PKA-dependent Mechanism Research Articles

Chlorogenic Acid Activates CFTR-Mediated Cl- Secretion in Mice and Humans: Therapeutic Implications for Chronic Rhinosinusitis.

Salubrious effects of the green coffee bean are purportedly secondary to high concentrations of chlorogenic acid. Chlorogenic acid has a molecular structure similar to bioflavonoids that activate transepithelial Cl(-) transport in sinonasal epithelia. In contrast to flavonoids, the drug is freely soluble in water. The objective of this study is to evaluate the Cl(-) secretory capability of chlorogenic acid and its potential as a therapeutic activator of mucus clearance in sinus disease. Basic research. Laboratory. Chlorogenic acid was tested on primary murine nasal septal epithelial (MNSE) (CFTR(+/+) and transgenic CFTR(-/-)) and human sinonasal epithelial (HSNE) (CFTR(+/+) and F508del/F508del) cultures under pharmacologic conditions in Ussing chambers to evaluate effects on transepithelial Cl(-) transport. Cellular cyclic adenosine monophosphate (cAMP), phosphorylation of the CFTR regulatory domain (R-D), and CFTR mRNA transcription were also measured. Chlorogenic acid stimulated transepithelial Cl(-) secretion (change in short-circuit current [ΔISC = µA/cm(2)]) in MNSE (13.1 ± 0.9 vs 0.1 ± 0.1; P < .05) and HSNE (34.3 ± 0.9 vs 0.0 ± 0.1; P < .05). The drug had a long duration until peak effect at 15 to 30 minutes after application. Significant inhibition with INH-172 as well as absent stimulation in cultures lacking functional CFTR suggest effects are dependent on CFTR-mediated pathways. However, the absence of elevated cellular cAMP and phosphorylation the CFTR R-D indicates chlorogenic acid does not work through a PKA-dependent mechanism. Chlorogenic acid is a water-soluble agent that promotes CFTR-mediated Cl(-) transport in mouse and human sinonasal epithelium. Translating activators of mucociliary transport to clinical use provides a new therapeutic approach to sinus disease. Further in vivo evaluation is planned.

MARK/Par1 Kinase Is Activated Downstream of NMDA Receptors through a PKA-Dependent Mechanism.

The Par1 kinases, also known as microtubule affinity-regulating kinases (MARKs), are important for the establishment of cell polarity from worms to mammals. Dysregulation of these kinases has been implicated in autism, Alzheimer’s disease and cancer. Despite their important function in health and disease, it has been unclear how the activity of MARK/Par1 is regulated by signals from cell surface receptors. Here we show that MARK/Par1 is activated downstream of NMDA receptors in primary hippocampal neurons. Further, we show that this activation is dependent on protein kinase A (PKA), through the phosphorylation of Ser431 of Par4/LKB1, the major upstream kinase of MARK/Par1. Together, our data reveal a novel mechanism by which MARK/Par1 is activated at the neuronal synapse.

Effects of monocrotophos pesticide on steroidogenesis and transcription of steroidogenic enzymes in rainbow trout RTG-2 cells involving the protein kinase A signal pathway

Monocrotophos (MCP) pesticide, listed as a UNEP Prior Informed Consent chemical, has been proved to exert toxic effects on the reproductive system of teleost fishes by changing the balance of sex steroid hormones. To investigate the effects of MCP on steroidogenesis in vitro, the rainbow trout (Oncorhynchus mykiss) gonadal cell line RTG-2 was exposed to different MCP concentrations for 48h. The levels of 17β-estradiol (E2) and testosterone in the medium were measured by radioimmunoassay and the expression of steroidogenic acute regulatory protein and cytochrome P450 enzymes CYP11A1, CYP17, and CYP19A was detected by quantitative real-time PCR. The results showed that 1.0 and 10.0μg/L MCP pesticide induced E2 levels and promoted steroidogenic enzyme expression. The possible mechanisms of MCP steroidogenic activity were investigated using inhibitors of protein kinase A (PKA) and protein kinase C. The PKA inhibitor H-89 abrogated the 10.0μg/L MCP-induced transcriptional up-regulation of steroidogenic enzymes, suggesting an involvement of PKA-dependent mechanism in the disruption of steroidogenesis by the MCP pesticide in rainbow trout RTG-2 cells.

Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.

