Role of Epac and protein kinase A in thyrotropin-induced gene expression in primary thyrocytes

cAMP pathway activation by thyrotropin (TSH) induces differentiation and gene expression in thyrocytes. We investigated which partners of the cAMP cascade regulate gene expression modulations: protein kinase A and/or the exchange proteins directly activated by cAMP (Epac). Human primary cultured thyrocytes were analysed by microarrays after treatment with the adenylate cyclase activator forskolin, the protein kinase A (PKA) activator 6-MB-cAMP and the Epac-selective cAMP analog 8-pCPT-2'-O-Me-cAMP (007) alone or combined with 6-MB-cAMP. Profiles were compared to those of TSH. Cultures treated with the adenylate cyclase- or the PKA activator alone or the latter combined with 007 had profiles similar to those induced by TSH. mRNA profiles of 007-treated cultures were highly distinct from TSH-treated cells, suggesting that TSH-modulated gene expressions are mainly modulated by cAMP and PKA and not through Epac in cultured human thyroid cells. To investigate whether the Epac-Rap-RapGAP pathway could play a potential role in thyroid tumorigenesis, the mRNA expressions of its constituent proteins were investigated in two malignant thyroid tumor types. Modulations of this pathway suggest an increased Rap pathway activity in these cancers independent from cAMP activation.     

MAP kinase activation by mu opioid receptor involves phosphatidylinositol 3-kinase but not the cAMP/PKA pathway.

The involvement of protein kinases was studied in mu opioid receptor activation of mitogen-activated protein (MAP) kinase using cells transfected with the receptor clone. The cAMP/protein kinase A (PKA) pathway is known to be the major biochemical pathway for mu opioid receptor signaling. However, our data showed that stimulating adenylyl cyclase or activating PKA had no effect on mu receptor enhancement of MAP kinase activity, suggesting that the cAMP/PKA pathway is not involved in mediating the mu receptor activation of MAP kinase. Inhibition of phosphatidylinositol (PI) 3-kinase reduced mu receptor enhancement of MAP kinase activity, suggesting PI 3-kinase involvement. Together, these results show that cross-talk between the mu opioid receptor and the MAP kinase cascade is not mediated by the cAMP/PKA pathway, but involves PI 3-kinase.     

Dissection of the signaling mechanism for capsule detachment of lipid droplets in rat adrenocortical cells

In a previous study, we used a monoclonal antibody, A2, to demonstrate the presence of the lipid droplet-specific capsule in adrenocortical cells and the stimulation of steroid secretion with adrenocorticotrophic hormone (ACTH), resulting in the detachment of this capsule from the droplet surface into the cytosol. To investigate the signaling pathway for this event, we tested the role of adenylate cyclase, cAMP, and protein kinases A and C (PKA and PKC) in this response. ACTH-induced decapsulation of lipid droplets was blocked by either adenylate cyclase inhibitor or PKA inhibitor and stimulated by Bt₂cAMP. We conclude that the signaling mechanism involved in lipid droplet decapsulation is the cascade consisting of adenylate cyclase activation, cAMP elevation, and subsequent PKA activation. Furthermore, the cytosolic detached capsular protein was able to relocate to the lipid droplet surface on cessation of ACTH or Bt₂cAMP stimulation. In addition to PKA-mediated decapsulation, inhibition of PKC by calphostin C alone was enough to induce decapsulation, a process that was independent of PKA activity, whereas activation of PKC could prevent Bt₂cAMP-induced decapsulation. A cAMP radioimmunoassay also confirmed that ACTH caused a marked increase in intracellular levels of cAMP, while PMA or calphostin C caused no significant changes. We conclude that PKA and PKC are reciprocally operated to regulate the decapsulation of lipid droplets, the same mechanism adopted in steroidogenesis. A time-course study also indicates that decapsulation of lipid droplets was accompanied by detectable changes in the size and the area of lipid droplets upon the stimulation of Bt₂cAMP or calphostin C, implying a possible coupling between the capsule detachment and steroidogenesis. J. Cell. Biochem. 65:67–74. © 1997 Wiley-Liss, Inc.     

