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PubMed2006

cAMP sensor Epac as a determinant of ATP-sensitive potassium channel activity in human pancreatic beta cells and rat INS-1 cells.

The Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic beta cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide-binding fold-1 (NBF-1) of the beta-cell sulphonylurea receptor-1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit beta-cell ATP-sensitive K+ channels (K(ATP) channels; a hetero-octomer of SUR1 and Kir6.2). If so, Epac-mediated inhibition of K(ATP) channels might explain prior reports that cAMP-elevating agents promote beta-cell depolarization, Ca2+ influx and insulin secretion. Here we report that Epac-selective cAMP analogues (2'-O-Me-cAMP; 8-pCPT-2'-O-Me-cAMP; 8-pMeOPT-2'-O-Me-cAMP), but not a cGMP analogue (2'-O-Me-cGMP), inhibit the function of K(ATP) channels in human beta cells and rat INS-1 insulin-secreting cells. Inhibition of K(ATP) channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6-Bnz-cAMP, a cAMP analogue that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac-selective cAMP analogues at K(ATP) channels are mimicked by a cAMP agonist (8-Bromoadenosine-3', 5'-cyclic monophosphorothioate, Sp-isomer, Sp-8-Br-cAMPS), but not a cAMP antagonist (8-Bromoadenosine-3', 5'-cyclic monophosphorothioate, Rp-isomer, Rp-8-Br-cAMPS), and are abrogated following transfection of INS-1 cells with a dominant-negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 coimmunoprecipitate with full-length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of K(ATP) channel activity in pancreatic beta cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

cAMP-independent effects of 8-(4-parachlorophenylthio)-cyclic AMP on spike duration and membrane currents in pleural sensory neurons of Aplysia.

1. The serotonergic modulation of pleural sensory neurons in Aplysia is mediated via two second messenger systems: the adenosine cyclic monophosphate/protein kinase A (cAMP/PKA) and diacylglycerol/protein kinase C systems. Often membrane permeable derivatives of cAMP, such as 8-(4-parachlorophenylthio)-cAMP (pcpt-cAMP), have been used to investigate the role of cAMP/PKA in modulating sensory neurons. In light of recent findings that pcpt-cAMP may have cAMP-independent actions, we have reexamined the effects of pcpt-cAMP on the action potential and membrane currents of the sensory neurons. 2. Although pcpt-cAMP (500 microM to 1 mM) and serotonin (5-HT; 10 microM) induced comparable measures of spike broadening (an average increase above baseline of 29 and 40%, respectively), the broadening produced by the two was qualitatively different. Serotonin-induced broadening developed slowly over 9-12 min, was most prominent during later phases of the spike repolarization, and reduced the spike afterhyperpolarization. In contrast, pcpt-cAMP-induced broadening developed rapidly, was rather uniform throughout the repolarization phase of the spike, delayed the peak of the action potential, and increased the afterhyperpolarization. 3. Preexposure of sensory neurons to 5-HT did not occlude further spike broaden by subsequent application of pcpt-cAMP. Indeed the effects of the two were additive. In addition, the effects of pcpt-cAMP were not mimicked by another analogue of cAMP, 8-bromo-cAMP. Interestingly, most of the effects of pcpt-cAMP on the action potential were mimicked by 8-(4-parachlorophenyl-thio)-guanosine cyclic monophosphate (pcpt-cGMP), but not by 8-bromo-cGMP. 4. During voltage-clamp pulses to 20 mV, pcpt-cAMP reduced the membrane current throughout the voltage-clamp pulse, which was qualitatively different from the modulation of the membrane current by 5-HT. In addition, the pcpt-cAMP-induced reduction in the membrane current at the beginning of the pulse was much greater than that induced by 5-HT. Moreover, preexposure of sensory neurons to 5-HT did not occlude further reduction in the membrane current by subsequent application of pcpt-cAMP. 5. These results suggest that pcpt-cAMP has some mechanisms of action that are not shared by 5-HT or cAMP but are shared by pcpt-cGMP. In addition, these findings provide further evidence that results obtained with this compound should be interpreted with caution.

Animals↗
PubMed1990

Regulation of the two forms of glycogen phosphorylase by cAMP and its analogs in Dictyostelium discoideum.