Bile acid signaling is a critical regulator of glucose and energy metabolism, mainly through the nuclear receptor FXR and the G protein-coupled receptor TGR. The purpose of the present study was to investigate whether dual activation of FXR and TGR5 plays a significant role in the prevention of atherosclerosis progression. To evaluate the effects of bile acid signaling in atherogenesis, ApoE−/− mice and LDLR−/− mice were treated with an FXR/TGR5 dual agonist (INT-767). INT-767 treatment drastically reduced serum cholesterol levels. INT-767 treatment significantly reduced atherosclerotic plaque formation in both ApoE−/− and LDLR−/− mice. INT-767 decreased the expression of pro-inflammatory cytokines and chemokines in the aortas of ApoE−/− mice through the inactivation of NF-κB. In addition, J774 macrophages treated with INT-767 had significantly lower levels of active NF-κB, resulting in cytokine production in response to LPS through a PKA dependent mechanism. This study demonstrates that concurrent activation of FXR and TGR5 attenuates atherosclerosis by reducing both circulating lipids and inflammation.

Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation.

Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

Reduction of glutamate release probability and the number of releasable vesicles are required for suppression of glutamatergic transmission by β1-adrenoceptors in the medial prefrontal cortex

The prefrontal cortex (PFC) plays a critical role in cognitive functions, including working memory, attention regulation and behavioral inhibition. Microinjection of β1-adrenoceptor (β1-AR) agonist into the PFC has been shown to impair PFC cognitive function. However, the underlying cellular and molecular mechanisms have not been determined yet. In the present study, we tested the hypothesis that β1-AR mediated modulation of excitatory synaptic transmission contributes to PFC dysfunction. We found that 1) the β1-AR agonist Dobutamine (Dobu) suppressed the amplitude evoked excitatory postsynaptic currents (eEPSCs). 2) Dobu induced a significant suppression of the frequency and amplitude of miniature EPSCs (mEPSCs). 3) Dobu-suppressed glutamate release was mediated via decreasing release probability and the number of releasable vesicles. 4) Dobu suppressed inward currents evoked by puff application of glutamate or NMDA via postsynaptic PKA-dependent pathway. The present study indicates that β1-AR activation suppresses excitatory synaptic transmission in medial PFC (mPFC) via both pre- and post-synaptic PKA-dependent mechanisms. Our results may provide a cellular and molecular mechanism that helps explain β1-AR-induced PFC dysfunction.

Inhibitory Effects of Capsaicin on Voltage-Gated Potassium Channels by TRPV1-Independent Pathway

Previously we observed that capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor activator, inhibited transient potassium current (IA) in capsaicin-sensitive and capsaicin-insensitive trigeminal ganglion (TG) neurons from rats. It suggested that the inhibitory effects of capsaicin on IA have two different mechanisms: TRPV1-dependent and TRPV1-independent pathways. The main purpose of this study is to further investigate the TRPV1-independent effects of capsaicin on voltage-gated potassium channels (VGPCs). Whole cell patch-clamp technique was used to record IA and sustained potassium current (IK) in cultured TG neurons from trpv1 knockout (TRPV1(-/-)) mice. We found that capsaicin reversibly inhibited IA and IK in a dose-dependent manner. Capsaicin (30 μM) did not alter the activation curve of IA and IK but shifted the inactivation-voltage curve to hyperpolarizing direction, thereby increasing the number of inactivated VGPCs at the resting potential. Administrations of high concentrations capsaicin, no use-dependent block, and delay of recovery time course were found on IK and IA. Moreover, forskolin, an adenylate cyclase agonist, selectively decreased the inhibitory effects of IK by capsaicin, whereas none influenced the inhibitions of IA. These results suggest that capsaicin inhibits the VGPCs through TRPV1-independent and PKA-dependent mechanisms, which may contribute to the capsaicin-induced nociception.

