Sirtuin 6 ameliorates arthritis through modulating cyclic AMP-responsive element binding protein/CCN1/cyclooxygenase 2 pathway in osteoblasts.

INTRODUCTION: CCN1 is an immediate-early gene product pivotal for arthritis progression. We have previously shown that sirtuin 6 (SIRT6) inhibited hypoxia-induced CCN1 expression in osteoblasts. Herein we examined the contribution of cyclic AMP-responsive element binding protein (CREB)/CRE to this suppressive action and the influence of CCN1 on cyclooxygenase (COX) 2 synthesis. MATERIALS AND METHODS: MC3T3-E1 murine osteoblasts were cultured under normoxia (21% oxygen) or hypoxia (2% oxygen). Expressions of CCN1, phospho-CREB (Ser133), COX2 and relevant kinases were assessed by Western blot. SIRT6 was overexpressed in cultured osteoblasts and arthritic joints by a lentiviral-based technique. Activities of CCN1 gene promoter constructs were examined by luciferase reporter assay. Interaction between CREB and CCN1 promoter was assessed by chromatin immunoprecipitation (ChIP). Collagen-induced arthritis (CIA) was established in 20 rats to evaluate the effects of SIRT6 therapy on osteoblastic expressions of phospho-CREB, CCN1 and COX2. RESULTS: SIRT6 suppressed hypoxia-enhanced CCN1 expression and CREB phosphorylation. Attenuation of calcium/calmodulin-dependent protein kinase II (CaMKII) may be responsible for SIRT6-induced CREB inhibition. CRE at - 286 bp upstream of the ATG start codon was essential for CCN1 expression under hypoxia and SIRT6 reduced hypoxia-stimulated CREB/CRE interaction. Forced expression of CREB rescued SIRT6-suppressed CCN1 synthesis. CCN1 induced COX2 expression in osteoblasts. In rat CIA, the therapeutic effect of SIRT6 was accompanied by decreases in osteoblastic expressions of phospho-CREB, CCN1 and COX2. CONCLUSION: Our study indicated that the benefits of SIRT6 to inflammatory arthritis and bone resorption are at least partially derived from its modulation of CREB/CCN1/COX2 pathway in osteoblasts.

Cyclic adenosine monophosphate (cAMP) analog and phosphodiesterase inhibitor (IBMX) ameliorate human sperm capacitation and motility.

OBJECTIVES: Human sperm quality is decreasing progressively. One of the foremost reasons for infertility is the failure in sperm capacitation. We examined the influence of a cAMP (cyclic-adenosine mono phosphate analog)+IBMX (3-isobutyl-1-methylxanthine) on the motility and capacitation rate of human sperm over time. MATERIAL AND METHODS: Samples were gotten from 20 asthenozoospermic infertile patients referring to the Academic Center for Education, Culture and Research unit of the infertility research center, Qom, Iran. Samples were processed with a Density Gradient Centrifuging. Spermatozoa were divided into 4 groups: control, experimental 1, 2 and 3 (E1, E2, E3) based on the dose/time schedules (cAMP 5mmol+IBMX 0.2mmol/2, 4, and 6h, respectively). The computer-assisted sperm analysis and chlortetracycline assays were used to measure sperm motility and capacitation. RESULTS: After incubation with a cAMP analog and IBMX, the levels of progressive motile sperms considerably improved in all experimental groups compared to the control group (E1=18.89±7.1, E2=30±9.7, E3=26.3±9.6 vs Control=10.28±6.2, P<0.05) especially in E2 group (P<0.05), indicating a greater effect of db cAMP (5mmol) and IBMX (0.2mmol) for 4h compared to the same doses at 2 and 6h. Also, non-progressive motile sperms significantly decreased in E2 group compared to the other groups (P<0.05). Moreover, both patterns C and B were substantially improved in all experimental groups especially in E2 group (P<0.05). CONCLUSION: Our findings support that the supplementation of sperm with db cAMP+IBMX specially for 4h, could be useful for men with asthenozoospermia to improve the success of assisted reproductive technology.

Effects of Hangeshashinto on the nasal physiological function: An in vitro study.

