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PKA
Kinds of PKA Terms modified by PKA Selected AbstractsImpaired cardiac functional reserve in type 2 diabetic db/db mice is associated with metabolic, but not structural, remodellingACTA PHYSIOLOGICA, Issue 1 2010A. Daniels Abstract Aim:, To identify the initial alterations in myocardial tissue associated with the early signs of diabetic cardiac haemodynamic dysfunction, we monitored changes in cardiac function, structural remodelling and gene expression in hearts of type 2 diabetic db/db mice. Methods:, Cardiac dimensions and function were determined echocardiographically at 8, 12, 16 and 18 weeks of age. Left ventricular pressure characteristics were measured at 18 weeks under baseline conditions and upon dobutamine infusion. Results:, The db/db mice were severely diabetic already at 8 weeks after birth, showing elevated fasting blood glucose levels and albuminuria. Nevertheless, echocardiography revealed no significant changes in cardiac function up to 18 weeks of age. At 18 weeks of age, left ventricular pressure characteristics were not significantly different at baseline between diabetic and control mice. However, dobutamine stress test revealed significantly attenuated cardiac inotropic and lusitropic responses in db/db mice. Post-mortem cardiac tissue analyses showed minor structural remodelling and no significant changes in gene expression levels of the sarcoplasmic reticulum calcium ATPase (SERCA2a) or ,1-adrenoceptor (,1-AR). Moreover, the phosphorylation state of known contractile protein targets of protein kinase A (PKA) was not altered, indicating unaffected cardiac ,-adrenergic signalling activity in diabetic animals. By contrast, the substantially increased expression of uncoupling protein-3 (UCP3) and angiopoietin-like-4 (Angptl4), along with decreased phosphorylation of AMP-activated protein kinase (AMPK) in the diabetic heart, is indicative of marked changes in cardiac metabolism. Conclusion:, db/db mice show impaired cardiac functional reserve capacity during maximal ,-adrenergic stimulation which is associated with unfavourable changes in cardiac energy metabolism. [source] Orexins/hypocretins control bistability of hippocampal long-term synaptic plasticity through co-activation of multiple kinasesACTA PHYSIOLOGICA, Issue 3 2010O. Selbach Abstract Aim:, Orexins/hypocretins (OX/Hcrt) are hypothalamic neuropeptides linking sleep,wakefulness, appetite and neuroendocrine control. Their role and mechanisms of action on higher brain functions, such as learning and memory, are not clear. Methods:, We used field recordings of excitatory post-synaptic potentials (fEPSP) in acute mouse brain slice preparations to study the effects of orexins and pharmacological inhibitors of multiple kinases on long-term synaptic plasticity in the hippocampus. Results:, Orexin-A (OX-A) but not orexin-B (OX-B) induces a state-dependent long-term potentiation of synaptic transmission (LTPOX) at Schaffer collateral-CA1 synapses in hippocampal slices from adult (8- to 12-week-old) mice. In contrast, OX-A applied to slices from juvenile (3- to 4-week-old) animals causes a long-term depression (LTDOX) in the same pathway. LTPOX is blocked by pharmacological inhibition of orexin receptor-1 (OX1R) and plasticity-related kinases, including serine/threonine- (CaMKII, PKC, PKA, MAPK), lipid- (PI3K), and receptor tyrosine kinases (Trk). Inhibition of OX1R, CaMKII, PKC, PKA and Trk unmasks LTDOX in adult animals. Conclusion:, Orexins control not only the bistability of arousal states and threshold for appetitive behaviours but, in an age- and kinase-dependent manner, also bidirectional long-term synaptic plasticity in the hippocampus, providing a possible link between behavioural state and memory functions. [source] Protein kinase A modulates A-type potassium currents of larval zebrafish (Danio rerio) white muscle fibresACTA PHYSIOLOGICA, Issue 2 2009C. A. Coutts Abstract Aims:, Potassium (K+) channels are involved in regulating cell excitability and action potential shape. To our knowledge, very little is known about the modulation of A-type K+ currents in skeletal muscle fibres. Therefore, we sought to determine whether K+ currents of zebrafish white skeletal muscle were modulated by protein kinase A (PKA). Methods:, Pharmacology and whole-cell patch clamp were used to examine A-type K+ currents and action potentials associated with zebrafish white skeletal muscle fibres. Results:, Activation of PKA by a combination of forskolin + 3-isobutyl-1-methylxanthine (Fsk + IBMX) decreased the peak current density by ,60% and altered the inactivation kinetics of A-type K+ currents. The specific PKA inhibitor H-89 partially blocked the Fsk + IBMX-induced reduction in peak current density, but had no effect on the change in decay kinetics. Fsk + IBMX treatment did not shift the activation curve, but it significantly reduced the slope factor of activation. Activation of PKA by Fsk + IBMX resulted in a negative shift in the V50 of inactivation. H-89 prevented all Fsk + IBMX-induced changes in the steady-state properties of