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PubMedDate not supplied

The effects of cAMP on the excitability and responses of defensive behavior command neurons in the common snail evoked by sensory stimuli.

Experiments on snails showed that extracellular application of dibutyryl-cAMP (db-cAMP) or intracellular application of cAMP for 30 min evoked increases in excitability and synaptic facilitation in responses to sensory stimulation of defensive behavior command neurons LP11 and RP11. Excitability increased 45-60 min after the start of addition of db-cAMP or cAMP and remained elevated until the end of the experiment (3-4 h). Synaptic facilitation started 50-60 min after the onset of extracellular application of db-cAMP and remained detectable in the responses of neurons to tactile stimulation of the head for 1 h and to application of dilute quinine solution for 2-4 h. Application of db-cAMP produced no changes in responses to tactile stimulation of the foot or mantle ridge. Intracellular injection of cAMP induced facilitation of neuron responses only to weak quinine solutions. The responses of neurons to tactile stimulation of the head, foot, and mantle ridge did not change after injections of cAMP. It is suggested that cAMP is involved in the mechanisms controlling the excitability of neurons LP11 and RP11. In addition, cAMP is selectively involved in the postsynaptic mechanism inducing the transient stage of long-term facilitation of synaptic "inputs," which mediates excitation evoked by chemical stimuli. This set of effects of cAMP is similar to effects arising during the development of nociceptive sensitization and in response to serotonin.

Animals↗
PubMed2001

Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes.

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) through the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet understood. This stimulation by cAMP could not be reproduced with the rat or Xenopus ENaC expressed in Xenopus oocyte. Recently, it was shown that the alpha-subunit cloned from the guinea-pig colon (alpha gp) could confer the ability to be activated by the membrane-permeant cAMP analogue 8-chlorophenyl-thio-cAMP (cpt-cAMP) to channels produced by expression of alpha gp, beta rat and gamma rat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment, endogenous production of cAMP by activation of coexpressed beta adrenergic receptors, or intracellular perfusion with cAMP did not increase the amiloride-sensitive Na current, even though these maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance regulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increased alpha gp, beta rat and gamma rat ENaC activity but had no effect on CFTR. Swapping intracellular domains between the cpt-cAMP-sensitive alpha gp and the cpt-cAMP-resistant alpha rat-subunit showed that neither the N-terminal nor the C-terminal of alpha ENaC was responsible for the effect of cpt-cAMP. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP/PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by alpha gp and beta gamma rat by interacting with the extracellular part of the protein.

Amiloride↗
PubMed2001

8-Cl-adenosine is an active metabolite of 8-Cl-cAMP responsible for its in vitro antiproliferative effects on CHO mutants hypersensitive to cytostatic drugs.

8-Cl-cAMP has been undergoing clinical trials as a potential chemotherapy agent, but there is much discussion in the literature as to whether the active agent is 8-Cl-cAMP itself, or its major metabolite, 8-Cl-adenosine. 8-Cl-cAMP is susceptible to the action of serum enzymes such as phosphodiesterases, and its metabolism when administered to cancer patients raises questions as to the mechanism of action of 8-Cl-cAMP. The stability of 8-Cl-cAMP when incubated with serum, and the effects of both 8-Cl-cAMP and 8-Cl-adenosine on the proliferation of variant lines of CHO cells hypersensitive to 8-Cl-cAMP were investigated. A solid-phase extraction (SPE) purification protocol and the HPLC method previously developed were used to determine 8-Cl-cAMP and 8-Cl-adenosine. Heat treatment of serum inactivated the enzymes in the culture medium responsible for activating 8-Cl-cAMP. Under these conditions 8-Cl-cAMP remained stable and there were no traces of its metabolite, 8-Cl-adenosine. Cell culture experiments showed that 8-Cl-cAMP only affected cell growth in medium that contained untreated serum. In contrast, 8-Cl-adenosine was shown to be growth inhibitory in medium containing either heat-treated or untreated serum. HPLC analysis of the culture medium from the cell culture experiments supported the hypothesis that 8-Cl-cAMP was only effective in inhibiting cell growth after metabolism to 8-Cl-adenosine. Thus further studies of this drug and its mechanism of action should focus on 8-Cl-adenosine.