OBJECTIVE: Hangeshashinto is a Japanese Kampo medicine applied for the treatment of oral mucositis and gastroenteritis. Hangeshashinto exhibits broad-spectrum antibacterial activity and suppresses prostaglandin (PG)E2 production in the mucosa and has the ability to improve the inflammatory condition. In addition to these effects, because cAMP, a composition of Hangeshashinto, facilitates ciliary beat, Hangeshashinto could also improve the physiological function of the nasal mucosa, consist of ciliated epithelium, but details were unknown. METHODS: This study was aimed to investigate the effects of Hangeshashinto on the nasal mucosa. Healthy nasal mucosal sections were collected from the nasal septum of ten Japanese white rabbits, placed in a collagen dish for tissue culture, and rinsed with two different concentrations of Hangeshashinto solution (1.0%, n = 10 and 2.5%, n = 10) and cAMP solution (50µM, n=10 and 100 µM, n=10) or saline (control, n = 10). Ciliary beat frequency (CBF) as a physiological function of the nasal mucosa was recorded at 1, 3 and 7 days after rinsing, and histological evaluation of epithelial damage was performed at 7 days after rinsing. RESULTS: CBF in the 1.0% but not in the 2.5% Hangeshashinto group, increased at 3 and 7 days compared with that in the control group (p < 0.05). This trend was also observed in the CBF in the 100 µM cAMP group, significant difference was not observed between the CBF of the 1.0% Hangeshashinto group and the 100 µM cAMP group at 1, 3 and 7 days after rinsing (p > 0.05). Histological score only in the 2.5% Hangeshashinto group was lower than that in the control group (p < 0.05), while a significant decline was not observed in the other groups compared to that in the control group (p > 0.05). CONCLUSION: Our results suggest that 1.0% Hangeshashinto solution facilitates the physiological function of the nasal mucosa by promoting ciliary functions without histological damage of cilia epithelium. When applied with the appropriate concentration, Hangeshashinto could have ability to improve the physiological functions of the nasal mucosal epithelium.

Fastigial nucleus electrostimulation promotes axonal regeneration after experimental stroke via cAMP/PKA pathway.

Axon regeneration after cerebral ischemia in mammals is inadequate to restore function, illustrating the need to design better strategies for improving outcomes. Improvement of axon regeneration has been achieved through fastigial nucleus electrostimulation (FNS) in animal researches. However, the mechanisms underlying this neuroprotection remain poorly understood. Increasing the levels of the second messenger cyclic AMP (cAMP) enhances axon regeneration, making it an excellent candidate molecule that has therapeutic potential. In the present study, we examined the expression of cAMP signaling in ischemic brain tissues following focal cerebral ischemia. Adult rats were subjected to ischemia induced by middle cerebral artery occlusion (MCAO). A dipolar electrode was placed into the cerebellum to stimulate the cerebellar fastigial nucleus for 1 h after ischemia. Neurological deficits and the expressions of cAMP, PKA (protein kinase A) and ROCK (Rho-kinase) were determined. Axonal regeneration was measured by upregulation of growth-associated protein 43 (GAP43). The data indicated that FNS significantly enhanced axonal regeneration and motor function recovery after cerebral ischemia. FNS also significantly increased cAMP and PKA levels after ischemic brain injury. All the beneficial effects of FNS were blocked by Rp-cAMP, an antagonist of PKA. Our research suggested that the axonal regeneration conferred by FNS was likely achieved via the regulation of cAMP/PKA pathway.

Modulation of thalamocortical oscillations by TRIP8b, an auxiliary subunit for HCN channels.

Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels have important functions in controlling neuronal excitability and generating rhythmic oscillatory activity. The role of tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) in regulation of hyperpolarization-activated inward current, I h, in the thalamocortical system and its functional relevance for the physiological thalamocortical oscillations were investigated. A significant decrease in I h current density, in both thalamocortical relay (TC) and cortical pyramidal neurons was found in TRIP8b-deficient mice (TRIP8b-/-). In addition basal cAMP levels in the brain were found to be decreased while the availability of the fast transient A-type K+ current, I A, in TC neurons was increased. These changes were associated with alterations in intrinsic properties and firing patterns of TC neurons, as well as intrathalamic and thalamocortical network oscillations, revealing a significant increase in slow oscillations in the delta frequency range (0.5-4 Hz) during episodes of active-wakefulness. In addition, absence of TRIP8b suppresses the normal desynchronization response of the EEG during the switch from slow-wave sleep to wakefulness. It is concluded that TRIP8b is necessary for the modulation of physiological thalamocortical oscillations due to its direct effect on HCN channel expression in thalamus and cortex and that mechanisms related to reduced cAMP signaling may contribute to the present findings.

Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation.

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor - GSNORi - or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

Costunolide and dehydrocostuslactone combination treatment inhibit breast cancer by inducing cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 pathway.

Our previous studies demonstrated that volatile oil from saussurea lappa root (VOSL), rich in two natural sesquiterpene lactones, costunolide (Cos) and dehydrocostuslactone (Dehy), exerts better anti-breast cancer efficacy and lower side effects than Cos or Dehy alone in vivo, however, their anti-cancer molecular mechanisms were still unknown. In this study, we investigated the underlying mechanisms of Cos and Dehy combination treatment (CD) on breast cancer cells through proteomics technology coupled with Western blot validation. Ingenuity Pathways Analysis (IPA) results based on the differentially expressed proteins revealed that both VOSL and CD affect the 14-3-3-mediated signaling, c-Myc mediated apoptosis signaling and protein kinase A (PKA) signaling. Western blot coupled with cell cycle and apoptosis analysis validated the results of proteomics analysis. Cell cycle arrest and apoptosis were induced in a dose-dependent manner, and the expressions of p53 and p-14-3-3 were significantly up-regulated, whereas the expressions of c-Myc, p-AKT, p-BID were significantly down-regulated, furthermore, the ratio of BAX/BCL-2 were significantly increased in breast cancer cells after CD and VOSL treatment. The findings indicated that VOSL and CD could induce breast cancer cell cycle arrest and apoptosis through c-Myc/p53 and AKT/14-3-3 signaling pathways and may be novel effective candidates for breast cancer treatment.

EXPRESS: F-actin links Epac-PKC signaling to purinergic P2X3 receptors sensitization in dorsal root ganglia following inflammation.

Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund's adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund's adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus.

BACKGROUND: Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression. RESULTS: The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress. CONCLUSION: Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.

Neuroprotective role of prostaglandin PGE2 EP2 receptor in hemin-mediated toxicity.

Heme (Fe(2+) protoporphyrin IX) and hemin (Fe(3+)), the prosthetic group of hemoprotein, are cytotoxic due to their ability to contribute to the production of reactive oxygen species, increased intracellular calcium levels, and stimulate glutamate-mediated excitotoxicity. Previous work by our group showed that blockade of the prostaglandin E2 (PGE2)-EP1 receptor reduced hemin-induced cytotoxicity in primary cortical neuronal cultures. However, the role of the prostaglandin E2 (PGE2)-EP2 receptor in hemin neurotoxicity remains unclear. Activation of the EP2 receptor in neurons results in increased cyclic AMP (cAMP) and protein kinase A signaling; therefore, we hypothesized that the activation of the EP2 receptor decreases hemin neurotoxicity. Using postnatal primary cortical neurons cultured from wildtype-control (WT) and EP2(-/-) mice, we investigated the role of the EP2 receptor in hemin neurotoxicity by monitoring cell survival with the Calcein-AM live-cell and lactate dehydrogenase assays. MitoTracker staining was also performed to determine how mitochondria were affected by hemin. Hemin neurotoxicity in EP2(-/-) neurons was 37.2 ± 17.0% greater compared to WT neurons. Of interest, cotreatment with the EP2 receptor agonist, butaprost (1 and 10 µM), significantly attenuated hemin neurotoxicity by 55.7 ± 21.1% and 60.1 ± 14.8%, respectively. To further investigate signaling mechanisms related to EP2 receptor mediating cytoprotection, neurons were cotreated with hemin and activators/inhibitors of both the cAMP-protein kinase A/exchange protein directly activated by cAMP (Epac) pathways. Forskolin, a cAMP activator, and 8-pCPT-cAMP, an Epac activator, both attenuated hemin neurotoxicity by 78.8 ± 22.2% and 58.4 ± 9.8%, respectively, as measured using the lactate dehydrogenase assay. Together, the results reveal that activation of the EP2 receptor is protective against hemin neurotoxicity in vitro and these findings suggest that neuroprotection occurs through the cAMP-Epac pathway in neuronal cultures. Therefore, activation of the EP2 receptor could be used to minimize neuronal damage following exposure to supraphysiological levels of hemin.