We have recently reported the existence of two forms of glycogen phosphorylase (1,4-alpha-D-glucan: orthophosphate-alpha-glucosyltransferase; EC 2.4.1.1) in Dictyostelium discoideum. During development the activity of the glycogen phosphorylase b form decreased as the activity of the a form increased. The total phosphorylase activity remained constant. The physical and kinetic properties of the Dictyostelium enzyme were similar to those of the mammalian enzyme. In mammals, cAMP regulates the conversion of the two forms by a cAMP dependent protein kinase (cAMPdPK). We report here that if cAMP is added to a single cell suspension, the Dictyostelium phosphorylase activity becomes independent of 5'AMP and a 104 kd peptide appears. We also show the effect of several cAMP analogs on the phosphorylase activity in these single-cell suspensions. The cAMP analogs were selected on the basis of their affinities for the membrane-bound cAMP receptor or the cytoplasmic cAMPdPK. We found that relatively low levels, 100 microM, of cAMP or 2'd-cAMP added to aggregation-competent cells in shaking culture caused a loss of phosphorylase b activity and the appearance of phosphorylase a activity. The analog, 2'd-cAMP, has a high affinity for the cAMP receptor but a low affinity for the cAMPdPK. Two other analogs, Bt2-cAMP and 8-Br-cAMP, which have low affinities for the cAMP receptor but high affinities for the cAMPdPK, required high levels (500 microM) for 'b' to 'a' conversion. cDNAs to three cAMP-regulated genes--PL3, D11, and D3--were used as controls in the above experiments. In order to determine if intracellular levels of cAMP were involved in the regulation of phosphorylase activity, both the phosphorylase and the PL3, D11 and D3 mRNA levels were examined in cells suspended in a glucose/albumin mixture--a medium in which adenylate cyclase is inhibited. Under these conditions, neither gene regulation nor a change in the phosphorylase b to a activity occurred in response to added extra cellular cAMP. The results suggest that an intracellular increase in cAMP is involved in the regulation of the two forms of glycogen phosphorylase in Dictyostelium.

Albumins↗
PubMed1986

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens).

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Escherichia coli cAMP receptor protein: evidence for three protein conformational states with different promoter binding affinities.

Cyclic AMP receptor protein (CRP) from Escherichia coli is assumed to exist in two states, namely, those represented by the free protein and that of the ligand-protein complex. To establish a quantitative structure-function relation between cAMP binding and the cAMP-induced conformational changes in the receptor, protein conformational change was quantitated as a function of cAMP concentration up to 10 mM. The protein conformation was monitored by four different methods at pH 7.8 and 23 degrees C, namely, rate of proteolytic digestion by subtilisin, rate of chemical modification of Cys-178, tryptophan fluorescence, and fluorescence of the extrinsic fluorescence probe 8-anilino-1-naphthalenesulfonic acid (ANS). Each of these techniques reveals a biphasic dependence of protein conformation on cAMP concentration. At low cAMP concentrations ranging from 0 to 200 microM, the rates of proteolytic digestion and that of Cys-178 modification increase, whereas the fluorescence intensity of the ANS-protein complex is quenched, and there is no change in the fluorescence intensity of the tryptophan residues in the protein. At higher cAMP concentrations, the rates of proteolytic and chemical modification of the protein decrease, while the fluorescence intensity of the ANS-protein complex is further quenched but there is an increase in the intensity of tryptophan fluorescence. These results show unequivocally that there are at least three conformational states of the protein. The association constants for the formation of CRP-cAMP and CRP-(cAMP)2 complexes derived from conformational studies are in good agreement with those determined by equilibrium dialysis, nonequilibrium dialysis, and ultrafiltration. Therefore, the simplest explanation would be that the protein exhibits three conformational states, free CRP and two cAMP-dependent states, which correspond to the CRP-cAMP and CRP-(cAMP)2 complexes. The binding properties of CRP-cAMP and CRP-(cAMP)2 to the lac promoter were studied by using the gel retardation technique. At a high concentration of cAMP which favors the formation of the CRP-(cAMP)2 complex, binding of the protein to DNA is decreased. This, together with conformational data, strongly suggests that only the CRP-cAMP complex is active in specific DNA binding whereas CRP and CRP-(cAMP)2 are not.