2-Chloroadenosine↗
PubMed2004

Hypotonic cell swelling stimulates permeability to cAMP in a rat colonic cell line.

This study characterized the membrane permeability to cAMP in a cell line derived from the rat colon (CC531(mdr+)) by comparison of fluxes of 3H-cAMP, 3H-8-bromo-cAMP, 3H-taurine, 3H-adenosine and 3H-5'AMP under various experimental conditions including cell membrane depolarization and hypotonic cell swelling. Cell volume was modified by changing the osmolality and composition of the extracellular medium. Incubation in iso- and hypotonic KCl media induced graded increases in cell volume and stable activation of volume-sensitive channels that was reflected in an increased efflux of 3H-taurine. Incubation in hypotonic KCl solution also enhanced the efflux of 3H-8-Br-cAMP (a non-hydrolysable analogue of cAMP). Both the efflux of 3H-taurine and of 3H-8-Br-cAMP were inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 100 microM) suggesting the involvement of volume-sensitive anion channels. To gain further insight into the route mediating cAMP permeability, the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine were determined over short (5-min) periods. Uptakes of these substrates demonstrated close similarities: comparable increases were observed that correlated with the increases in cell volume in iso- and hypoosmotic KCl media; they were inhibited strongly by NPPB (100 microM) and metabolic inhibitors (deoxyglucose, 20 mM together with the mitochondrial uncoupler carbonylcyanide p-(trifluoromethoxy)phenylhydrazone, FCCP, 10 microM) while barely reduced by dipyridamole (100 microM) and they were not affected by adenosine (1 mM). In contrast, the uptakes of 3H-adenosine and 3H-5'AMP had strikingly different properties; they were insensitive to cell swelling; barely inhibited by NPPB (100 microM) and metabolic inhibitors (deoxyglucose and FCCP) while strongly reduced by dipyridamole (100 micro M). Unlike the uptakes of 3H-cAMP, 3H-8-Br-cAMP and 3H-taurine, the uptakes of 3H-adenosine and 3H-5'AMP were reduced in Na(+)-free media, suggesting the presence in this cell line of two different adenosine carriers, one sodium-dependent and one sodium-independent. Taken together the present data show that in this rat colonic cell line, cAMP permeability is increased by cell swelling in hypotonic KCl medium and inhibited by NPPB and metabolic inhibitors. The similarity of these characteristics to those of taurine permeability suggests the involvement of a volume-sensitive anion pathway.

Animals↗
PubMed1997

Effect of cAMP and ATP on the hyperpolarization-activated current in mouse dorsal root ganglion neurons.

In mouse dorsal root ganglion (DRG) neurons the activation curve of the hyperpolarization-activated current (Ih) shifted towards depolarized potentials when cAMP was present in the pipette. The relation between the midpoint potential and cAMP concentration could be described by a Hill function with a half-maximal concentration of 0.55 microM cAMP, reflecting a direct action of cAMP on the channel. With 5 mM ATP and a saturating concentration of cAMP an additional shift of the midpoint potential is observed which can be explained by phosphorylation. Application of Rp-cAMPS and Sp-cAMPS support the hypothesis of both a phosphorylation pathway and a direct effect exhibited by these molecules. The bell-shaped curves, relating the time constants for the slow and fast current components to the voltage, shifted towards positive membrane potentials when cAMP and ATP were in the pipette. The fully activated Ih/voltage relation and the reversal potential were not dependent on the presence of cAMP or ATP in the pipette. The mean resting membrane potential of -59 mV, using the perforated-patch configuration, hyperpolarized in the presence of extracellular CsCl. In the whole-cell configuration the resting membrane potential was significantly more negative at 0 microM cAMP (-61 mV) than at 100 microM cAMP (-57 mV). Thus, the activation of Ih, regulated by both the intracellular cAMP and the ATP concentration, may influence the excitability of DRG neurons.