Regulation of tyrosine phosphatase STEP61 by protein kinase A during motor skill learning in mice.

Recently, striatal-enriched protein tyrosine phosphatase (STEP) and its upstream regulator protein kinase A (PKA) have been suspected to play a role in the intracellular mechanisms of fear conditioning and spatial memory. However, whether they contribute to the learning and memory of motor skills is totally unknown. In this study, we have investigated the role of STEP and PKA activities during motor skill learning associated with the accelerating rotarod task. We observed that learning the rotarod task differentially modulated the levels of phosphorylated STEP61 at serine 221, a site directly regulated by PKA, in the hippocampus, motor cortex and striatum. In a second set of experiments, we have pharmacologically inhibited PKA by the injection of Rp-cAMPS directly into the dorsal striatum of mice before rotarod trainings. PKA phosphorylation of STEP prevents the dephosphorylation of STEP substrates, whereas inhibition of PKA promotes STEP activity. Striatal PKA inhibitions dose-dependently impaired mice performances on the accelerating rotarod task. General motor abilities testing revealed an intact motor control in mice treated with 5 and 20 µg of Rp-cAMPS, but not at the highest dose of 40 µg. This suggested that motor learning was selectively affected by PKA inhibition at lower doses. Most notably, striatal inhibition of PKA reduced the levels of phosphorylated STEP61 at serine 221. Our data support that inactivation of STEP61 by the PKA activity is part of the molecular process associated with motor skill learning.

Epac activator critically regulates action potential duration by decreasing potassium current in rat adult ventricle.

Sympathetic stimulation is an important modulator of cardiac function via the classic cAMP-dependent signaling pathway, PKA. Recently, this paradigm has been challenged by the discovery of a family of guanine nucleotide exchange proteins directly activated by cAMP (Epac), acting in parallel to the classic signaling pathway. In cardiac myocytes, Epac activation is known to modulate Ca(2+) cycling yet their actions on cardiac ionic currents remain poorly characterized. This study attempts to address this paucity of information using the patch clamp technique to record action potential (AP) and ionic currents on rat ventricular myocytes. Epac was selectively activated by 8-CPT-AM (acetoxymethyl ester form of 8-CPT). AP amplitude, maximum depolarization rate and resting membrane amplitude were unaltered by 8-CPT-AM, strongly suggesting that Na(+) current and inward rectifier K(+) current are not regulated by Epac. In contrast, AP duration was significantly increased by 8-CPT-AM (prolongation of duration at 50% and 90% of repolarization by 41±10% and 43±8% respectively, n=11). L-type Ca(2+) current density was unaltered by 8-CPT-AM (n=16) so this cannot explain the action potential lengthening. However, the steady state component of K(+) current was significantly inhibited by 8-CPT-AM (-38±6%, n=15), while the transient outward K(+) current was unaffected by 8-CPT-AM. These effects were PKA-independent since they were observed in the presence of PKA inhibitor KT5720. Isoprenaline (100nM) induced a significant prolongation of AP duration, even in the presence of KT5720. This study provides the first evidence that the cAMP-binding protein Epac critically modulates cardiac AP duration by decreasing steady state K(+) current. These observations may be relevant to diseases in which Epac is upregulated, like cardiac hypertrophy.

Direct regulation of the proglucagon gene by insulin, leptin, and cAMP in embryonic versus adult hypothalamic neurons.