K+ currents. Application of Fsk + IBMX increased action potential amplitude, but had no significant effect on action potential threshold, half width or recovery rate, when fibres were depolarized with single pulses, paired pulses or with high-frequency stimuli. Conclusion:, PKA modulates the A-type K+ current in zebrafish skeletal muscle and affects action potential properties. Our results provide new insights into the role of A-type K+ channels in muscle physiology. [source] Protein kinases A and C stimulate the Na+ active transport in frog skeletal muscle without an appreciable change in the number of sarcolemmal Na+ pumpsACTA PHYSIOLOGICA, Issue 4 2005R. A. Venosa Abstract Aim:, The activation of both protein kinases A (PKA) and protein kinases C (PKC) in some cell types increases and in others reduces active Na+ efflux. These effects have been ascribed to either a change in the rate of ionic translocation by a fixed number of Na+ pumps or, a change in the number of plasma membrane pumps. The purpose of the present experiments was to study the effect of activating PKA and PKC on the Na+ extrusion by the Na+ pump in frog skeletal muscle. Methods:, Na+ (22Na+) fluxes and ouabain (3H-ouabain) binding were measured in frog sartorius muscles. Results:, Both activation of PKA and PKC increased the active Na+ extrusion by a factor of two; these effects were not additive. Ouabain binding experiments indicated that the pump stimulation by activation of these kinases is not associated with any significant increase in the number of plasma membrane pumps. Stimulation of the active Na+ efflux by protein kinase activation (no change in the number of sarcolemmal pumps) and by hypotonicity (increase in the number of pumps) could be elicited in the same preparation and they were additive. Conclusion:, It is concluded that in frog skeletal muscle fibres, (1) activation of both PKA and PKC stimulate the Na+ pump by increasing its rate of ionic translocation; and (2) two modes of Na+ active transport (with and without an increase in the number of pumps) are operative, and can be at work simultaneously, a phenomenon to be reckoned with. [source] AKAP-independent localization of type-II protein kinase A to dynamic actin microspikesCYTOSKELETON, Issue 9 2009Robert L. Rivard Abstract Regulation of the cyclic AMP-dependent protein kinase (PKA) in subcellular space is required for cytoskeletal dynamics and chemotaxis. Currently, spatial regulation of PKA is thought to require the association of PKA regulatory (R) subunits with A-kinase anchoring proteins (AKAPs). Here, we show that the regulatory RII, subunit of PKA associates with dynamic actin microspikes in an AKAP-independent manner. Both endogenous RII, and a GFP-RII, fusion protein co-localize with F-actin in microspikes within hippocampal neuron growth cones and the leading edge lamellae of NG108-15 cells. Live-cell imaging demonstrates that RII,-associated microspikes are highly dynamic and that the coupling of RII, to actin is tight, as the movement of both actin and RII, are immediately and coincidently stopped by low-dose cytochalasin D. Importantly, co-localization of RII, and actin in these structures is resistant to displacement by a cell-permeable disrupter of PKA-AKAP interactions. Biochemical fractionation confirms that a substantial pool of PKA RII, is associated with the detergent-insoluble cytoskeleton and is resistant to extraction by a peptide inhibitor of AKAP interactions. Finally, mutation of the AKAP-binding domain of RII, fails to disrupt its association with actin microspikes. These data provide the first demonstration of the physical association of a kinase with such dynamic actin structures, as well as the first demonstration of the ability of type-II PKA to localize to discrete subcellular structures independently of canonical AKAP function. This association is likely to be important for microfilament dynamics and cell migration and may prime the investigation of novel mechanisms for localizing PKA activity. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Protein kinase A RII-like (R2D2) proteins exhibit differential localization and AKAP interaction,CYTOSKELETON, Issue 7 2008Amy E. Hanlon Newell Abstract A-kinase anchoring proteins (AKAPs) bind to protein kinase A (PKA) via an amphipathic helix domain that interacts with a dimerization/docking domain on the regulatory (R) subunit of PKA. Four other mammalian proteins (ROPN1, ASP, SP17, and CABYR) also contain a highly conserved RII dimerization/docking (R2D2) domain, suggesting all four proteins may interact with all AKAPs in a manner similar to RII. All four of these proteins were originally detected in the flagellum of mammalian sperm. In this report, we demonstrate that all four R2D2 proteins are expressed in a wide variety of tissues and three of the proteins SP17, CABYR, and ASP are located in motile cilia of human bronchus and fallopian tubes. In addition, we detect SP17 in primary cilia. We also provide evidence that ROPN1 and ASP bind to a variety of AKAPs and this interaction can be disrupted with anchoring inhibitor peptides. The interaction of SP17 and CABYR with AKAPs appears to be much more limited. None of the R2D2 proteins appears to bind cAMP, a fundamental characteristic of the regulatory subunits of PKA. These observations suggest that R2D2 proteins utilize docking interactions with AKAPs to accomplish their function of regulating cilia and flagella. Based on location, affinity for AKAPs and lack of affinity for cAMP, it appears that each R2D2 protein has a unique role in this process. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Sox genes regulate type 2 collagen expression in avian neural crest cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2006Takashi Suzuki Neural crest cells give rise to a wide variety of cell types, including cartilage cells in the cranium and neurons and glial cells in the peripheral nervous system. To examine the relationship of cartilage differentiation and neural crest differentiation, we examined the expression of Col2a1, which encodes type 2 collagen often used as a cartilage marker, and compared it with the expression of Sox transcription factor genes, which are involved in neural crest development and chondrogenesis. We found that Col2a1 is expressed in many neural crest-derived cell types along with combinations of Sox9, Sox10 and LSox5. Overexpression studies reveal the activation of Col2a1 expression by Sox9 and Sox10, and cross-regulation of these Sox genes. Luciferase assay indicates a direct activation of the Col2a1 enhancer/promoter both by Sox9 and Sox10, and this activation is further enhanced by cAMP-dependent kinase (PKA) signaling. Our study suggests that the regulatory mechanisms are similar in cartilage and neural crest differentiation. [source] Local activation of protein kinase A inhibits morphogenetic movements during Xenopus gastrulationDEVELOPMENTAL DYNAMICS, Issue 1 2003Byung-Ho Song Abstract cAMP-dependent protein kinase (PKA) has various biological roles in many organisms. However, little is known about its role in the developmental processes of vertebrates. In this study, we describe the functional analysis of PKA during gastrulation movements in Xenopus laevis. Overexpression of constitutively active PKA (cPKA) in the dorsal equatorial region of the embryo affects morphogenetic movement during gastrulation. We also show that intrinsic differences of PKA activities along the dorsoventral axis are set up and the level of PKA activity on the dorsal region is lower than that on the ventral region from late blastula to gastrula stages. In addition, PKA activation in animal explants inhibits activin-induced elongation. In cPKA-injected embryos, there were no changes in the expressions of markers involved in mesoderm specification, although the correct expression domains of these genes were altered. The effects of PKA activation can be restored by coexpression of PKI, a pseudosubstrate of PKA. We further analyzed the effects of PKA activation on the behavior of migratory gastrulating cells in vitro. Expression of cPKA in head mesoderm cells causes less polarized and/or randomized migration as demonstrated by a directional cell migration assay. Finally, we show that RhoA GTPase lies downstream of PKA, affecting activin-induced convergent extension movements. Taken together, these results suggest that overexpressed PKA can modulate a pathway responsible for morphogenetic movements during Xenopus gastrulation. Developmental Dynamics 227:91,103, 2003. © 2003 Wiley-Liss, Inc. [source] Activation of receptors negatively coupled to adenylate cyclase is required for induction of long-term synaptic depression at Schaffer collateral-CA1 synapsesDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2006Linda A. Santschi Abstract Chemical LTD (CLTD) of synaptic transmission is triggered by simultaneously increasing presynaptic [cGMP] while inhibiting PKA. Here, we supply evidence that class II, but not III, metabotropic glutamate receptors (mGluRs), and A1 adenosine receptors, both negatively coupled to adenylate cyclase, play physiologic roles in providing PKA inhibition necessary to promote the induction of LTD at Schaffer collateral-CA1 synapses in hippocampal slices. Simultaneous activation of group II mGluRs with the selective agonist (2S,2,R,3,R)-2-(2,,3,-dicarboxy-cyclopropyl) glycine (DCGIV; 5 ,M), while raising [cGMP] with the type V phosphodiesterase inhibitor, zaprinast (20 ,M), resulted in a long-lasting depression of synaptic strength. When zaprinast (20 ,M) was combined with a cell-permeant PKA inhibitor H-89 (10 ,M), the need for mGluR IIs was bypassed. DCGIV, when combined with a "submaximal" low frequency stimulation (1 Hz/400 s), produced a saturating LTD. The mGluR II selective antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 5 ,M), blocked induction of LTD by prolonged low frequency stimulation (1 Hz/900 s). In contrast, the mGluR III selective receptor blocker, (RS)-a-Cyclopropyl-[3- 3H]-4-phosphonophenylglycine (CPPG; 10 ,M), did not impair LTD. The selective adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 100 nM), also blocked induction of LTD, while the adenosine A1 receptor agonist N6 -cyclohexyl adenosine (CHA; 50 nM) significantly enhanced the magnitude of LTD induced by submaximal LFS and, when paired with zaprinast (20 ,M), was sufficient to elicit CLTD. Inhibition of PKA with H-89 rescued the expression of LTD in the presence of either EGLU or DPCPX, confirming the hypothesis that both group II mGluRs and A1 adenosine receptors enhance the induction of LTD by inhibiting adenylate cyclase and reducing PKA activity. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2006 [source] Parathyroid hormone stimulates the endothelial expression of vascular endothelial growth factorEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2008G. Rashid ABSTRACT Background, We showed previously that parathyroid hormone (PTH) may stimulate the endothelial expression of pro-atherosclerotic and pro-inflammatory markers. Considering the impact of PTH on vasculature, we decided to evaluate its effect on mRNA and intra-cellular protein expressions of endothelial vascular endothelial growth factor (VEGF) taking into account that VEGF may play a role in the pathogenesis of endothelial dysfunctions. Materials and methods, Human umbilical vein cords endothelial cells (HUVEC) were stimulated for 24 h with 10,12,10,10 mol L,1 PTH. The VEGF-165 mRNA expression (critical in stimulating endothelial cell proliferation) was evaluated by RT/PCR and the intra-cellular VEGF protein expression by flow cytometry. The pathways by which PTH may have an effect on VEGF expression were also evaluated. Results, PTH (10,10 mol L,1) significantly increased VEGF-165 mRNA expression (P < 0·05). The addition of 50 nmol L,1 protein kinase C (PKC) and 10 µmol L,1 protein kinase A (PKA) inhibitors significantly reduced the VEGF-165 mRNA expression (P = 0·01). We also examined whether nitric oxide (NO) may be involved in the PTH-induced stimulation of VEGF-165 expression. Pre-treatment of the cells with 200 µmol L-nitro arginine methyl ester (L-NAME, NO synthase inhibitor) was found to inhibit VEGF-165 mRNA expression (P = 0·006). VEGF protein could not be detected in the medium of HUVEC but it was present in the cell cytoplasm. PTH had no significant effect on cytoplasmatic VEGF protein expression. Conclusion, The stimulatory effect of PTH on endothelial VEGF-165 mRNA expression is partly through PKC and PKA pathways and is also NO dependent. [source] Role for cAMP-protein kinase A signalling in augmented neutrophil adhesion and chemotaxis in sickle cell diseaseEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 4 2007Andreia A. Canalli Abstract The significance of the leukocyte in sickle cell disease (SCD) pathophysiology is becoming increasingly recognised; we sought to examine whether the chemotactic properties of neutrophils of SCD individuals may be altered and, further, to better understand the signalling events that mediate altered SCD neutrophil function. Adhesion to immobilised fibronectin (FN) and chemotaxis of control and SCD neutrophils were assessed using in vitro static adhesion assays and 96-well chemotaxis chamber assays. Adhesion assays confirmed a significantly higher basal adhesion of SCD neutrophils to FN, compared with control neutrophils. Chemotaxis assays established, for the first time, that SCD neutrophils demonstrate greater spontaneous migration and, also, augmented migration in response to IL-8, when compared with control neutrophils. Co-incubation of SCD neutrophils with KT5720 (an inhibitor of PKA) abrogated increased basal SCD neutrophil adhesion, spontaneous chemotaxis and IL-8-stimulated chemotaxis. Stimulation of SCD neutrophils with IL-8 also significantly augmented SCD neutrophil adhesion to FN with a concomitant increase in cAMP levels and this increase in adhesion was abolished by KT5720. Interestingly, the adhesive properties of neutrophils from SCD individuals on hydroxyurea therapy were not significantly altered and results indicate that a reduction in intracellular cAMP may contribute to lower the adhesive properties of these cells. Data indicate that up-regulated cAMP signalling plays a significant role in the altered adhesive and migratory properties in SCD neutrophils. Such alterations may have important implications for the pathophysiology of the disease and the cAMP-PKA pathway may represent a therapeutic target for the abrogation of altered leukocyte function. [source] Pituitary adenylyl cyclase-activating polypeptide controls the proliferation of retinal progenitor cells through downregulation of cyclin D1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2010Brian Njaine Abstract During retinal development, cell proliferation and exit from the cell cycle must be precisely regulated to ensure the generation of the appropriate numbers and proportions of the various retinal cell types. Previously, we showed that pituitary adenylyl cyclase-activating polypeptide (PACAP) exerts a neuroprotective effect in the developing retina of rats, through the cAMP,cAMP-dependent protein kinase (protein kinase A) (PKA) pathway. Here, we show that PACAP also regulates the proliferation of retinal progenitor cells. PACAP, PACAP-specific