Calcium-stimulated adenylyl cyclase activity is critical for hippocampus-dependent long-term memory and late phase LTP.

It is hypothesized that Ca2+ stimulation of calmodulin (CaM)-activated adenylyl cyclases (AC1 or AC8) generates cAMP signals critical for late phase LTP (L-LTP) and long-term memory (LTM). However, mice lacking either AC1 or AC8 exhibit normal L-LTP and LTM. Here, we report that mice lacking both enzymes (DKO) do not exhibit L-LTP or LTM. To determine if these defects are due to a loss of cAMP increases in the hippocampus, DKO mice were unilaterally cannulated to deliver forskolin. Administration of forskolin to area CA1 before training restored normal LTM. We conclude that Ca2+-stimulated adenylyl cyclase activity is essential for L-LTP and LTM and that AC1 or AC8 can produce the necessary cAMP signal.     

Modulation on tetrodotoxin-resistant sodium current of loureirin B in rat dorsal root ganglion neurons via cyclic AMP–dependent protein kinase A

To search the modulation mechanism of loureirin B, a flavonoid is extracted from Dracaena cochinchinensis, on tetrodotoxin-resistant (TTX-R) sodium channel in dorsal root ganglion (DRG) neurons of rats. Experiments were carried out based on patch-clamp technique and molecular biological methods. We observed the time-dependent inhibition of loureirin B on TTX-R sodium currents in DRG neurons and found that neither occupancy theory nor rate theory could well explain the time-dependent inhibitory effect of loureirin B on TTX-R sodium currents. It suggested that a second messenger-mediated signaling pathway may be involved in the modulation mechanism. So the cyclin AMP (cAMP) level of the DRG neurons before and after incubation with loureirin B was tested by ELISA Kit. Results showed that loureirin B could increase the cAMP level and the increased cAMP was caused by the enhancement of adenylate cyclase (AC) induced by loureirin B. Immunolabelling experiments further confirmed that loureirin B can promote the production of PKA in DRG neurons. In the presence of the PKA inhibitor H-89, the inhibitory effect of loureirin B on TTX-R sodium currents was reversed. Forskolin, a tool in biochemistry to raise the levels of cAMP, also could reduce TTX-R sodium currents similar to that of loureirin B. These studies demonstrated that loureirin B can modulate the TTX-R sodium channel in DRG neurons via an AC/cAMP/PKA pathway involving the activation of AC and PKA, which also can be used to explain the other pharmacological effects of loureirin B.     

Novel mechanisms in nutrient activation of the yeast protein kinase A pathway

In yeast the Protein Kinase A (PKA) pathway can be activated by a variety of nutrients. Fermentable sugars, like glucose and sucrose, trigger a spike in the cAMP level, followed by activation of PKA and phosphorylation of target proteins causing a.o. mobilization of reserve carbohydrates, repression of stress-related genes and induction of growth-related genes. Glucose and sucrose are sensed by a G-protein coupled receptor system that activates adenylate cyclase and also activates a bypass pathway causing direct activation of PKA. Addition of other essential nutrients, like nitrogen sources or phosphate, to glucose-repressed nitrogen- or phosphate-starved cells, also triggers rapid activation of the PKA pathway. In these cases cAMP is not involved as a second messenger. Amino acids are sensed by the Gap1 transceptor, previously considered only as an amino acid transporter. Recent results indicate that the amino acid ligand has to induce a specific conformational change for signaling. The same amino acid binding site is involved in transport and signaling. Similar results have been obtained for Pho84 which acts as a transceptor for phosphate activation of the PKA pathway. Ammonium activation of the PKA pathway in nitrogen-starved cells is mediated mainly by the Mep2 transceptor, which belongs to a different class of transporter proteins. Hence, different types of sensing systems are involved in control of the yeast PKA pathway by nutrients.     