The proglucagon gene is expressed not only in the pancreas and intestine but also in the hypothalamus. Proglucagon-derived peptides have emerged as potential regulators of energy homeostasis. Whether leptin, insulin, or cAMP activation controls proglucagon gene expression in the hypothalamus is not known. A key reason for this has been the inaccessibility of hypothalamic proglucagon-expressing neurons and the lack of suitable neuronal cell lines. Herein we describe the mechanisms involved in the direct regulation of the proglucagon gene by insulin, leptin, and cAMP in hypothalamic cell models. Insulin, through an Akt-dependent manner, significantly induced proglucagon mRNA expression by 70% in adult-derived mHypoA-2/10 neurons and significantly suppressed it by 45% in embryonic-derived mHypoE-39 neurons. Leptin, via the Janus kinase-2/ signal transducer and activator of transcription-3 pathway, caused an initial increase by 66 and 43% at 1 h followed by a decrease by 45 and 34% at 12 h in mHypoA-2/10 and mHypoE-39 cells, respectively. Furthermore, cAMP activation by forskolin up-regulated proglucagon expression by 87% in mHypoE-39 neurons and increased proglucagon mRNA, through Epac activation, in the mHypoE-20/2 neurons. Specific regions of the proglucagon promoter were regulated by cAMP signaling, as determined by transient transfections, whereas mRNA stability assays demonstrate that insulin and leptin increase proglucagon mRNA stability in the adult cells. These findings suggest that insulin, leptin, and cAMP act directly, but differentially, on specific hypothalamic neurons to regulate proglucagon gene expression. Because proglucagon-derived peptides are potential regulators of energy homeostasis, an understanding of hypothalamic proglucagon neurons is important to further expand our knowledge of alternative feeding circuits.

Low-molecular-weight compounds having neurotrophic activity in cultured PC12 cells and neurons.

Recent reports have indicated that some low-molecular-weight compounds mimic neurotrophic factors inducing neurite outgrowth and neuroprotection. Carnosic acid (CA) promotes neurite outgrowth through the activation of Nrf2 in PC12 cells. CA also protects neurons via the keap/Nrf2 transcriptional pathway from oxidative stress. Forskolin-induced neurite outgrowth is mediated by activation of the PKA signalling pathway and this PKA-mediated neurite outgrowth is achieved by the expression of nur77 in PC12 cells. In addition, forskolin at its low concentration is closely related to the cAMP-induced protective function against L-DOPA-induced cytotoxicity in PC12 cells. A HDAC inhibitor trichostatin A (TSA) increases neurite length via p53 acetylation in rat cultured cerebellar granule neurons and in cerebral cortical neurons, and also protects neurons against glutathione depletion-induced oxidative stress. Recently, it was revealed that Nrf2 and p53 bind to CBP/p300 directly, and Nur77 is acetylated in vivo and in vitro by CBP/p300. Acetylation of Nrf2, p53 and Nur77 by CBP/p300 may constitute a novel similar regulatory mechanism for low-molecular-weight compounds with neurotrophic activities.

8-Phenylthio-adenines stimulate the expression of steroid hydroxylases, Cav3.2 Ca²âº channels, and cortisol synthesis by a cAMP-independent mechanism.

The regulation of cortisol synthesis and the expression of genes coding for steroidogenic proteins by 8-substituted cAMP and 8-substituted adenine derivatives were studied in bovine adrenal zona fasciculata (AZF) cells. At concentrations of 10-50 µM, 8-(4-chlorophenylthio)-cAMP (8CPT-cAMP), but not the poorly hydrolyzable Sp-8CPT-cAMP, stimulated large increases in cortisol synthesis and CYP17 mRNA expression. Of the three Epac (exchange protein activated by cAMP)-specific cAMP analogs, 8CPT-2'-OMe-cAMP, but not 8HPT-2'-OMe-cAMP or 8MeOPT-2'-OMe-cAMP, induced mRNAs for CYP17 and CYP11a1 steroid hydroxylases and stimulated cortisol synthesis. 8-Substituted adenine derivatives (10-200 µM), including 8PT-adenine, 8MeOPT-adenine, and 8CPT-adenine, stimulated similar large, concentration-dependent, and reversible increases in cortisol synthesis and steroid hydroxylase gene expression, whereas 8Br-adenine was ineffective. The phenylthio-adenine derivatives produced additive effects on cortisol synthesis when applied to AZF cells in combination with 8Br-cAMP. In contrast, no additivity was observed for these three compounds when used in combination with ACTH. 8PT-adenine did not activate PKA or inhibit DNA synthesis by AZF cells. 8PT-adenine-stimulated cortisol secretion and CYP17 steroid hydroxylase mRNA expression were potently inhibited by diphenyl-butylpiperidine T-type Ca(2+) antagonists. In AZF cells, 8PT-adenine and 8MeOPT-adenine induced the expression of both CACNA1H mRNA and associated Ca(v)3.2 Ca(2+) current. These results indicate that 8-chloro (but not 8-hydroxy- or 8-methoxy-)-phenylthio-cAMP analogs are converted to an active metabolite, 8CPT-adenine, that induces the expression of genes coding for steroidogenic proteins in bovine AZF cells. Other PT-adenine analogs also potently stimulate cortisol synthesis through the same unidentified signaling pathway that requires the expression of functional Ca(v)3.2 Ca(2+) channels. These phenylthio-adenine compounds and ACTH may stimulate cortisol synthesis through the same cAMP-independent mechanism.