Binding Sites↗
PubMed1997

Activation of cGMP-stimulated phosphodiesterase by nitroprusside limits cAMP accumulation in human platelets: effects on platelet aggregation.

cGMP enhances cAMP accumulation in platelets via cGMP-inhibited phosphodiesterase (PDE3) [Maurice and Haslam (1990) Mol. Pharmacol. 37, 671-681]. However, cGMP might also limit cAMP accumulation by activating cGMP-stimulated phosphodiesterase (PDE2). We therefore evaluated the role of PDE2 in human platelets by using erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to inhibit this enzyme selectively. IC50 values for the inhibition of platelet PDE2 by EHNA, with 10 microM cAMP as substrate in the absence and in the presence of 1 microM cGMP, were 15 and 3 microM respectively. Changes in platelet cyclic [3H]nucleotides were measured after prelabelling with [3H]adenine and [3H]guanine. Nitroprusside (NP) caused concentration-dependent increases in [3H]cGMP and a biphasic increase in [3H]cAMP, which was maximal at 10 microM (49+/-6%) and smaller at 100 microM (32+/-6%) (means+/-S.E.). In the presence of EHNA (20 microM), which had no effects alone, NP caused much larger increases in platelet [3H]cAMP (125+/-14% at 100 microM). EHNA also enhanced [3H]cGMP accumulation at high NP concentrations. In accord with these results, EHNA markedly potentiated the inhibition of thrombin-induced platelet aggregation by NP. The roles of cAMP and cGMP in this effect were investigated by using 2', 5'-dideoxyadenosine to inhibit adenylate cyclase. This compound decreased the accumulation of [3H]cAMP but not that of [3H]cGMP, and diminished the inhibition of platelet aggregation by NP with EHNA. We conclude that much of the effect of NP with EHNA is mediated by cAMP. Lixazinone (1 microM), a selective inhibitor of PDE3, increased platelet [3H]cAMP by 177+/-15%. This increase in [3H]cAMP was markedly inhibited by NP; EHNA blocked this effect of NP. Parallel studies showed that NP suppressed the inhibition of platelet aggregation by lixazinone. EHNA enhanced the large increases in [3H]cAMP seen with 20 nM prostacyclin (PGI2), but had no effect with 1 nM PGI2. NP and 1 nM PGI2 acted synergistically to increase [3H]cAMP, an effect attributable to the inhibition of PDE3 by cGMP; EHNA greatly potentiated this synergism. In contrast, NP decreased the [3H]cAMP accumulation seen with 20 nM PGI2, an effect that was blocked by EHNA. The results show that, provided that cGMP is present, PDE2 plays a major role in the hydrolysis of low cAMP concentrations and restricts any increases in cAMP concentration and decreases in platelet aggregation caused by the inhibition of PDE3. At high cAMP, PDE2 plays the major role in cAMP breakdown, whether cGMP is present or not.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1995

cAMP-associated inhibition of phenobarbital-inducible cytochrome P450 gene expression in primary rat hepatocyte cultures.

The effects of elevated intracellular cyclic adenosine monophosphate (cAMP) in regulating phenobarbital (PB)-inducible gene expression in primary rat hepatocyte cultures were investigated. Cells were exposed to various concentrations (0.1-100 microM) of cAMP analogs and/or activators of intracellular cAMP-dependent pathways. Effects of these treatments were assessed either using a 1-h pulse prior to PB (100 microM) exposure or in conjunction with PB during a 24-h exposure period. PB-inducible responses were measured in hepatocytes by hybridization to cytochrome P450 (CYP) CYP2B1, CYP2B2, and CYP3A1 mRNAs. The cAMP analogs, 8-bromo-cAMP, 8-(4-chlorophenylthio)-cAMP, dibutyryl cAMP, and (Sp)-5,6-DCl-cBiMPS ((Sp)-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate), and the activators of adenylate cyclase, forskolin and glucagon, dramatically inhibited PB-mediated induction of CYP2B1 and CYP2B2 in a concentration-dependent manner. A similar inhibition of PB-induced CYP3A1 mRNA levels was effected by the cAMP analogs and glucagon. The phosphodiesterase inhibitors isobutylmethylxanthine and RO 201724 potentiated the cAMP responses. Increasing the concentration of PB (0.05-1.00 mM) did not alleviate the cAMP-mediated repression. A requirement for protein kinase A (PKA) was demonstrated by the use of (Sp)-cAMPS, a highly specific activator of PKA, whereas the inactive diastereoisomer, (Rp)-cAMPS, was ineffective in modulating PB induction. The response to cAMP was specific since elevated intracellular cAMP levels did not perturb beta-naphtholflavone-mediated induction of CYP1A1, CYP1A2, microsomal epoxide hydrolase, or dexamethasone-mediated induction of CYP3A1 gene expression. Nor did elevated intracellular cAMP modulate the liver-selective albumin gene expression levels. The results of the present study demonstrated striking inhibition of PB-mediated CYP gene induction by cAMP and PKA activators, indicating a negative regulatory role for the cAMP signal transduction pathway on PB gene induction.

