Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “CAMPS”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 685 records · Page 38Linked to original sources
PubMed1995

8-Chloro-cAMP induces apoptotic cell death in a human mammary carcinoma cell (MCF-7) line.

8-Cl-cAMP and 8-NH2-cAMP induced MCF-7 cell death. The type(s) of cell death were studied in more detail and compared with the cell death type (apoptosis) induced by okadaic acid, an inhibitor of serine/threonine phosphatases. By morphological criteria dying cells showed loss of cell-cell interactions and microvilli, condensation of nuclear chromatin and segregation of cytoplasmic organelles. By in situ nick end-labelling, using digoxigenin-conjugated dUTP as probe, a large fraction of 8-Cl-cAMP, 8-NH2-cAMP and 8-Cl-adenosine-exposed cells stained positively in the advanced stages of death. In the early phase of chromatin condensation the cells stained negatively. Specific (internucleosomal) DNA fragmentation was not observed. The MCF-7 cell death induced by 8-Cl-cAMP and 8-NH2-cAMP was not mediated by activation of the cAMP kinase since more stable cAMP analogues (8-CPT-cAMP and N6-benzoyl-cAMP) or forskolin failed to induce death. Furthermore, 8-Cl-cAMP action was counteracted by adenosine deaminase and 3-isobutyl-1-methylxanthine, and mimicked by 8-Cl-adenosine, a major metabolite of 8-Cl-cAMP. It is concluded that 8-Cl- and 8-NH2-cAMP can induce morphological and biochemical effects resembling apoptotic cell death in MCF-7 cells through their conversion into potent cytotoxic metabolite(s).

1-Methyl-3-isobutylxanthine↗
PubMed2001

cAMP stimulation of vasopressin and oxytocin release and regulation of vasopressin mRNA stability: role of auto-facilitation.

The effects of cycloheximide and actinomycin on 8-bromo-cAMP (8-Br-cAMP) stimulated vasopressin and oxytocin release from the posterior pituitary and vasopressin mRNA content of the supraoptic nucleus were studied with perifused explants of the hypothalamo-neurohypophyseal system. 8-Br-cAMP stimulated vasopressin and oxytocin release from the explant for up to 6 h. Inhibition of protein synthesis by cycloheximide completely suppressed the response to 8-Br-cAMP. When gene transcription was inhibited by actinomycin, vasopressin release was stimulated by 8-Br-cAMP for approximately 2 h, but the response was not sustained. Vasopressin mRNA content was not changed by 8-Br-cAMP in the absence or presence of cycloheximide, but it was significantly decreased by simultaneous exposure to 8-Br-cAMP and actinomycin. Actinomycin alone did not change vasopressin mRNA content. Since other studies have demonstrated that cAMP stimulates vasopressin gene transcription, and since vasopressin mRNA content reflects the balance between gene transcription and mRNA degradation, the effect of actinomycin and 8-Br-cAMP on vasopressin mRNA content suggests that 8-Br-cAMP also decreased vasopressin mRNA stability and thereby induced a rapid turnover of vasopressin mRNA. The effects of cycloheximide and actinomycin on vasopressin and oxytocin release suggest that ongoing protein synthesis is required for stimulation of hormone release. Since the posterior pituitary hormone stores are not depleted with a stimulus for release that is even more potent than cAMP, it is possible that cycloheximide and actinomycin depleted smaller pools of the peptides such as those responsible for intranuclear vasopressin and oxytocin release. Further evidence that intranuclear release of vasopressin and oxytocin is a prerequisite for cAMP stimulation of vasopressin and oxytocin release was obtained by demonstrating that d(CH2)5-D-Tyr(Me)VAVP, a potent combined V1a/V2/oxytocin receptor antagonist blocked stimulation of vasopressin and oxytocin release by 8-Br-cAMP.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

cAMP analogs promote survival and neurite outgrowth in cultures of rat sympathetic and sensory neurons independently of nerve growth factor.