receptor (PAC1), and the receptors activated by both PACAP and vasoactive intestinal peptide (VIP), VPAC1 and VPAC2, are expressed during embryonic and postnatal development of the rat retina. Treatment of retinal explants with PACAP38 reduced the incorporation of [3H]thymidine as well as the number of 5-bromo-2,-deoxyuridine-positive and cyclin D1-positive cells. Pharmacological experiments indicated that PACAP triggers this antiproliferative effect through the activation of both PAC1 and VPACs, and the cAMP,PKA pathway. In addition, PACAP receptor activation decreased both cyclin D1 mRNA and protein content. Altogether, the data support the hypothesis that PACAP is a cell-extrinsic regulator with multiple roles during retinal development, including the regulation of proliferation in a subpopulation of retinal progenitor cells. [source] Depolarization promotes GAD 65-mediated GABA synthesis by a post-translational mechanism in neural stem cell-derived neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2008Nidhi Gakhar-Koppole Abstract Neuronal activity regulates neurogenesis and neuronal differentiation in the mammalian brain. The commencement of neurotransmitter expression establishes the neuronal phenotype and enables the formation of functional connectivity between neurons. In addition, release of neurotransmitters from differentiating neurons may modulate the behaviour of neural precursors. Here, we show that neuronal activity regulates ,-aminobutyric acid (GABA) expression in neurons generated from stem cells of the striatum and adult subventricular zone (SVZ). Differentiating neurons display spontaneous Ca2+ events, which are voltage-gated calcium channel (VGCC) dependent. Depolarization increases both the frequency of Ca2+ transients and the amount of Ca2+ influx in differentiating neurons. We show that depolarization-dependent GABA expression is regulated by the amplitude and not by the frequency of Ca2+ influx. Brief activation of VGCCs leads to Ca2+ influx that in turn promotes a rapid expression of GABA. Depolarization-dependent GABA expression does not require changes in gene expression. Instead, it involves cAMP-dependent protein kinase (PKA) and Ca2+ and phospholipid-dependent protein kinase (PKC) signalling. Activity increases the number of glutamic acid decarboxylase (GAD) 65-immunoreactive neurons in a PKA-dependent manner, without altering the expression of GAD 65, suggesting that depolarization promotes recruitment of GAD 65 by a post-translational mechanism. In line with this, depolarization does not permanently increase the expression of GABA in neurons derived from neural stem cells of the embryonic striatum, cortex and adult SVZ. Thus, neuronal activity does not merely accelerate neuronal differentiation but it may alter the mechanism of GABA synthesis in newly generated neurons. [source] Metaplasticity of the late-phase of long-term potentiation: a critical role for protein kinase A in synaptic taggingEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2006Jennie Z. Young Abstract The late-phase of long-term potentiation (L-LTP) in hippocampal area CA1 requires gene expression and de novo protein synthesis but it is expressed in an input-specific manner. The ,synaptic tag' theory proposes that gene products can only be captured and utilized at synapses that have been ,tagged' by previous activity. The mechanisms underlying synaptic tagging, and its activity dependence, are largely undefined. Previously, we reported that low-frequency stimulation (LFS) decreases the stability of L-LTP in a cell-wide manner by impairing synaptic tagging. We show here that a phosphatase inhibitor, okadaic acid, blocked homosynaptic and heterosynaptic inhibition of L-LTP by prior LFS. In addition, prior LFS homosynaptically and heterosynaptically impaired chemically induced synaptic facilitation elicited by forskolin/3-isobutyl-1-methylxanthine, suggesting that there is a cell-wide dampening of cAMP/protein kinase A (PKA) signaling concurrent with phosphatase activation. We propose that prior LFS impairs expression of L-LTP by inhibiting synaptic tagging through its actions on the cAMP/PKA pathway. In support of this notion, we show that hippocampal slices from transgenic mice that have genetically reduced hippocampal PKA activity display impaired synaptic capture of L-LTP. An inhibitor of PKA, KT-5720, also blocked synaptic capture of L-LTP. Moreover, pharmacological activation of the cAMP/PKA pathway can produce a synaptic tag to capture L-LTP expression, resulting in persistent synaptic facilitation. Collectively, our results show that PKA is critical for synaptic tagging and for input-specific L-LTP. PKA-mediated signaling can be constrained by prior episodes of synaptic activity to regulate subsequent L-LTP expression and perhaps control the integration of multiple synaptic events over time. [source] Somatodendritic autoreceptor regulation of serotonergic neurons: dependence on l -tryptophan and tryptophan hydroxylase-activating kinasesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2005Rong-Jian Liu Abstract The somatodendritic 