Albumins↗
PubMed1991

Cyclic AMP antagonist Rp-cAMPS inhibits amylase exocytosis from saponin-permeabilized parotid acini.

Rp-cAMPS, the Rp-diastereomer of adenosine 3',5'-phosphorothioate, is often referred to as a cAMP antagonist, since it binds to the regulatory subunit of cAMP-dependent protein kinase without dissociation of free catalytic subunits. To evaluate the role of cAMP-dependent protein kinase in amylase exocytosis, we examined the effect of Rp-cAMPS on amylase release from rat parotid acini. Rp-cAMPS did not stimulate amylase release from saponin-permeabilized parotid acini, whereas its Sp-isomer strongly evoked amylase release. Rp-cAMPS dose-dependently inhibited amylase release stimulated by Sp-cAMPS. In the presence of Rp-cAMPS, the dose-response curve of Sp-cAMPS was shifted to the right. The inhibitory effect of Rp-cAMPS on isoproterenol-induced amylase release was not detected in intact acini, but was clearly observed in the permeabilized ones. Rp-cAMPS markedly inhibited protein phosphorylation evoked by Sp-cAMPS, indicating that Rp-cAMPS prevents the dissociation of cAMP-dependent protein kinase. These results, taken together with synergistic increase in amylase release by the combination of site-selective cAMP analogues [T. Takuma (1990) J. Biochem. 108, 99-102], suggest that cAMP-dependent protein kinase is involved in the exocytosis of amylase from parotid acini.

Amylases↗
PubMed1989

Shigellosis outbreaks at summer camps for the mentally retarded in New York State.

During 1987, four New York State summer camps for the mentally retarded and developmentally disabled experienced outbreaks of Shigella sonnei gastroenteritis. Cases occurred in 150 of 286 (attack rate (AR) = 52%), 167 of 295 (AR = 57%), and 25 of 114 (AR = 22%) persons in three camps, respectively; a fourth camp reported eight cases. Epidemiologic investigation suggested point-source foodborne outbreaks in two camps, while person-to-person spread appeared to predominate in the other two. Numerous secondary cases were identified among contacts outside the facilities in the second and third camps. To quantify the extent of the problem, the authors reviewed data on 77 infectious disease outbreaks (12 in camps for the handicapped and 65 in camps for the nonhandicapped) that occurred in the 12,484 registered camp sessions (316 for the handicapped and 12,168 for the nonhandicapped) held during the six-year period 1982-1987. Camps for the handicapped demonstrated approximately a seven times greater risk for all types of infectious disease outbreaks (AR = 38 outbreaks/1,000 camp sessions vs. five outbreaks/1,000 camp sessions; relative risk (RR) = 7.1, 95% confidence interval (CI) 3.9-13.0), including those of gastrointestinal etiology (RR = 8.6, 95% CI 4.4-16.8) and those due to Shigella (RR = infinity). Large camp size (RR = 2.3, 95% CI 1.3-4.1) and long duration of camp sessions (RR = 3.9, 95% CI 1.3-11.6) also increased the risk for outbreaks; however, this risk was predominantly in the camps for the nonhandicapped--other factors relating to personal hygiene and close camper-staff contact were probably more important in the camps for the handicapped. These outbreaks demonstrate the impact of shigellosis at summer camps for the mentally retarded and the need for early preventive action.

Adult↗
PubMed1991

Cell-cell contact mediates cAMP secretion in Dictyostelium discoideum.