Nerve growth factor (NGF) is a neurotrophic agent for sympathetic and embryonic sensory neurons both in vivo and in vitro. We report here that the membrane-permeant cAMP analogs, 8-(4-chlorophenylthio)-cAMP and 8-bromo-cAMP, can replace NGF in promoting long-term survival and neurite outgrowth in cultures of rat neonatal sympathetic and embryonic sensory neurons. N6-substituted analogs, including the more commonly used N6,O2'-dibutyryl-cAMP, are less efficacious. Additivity and switching experiments indicate that the cAMP analogs affect the same neuronal population as that maintained by NGF. However, unlike NGF, the cAMP analogs do not evoke somatic hypertrophy. Moreover, studies with sympathetic neurons reveal that the neurotrophic actions of the cAMP analogs, but not of NGF, are blocked by the axial diastereoisomer of adenosine 3',5'-phosphorothioate, a competitive cAMP antagonist. Thus, the mechanism by which cAMP analogs promote neuronal survival and differentiation appears to involve activation of cAMP-dependent protein kinases, whereas, in contrast, the same effects of NGF neither require nor are mediated by such a pathway. Furthermore, the different efficacies observed with N6- and C8-substituted cAMP analogs suggest that this neurotrophic pathway may involve differential activation of the regulatory subunits of cAMP-dependent protein kinases. The presence of this parallel, cAMP-responsive, neurotrophic pathway in at least two types of NGF-responsive neurons may be developmentally important and has the potential to be exploited for the treatment of injuries or diseases affecting these and possibly other nerve cells.

Animals↗
PubMed2000

The structure of the T127L/S128A mutant of cAMP receptor protein facilitates promoter site binding.

The x-ray crystal structure of the cAMP-ligated T127L/S128A double mutant of cAMP receptor protein (CRP) was determined to a resolution of 2.2 A. Although this structure is close to that of the x-ray crystal structure of cAMP-ligated CRP with one subunit in the open form and one subunit in the closed form, a bound syn-cAMP is clearly observed in the closed subunit in a third binding site in the C-terminal domain. In addition, water-mediated interactions replace the hydrogen bonding interactions between the N(6) of anti-cAMP bound in the N-terminal domains of each subunit and the OH groups of the Thr(127) and Ser(128) residues in the C alpha-helix of wild type CRP. This replacement induces flexibility in the C alpha-helix at Ala(128), which swings the C-terminal domain of the open subunit more toward the N-terminal domain in the T127L/S128A double mutant of CRP (CRP*) than is observed in the open subunit of cAMP-ligated CRP. Isothermal titration calorimetry measurements on the binding of cAMP to CRP* show that the binding mechanism changes from an exothermic independent two-site binding mechanism at pH 7.0 to an endothermic interacting two-site mechanism at pH 5.2, similar to that observed for CRP at both pH levels. Differential scanning calorimetry measurements exhibit a broadening of the thermal denaturation transition of CRP* relative to that of CRP at pH 7.0 but similar to the multipeak transitions observed for cAMP-ligated CRP. These properties and the bound syn-cAMP ligand, which has only been previously observed in the DNA bound x-ray crystal structure of cAMP-ligated CRP by Passner and Steitz (Passner, J. M., and Steitz, T. A. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2843-2847), imply that the cAMP-ligated CRP* structure is closer to the conformation of the allosterically activated structure than cAMP-ligated CRP. This may be induced by the unique flexibility at Ala(128) and/or by the bound syn-cAMP in the hinge region of CRP*.

Bacterial Proteins↗
PubMed2006

A model for agonism and antagonism in an ancient and ubiquitous cAMP-binding domain.

The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R(p))-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S(p))-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R(p))- and (S(p))-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S(p))-cAMPS agonist from the (R(p))-cAMPS antagonist. Based on these data, we have proposed a model for the (R(p)/S(p))-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile(163) and Asp(170), respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.

Allosteric Site↗
PubMed1997

The relationship between external glucose concentration and cAMP levels inside Escherichia coli: implications for models of phosphotransferase-mediated regulation of adenylate cyclase.