GLP-1 promotes mitochondrial metabolism in vascular smooth muscle cells by enhancing endoplasmic reticulum–mitochondria coupling

Incretin GLP-1 has important metabolic effects on several tissues, mainly through the regulation of glucose uptake and usage. One mechanism for increasing cell metabolism is modulating endoplasmic reticulum (ER)–mitochondria communication, as it allows for a more efficient transfer of Ca2+ into the mitochondria, thereby increasing activity. Control of glucose metabolism is essential for proper vascular smooth muscle cell (VSMC) function. GLP-1 has been shown to produce varied metabolic actions, but whether it regulates glucose metabolism in VSMC remains unknown. In this report, we show that GLP-1 increases mitochondrial activity in the aortic cell line A7r5 by increasing ER–mitochondria coupling. GLP-1 increases intracellular glucose and diminishes glucose uptake without altering glycogen content. ATP, mitochondrial potential and oxygen consumption increase at 3h of GLP-1 treatment, paralleled by increased Ca2+ transfer from the ER to the mitochondria. Furthermore, GLP-1 increases levels of Mitofusin-2 (Mfn2), an ER-mitochondria tethering protein, via a PKA-dependent mechanism. Accordingly, PKA inhibition and Mfn2 down-regulation prevented mitochondrial Ca2+ increases in GLP-1 treated cells. Inhibiting both Ca2+ release from the ER and Ca2+ entry into mitochondria as well as diminishing Mfn2 levels blunted the increase in mitochondrial activity in response to GLP-1. Altogether, these results strongly suggest that GLP-1 increases ER–mitochondria communication in VSMC, resulting in higher mitochondrial activity.

Tauroursodeoxycholic acid exerts anti-apoptotic effects by a MKP-1- and PKA-dependent mechanism in rat liver

Ursodeoxycholic acid, which in vivo is converted to its taurine conjugate tauroursodeoxycholic acid (TUDC), is a mainstay for the treatment of cholestatic liver disease. TUDC is also effective in protecting hepatocytes for apoptosis induced by hydrophobic bile salts. The aim of the study was to elucidate the underlying signaling events.

Aging Increases Cardiac L-Type Calcium Channel Current of Ventricular Myocytes from Male Hearts via a PKA-Dependent Mechanism

Consistent findings of the aging heart include diastolic dysfunctions and attenuated β-adrenergic receptor (β-AR) stimulation of heart rate and contractility. However, variable results were reported for the impact of aging on L-type Ca channel (LTCC) function, the trigger of excitation-contraction. In this study, we evaluated the impact of aging and gender on LTCC of ventricular myocytes (VM).VMs were isolated from LV of young and old mice (C57BL/6) hearts of both gender and processed for whole-cell voltage clamp recording to measure LTCC current. VMs from old male showed increased LTCC current density compared to young VMs. LTCC peak current density were −5.7 ± 0.3 pA/pF (n=33) and −12.6 ± 1.7 pA/pF (n = 21) (P < 0.01), for young and old, respectively. Application of PKI, a selective PKA inhibitor, reseted LTCC current density of old VMs to levels comparable to young VMs. PKI application decreased LTCC peak current density by 15±7% and 59±5% (P < 0.05) in young and old VMs, respectively. Lastly, we tested the impact of aging on β-AR stimulation of LTCC. Relative to young controls, β-AR stimulation of LTCC was attenuated in old male but not in female VMs. Also, in VMs from female hearts, we observed no significant age-dependent effects on LTCC.Our novel findings show that LTCC activity is regulated in an age- and gender-dependent manner via a PKA-dependent mechanism. This may underlie the diastolic dysfunction and attenuated β-AR stimulation of cardiac contractility observed in the aging heart. Our results suggest that this age-dependent attenuation of the β-AR/c-AMP/PKA stimulation of LTCC may ultimately decrease the cardiac reserve and increase the susceptibility to heart failure. Efforts to better understand the structural and functional changes associated with aging will lead to age-appropriate successful therapeutic interventions.

Cell-to-cell coordination for the spontaneous cAMP oscillation inDictyostelium

We propose a new cellular dynamics scheme for the spontaneous cAMP oscillations in Dictyostelium discoideum. Our scheme seamlessly integrates both receptor dynamics and G-protein dynamics into our previously developed cellular dynamics scheme. Extensive computer simulation studies based on our new cellular dynamics scheme were conducted in mutant cells to evaluate the molecular network. The validity of our proposed molecular network as well as the controversial PKA-dependent negative feedback mechanism was supported by our simulation studies. Spontaneous cAMP oscillations were not observed in a single mutant cell. However, multicellular states of various mutant cells consistently initiated spontaneous cAMP oscillations. Therefore, cell-to-cell coordination via the cAMP receptor is essential for the robust initiation of spontaneous cAMP oscillations.