Adenosine Triphosphate↗
PubMed1997

"Voltage-activated Ca release" in rabbit, rat and guinea-pig cardiac myocytes, and modulation by internal cAMP.

It is widely believed that Ca release from the sarcoplasmic reticulum (SR) in heart muscle is due to "Ca-induced Ca-release" (CICR), triggered by transmembrane Ca entry. However, in intact guinea-pig cells or cells dialysed with cAMP there may be an additional mechanism - SR release may be activated directly by membrane depolarisation without Ca entry. The first objective of the present study was to investigate whether this "voltage-activated Ca release" (VACR) mechanism is present across species such as rabbit, rat and guinea-pig. The second objective was to characterise the dependence of a VACR mechanism on internal [cAMP]. Membrane current was measured with the whole-cell patch-clamp technique, intracellular [Ca] was monitored with Fura-2 (or a combination of Fluo-3/SNARF-1). Rapid changes of superfusate (within 100 ms) were made using a system which maintained cell temperature at 37 degrees C. We used a train of conditioning pulses to ensure a standard SR load before each test pulse. In rabbit myocytes dialysed with 100 microM cAMP, 89.6 +/- 7.0% of the control intracellular Ca (Cai) transient was still elicited by depolarisation during a switch to 5 mM Ni, which blocked pathways for Ca entry. This suggested that rabbit myocytes possess a VACR mechanism. The percentage of control Cai transient elicited by depolarisation in the presence of 5 mM Ni (i.e. magnitude of VACR) increased in a graded fashion with the pipette [cAMP] between zero and 100 microM. In rat myocytes dialysed with 50 microM cAMP, 64.4 +/- 6.2% of SR release was activated by depolarisation in the presence of 5 mM Ni, suggesting the presence of a VACR mechanism. The extent to which VACR triggered SR release increased with the pipette [cAMP] between zero and 50 microM. In guinea-pig myocytes dialysed with 100 microM cAMP, 74.6 +/- 3.6% of the control Cai transient was elicited by depolarisation in the presence of 5 mM Ni. The degree to which VACR triggered SR release was also graded with the pipette [cAMP] between zero and 100 microM. It therefore appears that each of the three species might possess a VACR mechanism which can be modulated by the internal [cAMP]. This may reflect an effect of cAMP to phosphorylate key proteins involved in excitation-contraction coupling. Under normal physiological conditions with a basal [cAMP] between 2 and 20 microM, VACR may play a role in triggering SR release. The role of VACR may increase under conditions which increase internal [cAMP].

Animals↗
PubMed2001

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel.

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 microM and 5 microM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.

Adenylyl Cyclases↗
PubMed1991

A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response.

A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1980

Different effects of cAMP and cGMP derivatives on the activity of an identified neuron: biochemical and electrophysiological analysis.