5-HT1A autoreceptor has been considered a major determinant of the output of the serotonin (5-HT) neuronal system. However, recent studies in brain slices from the dorsal raphe nucleus have questioned the relevance of 5-HT autoinhibition under physiological conditions. In the present study, we found that the difficulty in demonstrating 5-HT tonic autoinhibition in slice results from in vitro conditions that are unfavorable for sustaining 5-HT synthesis. Robust, tonic 5-HT1A autoinhibition can be restored by reinstating in vivo 5-HT synthesizing conditions with the initial 5-HT precursor l -tryptophan and the tryptophan hydroxylase co-factor tetrahydrobiopterin (BH4). The presence of tonic autoinhibition under these conditions was revealed by the disinhibitory effect of a low concentration of the 5-HT1A antagonist WAY 100635. Neurons showing an autoinhibitory response to l -tryptophan were confirmed immunohistochemically to be serotonergic. Once conditions for tonic autoinhibition had been established in raphe slice, we were able to show that 5-HT autoinhibition is critically regulated by the tryptophan hydroxylase-activating kinases calcium/calmodulin protein kinase II (CaMKII) and protein kinase A (PKA). In addition, at physiological concentrations of l -tryptophan, there was an augmentation of 5-HT1A receptor-mediated autoinhibition when the firing of 5-HT cells activated with increasing concentrations of the ,1 adrenoceptor agonist phenylephrine. Increased calcium influx at higher firing rates, by activating tryptophan hydroxylase via CaMKII and PKA, can work together with tryptophan to enhance negative feedback control of the output of the serotonergic system. [source] SHORT COMMUNICATION Learning-induced reduction in post-burst after-hyperpolarization (AHP) is mediated by activation of PKCEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2002Yaron Seroussi Abstract We studied the role of protein kinase C (PKC) and protein kinase A (PKA) in mediating learning-related long lasting reduction of the post-burst after-hyperpolarization (AHP) in cortical pyramidal neurons. We have shown previously that pyramidal neurons in the rat piriform (olfactory) cortex from trained (TR) rats have reduced post-burst AHP for 3 days after odour-discrimination learning, and that this reduction is due to decreased conductance of calcium-dependent potassium current. In the present study, we examined whether this long-lasting reduction in AHP is mediated by second messenger systems. The broad-spectrum kinase inhibitor, H7, increased the AHP in neurons from TR rats, but not in neurons from pseudo-trained (pseudo-TR) and naive rats. Consequently, the difference in AHP amplitude between neurons from TR and control animals was diminished. This effect was also obtained by application of the specific PKC inhibitor, GF-109203x. The PKC activator, 1-Oleoyl-2-acetyl- sn -glycerol (OAG), significantly reduced the AHP in neurons from naive and pseudo-TR rats, but not in neurons from TR rats, so that the difference between the groups was abolished. The PKA-specific inhibitor, H-89, increased the AHP in neurons from all groups to a similar extent, and the difference in AHP amplitude between neurons from TR rats and neurons from controls was maintained. We suggest that while the post-burst AHP in piriform cortex pyramidal neurons is modulated by both PKC and PKA, a PKC-dependent process maintains the learning-related reduction of the AHP in these cells. [source] Dopaminergic signalling in the rodent neonatal suprachiasmatic nucleus identifies a role for protein kinase A and mitogen-activated protein kinase in circadian entrainmentEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2002Irina L. Schurov Abstract The circadian clock of the suprachiasmatic nuclei (SCN) of perinatal rodents is entrained by maternally derived cues. The SCN of neonatal Syrian hamsters express high-affinity D1 dopamine receptors, and the circadian activity,rest cycle of pups can be entrained by maternal injection of dopaminergic agonists. The present study sought to characterize the intracellular pathways mediating dopaminergic signalling in neonatal rodent SCN. Both dopamine and the D1 agonist SKF81297 caused a dose-dependent increase in phosphorylation of the transcriptional regulator Ca2+/cyclic AMP response element (CRE) binding protein (CREB) in suprachiasmatic GABA-immunoreactive (-IR) neurons held in primary culture. The D1 antagonist SCH23390 blocked this effect. Dopaminergic induction of pCREB-IR in GABA-IR neurons was also blocked by a protein kinase A (PKA) inhibitor, 5,24, and by the MAPK inhibitor, PD98059, whereas KN-62, an inhibitor of Ca2+/calmodulin-dependent (CAM) kinase II/IV was ineffective. Treatment with NMDA increased the level of intracellular Ca2+ in the cultured primary SCN neurons in Mg2+ -free medium, but SKF81297 did not. Blockade of CaM kinase II/IV with KN-62 inhibited glutamatergic induction of pCREB-IR in GABA-IR neurons, whereas 5,24 was ineffective, confirming the independent action of Ca2+ - and cAMP-mediated inputs on pCREB. SKF81297 caused an increase in