Cyclic adenosine 3':5' monophosphate (cAMP) and cell-cell contact regulate developmental gene expression in Dictyostelium discoideum. Developing D. discoideum amoebae synthesize and secrete cAMP following the binding of cAMP to their surface cAMP receptor, a response called cAMP signaling. We have demonstrated two responses of developing D. discoideum amoebae to cell-cell contact. Cell-cell contact elicits cAMP secretion and alters the amount of cAMP secreted in a subsequent cAMP signaling response. Depending upon experimental conditions, bacterial-amoebal contact and amoebal-amoebal contact can enhance or diminish the amount of cAMP secreted during a subsequent cAMP signaling response. We have hypothesized that cell-cell contact regulates D. discoideum development by altering cellular and extracellular levels of cAMP. To begin testing this hypothesis, these responses were further characterized. The two responses to cell-cell contact are independent, i.e., they can each occur in the absence of the other. The responses to cell-cell contact also have unique temperature dependences when compared to each other, cAMP signaling, and phagocytosis. This suggests that these four responses have unique steps in their transduction mechanisms. The secretion of cAMP in response to cell-cell contact appears to be a non-specific response; contact between D. discoideum amoebae and Enterobacter aerogenes, latex beads, or other amoebae elicits cAMP secretion. Despite the apparent similarities of the effects of bacterial-amoebal and amoebal-amoebal contact on the cAMP signaling response, this contact-induced response appears to be specific. Latex beads addition does not alter the magnitude of a subsequent cAMP signaling response.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed2001

8-Cl-cAMP induces cell cycle-specific apoptosis in human cancer cells.

8-Cl-cyclic adenosine monophosphate (8-Cl-cAMP) has been known to induce growth inhibition and differentiation in a variety of cancer cells by differential modulation of protein kinase A isozymes. To understand the anticancer activity of 8-Cl-cAMP further, we investigated the effect of 8-Cl-cAMP on apoptosis in human cancer cells. Most of the tested human cancer cells exhibited apoptosis upon treatment with 8-Cl-cAMP, albeit with different sensitivity. Among them, SH-SY5Y neuroblastoma cells and HL60 leukemic cells showed the most extensive apoptosis. The effect of 8-Cl-cAMP was not reproduced by other cAMP analogues or cAMP-elevating agents, showing that the effect of 8-Cl-cAMP was not caused by simple activation of protein kinase A (PKA). However, competition experiments showed that the binding of 8-Cl-cAMP to the cAMP receptor was essential for the induction of apoptosis. After the treatment of 8-Cl-cAMP, cells initially accumulated at the S and G2/M phases of the cell cycle and then apoptosis began to occur among the population of cells at the S/G2/M cell cycle phases, indicating that the 8-Cl-cAMP-induced apoptosis is closely related to cell cycle control. In support of this assumption, 8-Cl-cAMP-induced apoptosis was blocked by concomitant treatment with mimosine, which blocks the cell cycle at early S phase. Interestingly, 8-Cl-cAMP did not induce apoptosis in primary cultured normal cells and non-transformed cell lines, showing that 8-Cl-cAMP-induced apoptosis is specific to transformed cells. Taken together, our results show that the induction of apoptosis is one of the mechanisms through which 8-Cl-cAMP exerts anticancer activity.

3T3 Cells↗
PubMed1996

Active site mutations define the pathway for the cooperative activation of cAMP-dependent protein kinase.