Eight-position substituted cAMP and cGMP derivatives, and phosphodiesterase inhibitors, modify endogenous 'bursting' activity in Aplysia neuron R15. Several different patterns of activity were elicited depending on the agent used. 8-Benzylthio-cAMP or 8-parachlorophenylthio-cAMP, at concentrations between 5 muM and 0.3 mM, markedly enhanced the depth and duration of the interburst hyperpolarization, and in some cells bursting was inhibited completely. In contrast, 8-parachlorophenyl-thio-cGMP treatment led to some depolarization and to the appearance of long slow bursts, with little effect on the interburst phase. When the parachlorophenylthio-derivatives of cAMP and cGMP were added together at equal concentrations, a pattern consisting of long bursts interrupted by long and deep interburst hyperpolarizations was observed. This pattern could also be elicited by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX). IBMX inhibited cAMP and cGMP phosphodiesterases and caused both cAMP and cGMP to accumulate in intact ganglia and in individual identified neuronal cell bodies including that of R15. Another phosphodiesterase inhibitor, Ro 7-2956, was a more potent inhibitor of cAMP than of cGMP phosphodiesterase; Ro 7-2956 also modified bursting activity, and seemed to enhance preferentially the interburst hyperpolarization. At high concentrations the 8-substituted cAMP and cGMP derivatives also inhibited cAMP and cGMP phosphodiesterases. The 8-parachlorophenylthio-derivatives of cAMP and cGMP were indistinguishable from each other in this assay, and thus phosphodiesterase inhibition cannot be responsible for their differential effects on bursting activity. The derivatives stimulated protein kinase activity in Aplysia ganglion homogenates, as measured by the incorporation of 32P from ATP into histone. IBMX and Ro 7-2956 had no detectable effect on protein kinase activity. The concentrations of cAMP and cGMP derivatives required for protein kinase activation (10(-8)M-10(-6)M) were much lower than those required for phosphodiesterase inhibition (10(-5)M-10(-3)M). Thus, differential protein phosphorylation is more likely to be responsible for the effects of cAMP and cGMP derivatives on neuron R15 bursting activity than is differential phosphodiesterase inhibition.

1-Methyl-3-isobutylxanthine↗
PubMed1992

The effects of E and F prostaglandins on ovarian cAMP production and follicular contraction in the brook trout (Salvelinus fontinalis).

The effects of PGE1, PGE2, PGF2 alpha (2 micrograms/ml), and forskolin (10 microM) on follicle contraction of brook trout (salvelinus fontinalis) were studied by measuring the weight loss of punctured follicles during in vitro incubation. PGE1, PGE2, and forskolin significantly inhibited spontaneous contraction of follicles. In contrast, PGF2 alpha significantly increased the weight loss of punctured follicles. The results indicate that PGs could influence ovulation through their effects on follicle wall contraction. The effects of PGs and forskolin on cAMP production in follicles were also investigated by incubating intact follicles (without extrafollicular tissue), follicle walls, and denuded oocytes at pregerminal vesicle breakdown (preGVBD) and preovulatory (preOV) stages in vitro with PGE1, PGE2, PGF2 alpha (0.2 and 2 micrograms/ml), or forskolin (10 microM). At the end of incubation, cAMP content in incubation medium and follicular tissue was assayed by a specific protein-binding assay. Intact follicles in control groups contained high amounts of cAMP at both stages, but released significantly more cAMP into the medium at the preGVBD stage (P < 0.05). Forskolin stimulated significantly higher levels of cAMP in incubation medium at either stage. At the preGVBD stage, significantly higher cAMP levels were measured in the medium with higher dose of PGE1 at 1 and 4 hr and with higher dose of PGE2 at 1, 2, and 4 hr. Significantly higher cAMP levels were also measured in incubates with high dose of PGE1 at 2 and 4 hr and with high doses of PGE2 at 2, 4, and 16 hr at the preOV stage. However, follicular cAMP levels were significantly elevated only by forskolin at both stages. Experiments incubating only follicle walls or denuded oocytes demonstrated that most of the medium cAMP measured in preGVBD intact follicle incubates was derived from denuded oocytes, and that the stage difference in cAMP release within intact follicle incubates was due to a stage difference in cAMP release from denuded oocytes. In addition, follicle walls appeared to be more sensitive to forskolin and PG stimulations than denuded oocytes. The stimulation of cAMP accumulation in the medium by PGEs may be significant since both forskolin and PGEs have been shown to inhibit brook trout ovulation in vitro (F. W. Goetz, D. C. Smith, and S. P. Krickle (1982). Gen. Comp. Endocrinol. 48, 154-160) and follicle contraction in the present study.

Animals↗
PubMed1985

Regulation of 2-5 A phosphodiesterase activity by cAMP-dependent phosphorylation: mechanism and biological role.