The concentration of glucose in the medium influences the regulation of cAMP levels in Escherichia coli. Growth in minimal medium with micromolar glucose results in 8- to 10-fold higher intracellular cAMP concentrations than observed during growth with excess glucose. Current models would suggest that the difference in cAMP levels between glucose-rich and glucose-limited states is due to altered transport flux through the phosphoenolpyruvate: glucose phosphotransferase system (PTS), which in turn controls adenylate cyclase. A consequence of this model is that cAMP levels should be inversely related to the saturation of the PTS transporter. To test this hypothesis, the relationship between external glucose concentration and cAMP levels inside E. coli were investigated in detail, both through direct cAMP assay and indirectly through measurement of expression of cAMP-regulated genes. Responses were followed in batch, dialysis and glucose-limited continuous culture. A sharp rise in intracellular cAMP occurred when the nutrient concentration in minimal medium dropped to approximately 0.3 mM glucose. Likewise, addition of > 0.3 mM glucose, but not < 0.3 mM glucose, sharply reduced the intracellular cAMP level of starving bacteria. There was no striking shift in growth rate or [14C] glucose assimilation in bacteria passing through the 0.5 to 0.3 mM concentration threshold influencing cAMP levels, suggesting that neither metabolic flux nor transporter saturation influenced the sensing of nutrient levels. The (IIA/IIBC)Glc PTS is 96-97% saturated at 0.3 mM glucose so these results are not easily reconcilable with current models of cAMP regulation. Aside from the transition in cAMP levels initiated above 0.3 mM, a second shift occurred below 1 muM glucose. Approaching starvation, well below saturation of the PTS, cAMP levels either increased or decreased depending on unknown factors that differ between common E. coli K-12 strains.

Adenylyl Cyclases↗
PubMed1984

Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants.

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.

Adenylyl Cyclases↗
PubMed1994

(Rp)- and (Sp)-8-piperidino-adenosine 3',5'-(cyclic)thiophosphates discriminate completely between site A and B of the regulatory subunits of cAMP-dependent protein kinase type I and II.

8-Piperidino-cAMP has been shown to bind with high affinity to site A of the regulatory subunit of cAMP-dependent protein kinase type I (AI) whereas it is partially excluded from the homologous site (AII) of isozyme II [Ogreid, D., Ekanger, R., Suva, R. H., Miller, J. P., and Døskeland, S. O. (1989), Eur. J. Biochem. 181, 19-31]. To further increase this selectivity, the (Rp)- and (Sp)-diastereoisomers of 8-piperidino-cAMP[S] were synthesized and analyzed for their potency to inhibit binding of [3H]cAMP to site A and site B from type I (rabbit skeletal muscle) and type II (bovine myocardium) cAMP-dependent protein kinases. (Sp)-8-Piperidino-cAMP[S] showed an enhanced relative affinity for site AI, thus being by far the best A-selective compound (more than 100-fold) tested for this isozyme. In contrast, the (Rp)-isomer was less selective for AI than 8-Piperidino-cAMP itself. The reduction in affinities for BII, compared to 8-piperidino-cAMP, were 10-fold and 50-fold for the (Sp)- and (Rp)-isomer, respectively. Both isomers were almost completely excluded from AII, with affinities about 1000-fold lower than 8-piperidino-cAMP itself. The (Rp)-isomer selected BII with an affinity about 10,000 times higher than for AII, whereas the (Sp)-isomer showed a preference of about 70,000-fold in favour of BII. 8-Piperidino-cAMP as well as its (Sp)-isomer activated both types of holoenzyme protein kinases whereas the (Rp)-isomer acted as an antagonist of cAMP-induced activation. The study concludes that the combination of piperidino- and exocyclic sulfur substitutions generate cAMP analogs that completely discriminate between site A and B of cAMP-dependent protein kinases.

Binding Sites↗
PubMed2000

Diverse roles of intracellular cAMP in early synaptic modifications in the rat visual cortex.