Role of Tyrosine Phosphorylation in the Muscarinic Activation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

Activation of adenosine A2A receptor reduces osteoclast formation via PKA‐ and ERK1/2‐mediated suppression of NFκB nuclear translocation

We previously reported that adenosine, acting at adenosine A(2A) receptors (A(2A)R), inhibits osteoclast (OC) differentiation in vitro (A(2A)R activation OC formation reduces by half) and in vivo. For a better understanding how adenosine A(2A)R stimulation regulates OC differentiation, we dissected the signalling pathways involved in A(2A)R signalling. OC differentiation was studied as TRAP+ multinucleated cells following M-CSF/RANKL stimulation of either primary murine bone marrow cells or the murine macrophage line, RAW264.7, in presence/absence of the A(2A)R agonist CGS21680, the A(2A)R antagonist ZM241385, PKA activators (8-Cl-cAMP 100 nM, 6-Bnz-cAMP) and the PKA inhibitor (PKI). cAMP was quantitated by EIA and PKA activity assays were carried out. Signalling events were studied in PKA knockdown (lentiviral shRNA for PKA) RAW264.7 cells (scrambled shRNA as control). OC marker expression was studied by RT-PCR. A(2A)R stimulation increased cAMP and PKA activity which and were reversed by addition of ZM241385. The direct PKA stimuli 8-Cl-cAMP and 6-Bnz-cAMP inhibited OC maturation whereas PKI increased OC differentiation. A(2A)R stimulation inhibited p50/p105 NFκB nuclear translocation in control but not in PKA KO cells. A(2A)R stimulation activated ERK1/2 by a PKA-dependent mechanism, an effect reversed by ZM241385, but not p38 and JNK activation. A(2A)R stimulation inhibited OC expression of differentiation markers by a PKA-mechanism. A(2A)R activation inhibits OC differentiation and regulates bone turnover via PKA-dependent inhibition of NFκB nuclear translocation, suggesting a mechanism by which adenosine could target bone destruction in inflammatory diseases like rheumatoid arthritis.

Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 is Expressed inOsteoblasts and Regulated by PTH

Bone mass is dependent on osteoblast proliferation, differentiation and life-span of osteoblasts. Parathyroid hormone (PTH) controls osteoblast cell cycle regulatory proteins and suppresses mature osteoblasts apoptosis. Intermittent administration of PTH increases bone mass but the mechanism of action are complex and incompletely understood. Cell Cycle and Apoptosis Regulatory Protein (CARP)-1 (aka CCAR1) is a novel transducer of signaling by diverse agents including cell growth and differentiation factors. To gain further insight into the molecular mechanism, we investigated involvement of CARP-1 in PTH signaling in osteoblasts. Immunostaining studies revealed presence of CARP-1 in osteoblasts and osteocytes, while a minimal to absent levels were noted in the chondrocytes of femora from 10 to 12-week old mice. Treatment of 7-day differentiated MC3T3-E1 clone-4 (MC-4) mouse osteoblastic cells and primary calvarial osteoblasts with PTH for 30min to 5h followed by Western blot analysis showed 2- to 3-fold down-regulation of CARP-1 protein expression in a dose- and time-dependent manner compared to the respective vehicle treated control cells. H-89, a Protein Kinase A (PKA) inhibitor, suppressed PTH action on CARP-1 protein expression indicating PKA-dependent mechanism. PMA, a Protein Kinase C (PKC) agonist, mimicked PTH action, and the PKC inhibitor, GF109203X, partially blocked PTH-dependent downregulation of CARP-1, implying involvement of PKC. U0126, a Mitogen-Activated Protein Kinase (MAPK) Kinase (MEK) inhibitor, failed to interfere with CARP-1 suppression by PTH. In contrast, SB203580, p38 inhibitor, attenuated PTH down-regulation of CARP-1 suggesting that PTH utilized an Extracellular Signal Regulated Kinase (ERK)-independent but p38 dependent pathway to regulate CARP-1 protein expression in osteoblasts. Immunofluorescence staining of differentiated osteoblasts further revealed nuclear to cytoplasmic translocation of CARP-1 protein following PTH treatment. Collectively, our studies identified CARP-1 for the first time in osteoblasts and suggest its potential role in PTH signaling and bone anabolic action.

