cAMP-PKA Signal Transduction Pathway Research Articles

Effects of liver depression and psychological stress on human uterine leiomyoma cells by an AR–cAMP–PKA signal transduction pathway

ObjectiveBased on the emotional theory of Traditional Chinese Medicine, and combined with the modern medicine theory of psychological stress, a research model of human uterine leiomyoma cells (ULM) was cultured in vitro to determine the effectiveness of adrenergic receptor (AR) agonists in human ULM cell growth. In addition, we studied the functional influence of “liver depression and psychological stress theory” on fibroid formation by intervening in the AR–cAMP–PKA signaling pathway. The intention was to establish a new method to prevent and cure fibroids through “liver depression and psychological stress theory” and provide an experimental basis for the Traditional Chinese Medicine emotional theory. Materials and methodsPrimary human ULM cells were enriched by collagenase digestion. Immunohistochemistry and hematoxylin and eosin (HE) staining were used for cytological identification. Using this model, we studied intervention using specific AR agonists on ULM cells to observe the influence of “liver depression and psychological stress theory” on estrogen receptor (ER), progesterone receptor (PR), vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF). ResultsNorepinephrine (NE) and epinephrine (E) are adrenergic receptor agonists. They promoted ULM cell proliferation and increased the levels of ER, PR, VEGF and FGF. In contrast, isoproterenol (ISO) inhibited ULM cell proliferation and decreased the levels of ER, PR, VEGF and FGF. The protein expression of cAMP and PKA in ULM cells was reduced and the levels of ER, PR, VEGF and FGF were increased when co-treatment with the α-AR blocker (phentolamine). The β-AR blocker (metoprolol) displayed an opposite effect. ConclusionsAR agonists modulated ER, PR, VEGF and FGF levels in ULM cells in an AR–cAMP–PKA-dependent signaling pathways to influence fibroid occurrence and development.

Role of cAMP-PKA signal transduction pathway in lidocaine-induced up-regulation of expression of surfactant protein-A in rat alveolar epithelial type II cells

Objective To evaluate the role of cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal transduction pathway in lidocaine-induced up-regulation of the expression of surfactant protein-A (SP-A) in rat alveolar epithelial type Ⅱ cells (AEC Ⅱ).Methods Healthy male Sprague-Dawley rats were sacrificed and AEC Ⅱ were isolated,purified and incubated in 24-well culture plates (100μd/hole) with density of 1 × 106/ml.After being incubated for 2 h,the culture medium was replaced with serum-free medium DMEM.The cells were randomly divided into 4 groups (n =48 each):control group (group C),forskolin (adenylate cyclase agonist) group (group F),lidocaine 200 μg/ml group (group L),and PKA inhibitor H89 + L group (group P+ L).Forskolin 10 μmol/ml was added to DMEM in group F.Lidocaine 200 μg/ml was added to DMEM in group L.H89 10μnol/ ml was added to DMEM and AEC Ⅱ were incubated for 10 min,and then lidocaine 200 μg/ml was added in group P + L.At 6,12 and 24 h of incubation (T1-3),cAMP content and PKA activity (using ELISA),and expression of SP-A mRNA (by real-time fluorescent quantitative PCR) and SP-A (by Western blot) were measured.Results Compared with group C,the expression of SP-A mRNA and SP-A was significantly upregulated,and cAMP level and PKA activity were increased at T1-3 in group F and at T2,3 in group L.Compared with group L,the expression of SP-A and SP-A mRNA was down-regulated,PKA activity was decreased,and no significant change was found in cAMP level at T1-3 in group P + L.Conclusion Lidocaine can up-regulate the expression of SP-A in AEC Ⅱ of rats through activating cAMP-PKA signal transduction pathway. Key words: Lidocaine; Cyclic AMP; Cyclic AMP-dependent protein kinases; Epithelial cells; Pulmonary alveoli; Pulmonary surfactant-associated protein A

Regulation of aquaporin 3 protein expression in amnion epithelial cells through cAMP-PKA signal pathway