1. The effects of increasing intracellular cAMP concentration were studied using photolysis of caged-cAMP in layer II/III neurons recorded intracellularly in visual cortex slices. The recorded neurons exhibited either after-hyperpolarization (AHP) or after-depolarization (ADP) in response to depolarizing current injection. Depending on which afterpotential appeared, the effects of photolysis differed. 2. In ADP-generating neurons, photolysis of caged-cAMP induced long-lasting depression of postsynaptic potentials (PSPs) evoked by grey matter (GM) stimulation, without altering the size of the ADP. In AHP-generating neurons, photolysis induced long-lasting potentiation of GM-evoked PSPs, with the size of the AHP reduced in the same time course. White matter (WM)-evoked PSPs showed no change. 3. Extracellular application of bromo-cAMP depressed both GM- and WM-evoked PSPs in ADP- and AHP-generating neurons. This depression may be due to presynaptic effects of cAMP, since photolysis-evoked postsynaptic increase in cAMP concentration never induced depression of PSPs in AHP-generating neurons. This depression was reversible but continued until bromo-cAMP was washed out, while ADP and AHP in the postsynaptic neurons were depressed only temporarily and returned to the pre-application level even in the continued presence of bromo-cAMP. 4. Bromo-cAMP was applied following photolysis of caged-cAMP. In the neurons in which the photolysis potentiated GM-evoked PSPs this potentiation was cancelled out by bromo-cAMP (depotentiation). In the other neurons, PSPs were depressed only reversibly. 5. Thus, a postsynaptic increase in cAMP concentration exerts more diverse effects on synaptic plasticity than thus far reported, depending on the difference in neuronal intrinsic excitability and probably on how much, or the way in which, cAMP concentration is increased.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

Failure of insulin to antagonize cAMP-mediated glycogenolysis in rat ventricular cardiomyocytes.

Isolated rat ventricular cardiomyocytes were used to study the effects of insulin on glycogen metabolism in cells treated with various agents that activate adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. Incubation of myocytes with isoproterenol produced a rapid concentration-dependent increase in cAMP concentration, cAMP-dependent protein kinase activity, and phosphorylase activity and a simultaneous decrease in the glycogen synthase activity ratio. Various cAMP analogues also produced a concentration-dependent increase in phosphorylase activity and a decline in the glycogen synthase activity ratio. Incubation of cells with insulin produced no change in basal phosphorylase activity but produced a rapid 40% increase in the glycogen synthase activity ratio. Inclusion of insulin in cell incubations containing increasing concentrations of isoproterenol did not modify the increases in cAMP concentration, protein kinase activity, or phosphorylase activity. Insulin also did not antagonize the ability of any of the cAMP analogues tested to activate phosphorylase, irrespective of the suitability of the particular cAMP analogue as a substrate for cAMP phosphodiesterases. The failure of insulin to antagonize the glycogenolytic effects of isoproterenol or cAMP analogues was paralleled by its failure to activate low-Km phosphodiesterase activity, but the cAMP analogue, 8-parachlorophenylthio-cAMP produced a small reproducible activation of the low-Km enzyme. In contrast to hepatocytes and adipocytes, where some effects of insulin appear to be due to activation of the phosphodiesterase and hydrolysis of cAMP, the effects in cardiomyocytes appear to be independent of an insulin-sensitive phosphodiesterase or of the effects on other components of the cAMP cascade.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

cAMP can raise or lower cardiac actomyosin ATPase activity depending on alpha-adrenergic activity.