cAMP-dependent protein kinase (cAPK) is a heterotetramer containing two regulatory (R) and two catalytic (C) subunits. Each R-subunit contains two tandem cAMP-binding domains, and activation of cAPK is mediated by the cooperative, high affinity binding of cAMP to these two domains. Mutant R-subunits containing one intact high affinity cAMP-binding site and one defective site were used to define the pathway for activation and to delineate the unique roles that each cAMP-binding domain plays. Two mutations were introduced by replacing the essential Arg in each cAMP-binding site with Lys (R209K in Site A and R333K in Site B). Also, the double mutant (R209/333K) was constructed. Analysis of cAMP binding and dissociation and the apparent constants for holoenzyme activation and R- and C-subunit interaction, measured by analytical gel filtration and surface plasmon resonance, established the following: (1) For rR(R209K), occupancy of Site B is not sufficient to activate the holoenzyme; the low affinity Site A must also be occupied. In rR(R333K), Site A retains its high affinity for cAMP, but Site A cannot bind until the low affinity Site B is occupied. Thus, both mutants, for different reasons, have similar Ka's for activation that are approximately 20-fold higher than that of the wild-type holoenzyme. The double mutant with two defective sites is no worse than either single mutant. (2) Kinetic analysis of cAMP binding showed that the mutation in Site A or B abolishes high affinity cAMP binding to that site and slightly weakens the affinity of the adjacent site for cAMP. (3) In the presence of MgATP, both mutants rapidly form a stable holoenzyme even in the presence of cAMP in contrast to the wild-type R where holoenzyme forms slowly in vitro and requires dialysis. Regarding the mechanism of activation based on these and other mutants and from kinetic data, the following conclusions are reached: Site A provides the major contact site with the C-subunit; Site B is not essential for holoenzyme formation. Occupancy of Site A by cAMP mediates dissociation of the C-subunit. Site A is inaccessible to cAMP in the full length holoenzyme, while Site B is fully accessible. Access of cAMP to Site A is mediated by Site B. Thus Site B not only helps to shield Site A, it also provides the specific signal that "opens up" Site A. Finally, a nonfunctional Site A in the holoenzyme prevents stable binding of cAMP to Site B in the absence of subunit dissociation.

Amino Acid Sequence↗
PubMed1989

Induction of megakaryocytic differentiation and modulation of protein kinase gene expression by site-selective cAMP analogs in K-562 human leukemic cells.

Two classes (site 1- and site 2-selective) of cAMP analogs, which either alone or in combination demonstrate a preference for binding to type II rather than type I cAMP-dependent protein kinase isozyme, potently inhibit growth in a spectrum of human cancer cell lines in culture. Treatment of K-562 human leukemic cells for 3 days with 30 and 10 microM 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP) (site 1-selective) resulted in 60% and 20% growth inhibition, respectively (with over 90% viability). N6-Benzyl-cAMP (site 2-selective) (30 microM) treatment resulted in 20% growth inhibition by day 3. When 8-Cl-cAMP (10 microM) and N6-benzyl-cAMP (30 microM) were both added, growth was almost completely arrested. The growth inhibition was accompanied by megakaryocytic differentiation in K-562 cells. The untreated control cells expressed little or no detectable levels of glycoprotein IIb-IIIa surface antigen complex. 8-Cl-cAMP (30 microM) treatment for 3 days substantially increased the antigen expression, while N6-benzyl-cAMP caused little or no change in the antigen expression. When cells were treated with 8-Cl-cAMP in combination with N6-benzyl-cAMP, antigen expression was synergistically enhanced, and cells demonstrated megakaryocyte morphology. By Northern blotting, we examined the mRNA levels of the type I and type II protein kinase regulatory subunits (RI alpha and RII beta), the catalytic subunit, and c-myc during 8-Cl-cAMP treatment. The steady-state level of RII beta cAMP receptor mRNA sharply increased within 1 hr of treatment and remained elevated for 3 days, while that of the RI alpha receptor markedly decreased to below control level within 6 hr and remained low during treatment. However, 8-Cl-cAMP did not affect the mRNA level of the catalytic subunit. 8-Cl-cAMP treatment also brought about a rapid decrease in c-myc mRNA. Thus, differential regulation of cAMP receptor genes is an early event in cAMP-induced differentiation and growth control of K-562 leukemia cells.

Antigens, Surface↗
PubMed1989

Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity.

The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.

Adipose Tissue↗
PubMed1996

Role of cAMP in the regulation of hepatocytic autophagy.