Megadose vitamin C suppresses sulfoconjugation in human colon carcinoma cell line Caco-2.

Intake of high doses of vitamin C has known to modulate sulfoconjugation of drugs in the intestine, but the underlying mechanisms for this effect remain to be elucidated. In the present study, we investigated the effects of vitamin C (l-ascorbic acid (AA)) on sulfation of 1-naphthol using Caco-2 cells, a model of human intestinal cells. We found that high dose of AA inhibited the accumulation of 1-naphthyl sulfate in Caco-2 culture medium within 24h in a dose-dependent manner (IC(50)=42 mM). Dehydroascorbic acid (DA), an oxidized form of AA, showed no inhibition. AA did not inhibit the in vitro sulfotransferase (SULT) activity toward 1-naphthol, whereas it reduced the expression of genes belonging to SULT1A family, SULT1A1 and SULT1A3. DA showed no effect on SULT1A gene expression. Consistent with the reduction in gene expression, AA reduced the cytosolic SULT activity towards 1-naphthol in the AA-treated Caco-2 cells. In addition, cAMP exerted an additive effect on AA-mediated repression of SULT1A gene expression. Our results suggest that megadose AA suppresses sulfoconjugation in the intestine mainly by downregulating the expression of SULT1A genes.

Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin-sensitive adenylyl cyclase.

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca(2+)/CaM. AC-AplC is most similar to the Ca(2+)-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca(2+), independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca(2+)-inhibited AC in SNs, together with a Ca(2+)/CaM-stimulated AC, would enhance the associative requirement for coincident Ca(2+) influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.

Facilitation of ß-cell K(ATP) channel sulfonylurea sensitivity by a cAMP analog selective for the cAMP-regulated guanine nucleotide exchange factor Epac.

Clinical studies demonstrate that combined administration of sulfonylureas with exenatide can induce hypoglycemia in type 2 diabetic subjects. Whereas sulfonylureas inhibit ß-cell K(ATP) channels by binding to the sulfonylurea receptor-1 (SUR1), exenatide binds to the GLP-1 receptor, stimulates ß-cell cAMP production and activates both PKA and Epac. In this study, we hypothesized that the adverse in vivo interaction of sulfonylureas and exenatide to produce hypoglycemia might be explained by Epac-mediated facilitation of K(ATP) channel sulfonylurea sensitivity. We now report that the inhibitory action of a sulfonylurea (tolbutamide) at K(ATP) channels was facilitated by 2'-O-Me-cAMP, a selective activator of Epac. Thus, under conditions of excised patch recording, the dose-response relationship describing the inhibitory action of tolbutamide at human ß-cell or rat INS-1 cell K(ATP) channels was left-shifted in the presence of 2'-O-Me-cAMP, and this effect was abolished in INS-1 cells expressing a dominant-negative Epac2. Using an acetoxymethyl ester prodrug of an Epac-selective cAMP analog (8-pCP T-2'-O-Me-cAMP-AM), the synergistic interaction of an Epac activator and tolbutamide to depolarize INS-1 cells and to raise [Ca²(+)](i) was also measured. This effect of 8-pCP T-2'-O-Me-cAMP-AM correlated with its ability to stimulate phosphatidylinositol 4,5-bisphosphate hydrolysis that might contribute to the changes in K(ATP) channel sulfonylurea-sensitivity reported here. On the basis of such findings, we propose that the adverse interaction of sulfonylureas and exenatide to induce hypoglycemia involves at least in part, a functional interaction of these two compounds to close K(ATP) channels, to depolarize ß-cells and to promote insulin secretion.