Adenosine 3',5'-cyclic monophosphate (cAMP) or beta-adrenergic stimulation has been shown to increase actomyosin adenosinetriphosphatase (ATPase) activity in cardiac muscle. Because the major catecholamine transmitters have both alpha- and beta-adrenergic activity, the possibility of a role for alpha-adrenergic stimulation in the regulation of ATPase activity has been investigated. Histochemical measurement of actomyosin ATPase activity in quickly frozen rat hearts has been used as the assay of enzymatic function of the contractile proteins. The dose-response curve of ATPase activity to cAMP shows an increase in ATPase activity at a threshold concentration of 0.01 microM, a peak effect at 0.5-1.0 microM, and a decline beyond 1.5 microM to a level below control at 10 microM cAMP. Kinetic studies varying ATP concentration from 0.5 to 10 mM indicated the existence of multiple forms of actomyosin ATPase activity in the absence of cAMP and only one form with a higher maximum velocity in the presence of 1 microM cAMP. Apparently cAMP raises the enzymatic activity of the individual actomyosin molecule rather than increasing the number of active molecules. The addition of an alpha-adrenergic blocker had no significant effect in the absence of added cAMP, but in the presence of the cyclic nucleotide, 1 microM prazosin always produced a negative effect on ATPase activity. Over the entire range of 0.01-10 microM, cAMP lowered ATPase activity when the alpha-adrenergic antagonist was present. The integrity of the cAMP regulatory system was sensitive to the tissue oxygen tension at the time the heart was quickly frozen. At certain oxygen tension, the stimulatory component of the cAMP regulation was observed without any inhibitory component, suggesting that there are two relatively independent parts of the regulatory mechanism, an inhibitory and a stimulatory. In the presence of gamma-labeled [32P]ATP, 32P was incorporated into several proteins, including the inhibitory subunit of troponin (TNI), C protein, and the regulatory light chain of myosin. cAMP (1 microM) caused an increase in 32P labeling of TNI and C protein. The addition of prazosin with cAMP caused a decrease in the overall level of phosphorylation with specific dephosphorylation of C protein and TNI, the former to a degree similar to the decrease in actomyosin ATPase activity, the latter to a greater degree. These results indicate that alpha-adrenergic activity modulates the balance between kinase and phosphatase activity in the presence of cAMP, probably by inhibiting phosphatase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗
PubMed1985

Effects of calcium on the vasopressin-sensitive cAMP metabolism in medullary tubules.

The modulatory effect of Ca on [Arg8]vasopressin-dependent (AVP) cAMP metabolism was studied in medullary collecting tubules (MCT) and medullary ascending limbs (MAL) microdissected from rat kidney. In MCT segments incubated in vitro with AVP, the accumulation of cAMP was enhanced (delta +59%) when Ca was omitted from the incubation medium compared with a medium with 2 mM of ionized calcium (Ca2+). Ionophore A23187 caused a decrease in AVP-stimulated cAMP accumulation in MCT in the presence of 2 mM Ca2+ but not in a Ca2+-free medium. Diltiazem and verapamil enhanced the AVP-stimulated cAMP accumulation in MCT; PTH had no detectable effect. A23187 caused a dose-dependent inhibition of cAMP accumulation stimulated by AVP with forskolin in both MCT and in MAL. However, in MAL the A23187 concentration needed for half-maximum inhibition (6.3 X 10(-6) M) was higher than for MCT (3.9 X 10(-7) M). The maximum inhibition in MAL (-65%) was less than in MCT (-97%). In the presence of 3-isobutyl-1-methylxanthine, AVP-stimulated cAMP accumulation was inhibited by A23187 in MCT (-45%) but not in MAL. Naproxen or ibuprofen did not relieve the inhibitory action of A23187 in MCT. Added Ca2+ inhibited the AVP-stimulated adenylate cyclase in MCT and MAL (half-maximum approximately equal to 5 X 10(-4) M Ca2+) and stimulated cAMP phosphodiesterase (cAMP-PDIE) in both MCT and in MAL (half-maximum approximately equal to 9 X 10(-5) M Ca2+). Incubation of MCT and MAL with A23187 decreased (-50%) the content of ATP. Results suggest that increased influx of extracellular Ca2+ inhibits the AVP-stimulated cAMP accumulation in MCT and to a much lesser degree in MAL. Deceased cAMP accumulation in MCT is probably due to both stimulation of cAMP-PDIE and the inhibition of adenylate cyclase, whereas in MAL it is due to stimulation of cAMP-PDIE. The results suggest that Ca2+ influx exhibits a negative modulatory effect on AVP-dependent cAMP metabolism mainly in MCT.

1-Methyl-3-isobutylxanthine↗
PubMed2005

PKA-dependent and PKA-independent pathways for cAMP-regulated exocytosis.