To assess the role of cAMP in the regulation of autophagy, we examined the effects of cAMP analogues and cAMP-elevating agents on freshly isolated rat hepatocytes, using electroinjected [3H]raffinose as an autophagy probe. Glucagon was found to stimulate, inhibit or have no effect on autophagy, depending on the inclusion of metabolites like pyruvate (which caused ATP depletion and autophagy suppression) and amino acids (a complete mixture that antagonized pyruvate) in the incubation medium. Inhibition was also observed with theophylline, a cAMP-elevating inhibitor of cyclic nucleotide phosphodiesterases, and with the adenylyl cyclase activator deacetylforskolin. At low concentrations of deacetylforskolin, the inhibition could be abolished by amino acids. N6,2'-O-Dibutyryladenosine 3',5'-monophosphate (Bt2-cAMP) strongly inhibited both autophagic sequestration of [3H]raffinose and overall autophagic protein degradation; again, amino acids abolished the autophagy-inhibitory effect of low Bt2-cAMP concentrations. Several other cAMP analogues (8-thiomethyl-cAMP, N6-benzoyl-cAMP, (S)-5,6-dichloro-1-D-ribofuranosylbenzimidazole 3',5'-[thio]monophosphate, (S)-8-bromoadenosine 3',5'-[thio]monophosphate) inhibited autophagy as well. The effect of Bt2-cAMP was rapid, dose-dependent, reversible and did not require concomitant protein synthesis. Neither Bt2-cAMP nor deacetylforskolin reduced intracellular ATP levels or cell viability, ruling out inhibition of autophagy by non-specific cytotoxicity. The autophagy-inhibitory effect of Bt2-cAMP could be substantially antagonized (40-50%) by KT-5720, a specific inhibitor of the cAMP-dependent protein kinase A, and by the nonspecific protein kinase inhibitor K-252a. Somewhat surprisingly, KN-62 and KT-5926, allegedly specific inhibitors of Ca2+/calmodulin-dependent protein kinase II and myosin light chain kinase, respectively, were also Bt2-cAMP-antagonistic. These results suggest that cAMP regulates the early, sequestrational step of hepatocytic autophagy by a highly conditional, dual mechanism, inhibition being predominant under most conditions in freshly isolated hepatocytes, whereas stimulation reportedly predominates in vivo. The effect of cAMP is probably mediated by protein kinase A, but other protein kinases would appear to participate in the regulation of autophagic sequestration as well.

Animals↗
PubMed1983

cAMP-dependent protein kinase and protein phosphorylation in developing rat lung.

Protein kinase activity that is dependent on 3',5'-Cyclic adenosine monophosphate (cAMP-PK), [3H]cAMP binding, and cAMP-dependent protein phosphorylation were identified and partially characterized in cytosolic preparations of rat lung from day 18 of gestation to adulthood. Major cAMP-dependent phosphoproteins in lung preparations were compared to those in cytosol from purified Type II epithelial cells. Both Type I and Type II regulatory subunits of cAMP-PK were identified in fetal and adult lung. Inhibition of specific [3H]cAMP binding to lung cytosol (to the regulatory subunit of the cAMP-dependent protein kinase) followed the order of potency: cAMP greater than cGMP; adenosine, ADP, and ATP were inactive. Scatchard plots of saturation experiments with [3H]cAMP and lung cytosol were linear. Dissociation constant (KD) for cAMP binding was approximately 2-3 nM, and did not change significantly with age. In contrast, binding capacity varied significantly during development and age-related changes in binding capacity were associated with similar changes in cAMP-dependent histone kinase activity. Both [3H]cAMP binding and cAMP-dependent protein kinase activity decreased slightly before birth, reached maximal activity during the suckling period, and decreased in adulthood. cAMP enhanced histone kinase activity in rat lung cytosol at all ages studied, from day 18 of gestation to adulthood. cAMP also specifically enhanced phosphorylation of several endogenous cytosolic proteins that were identified by autoradiography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major proteins whose phosphorylation was selectively enhanced by cAMP or inhibited by protein kinase inhibitor were approximately Mr = 260,000, 240,000, 97,000, 56,000, 44,000, and 28,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

The effects of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on intracellular adenosine 3',5'-monophosphate (cAMP) levels and GH secretion in ovine and rat somatotrophs.