To investigate the expression of aquaporins-3 (AQP3) in amniotic epithelial cells regulated by cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signal pathway and to explore the mechanisms of its expression. The amniotic epithelial cells were collected from 30 patients who underwent elective caesarean sections at term with normal amniotic fluid volume and primarily cultured. The cultured cells were treated with (1)forskolin groups: different concentration (0, 2.5, 5, 50 or 100 µmol/L) of forskolin treated cells for 2 hours, and the optimal concentration of forskolin treated cells with different time (0, 1, 2, 10 or 20 hours); (2)SP-cAMP groups: different concentration (0, 2.5, 5, 50 or 100 µmol/L) of SP-cAMP treated cells for 2 hours, and the optimal concentration of SP-cAMP treated cells with different time (0, 1, 2, 10 or 20 hours); (3)H-89 groups: different concentration (0, 5, 10, 50 or 100 µmol/L) of H-89 treated cells for 2 hours, and the optimal concentration of H-89 treated cells with different time (0, 1, 2, 10 or 20 hours ). The level of intracellular cAMP and activity of PKA were detected by using ELISA, and immunohistochemistry was used to detect the localization of AQP3, the protein expression of total cAMP-response element binding protein (CREB) and phospho-CREB (p-CREB) and AQP3 were assessed by western blot analysis. Cell proliferation was assessed by cell counting kit-8 (CCK-8) assay. (1) The brown staining of AQP3 was detected in both cell membrane and cytoplasm in each group. (2) There was no significant change of the cell proliferation rate among groups with different concentration of forskolin, SP-cAMP and H-89 treatment (P > 0.05). (3) After different concentration of forskolin treated 2 hours, the expression of total CREB had no significant difference among them(P > 0.05). While the expression of cAMP level, PKA activity, p-CREB and AQP3 protein were significantly changed, which were higher in 2.5 µmol/L, 5 µmol/L, 50 µmol/L forskolin group when compared with 0 µmol/L (P < 0.05). Their expressions in 5 µmol/L forskolin group were higher than that in 2.5 µmol/L and 50 µmol/L (P < 0.05). The optimal forskolin concentration was 5 µmol/L. (4) After different concentration of SP-cAMP treated 2 hours, the expression of total CREB and cAMP level had no significant difference among them (P > 0.05), while the expression of PKA activity, p-CREB and AQP3 protein were significantly changed, which were higher in 5 µmol/L, 50 µmol/L SP-cAMP group when compared with 0 µmol/L (P < 0.05). Their expressions in 50 µmol/L SP-cAMP group were higher than that in 5 µmol/L (P < 0.05). The optimal SP-cAMP concentration was 50 µmol/L. (5) After different concentration of H-89 treated 2 hours, the expression of total CREB and cAMP level had no significant difference among them (P > 0.05), while the expression of PKA activity, p-CREB and AQP3 protein were significantly changed, which were lower in 10 µmol/L, 50 µmol/L and 100 µmol/L H-89 group when compared with 0 µmol/L (P < 0.05). Their expressions in 10 µmol/L H-89 group were lower than that in 50 µmol/L, 100 µmol/L (P < 0.05). The optimal H-89 concentration was 10 µmol/L. (6) p-CREB and AQP3 protein expression were significantly lower in 5 µmol/L forskolin combined 10 µmol/L H-89 incubating 2 hours group when compared with 5 µmol/L forskolin, but higher than that in 10 µmol/L H-89 treated group (P < 0.05). Total CREB was no significant difference among the three groups (P > 0.05). cAMP-PKA signal transduction pathway may regulate AQP3 protein expression in human amniotic epithelial cells.

A putative phospholipase C is involved in Pichia fermentans dimorphic transition

BackgroundPichia fermentans DiSAABA 726 is a dimorphic yeast that reversibly shifts from yeast-like to pseudohyphal morphology. This yeast behaves as a promising antagonist of Monilia spp. in the yeast-like form, but becomes a destructive plant pathogen in the pseudohyphal form thus raising the problem of the biological risk associated with the use of dimorphic yeasts as microbial antagonists in the biocontrol of phytopathogenic fungi. MethodsPichia fermentans DiSAABA 726 was grown in urea- and methionine-containing media in order to induce and separate yeast-like and pseudohyphal morphologies. Total RNA was extracted from yeast-like cells and pseudohyphae and retro-transcribed into cDNA. A rapid subtraction hybridization approach was utilized to obtain the cDNA sequences putatively over-expressed during growth on methionine-containing medium and involved in pseudohyphal transition. ResultsFive genes that are over-expressed during yeast-like/pseudohyphal dimorphic transition were isolated. One of these, encoding a putative phospholipase C, is involved in P. fermentans filamentation. In fact, while the inhibition of phospholipase C, by means of 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine (Et-18), is accompanied by a significant reduction of pseudohyphae formation in P. fermentans, the addition of exogenous cAMP fully restores pseudohyphal growth also in the presence of Et-18. ConclusionPhospholipase C is part of a putative “methionine sensing machinery” that activates cAMP-PKA signal transduction pathway and controls P. fermentans yeast-like/pseudohyphal dimorphic transition. General significancePhospholipase C is a promising molecular target for further investigations into the link between pseudohyphae formation and pathogenicity in P. fermentans.