Adenosine A2A receptor (A2AR) is a fine-tune regulator of the collagen1:collagen3 balance

Adenosine is a potent endogenous anti-inflammatory and immunosuppressive metabolite that is a potent modulator of tissue repair. However, the adenosine A2A receptor (A2AR)-mediated promotion of collagen synthesis is detrimental in settings such as scarring and scleroderma. The signaling cascade from A2AR stimulation to increased collagen production is complex and obscure, not least because cAMP and its downstream molecules PKA and Epac1 have been reported to inhibit collagen production. We therefore examined A2AR-stimulated signaling for collagen production by normal human dermal fibroblasts (NHDF). Collagen1 (Col1) and collagen3 (Col3) content after A2AR activation by CGS21680 was studied by western blotting. Contribution of PKA and Epac was analyzed by the PKA inhibitor PKI and by knockdowns of the PKA-Cα, -Cβ, -Cγ, Epac1, and Epac2. CGS21680 stimulates Col1 expression at significantly lower concentrations than those required to stimulate Col3 expression. A2AR stimulates Col1 expression by a PKA-dependent mechanism since PKA inhibition or PKA-Cα and -Cβ knockdown prevents A2AR-mediated Col1 increase. In contrast, A2AR represses Col3 via PKA but stimulates both Col1 and Col3 via an Epac2-dependent mechanism. A2AR stimulation with CGS21680 at 0.1μM increased Col3 expression only upon PKA blockade. A2AR activation downstream signaling for Col1 and Col3 expression proceeds via two distinct pathways with varying sensitivity to cAMP activation; more highly cAMP-sensitive PKA activation stimulates Col1 expression, and less cAMP-sensitive Epac activation promotes both Col1 and Col3 expression. These observations may explain the dramatic change in Col1:Col3 ratio in hypertrophic and immature scars, where adenosine is present in higher concentrations than in normal skin.

Differential Requirement for Protein Synthesis in Presynaptic Unmuting and Muting in Hippocampal Glutamate Terminals

Synaptic function and plasticity are crucial for information processing within the nervous system. In glutamatergic hippocampal neurons, presynaptic function is silenced, or muted, after strong or prolonged depolarization. This muting is neuroprotective, but the underlying mechanisms responsible for muting and its reversal, unmuting, remain to be clarified. Using cultured rat hippocampal neurons, we found that muting induction did not require protein synthesis; however, slow forms of unmuting that depend on protein kinase A (PKA), including reversal of depolarization-induced muting and forskolin-induced unmuting of basally mute synapses, required protein synthesis. In contrast, fast unmuting of basally mute synapses by phorbol esters was protein synthesis-independent. Further studies of recovery from depolarization-induced muting revealed that protein levels of Rim1 and Munc13-1, which mediate vesicle priming, correlated with the functional status of presynaptic terminals. Additionally, this form of unmuting was prevented by both transcription and translation inhibitors, so proteins are likely synthesized de novo after removal of depolarization. Phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB), a nuclear transcription factor, was elevated after recovery from depolarization-induced muting, consistent with a model in which PKA-dependent mechanisms, possibly including pCREB-activated transcription, mediate slow unmuting. In summary, we found that protein synthesis was required for slower, PKA-dependent unmuting of presynaptic terminals, but it was not required for muting or a fast form of unmuting. These results clarify some of the molecular mechanisms responsible for synaptic plasticity in hippocampal neurons and emphasize the multiple mechanisms by which presynaptic function is modulated.

Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases.

Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca(2+) during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP(2) in two ways: first, PIP(2) acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP(2) and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR.

Evidence for an interaction of neuronostatin with the orphan G protein-coupled receptor, GPR107

Neuronostatin, derived from the somatostatin preprohormone, is a recently described peptide that is produced by several tissues involved in cardiovascular regulation and metabolism, including the hypothalamus. Injection of neuronostatin into the lateral cerebroventricle led to a dose-related increase in mean arterial pressure (MAP) in rats. Any attempt to inhibit the production of neuronostatin would alter somatostatin production as well, making determination of the physiological relevance of the peptide's pharmacologic effects by compromise of production approaches impossible. Therefore, we employed an alternative approach to identify and compromise the production of the neuronostatin receptor. Because neuronostatin was shown to signal via a PKA-dependent mechanism, we hypothesized that the neuronostatin receptor was a G protein-coupled receptor (GPCR), in particular, one of the orphan GPCRs for which the ligand is unknown. Therefore, we screened neuronostatin-responsive tissues, including hypothalamus, heart, pancreatic α-cells, and the gastric tumor cell line KATOIII, for expression of orphan GPCRs. Four orphan GPCRs were expressed by all cell types, including GPR56 and GPR107. Knockdown of GPR107, but not GPR56 or GPR146, led to a loss of responsiveness to neuronostatin by KATOIII cells. Rats injected with siRNA directed against GPR107 (2 μg/day for 2 days) into the lateral cerebroventricle did not exhibit an increase in MAP in response to neuronostatin treatment. Rats with compromised GPR107 expression also displayed blunted reactivity in a baroreflex sensitivity test, indicating that GPR107 and neuronostatin may be important regulators of cardiovascular function. Thus, GPR107 is a promising candidate receptor for neuronostatin, and neuronostatin, interacting with GPR107, may play an important role in the central control of cardiovascular function.