The mechanism of action of GH-releasing peptide-6 (GHRP-6) and GHRP-2 on GH release was investigated in ovine and rat pituitary cells in vitro. In partially purified sheep somatotrophs, GHRP-2 and GH-releasing factor (GRF) increased intracellular cyclic AMP (cAMP) concentrations and caused GH release in a dose-dependent manner; GHRP-6 did not increase cAMP levels. An additive effect of maximal doses of GRF and GHRP-2 was observed in both cAMP and GH levels whereas combined GHRP-6 and GHRP-2 at maximal doses produced an additive effect on GH release only. Pretreatment of the cells with MDL 12,330A, an adenylyl cyclase inhibitor, prevented cAMP accumulation and the subsequent release of GH that was caused by either GHRP-2 or GRF. The cAMP antagonist, Rp-cAMP also blocked GH release in response to GHRP-2 and GRF. The cAMP antagonist did not prevent the effect of GHRP-6 on GH secretion whereas MDL 12,330A partially reduced the effect. An antagonist for the GRF receptor, [Ac-Tyr1,D-Arg2]-GRF 1-29, significantly diminished the effect of GHRP-2 and GRF on cAMP accumulation and GH release, but did not affect GH release induced by GHRP-6. Somatostatin prevented cAMP accumulation and GH release responses to GHRP-2, GRF and GHRP-6. Ca2+ channel blockade did not affect the cAMP increase in response to GHRP-2 or GRF but totally prevented GH release in response to GHRP-2, GRF and GHRP-6. These results indicated that GHRP-2 acts on ovine pituitary somatotrophs to increase cAMP concentration in a manner similar to that of GRF; this occurs even during the blockade of Ca2+ influx. GHRP-6 caused GH release without an increase in intracellular cAMP levels. GH release in response to all three secretagogues was reduced by somatostatin and was dependent upon the influx of extracellular Ca2+. The additive effect of GHRP-2 and GRF or GHRP-6 suggested that the three peptides may act on different receptors. In rat pituitary cell cultures, GHRP-6 had no effect on cAMP levels, but potentiated the effect of GRF on cAMP accumulation. The synergistic effect of GRF and GHRP-6 on cAMP accumulation did not occur in sheep somatotrophs. Whereas GHRP-2 caused cAMP accumulation in sheep somatotrophs, it did not do so in rat pituitary cells. These data indicate species differences in the response of pituitary somatotrophs to the GHRPs and this is probably due to different subtypes of GHRP receptor in rat or sheep.

Animals↗
PubMed1996

cAMP-signaling pathway acts in selective synergism with glucose or tolbutamide to increase cytosolic Ca2+ in rat pancreatic beta-cells.

cAMP and the insulinotropic peptides that raise cAMP glucose-dependently increase the cytosolic free Ca2+ concentration ([Ca2+]i) in pancreatic beta-cells, which is tightly linked to the potentiation of glucose-induced insulin release. We examined whether cAMP increases [Ca2+]i in specific cooperation only with glucose or also with other insulin secretagogues that act through different mechanisms. [Ca2+]i in single rat pancreatic beta-cells was measured by dual-wavelength fura-2 microfluorometry. In the presence of a stimulatory concentration of glucose (8.3 mmol/l) and the moderate elevation in [Ca2+]i induced by it, forskolin, an activator of adenylyl cyclase, or dibutyryl cAMP produced a marked additional increase in [Ca2+]i but was ineffective at the basal 2.8 mmol/l glucose. These cAMP-elevating agents also potentiated the effect of tolbutamide on [Ca2+]i. The cAMP-induced increase in [Ca2+]i was completely and selectively inhibited by a blocker of cAMP-dependent protein kinase A (PKA), and by nitrendipine, a blocker of the L-type Ca2+ channel. However, in the presence of high KCl and the [Ca2+]i elevation induced by it, a rise in cAMP failed to further increase [Ca2+]i, whereas BAY K8644, an agonist of L-type Ca2+ channels, evoked an additional increase in [Ca2+]i. Under low Na+ conditions, the [Ca2+]i response to cAMP was observed in the majority of the cells. In the cells in which glucose at 4.5-5 mmol/l was inadequate to increase [Ca2+]i, the glucose together with a rise in cAMP often increased [Ca2+]i. Likewise, tolbutamide and a rise in cAMP acted in concert to increase [Ca2+]i. Thus, cAMP left-shifted the concentration-[Ca2+]i response relationship for glucose and tolbutamide. In conclusion, the cAMP-PKA pathway acts in selective synergism with glucose and tolbutamide to initiate [Ca2+]i signals in pancreatic beta- cells. cAMP appears to regulate beta-cell sensitivity to glucose and tolbutamide. In contrast, cAMP fails to cooperate with high KCl to increase [Ca2+]i. It is suggested that cAMP acts mainly on a site that is more proximal but functionally linked to the L-type Ca2+ channel, thereby finally increasing Ca2+ influx through this channel.

Animals↗