CFTR-Adenylyl Cyclase I Association Responsible for UTP Activation of CFTR in Well-Differentiated Primary Human Bronchial Cell Cultures

Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca²⁺-activated chloride channel (CaCC) is activated by Ca²⁺ agonists like UTP. We found that most chloride current elicited by Ca²⁺ agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca²⁺ activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca²⁺ agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca²⁺ agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca²⁺/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1–CFTR association is responsible for Ca²⁺/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca²⁺ agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.     

Genome-wide expression analyses of gene regulation during early development of Dictyostelium discoideum

Using genome-wide microarrays, we recognized 172 genes that are highly expressed at one stage or another during multicellular development of Dictyostelium discoideum. When developed in shaken suspension, 125 of these genes were expressed if the cells were treated with cyclic AMP (cAMP) pulses at 6-min intervals between 2 and 6 h of development followed by high levels of exogenous cAMP. In the absence of cAMP treatment, only three genes, carA, gbaB, and pdsA, were consistently expressed. Surprisingly, 14 other genes were induced by cAMP treatment of mutant cells lacking the activatable adenylyl cyclase, ACA. However, these genes were not cAMP induced if both of the developmental adenylyl cyclases, ACA and ACR, were disrupted, showing that they depend on an internal source of cAMP. Constitutive activity of the cAMP-dependent protein kinase PKA was found to bypass the requirement of these genes for adenylyl cyclase and cAMP pulses, demonstrating the critical role of PKA in transducing the cAMP signal to early gene expression. In the absence of constitutive PKA activity, expression of later genes was strictly dependent on ACA in pulsed cells.     

Schizophrenia and reduced cyclic AMP production: evidence for the role of receptor-linked events

Reduced cyclic AMP (cAMP) production has been found in platelets of schizophrenic patients. cAMP is generated physiologically as a result of a series of steps beginning with receptor activation by a ligand, progressing through activation of the enzyme protein, adenylate cyclase. The deficit of cAMP found in the schizophrenic population may occur at any one, or at multiple steps in this cascade. The present study attempts to discriminate whether impaired adenylate cyclase itself was responsible for the cAMP deficit or whether abnormalities in receptor events or linkage are present in schizophrenics. The production of cAMP following direct stimulation of adenylate cyclase by NaF was contrasted with receptor mediated activation of adenylate cyclase by prostaglandin E1 (PGE1) in disrupted platelet preparations from schizophrenics and normal controls. cAMP formation stimulated by NaF was not different in platelets of schizophrenics as compared to controls, however, platelets of schizophrenics showed reduced response to PGE1 stimulation. The authors interpret these findings as evidence for a membrane associated abnormality of either receptor or receptor-adenylate cyclase linkage in the schizophrenias.     

Evidence for adenylate cyclase as a scaffold protein for Ras2–Ira interaction in Saccharomyces cerevisie

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.     

Regulation of melanosome movement by MAP kinase

Our objectives were to further characterize the signaling pathways in melatonin-induced aggregation in Xenopus melanophores, specifically to investigate a possible role of mitogen-activated protein kinase (MAPK). By Western blotting we found that melatonin activates MAPK, which precedes melanosome aggregation measured in a microplate reader. Activation of MAPK, tyrosine phosphorylation of a previously described 280-kDa protein, and melanosome aggregation are sensitive to PD98059, a selective inhibitor of MAPK kinase. The MAPK activation is also decreased by the adenylate cyclase stimulant forskolin. In summary, we found that MAPK is activated during melatonin-induced melanosome aggregation. Activation was decreased by an inhibitor of MAPK kinase, and by forskolin. In addition to inhibition of cyclic adenosine 3',5'-monophosphate (cAMP), reduction in protein kinase A activity (PKA), and activation of protein phosphatase 2A, we suggest that melatonin receptors activate the MAPK cascade and tyrosine phosphorylation of the 280-kDa protein. Although the cAMP/PKA signaling pathway is the most prominent, our data suggest that simultaneous activation of the MAPK cascade is of importance to obtain a completely aggregated state. This new regulatory mechanism of organelle transport by the MAPK cascade might be important in other eukaryotic cells.     