The results of the present study permit the explanation of one of the mechanisms of the interconnection between the regulatory systems of cAMP and 2-5A. cAMP-dependent regulation of 2'-PDE was found to involve phosphorylation of the specific protein inhibitor. Originally, a similar way of regulation of the enzyme activity was discovered for protein phosphatase I. This enzyme has a specific protein inhibitor type 1, which is phosphorylated by cAMP-dependent protein kinase and is activated by phosphorylation (18). It is interesting that the molecular weights of 2'-PDE protein inhibitor and of the inhibitor type 1 of protein phosphatase I are essentially the same. There is also a certain similarity between the above described mechanism and phosphorylation of the regulatory subunit of cAMP-dependent protein kinase type 2. The regulatory subunit can also act as a protein inhibitor of the enzyme and change its properties as a result of phosphorylation (19). The results obtained permit as well a more detailed explanation for cAMP-dependent inhibition of cell proliferation. Evidently, cAMP elevation causes activation of cAMP-dependent phosphorylation which, in turn, leads to the induction of 2-5A synthetase and inhibition of 2'-PDE. As a result of variations in the activities of these enzymes, the level of 2-5A rises. The latter brings about the changes characteristic of the resting state. They involve activation of RNase L and the succeeding acceleration of RNA hydrolysis, inhibition of protein synthesis and cell proliferation. The resting state is characterized by a rapid turnover of macromolecules due to their intensive degradation (20). The above described scheme suggested that the rapid turnover of RNA during inhibition of cell proliferation can be partially accounted for by activation of 2-5A-dependent RNase L. Thus, it can be thought that at least one of the mechanisms of the antiproliferative effect of cAMP-dependent phosphorylation of proteins involves cAMP-dependent elevation of intracellular 2-5A. Evidently, a number of properties of the resting cells are determined by the elevated content of 2-5A. Finally, it should be noted that the interconnection between the systems of cAMP and 2-5A is a multiple process. We have earlier demonstrated (12) that 2-5A activates cAMP phosphodiesterase in NIH 3T3 cell homogenates. These data suggest that the mutual regulation of cAMP and 2-5A levels involves the negative feedback mechanism (Fig. 8).

Adenine Nucleotides↗
PubMed1991

An hypothesis: phosphorylation fields as the source of positional information and cell differentiation--(cAMP, ATP) as the universal morphogenetic Turing couple.

It is hypothesized that (cAMP, ATP) is the elusive, universal Turing morphogenetic couple, which defies the second law of thermodynamics, i.e. the inexorable march towards homogeneity. cAMP and ATP can be distributed nonhomogeneously because the whole of the intermediary metabolism is so organized that they mutually satisfy the Turing bifurcation conditions upon nonlocalized application of an extracellular ligand, in particular a soluble peptide growth factor, which is nature's distinguished universal bifurcation parameter, acting homogeneously in space and removing the substrate inhibition from adenylate cyclase and thus triggering embryonic induction by triggering the (cAMP, ATP) Turing system. The hypothesis predicts that although the extracellular signal, the growth factor, is applied homogeneously, an organized "dissipative structure" will emerge spontaneously in the responding tissue; this "symmetry breaking" in a reaction-diffusion system occurs precisely in the manner envisaged by Turing, where (cAMP, ATP) constitutes the "reaction-diffusion system". This Turing bifurcation explicates the recent experiments where a differentiated embryoid emerges from the mere immersion of frog animal caps in an homogeneous growth factor solution, and similar experiments on chicks. The "metabolic" patterns found by Child and colleagues also reflect dissipative structures arising in a (cAMP, ATP) reaction-diffusion system when interpreted in the light of modern biochemistry: in particular, the localized glycogen depletion reflects localized cAMP; localized redox, respiratory or susceptibility activity reflects localized ATP. The dramatic collapse of organized structure found by Child and colleagues, for example, when Planaria or a section of it is exposed to an homogeneous environment of a narcotic solution, and the reemergence of structure upon return to water, are explained on the basis of the violation or satisfaction of the Turing bifurcation conditions with respect to (cAMP, ATP), respectively. cAMP is the "activator", ATP is the "inhibitor", and together they mutually satisfy the four activator-inhibitor inequalities, including the all-important autocatalytic cAMP production, as well as the lateral inhibition condition. The functional significance of gap junctions is to generate a multicellular purely reaction-diffusion system for (cAMP, ATP) as envisaged by Turing. It is emphasized that localization and pattern formation occur intracellularly in gap junction-coupled cells and not, as often suggested, extracellularly, the latter localization being too fragile to be maintained for long enough, and soon succumbing to the mixing effect of convection and movement. The activator-inhibitor property of (cAMP, ATP) means that the spatial distribution of cAMP and ATP could be not only nonhomogeneous but also of the same shape.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1989