Role of cAMP-PKA signal transduction pathway in the ischemic preconditioning-induced attenuation of ischemia-reperfusion injury In isolated rat hearts

Objective To investigate the role of cAMP-PKA signal transduction pathway in the ischemie pleeonditioning-induced attenuation of ischemia.1eperfusion(ITR)injury in isohted rat hearts.Methods Fifty healthy adult SD rats weighing 300-350 g were anesthetized with intraperitoneal(IP)pentobarbital 50 meg/kg.Their he.arts were excised and perfused in a Langendorff apparatus with 37℃ oxygenated(95%O2-5%CO2)modified K-H solution at a constant pressure of 70 cm H2O,and randomly divided into 5 groups(n=10 each):group Ⅰ I/R;group Ⅱ ischemic preconditioning(IPC);groupⅢH89(PKA inhibitor);group Ⅳ PDTC(NF-κB inhibitor)and group Ⅴ db-cAMP.The experiment started after 10 min stabilization.The isolated hearts were tinct perfuzed for 30 min.followed by 60 min ischemia and 30 min leperfusion in group I/R(Ⅰ).Group IPC(Ⅱ)w88 subjected to 3 episodes of 5 min ischemia at 5 min intervals before I/R.Group Ⅲ and Ⅳ received 5 min perfnsion withH89 10 μmol/L and PDTC 100 μmol/L 3 times at 5 min intervals respectively before I/R.Group Ⅴ was peffnsed with db-cAMP 200 μmol/L for 30 min before I/R.Left ventricular developed pressure(LVDP)and ± dp/dtu were measured at stabilization period and 10, 20, 30 rain of reperfusion. Coronary flow (CF) was measured at stabilization period and 30 min of reperfusion and activities of LDH and creafinc kinase (CK) in the coronary effluent were determined. The myocardial specimens were obtained at 30 rain of reperfusion for determination of NF-κB-DNA binding activity (by EMSA) and expression of TNF-κ mBNA (by RT-PCR ) and p-CBEB (Ser133) (by Western blot). Results Compared with 1/R group, NF-κB-DNA binding activity and TNF-α mRNA expression were significandy decreased, ± dp/dt and CF were significandy increased, CK and LHD activities in the coronary effluent were significantly decreased in group IPC and db-cAMP (group Ⅱ , Ⅴ ) and p-CREB (Ser133) expression was significantly increased in group IPC, PDTC and db-cAMP (group Ⅱ , Ⅳ,Ⅴ ). Compared with IPC group, NF-κB-DNA binding activity and TNF-α mBNA expression were significantly increased, ± dp/dtmax, LVDP and CF were significantly decreased, CK and LDH activities were significantly increased in group H89 and PDTC (group Ⅲ, Ⅳ ) and p-CREB (Ser133) expression was significantly decreased in group H89(group Ⅲ ). Conclusion lschemic preconditioning can attenuate I/R injury in isolated hearts by inhibition of NF-κB-DNA binding activity via cAMP-PKA signal transduction pathway which reduces gene transcription of inflammatory cytokine. Key words: Cyclic AMP; Cyclic AMP-dependent protein kinases; Ischemic preconditioning; Myocardial reperfusion injury

A novel mechanism linking corticotrophin‐releasing factor to ventral tegmental area dopamine neuron firing

A novel mechanism linking corticotrophin‐releasing factor to ventral tegmental area dopamine neuron firing

Protein Kinase A Regulates Growth, Sporulation, and Pigment Formation in Aspergillus fumigatus

Aspergillus fumigatus is an opportunistic human pathogenic fungus causing severe infections in immunocompromised patients. Cyclic AMP (cAMP) signal transduction plays an important role in virulence. A central component of this signaling cascade is protein kinase A (PKA), which regulates cellular processes by phosphorylation of specific target proteins. Here we describe the generation and analysis of A. fumigatus mutants expressing the gene encoding the catalytic subunit of PKA, pkaC1, under control of an inducible promoter. Strains overexpressing pkaC1 showed high PKA activity, reduced growth, sporulation deficiency, and formation of a dark pigment in the mycelium. These data indicate that cAMP-PKA signaling is involved in the regulation of important processes, such as growth, asexual reproduction, and biosynthesis of secondary metabolites. Furthermore, elevated PKA activity led to increased expression of the pksP gene. The polyketide synthase PksP is an essential enzyme for production of dihydroxynaphthalene-melanin in A. fumigatus and contributes to virulence. Our results suggest that increased pksP expression is responsible for pigment formation in the mycelium. Comparative proteome analysis of the pkaC1-overexpressing strain and the wild-type strain led to the identification of proteins regulated by the cAMP-PKA signal transduction pathway. We showed that elevated PKA activity resulted in activation of stress-associated proteins and of enzymes involved in protein biosynthesis and glucose catabolism. In contrast, proteins which were involved in nucleotide and amino acid biosynthesis were downregulated, as were enzymes involved in catabolism of carbon sources other than glucose.