Abstract 34: Apolipoprotein A-I Increases Insulin Secretion from β-cells via a Protein Kinase A Dependent Mechanism

Introduction: The progression to hyperglycaemia in type 2 diabetes is marked by β-cell insulin secretory dysfunction and cell loss. We have previously demonstrated that apolipoprotein (apo) A-I, the major protein constituent of high density lipoproteins (HDL) increases insulin expression and secretion from β-cells. Clinical data also suggests that pharmacological elevation of HDL levels is associated with improved glycemic control in patients with type 2 diabetes. With the current interest in HDL raising therapeutics, defining the mechanism by which apoA-I acts on insulin secretion is of importance. Objective: To elucidate the cell signalling events responsible for increasing insulin secretion from pancreatic β-cells treated with lipid-free apoA-I. Methods: Ins-1E (rat insulinoma) cells were pre-treated for 30 min with the Protein kinase A (PKA) specific inhibitor H89 (20 μM), soluble and transmembrane adenyl cyclase specific inhibitors (KH7, 30 μM and 2’5’ dideoxyadenosine, 50 μM, respectively) or vehicle control, then incubated for 1 h with lipid-free apoA-I (final concentration 1 mg/mL) under both basal (2.8 mM) and high (25 mM) glucose conditions. The insulin concentration in the culture supernatants was determined by radioimmunoassay and the cells were either lysed for protein analysis by western blotting or treated with 0.1 M HCl for determining cAMP by enzyme immunoassay. Results: Incubation of Ins-1E cells with apoA-I increased insulin secretion up to 3-fold. This increase was no longer apparent when the cells were pre-treated with H89. Incubation with apoA-I increased cAMP accumulation in Ins-1E cells 2.5-fold. This increase was totally inhibited when the cells were pre-incubated with 2’5’ dideoxyadenosine but not by KH7, indicating that transmembrane adenyl cyclase(s) are responsible for this response. ApoA-I also activated the small GTPase Cdc42, which may link cell surface apoA-I receptors with transmembrane adenyl cyclases. Conclusion: ApoA-I increases insulin secretion from pancreatic β-cells via a PKA-dependent mechanism involving transmembrane, but not soluble, adenyl cyclases and possibly Cdc42. This provides a possible explanation of the clinical observations that increased HDL may be beneficial in type 2 diabetes.

Underlying mechanisms of cAMP- and glucocorticoid-mediated inhibition of FasL expression in activation-induced cell death

Glucocorticoids (GCs) and cAMP-dependent signaling pathways exert diverse and relevant immune regulatory functions, including a tight control of T cell death and homeostasis. Both of these signaling molecules inhibit TCR-induced cell death and FasL expression, but the underlying mechanisms are still poorly understood. Therefore, to address this question, we performed a comprehensive screening of signaling pathways downstream of the TCR, in order to define which of them are targets of cAMP- and GC-mediated inhibition. We found that cAMP inhibited NF-κB and ERK pathways through a PKA-dependent mechanism, while Dexamethasone blocked TCR-induced NF-κB signaling. Although GCs and cAMP inhibited the induction of endogenous FasL mRNA expression triggered by TCR activation, they potentiated TCR-mediated induction of FasL promoter activity in transient transfection assays. However, when the same FasL promoter was stably transfected, the facilitatory effect of GCs and cAMP became inhibitory, thus resembling the effects on endogenous FasL mRNA expression. Hence, the endogenous chromatinization status known to occur in integrated or genomic vs. episomic DNA might be critical for proper regulation of FasL expression by cAMP and GCs. Our results suggest that the chromatinization status of the FasL promoter may function as a molecular switch, controlling cAMP and GC responsiveness and explaining why these agents inhibit FasL expression in T cells but induce FasL in other cell types.