Progesterone depression of norepinephrine-stimulated cAMP accumulation in hypothalamic slices.

The present experiments examined the effects of progesterone on adrenergic receptor coupling to adenylate cyclase in hypothalamic and preoptic area slices by monitoring norepinephrine (NE)-stimulated increases in cAMP accumulation. Progesterone treatment of estrogen-primed rats decreased NE-induced slice cAMP accumulation. The reduced cAMP response was estrogen-dependent since it was not demonstrable in slices from rats exposed to progesterone without prior estrogen priming. Neither generalized increases in phosphodiesterase activity nor decreases in the catalytic activity of adenylate cyclase could account for the reduced ability of NE to stimulate cAMP accumulation in hypothalamic slices. Moreover, the cAMP response to two other activators of adenylate cyclase, adenosine and vasoactive intestinal peptide, was not decreased in slices from rats treated with estrogen plus progesterone. Selective adrenergic agonists and antagonists were employed to determine which adrenergic receptors mediate cAMP accumulation in progesterone-exposed slices. Slice cAMP levels were elevated by the beta receptor agonist isoproterenol but not by alpha 1 (phenylephrine) or alpha 2 (clonidine) agonists. However, clonidine potentiated the effect of isoproterenol on slice cAMP formation whereas phenylephrine did not. Likewise, NE-stimulated cAMP accumulation was completely antagonized only by a combination of both beta (propranolol) and alpha 2 (yohimbine) antagonists. The data suggest that in slices from estrogen plus progesterone-treated rats, alpha 2 receptors contribute significantly to NE stimulation of cAMP accumulation. The overall depression of the cAMP response to NE in progesterone-exposed slices may involve a decrease of alpha 1 receptor facilitation of cAMP synthesis.

Animals↗
PubMed2006

Quantification of cAMP antagonist action in vitro and in living cells.

cAMP-dependent protein kinase (PKA) plays a key role in intracellular signalling. cAMP antagonists, acting as suppressors of PKA activity by preventing PKA-holoenzyme dissociation, have received increasing attention because of their potential use in diagnostics as well as for therapeutic purposes. A large number of cAMP analogs have been described over the last three decades and methodology has been established to monitor cAMP agonists action by either following enzymatic activity or holoenzyme dissociation. This is not the case for cAMP antagonists, where only a few substances have been demonstrated to exhibit effects in the low micromolar range, for example, Rp-8-Br-cAMPS. A main drawback in the development of new compounds is the lack of technologies to assess antagonist action in an in vitro situation as well as in living cells. Here we quantify the effect of several cAMP analogs applying three different biochemical/biophysical assay setups and one in-cell assay. This includes two methods monitoring subunit dissociation in a test tube, namely AlphaScreen, a bead-based proximity assay, and surface plasmon resonance, determining the association and dissociation patterns of the two PKA subunits in real time in response to antagonists. BRET(2), performed in living cells in a 96-well format, allows testing for the efficacy of membrane-permeable cAMP analogs based on a genetically engineered cAMP sensor. Using novel and established experimental strategies side by side, the action of cAMP and cAMP analogs was tested on type Ialpha PKA holoenzyme, thus generating methodology to screen drug libraries for potential cAMP antagonists with high accuracy, reproducibility as well as potential for automation.