Oxytocin receptors differentially signal via Gq and Gi proteins in pregnant and nonpregnant rat uterine myocytes: implications for myometrial contractility

Oxytocin (OT) receptors are important regulators of myometrial contractility. By using the activity of large conductance Ca2+-activated K+ (BKCa) channels as readout, we analyzed OT signaling in cells from nonpregnant (NPM) and pregnant (PM) rat myometrium in detail. In nystatin-perforated whole-cell patches from NPM cells, which leave the intracellular integrity intact, OT transiently increased BKCa-mediated outward currents (Iout). This OT-evoked Iout was caused by the Ca2+ transients in response to the Gq/11-mediated activation of phospholipase C and was inhibited by activation of protein kinase A (PKA). In an open-access whole-cell patch (OAP), the OT-induced transient rise in Iout was disrupted whereas the regulation of BKCa by the cAMP/PKA cascade remained intact. OT counteracted the isoprenaline, i.e. the beta-adrenoceptor/Gs-mediated effect in NPM cells measured in OAP. In contrast, OT further enhanced the beta-adrenoceptor/Gs-mediated effect on BKCa activity in PM cells. All OT effects in the OAP were mediated by pertussis toxin-sensitive Gi proteins and PKA. By quantitative real-time PCR and overexpression of the recombinant protein, we demonstrate that an up-regulation of the Gbetagamma-stimulated adenylyl cyclase II during pregnancy is most likely responsible for this switch. By studying the OT-evoked Iout in nystatin-perforated whole-cell patches of PM cells, we further detected that the OT receptor/Gibetagamma-mediated coactivation of adenylyl cyclase II enhanced the beta-adrenoceptor/Gs-induced suppression of the OT-evoked Ca2+ transients and thus diminishes and self-limits OT-induced contractility. The differential regulation of the PKA-mediated suppression of OT-evoked Ca2+ transients and BKCa activity likely supports uterine quiescence during pregnancy.     

Cross-talk between olfactory second messenger pathways.

The second messengers 3'-5'-cyclic-monophosphate (cAMP) and inositol 1,4,5-trisphosphate (InsP3) have been implicated in olfactory signal transduction in various species. The results of the present study provide evidence that the two olfactory second messenger pathways in rat olfactory neurons do not work independently but rather show a functional antagonism: whereas inhibition of phospholipase C (PLC) in isolated olfactory cilia by U-73122 led to an augmentation of odor-induced cAMP signaling, activation of the phosphoinositol pathway resulted in attenuation of odor-induced cAMP formation. Furthermore, this study indicates that elevated cAMP levels cause suppression of odor-induced InsP3 signaling, whereas inhibition of adenylate cyclase (AC) by cisN-(2-phenylcyclopentyl)azacylotridec-1-en-2-amine (MDL-12,330 A) results in potentiation of odor-induced InsP3 formation. Concerning the molecular mechanism involved in cross-interaction, the experimental data indicate that the observed antagonism of elevated cAMP is based on inhibition of PLC activation rather than on stimulation of InsP3 degradation. As blockage of the endogenous protein kinase A (PKA) prevented the inhibitory effect of cAMP, the suppression of odor-induced InsP3 signaling by cAMP may be mediated by a PKA-controlled reaction.     

Glucose and GLP-1 stimulate cAMP production via distinct adenylyl cyclases in INS-1E insulinoma cells

In β cells, both glucose and hormones, such as GLP-1, stimulate production of the second messenger cAMP, but glucose and GLP-1 elicit distinct cellular responses. We now show in INS-1E insulinoma cells that glucose and GLP-1 produce cAMP with distinct kinetics via different adenylyl cyclases. GLP-1 induces a rapid cAMP signal mediated by G protein–responsive transmembrane adenylyl cyclases (tmAC). In contrast, glucose elicits a delayed cAMP rise mediated by bicarbonate, calcium, and ATP-sensitive soluble adenylyl cyclase (sAC). This glucose-induced, sAC-dependent cAMP rise is dependent upon calcium influx and is responsible for the glucose-induced activation of the mitogen-activated protein kinase (ERK1/2) pathway. These results demonstrate that sAC-generated and tmAC-generated cAMP define distinct signaling cascades.