Stimulus-secretion coupling is an essential process in secretory cells in which regulated exocytosis occurs, including neuronal, neuroendocrine, endocrine, and exocrine cells. While an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) is the principal signal, other intracellular signals also are important in regulated exocytosis. In particular, the cAMP signaling system is well known to regulate and modulate exocytosis in a variety of secretory cells. Until recently, it was generally thought that the effects of cAMP in regulated exocytosis are mediated by activation of cAMP-dependent protein kinase (PKA), a major cAMP target, followed by phosphorylation of the relevant proteins. Although the involvement of PKA-independent mechanisms has been suggested in cAMP-regulated exocytosis by pharmacological approaches, the molecular mechanisms are unknown. Newly discovered cAMP-GEF/Epac, which belongs to the cAMP-binding protein family, exhibits guanine nucleotide exchange factor activities and exerts diverse effects on cellular functions including hormone/transmitter secretion, cell adhesion, and intracellular Ca(2+) mobilization. cAMP-GEF/Epac mediates the PKA-independent effects on cAMP-regulated exocytosis. Thus cAMP regulates and modulates exocytosis by coordinating both PKA-dependent and PKA-independent mechanisms. Localization of cAMP within intracellular compartments (cAMP compartmentation or compartmentalization) may be a key mechanism underlying the distinct effects of cAMP in different domains of the cell.

Action Potentials↗
PubMed1996

cAMP-mediated vascular protection in an orthotopic rat lung transplant model. Insights into the mechanism of action of prostaglandin E1 to improve lung preservation.

Prostaglandin E1 (PGE1) is often added to the donor pulmonary flush solution to enhance clinical lung preservation for transplantation. Although PGE1 is thought to act as a pulmonary vasodilator during the harvest period, the precise mechanism(s) of action whereby PGE1 enhances lung preservation is unknown. Because cAMP levels decline in endothelial and vascular smooth muscle cells exposed to hypoxia, we hypothesized that a PGE1-mediated increase in cAMP levels within the preserved lungs might improve pulmonary vascular homeostasis following lung transplantation. Rat lungs demonstrated a time-dependent decline in cAMP levels during hypothermic storage, with cAMP levels significantly increased by PGE1 supplementation (approximately 2-fold by 6 hours, P < .0005). To test whether augmenting cAMP levels may enhance lung preservation, experiments were performed using an orthotopic rat left lung transplant model. Compared with controls, supplementing the preservation solution with the membrane-permeable cAMP analogue dibutyryl-cAMP resulted in dose-dependent preservation enhancement, marked by reduced pulmonary vascular resistance (6.0-fold, P < .01), improved arterial oxygenation (3.0-fold, P < .01), reduced graft neutrophil infiltration (1.5-fold, P < .05), and improved recipient survival (7.0-fold, P < .005). Similar preservation enhancement was observed with another cAMP analogue (8-bromo-cAMP) or the phosphodiesterase inhibitor indolidan. Stimulating the cAMP second messenger system by PGE1 supplementation resulted in marked hemodynamic benefits and improved recipient survival, in parallel with reduced graft neutrophil infiltration, vascular permeability, and platelet deposition. These beneficial effects of PGE1 were abrogated by simultaneous administration of the cAMP-dependent protein kinase antagonist Rp-cAMPS. Although an arterial vasodilator (minoxidil) resulted in significant pulmonary vasodilation during harvest, it lacked other nonvasodilating effects of PGE1 and resulted in poor preservation. These data show that harvest vasodilation by itself is insufficient to enhance lung preservation and that PGE1 enhances lung preservation by stimulating the cAMP-dependent protein kinase and promoting non-vasodilatory mechanisms of pulmonary protection.

Alprostadil↗
PubMed2005

cAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum.

BACKGROUND: Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i. RESULTS: We analyzed Ca2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced. CONCLUSION: We conclude that there is a link between the second messengers cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2004

Membrane estrogen receptor-alpha levels in MCF-7 breast cancer cells predict cAMP and proliferation responses.