Effects of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) Hull Extracts on the Secretion of Progesterone and Estradiol In Vivo and In Vitro

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional Chinese medicine for dysfunction of the endocrine system. However, there have been few studies on the effects of adlay seed on the endocrine system. In the present study, both the in vivo and in vitro effects of methanolic extracts of adlay hull (AHM) on progesterone synthesis were studied. AHM was partitioned with four different solvents: water, 1-butanol, ethyl acetate, and n-hexane. Four fractions, namely, AHM-Wa (water fraction), AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction), and AHM-Hex (n-hexane fraction), were respectively obtained. Granulosa cells (GCs) were prepared from pregnant mare serum gonadotropin-primed immature female rats and were challenged with different reagents, including human chorionic gonadotropin (hCG; 0.5 IU/ml), 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP; 0.1 mM), forskolin (10 microM), 25-OH-cholesterol (10 microM), and pregnenolone (10 microM), in the presence or absence of AHM (100 microg/ml). The functions of steroidogenic enzymes, including protein expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc), protein kinase A (PKA), and aromatase activity, were investigated. The expression of StAR mRNA was also explored by using real-time reverse transcription-polymerase chain reaction. In the in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM (2 mg/ ml/kg). In the in vitro studies, AHM decreased progesterone and estradiol via inhibition of (i) the cAMP-PKA signal transduction pathway, (ii) cAMP accumulation, (iii) P450scc and 3beta-HSD enzyme activities, (iv) PKA, P450scc and StAR protein expressions and StAR mRNA expression, and (v) aromatase activity in rat GCs. These results suggest that AHM decreased the production of progesterone via mechanisms involving the inhibition of the cAMP pathway, enzyme activities, and the protein expressions of P450scc and StAR in rat GCs.

Downregulation of progesterone biosynthesis in rat granulosa cells by adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) bran extracts

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has long been used as a traditional Chinese medicine for dysfunctions of the endocrine system and inflammation conditions. However, the effect of adlay seed on the endocrine system has not yet been reported. In the present study, the effects and the mechanisms of methanolic extract of adlay bran (ABM) on progesterone synthesis in rat granulosa cell were studied. ABM was further partitioned with different solvents including water, 1-butanol, ethyl acetate and n-hexane. Four subfractions named ABM-Wa (water fraction), ABM-Bu (1-butanol fraction), ABM-EA (ethyl acetate fraction) and ABM-Hex (n-hexane fraction) were obtained. ABM-Bu was further fractionated using Diaion HP-20 resin column chromatography with gradient elution. Granulosa cells were prepared from pregnant mare serum gonadotropin-primed immature female rats and challenged with different reagents including human chorionic gonadotropin (hCG 0.5 IU/ml), forskolin (10 microM), 8-bromo-adenosine-3',5'-cyclic monophosphate (8-Br-cAMP, 1 mM), A23187 (10 microM), phorbol 12-myristate 13-acetate (PMA, 0.01 microM), 25-OH-cholesterol (0.1-10 microM) and pregnenolone (0.1-10 microM) in the presence or absence of ABM-Bu (100 microg/ml). The functions of steroidogenic enzyme including protein expression of the steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage enzyme (P450scc) protein were investigated. Expressions of both P450scc and StAR mRNA have also been explored. We found that ABM decreased progesterone production via an inhibition on (1) the cAMP-PKA and PKC signal transduction pathway, (2) P450scc and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzyme activity, (3) P450scc and StAR protein and mRNA expressions and (4) the phosphorylation of ERK1/2 in rat granulosa cells.