A rapid interference between glucocorticoids and cAMP-activated signalling in hypothalamic neurones prevents binding of phosphorylated cAMP response element binding protein and glucocorticoid receptor at the CRE-Like and composite GRE sites of thyrotrophin-releasing hormone gene promoter.

Glucocorticoids or cAMP increase, within minutes, thyrotrophin-releasing hormone (TRH) transcription in hypothalamic primary cultures, although this effect is prevented if cells are simultaneously incubated with both drugs. Rat TRH promoter contains a CRE site at -101/-94 bp and a composite GRE element (cGRE) at -218/-197 bp. Nuclear extracts of hypothalamic cells incubated with 8Br-cAMP or dexamethasone, and not their combination, bind to oligonucleotides containing the CRE or cGRE sequences. Adjacent to CRE are Sp/Krüppel response elements, and flanking the GRE half site, two AP1 binding sites. The present study aimed to identify the hypothalamic transcription factors that bind to these sites. We verified that the effects of glucocorticoid were not mimicked by corticosterone-bovine serum albumin. Footprinting and chromatin immunoprecipitation (ChIP) assays were used to examine the interaction of cAMP- and glucocorticoid-mediated regulation of TRH transcription at the CRE and cGRE regions of the TRH promoter. Nuclear extracts from hypothalamic cells incubated for 1 h with cAMP or glucocorticoids protected CRE. The GRE half site was recognised by nuclear proteins from cells stimulated with glucocorticoids and, for the adjacent AP-1 sites, by nuclear proteins from cells stimulated with cAMP or phorbol esters. Protection of CRE or cGRE was lost if cells were coincubated with dexamethasone and 8Br-cAMP. ChIP assays revealed phospho-CREB, c-Jun, Sp1, c-Fos and GR antibodies bound the TRH promoter of cells treated with cAMP or glucocorticoids; anti:RNA-polymerase II immunoprecipitated TRH promoter in a similar proportion as anti:pCREB or anti:GR. Recruitment of pCREB, SP1 or GR was lost when cells were exposed simultaneously to 8Br-cAMP and glucocorticoids. The data show that while pCREB and Sp1 bind to CRE-2, or GR to cGRE of the TRH promoter, the mutual antagonism between cAMP and glucocorticoid signalling, which prevent their binding to TRH promoter, could serve as a mechanism by which glucocorticoids rapidly suppress cAMP and noradrenaline-stimulated TRH transcription.

Enhancement of lipolytic responsiveness of adipocytes by novel plant extract in rat.

Subcutaneous adipocytes accumulate excess energy as triglycerides, but lipolytic response is less sensitive to catecholamines than visceral adipocytes. Obesity also induces catecholamine resistance of adipocytes. We have searched for crude drugs that could enhance the lipolytic response to noradrenalin. In this study, the lipolysis-promoting activities and action mechanisms of a novel plant extract from Hemerocallis fulva (HE) were investigated in isolated adipocytes from rat subcutaneous fat. HE exhibited no lipolysis-promoting activity alone but markedly promoted lipolysis when combined with noradrenaline; however, this synergistic activity was accompanied by no increase of intracellular cAMP production. This activity of HE was also observed when combined with cAMP analogue and was further enhanced by phosphodiesterase inhibitor. PKA inhibitor could reduce these activities of HE. These results indicate that HE is a novel lipolysis-promoting material that can sensitize the lipolytic response of adipocytes to catecholamine and suggest that HE can amplify the intra-cellular signaling pathway related to PKA or modify the other mechanism-regulating lipase activity. This characteristic material could contribute to improvement of adipose mobility in obesity-related disorder or in subcutaneous adiposity and to suppression of body fat accumulation.