Animals↗
PubMed2003

Yessotoxin, a novel phycotoxin, activates phosphodiesterase activity. Effect of yessotoxin on cAMP levels in human lymphocytes.

Yessotoxin (YTX) is a novel phycotoxin with an unknown mechanism of action that has been reported as cardiotoxic, when injected, but non-toxic if ingested orally. In this paper, we studied the effect of YTX on adenosine 3',5'-cyclic monophosphate (cAMP) pathway, since this pathway can be a cellular target to this toxin as happens in other diarrhetic toxins. We determined cAMP levels by enzymeimmunoassay and by using the cAMP dye recombinant fluorescein- and rhodamine-labeled protein kinase A, which increases their fluorescence when cAMP levels are increased. In the presence of YTX, and after a transient small increase, cAMP levels were decreased. This effect was Ca(2+) dependent since in a Ca(2+)-free medium YTX increased cAMP levels, but this event was reverted after addition of external calcium. YTX also reverted the increase of cAMP induced by the adenylyl cyclase activator forskolin. These variations in fluorescence units were confirmed when cAMP levels were measured by enzymeimmunoassay, YTX decreases cAMP from 52.81+/-3.66 to 44.53+/-4.5 fmol. Phosphodiesterase (PDE) IV inhibitors, rolipram or etazolate, did not modify the effect of YTX, however, when PDE IV was first inhibited no effect of YTX was observed. On the other hand, the PDE III inhibitor milrinone counteracted the effect of YTX, and a similar effect was observed with the unspecific PDE I inhibitor chlorpromazine. These results point to an effect of YTX on PDE activity. In the presence of YTX, the fluorescent PDE substrate Mant-cAMP, increased its rate of hydrolysis, the same as the PDE from bovine brain increased the hydrolysis of cAMP substrate. In addition, YTX increased interleukin-2 production, which indirectly confirms a decrease in cAMP. Although results show a very complex pattern of responses, due to the interactions and crosstalks between many systems, results suggest that YTX is a PDE activator in the presence of external Ca(2+).

Cyclic AMP↗
PubMed1999

Effects of ethanol on basal and adenosine-induced increases in beta-endorphin release and intracellular cAMP levels in hypothalamic cells.

Recently we have shown that the cAMP system is involved in ethanol-regulated beta-endorphin (beta-EP) release from rat hypothalamic neurons in primary cultures. The cascade of events that leads to activation of cAMP following ethanol treatment in hypothalamic beta-EP neurons is not apparent. In this study the role of adenosine, a cAMP regulator, in ethanol-regulated beta-EP release was determined by measuring the cellular incorporation of [3H]adenosine, intracellular cAMP levels and media immunoreactive (IR) beta-EP levels in cultures of rat hypothalamic cells following ethanol treatments in the presence and absence of an adenosine agonist and antagonist. Acute exposure to a 50 mM dose of ethanol for a period of 1 h increased media levels of IR-beta-EP and cellular contents of cAMP, but the ethanol treatment decreased [3H]adenosine uptake. Constant exposure to a 50 mM dose of ethanol for a period of 48 h, failed to alter media levels of IR-beta-EP, cell content of cAMP and [3H]adenosine uptake. The media level of IR-beta-EP was elevated following treatment with adenosine receptor agonist phenyl-isopropyl adenosine (PIA) and was reduced following treatment with adenosine receptor antagonist isobutylmethylxanthine (IBMX) or with adenosine uptake inhibitor adenosine deaminase. The level of cellular cAMP was also increased by PIA but was decreased by IBMX and adenosine deaminase. The stimulatory actions of the adenosine agonist PIA on IR-beta-EP release and on cAMP production were potentiated by simultaneous incubation with ethanol for 1 h. However, chronic ethanol exposure reduced PIA-induced IR-beta-EP release and cAMP production. Additionally, both IBMX and adenosine deaminase reduced ethanol-induced IR-beta-EP release and cAMP levels. These results suggest that ethanol inhibits adenosine uptake in IR-beta-EP neurons in the hypothalamus, thereby increasing extracellular levels of adenosine and leading to activation of membrane adenosine receptors, cAMP production and IR-beta-EP secretion from these neurons. Chronic ethanol desensitizes the adenosine-regulated cAMP production and IR-beta-EP release from hypothalamic neurons.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Effect of Rp diastereoisomer of adenosine 3',5' cyclic-monophosphothioate on the cAMP-dependent relaxation of smooth muscle.