The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To determine the importance of the cyclic AMP (cAMP) signaling pathway for virulence, the pkaC1 gene encoding a protein kinase A (PKA) catalytic subunit was cloned and characterized. Deletion of pkaC1 led to reduced conidiation and growth. PKA activity was not detectable in DeltapkaC1, DeltagpaB, and DeltaacyA mutant strains. gpaB and acyA encode a G protein alpha subunit involved in cAMP signal transduction and adenylate cyclase, respectively. Addition of cAMP led to PKA activity in crude extracts of both the DeltagpaB and DeltaacyA strains but not in crude extracts of the DeltapkaC1 strain. These findings provide evidence that PKAC1 represents the predominant form of PKA under the conditions tested, and GPAB and ACYA are members of the cAMP signaling cascade. Analysis of a pksPp-lacZ gene fusion indicated that the expression of the pathogenicity determinant-encoding pksP gene was reduced in DeltapkaC1 mutant strains compared with the expression of the gene fusion in the parental strain. In a low-dose murine inhalation model, conidia of both the DeltapkaC1 and DeltagpaB mutant strains were almost avirulent. Taken together, these findings indicate that the cAMP-PKA signal transduction pathway is required for A. fumigatus pathogenicity.

The cAMP–Protein Kinase A Signal Transduction Pathway Modulates Ethanol Consumption and Sedative Effects of Ethanol

Ethanol and other drugs of abuse modulate cAMP-PKA signaling within the mesolimbic reward pathway. To understand the role of the cAMP-PKA signal transduction in mediating the effects of ethanol, we have studied ethanol consumption and the sedative effects of ethanol in three lines of genetically modified mice. We report that mice with the targeted disruption of one Gsalpha allele as well as mice with reduced neuronal PKA activity have decreased alcohol consumption compared with their wild-type littermates. Genetic reduction of cAMP-PKA signaling also makes mice more sensitive to the sedative effects of ethanol, although plasma ethanol concentrations are unaffected. In contrast, mice with increased adenylyl cyclase activity resulting from the transgenic expression of a constitutively active form of Gsalpha in neurons within the forebrain are less sensitive to the sedative effects of ethanol. Thus, the cAMP-PKA signal transduction pathway is critical in modulating sensitivity to the sedative effects of ethanol as well as influencing alcohol consumption.

Role of Egr-1 in cAMP-dependent protein kinase regulation of the phenylethanolamine N-methyltransferase gene.

The molecular mechanism by which cAMP activates the rat phenylethanolamine N-methyltransferase (PNMT) gene was examined by transient transfection of the wild-type rat PNMT promoter-luciferase reporter gene construct pGL3RP893 into PC12 cells. Forskolin treatment (10 microM) of the transfected cells for 3--6 h maximally induced luciferase threefold. Induction by forskolin was mimicked by the cAMP analog, 8-Br-cAMP, and prevented in PC12 cells pretreated with the protein kinase A (PKA) inhibitor H-89 or co-transfected with an expression construct for PKI, a polypeptide inhibitor of PKA. Furthermore, forskolin did not activate the PNMT promoter when the 893 bp PNMT promoter-reporter gene construct was transfected into the PKA-deficient cell line, A126. Detailed examination of the forskolin responsiveness of PNMT constructs harboring > or = 60 bp and < 893 bp of PNMT promoter demonstrated that the cAMP-responsive element(s) lay between < 392 bp and > or =60 bp. Within this region of the promoter lies a functional binding element for Egr-1, a transcriptional activator of the PNMT gene. Forskolin treatment of PC12 cells also rapidly increased nuclear levels of Egr-1 and the catalytic subunit of PKA (PKA-C), with the rise in PKA-C preceding that of Egr-1. Mutation of the --165 bp Egr-1 site markedly decreased forskolin activation of the PNMT promoter. These findings demonstrate that the rat PNMT gene promoter can be activated via the cAMP-PKA signal transduction pathway, mediated by the immediate early gene transcription factor, Egr-1.

Regulation of ionic conductances and gene expression by hypoxia in an oxygen sensitive cell line.

We have shown that the PC12 cell line is an excellent model system for investigations of the molecular and cellular processes involved in O2-chemosensitivity. We have identified an O2-sensitive K channel in this cell line that mediates membrane depolarization, an increase in intracellular free Ca2+, and dopamine release during hypoxia. We also presented evidence which shows that expression of the gene for tyrosine hydroxylase, the rate-limiting enzyme in dopamine biosynthesis, is stimulated by reduced O2 tension in PC12 and type I carotid body cells. In addition, we have successfully identified the DNA sequences and trans-acting protein factors that regulate transcription of the TH gene during hypoxia. The mechanisms by which a reduction in O2 tension is transduced into alter cell function including increased gene expression remain unknown. Unpublished results from our laboratory show that the increased TH gene expression during hypoxia does not require activation of the cAMP-PKA signal transduction pathway. We propose that the increase in intracellular free Ca2+ that occurs as a result of membrane depolarization might play an important role. Preliminary findings from our laboratory show that blockade of the voltage operated Ca2+ channel or chelation of intracellular Ca2+ prevent full activation of the TH gene during hypoxia.