pERK-IR in SCN cells, and this was blocked by 5,24, indicative of activation of MAPK via D1/cAMP. These results demonstrate that dopaminergic signalling in the neonatal SCN is mediated via the D1-dependent activation of PKA and MAPK, and that this is independent of the glutamatergic regulation via Ca2+ and CaM kinase II/IV responsible for entrainment to the light/dark cycle. [source] Phosphorylation of voltage-gated ion channels in rat olfactory receptor neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2001Christian H. Wetzel Abstract In olfactory receptor neurons (ORNs), ligand,odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation. [source] Localization of the A kinase anchoring protein AKAP79 in the human hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2000Attila Sík Abstract The phosphorylation state of the proteins, regulated by phosphatases and kinases, plays an important role in signal transduction and long-term changes in neuronal excitability. In neurons, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and calcineurin (CN) are attached to a scaffold protein, A kinase anchoring protein (AKAP), thought to anchor these three enzymes to specific sites of action. However, the localization of AKAP, and the predicted sites of linked phosphatase and kinase activities, are still unknown at the fine structural level. In the present study, we investigated the distribution of AKAP79 in the hippocampus from postmortem human brains and lobectomy samples from patients with intractable epilepsy, using preembedding immunoperoxidase and immunogold histochemical methods. AKAP79 was found in the CA1, presubicular and subicular regions, mostly in pyramidal cell dendrites, whereas pyramidal cells in the CA3, CA2 regions and dentate granule cells were negative both in postmortem and in surgical samples. In some epileptic cases, the dentate molecular layer and hilar interneurons also became immunoreactive. At the subcellular level, AKAP79 immunoreactivity was present in postsynaptic profiles near, but not attached to, the postsynaptic density of asymmetrical (presumed excitatory) synapses. We conclude that the spatial selectivity for the action of certain kinases and phosphatases regulating various ligand- and voltage-gated channels may be ensured by the selective presence of their anchoring protein, AKAP79, at the majority of glutamatergic synapses in the CA1, but not in the CA2/CA3 regions, suggesting profound differences in signal transduction and long-term synaptic plasticity between these regions of the human hippocampus. [source] Regulation of MC1R signalling by G-protein-coupled receptor kinasesEXPERIMENTAL DERMATOLOGY, Issue 9 2004J. C. García-Borrón The melanocortin 1 receptor (MC1R) is a key regulator of melanocyte proliferation and differentiation and a major determinant of human skin phototype and skin cancer risk. Although the regulation of MC1R gene expression is fairly well understood, little is known about regulatory mechanisms acting at the protein level. In particular, no information is available on homologous desensitization of MC1R signalling. We studied MC1R and Mc1r desensitization and found that: 1) MC1R and Mc1r in melanoma cells undergo homologous desensitization, demonstrated by decreases in cAMP contents upon continuous exposure to agonists, 2) desensitization is not dependent on PKA, PKC, calcium mobilization or MAPKs but is agonist dose dependent, suggesting a role of receptor occupancy, 3) melanoma cells express two members of the GRK family of serine/threonine kinases, GRK2 and GRK6, 4. These kinases are expressed in normal melanocytes, 5) in cotransfection experiments performed with HEK 293T cells, GRK2 strongly impairs agonist-dependent signalling by MC1R or Mc1r, 6) expression of a dominant negative GRK2 mutant in melanoma cells increases their cAMP response to MC1R agonists, 7) cotransfection of HEK 293T cells with GRK6 and MC1R inhibits both basal and agonist-dependent signalling, and 8) cAMP production in agonist-stimulated melanoma cells is strongly impaired by enrichment with GRK6 following stable transfection. Therefore, GRK2 and GRK6 are key regulators of MC1R signalling and may be important determinants of normal and pathological skin pigmentation. [source] Enhancement of Ca2+ -regulated exocytosis by indomethacin in guinea-pig antral mucous cells: arachidonic acid accumulationEXPERIMENTAL PHYSIOLOGY, Issue 1 2006Shoko Fujiwara Ca2+ -regulated exocytosis is enhanced by an autocrine mechanism via the PGE2,cAMP pathway in antral mucous cells of guinea-pigs. The inhibition of the PGE2,cAMP pathway by H-89 (an inhibitor of protein kinase A, PKA) or aspirin (ASA, an inhibitor of cyclo-oxygenase, COX) decreased the frequency of ACh-stimulated exocytotic events by 60%. Indomethacin (IDM, an inhibitor of COX), however, decreased the frequency of ACh-stimulated exocytotic events only by 30%. Moreover, IDM increased the frequency of ACh-stimulated exocytotic events by 50% in H-89-treated or ASA-treated cells. IDM inhibits the synthesis of Prostaglandin (PGG/H) and (15R)-15-hydroxy-5,8,11 cis-13-trans-eicosatetraenoic