The effect of Rp diastereoisomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS) on relaxation elicited by histamine (1-100 microM), forskolin (1-60 microM), papaverine (1-100 microM), vinpocetine (1-100 microM), rolipram (0.1-1 mM), Sp-cAMPS (10-300 microM), 8-BrcAMP (10 microM - 1 mM) and 8-BrcGMP (3 microM - 1 mM) of the previous vanadate-induced contraction was assayed. The effect of Rp-cAMPS on the relaxing effect produced by forskolin, papaverine, vinpocetine, rolipram, Sp-cAMPS and 8-BrcAMP in KCl-induced tonic contraction was also assayed. Histamine, forskolin, papaverine, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP, but not vinpocetine, relaxed the vanadate-induced contractions in rat uterus incubated in medium lacking calcium plus EDTA in a concentration-dependent way. Rp-cAMPS (1-300 microM) had no effect on vanadate contraction. However, it antagonized the relaxation elicited by histamine and papaverine, but not that of forskolin, rolipram, Sp-cAMPS, 8-BrcAMP and 8-BrcGMP. Forskolin, papaverine, vinpocetine, rolipram and 8-BrcAMP, but not Sp-cAMPS, relaxed the KCl-induced contraction. Rp-cAMPS antagonized the relaxation elicited by forskolin, papaverine and vinpocetine, but not that of rolipram and 8-BrcAMP. Our results suggest that: a) Rp-cAMPS is an effective PKA inhibitor that could be used to study the involvement of cAMP on drug-induced response in smooth muscle, and b) the effects of Sp-cAMPS, 8-BrcAMP and rolipram were independent of the activation of protein kinases.

Animals↗
PubMed2003

Hypothalamic administration of cAMP agonist/PKA activator inhibits both schedule feeding and NPY-induced feeding in rats.

Following central administration, neuropeptides that decrease the level of cAMP induce feeding. Conversely, cAMP activating neuropeptides tend to elicit satiety. When the inhibitory effect of neuropeptide Y (NPY) on the hypothalamic cAMP production was blocked by pertussis toxin, the potent orexigenic effect of NPY was lost. These findings suggest that there may be a link between hypothalamic cAMP and the central regulation of food intake. In this report, we show that the injection of the membrane-permeable cAMP agonist, adenosine-3',5'-cyclic monophosphorothioate Sp-isomer (Sp-cAMP), into perifornical hypothalamus (PFH) significantly inhibited schedule-induced and NPY-induced food intake for up to 4h. This inhibitory effect was normalized within 24h. A taste aversion could not be conditioned to Sp-cAMP treatment, suggesting that the anorectic response was not due to malaise. Sp-cAMP administration significantly increased the active protein kinase A (PKA) activity in dorsomedial (DMH) and ventromedial (VMH), but not in lateral (LH) hypothalamus. Consistently, food deprivation lowered, while refeeding normalized endogenous cAMP content in DMH and VMH, but not in LH areas. No significant effect of adenosine-3',5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP, cAMP antagonist) was observed on hypothalamic PKA activity, schedule-induced, or NPY-induced food intake. These findings suggest that the increase in cAMP level and PKA activity in DMH and VMH areas may trigger a satiety signal.

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