INTRODUCTION: 17beta-estradiol (E2) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mERlow) and high (mERhigh) expression of the membrane form of estrogen receptor (mER)-alpha, and thus addressed the receptor subform involved in cAMP signaling. METHODS: MCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mERhigh (mER-alpha-enriched) and mERlow (mER-alpha-depleted) populations. Unique (compared with previously reported) incubation conditions at 4 degrees C were found to be optimal for demonstrating E2-induced cAMP production. Time-dependent and dose-dependent effects of E2 on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E2-induced cell proliferation were assessed using the crystal violet assay. RESULTS: We demonstrated a rapid and transient cAMP increase after 1 pmol/l E2 stimulation in mERhigh cells; at 4 degrees C these responses were much more reliable and robust than at 37 degrees C (the condition most often used). The loss of cAMP at 37 degrees C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mERhigh cells than in mERlow cells, implicating mER-alpha levels in the process. ICI172,780 blocked the E2-induced response and 17alpha-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E2 dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E2 concentrations in mERhigh cells. Proliferation of mERlow cells was stimulated over the whole range of E2concentrations, whereas the number of mERhigh cells was greatly decreased at concentrations above 1 nmol/l, suggesting that estrogen over-stimulation can lead to cell death, as has previously been reported, and that mER-alpha participates. E2-mediated activation of adenylyl cyclase and downstream participation of protein kinase A were shown to be involved in these responses. CONCLUSION: Rapid mER-alpha-mediated nongenomic signaling cascades generate cAMP and downstream signaling events, which contribute to the regulation of breast cancer cell number.

Breast Neoplasms↗
PubMed2006

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells.

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio). Apical application of forskolin (1 microM, 10 microM, 20 microM) and, in permeabilized cells, basolateral cAMP (20 microM, 50 microM, 100 microM) caused dose-dependent, albeit similar and simultaneous-increases in cAMP and ciliary beat frequency. However, decreases in cAMP preceded decreases in ciliary beat frequency, suggesting that either cellular cAMP decreases before ciliary cAMP or the dephosphorylation of target proteins by phosphatases occur at a rate slower than the rate of cAMP hydrolysis.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1998

Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy.

Islets undergo a number of upregulatory changes to meet the increased demand for insulin during pregnancy, including an increase in glucose-stimulated insulin secretion with a reduction in the stimulation threshold. Treatment with the lactogenic hormone prolactin (PRL) in vitro has been shown to induce changes in islets similar to those observed during pregnancy. We examined cAMP production in islets treated with PRL to determine if changes in cAMP are involved in the upregulation of insulin secretion. Insulin secretion and cAMP concentrations were measured from islets in response to a suprathreshold (6.8 mmol/l) or high (16.8 mmol/l) glucose concentration in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Insulin secretion increased by 2.1-, 5.0-, and 5.9-fold at the suprathreshold glucose concentration and by 1.6-, 2.3-, and 2.9-fold at the higher glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. After a similar pattern, cAMP metabolism increased by 1.2-, 1.6-, and 2.1-fold at the suprathreshold glucose concentration and by 1.2-, 1.7-, and 2.2-fold at the high glucose concentration after 1, 3, and 5 days of PRL treatment, respectively. The similar increases in insulin secretion and cAMP concentration suggest that changes in cAMP metabolism are involved in lactogen-induced upregulation of insulin secretion. To gain additional insight into the role of cAMP in the upregulation of islet function after lactogen treatment, we examined the relationship between changes in cAMP concentration and insulin secretion. Under all conditions (differing glucose concentrations and time periods), the increase in insulin release was directly proportional to the increase in cAMP. Thus increased glucose-stimulated insulin secretion from lactogen-treated islets could be accounted for by increased generation of cAMP and did not appear to require any further specific changes in intracellular processes mediated by cAMP. Because the PRL receptor is not directly involved in cAMP metabolism, the lactogen-induced increase in cAMP was most likely due to the increase in glucose metabolism that we have previously demonstrated in PRL-treated islets and in islets during pregnancy.

1-Methyl-3-isobutylxanthine↗