protein in (i) the cAMP-dependent initiation of transcription of the lac operon and (ii) the regulation of intracellular cAMP levels and the export of cAMP. We propose that the crp*-771 mutation has an indirect effect in relieving inducer exclusion in repressed or hypersensitive strains, in which the crp*-771 mutation allows the synthesis of inducer exclusion-sensitive transport systems to higher levels than the levels found in strains containing wild-type cAMP receptor protein.