acid (15R-HPETE), while ASA inhibits only the synthesis of PGG/H. Thus, IDM may accumulate arachidonic acid (AA). AACOCF3 or N -(p -amylcinnamoyl) anthranilic acid (ACA; both inhibitors of phospholipase A2, PLA2), which inhibits AA synthesis, decreased the frequency of ACh-stimulated exocytotic events by 60%. IDM, however, did not increase the frequency in AACOCF3 -treated cells. AA increased the frequency of ACh-stimulated exocytotic events in AACOCF3 - or ASA-treated cells, similar to IDM in ASA- and H-89-treated cells. Moreover, in the presence of AA, IDM did not increase the frequency of ACh-stimulated exocytotic events in ASA-treated cells. The PGE2 release from antral mucosa indicates that inhibition of PLA2 by ACA inhibits the AA accumulation in unstimulated and ACh-stimulated antral mucosa. The dose,response study of AA and IDM demonstrated that the concentration of intracellular AA accumulated by IDM is less than 100 nm. In conclusion, IDM modulates the ACh-stimulated exocytosis via AA accumulation in antral mucous cells. [source] Gene Transfer Strategies for the PhysiologistEXPERIMENTAL PHYSIOLOGY, Issue 6 2000Liang-Fong Wong Foreign genes can be introduced into whole animals using methods of germline transgenesis and somatic gene delivery. While germline transgenesis can generate useful animal models for genetic studies, it can be costly, time-consuming and requires the use of a large number of animals. An alternative means of gene transfer is to deliver genes to somatic cells using non-viral and viral technologies. Non-viral methods such as naked DNA injection, electroporation and liposome/cation lipid-mediated gene transfer are relatively inefficient. In contrast, viruses are effective vehicles that carry foreign genes into a cell rapidly and efficiently. Here we illustrate the usefulness of adenoviral vectors to express a potent and specific inhibitor of cAMP-dependent protein kinase (PKA) to study the role of cyclic 3,,5,-cyclic AMP (cAMP) in the osmotic regulation of the vasopressin gene in a transgenic rat model. The ability to modify endogenous systems within specific cells in a whole animal model allows gene effects to be studied with physiological relevance. The combination of molecular biology and integrative physiology is a powerful application that can aid in the elucidation of how gene function can translate into complex systems in an organism [source] Regulation of calpain B from Drosophila melanogaster by phosphorylationFEBS JOURNAL, Issue 17 2009László Kovács Calpain B is one of the two catalytically competent calpain (calcium-activated papain) isoenzymes in Drosophila melanogaster. Because structural predictions hinted at the presence of several potential phosphorylation sites in this enzyme, we investigated the in vitro phosphorylation of the recombinant protein by protein kinase A as well as by the extracellular signal-regulated protein kinases (ERK) 1 and 2. By MS, we identified Ser845 in the Ca2+ binding region of an EF-hand motif, and Ser240 close to the autocatalytic activation site of calpain B, as being the residues phosphorylated by protein kinase A. In the transducer region of the protease, Thr747 was shown to be the target of the ERK phosphorylation. Based on the results of three different assays, we concluded that the treatment of calpain B with protein kinase A and ERK1 and ERK2 kinases increases the rate of the autoproteolytic activation of the enzyme, together with the rate of the digestion of external peptide or protein substrates. Phosphorylation also elevates the Ca2+ sensitivity of the protease. The kinetic analysis of phosphorylation mimicking Thr747Glu and Ser845Glu calpain B mutants confirmed the above conclusions. Out of the three phosphorylation events tested in vitro, we verified the in vivo phosphorylation of Thr747 in epidermal growth factor-stimulated Drosophila S2 cells. The data obtained suggest that the activation of the ERK pathway by extracellular signals results in the phosphorylation and activation of calpain B in fruit flies. Structured digital abstract ,,MINT-7214239: ERK1 (uniprotkb:P40417) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214216, MINT-7214228: PKA (uniprotkb:P12370) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214325: CalpainB (uniprotkb:Q9VT65) cleaves (MI:0194) MAP2C (uniprotkb:P11137) by protease assay (MI:0435) ,,MINT-7214275: ERK2 (uniprotkb:P40417-2) phosphorylates (MI:0217) CalpainB (uniprotkb:Q9VT65) by protein kinase assay (MI:0424) ,,MINT-7214319: CalpainB (uniprotkb:Q9VT65) and CalpainB (uniprotkb:Q9VT65) cleave (MI:0194) by protease assay (MI:0435) [source] Stimulation of fibroblast proliferation by neokyotorphin requires Ca2+ influx and activation of PKA, CaMK II and MAPK/ERKFEBS JOURNAL, Issue 2 2007Olga V. Sazonova Neokyotorphin [TSKYR, hemoglobin ,-chain fragment (137,141)] has previously been shown to enhance fibroblast proliferation, its effect depending on cell density and serum level. Here we show the dependence of the effect of neokyotorphin on cell type and its correlation with the effect of protein kinase A (PKA) activator 8-Br-cAMP, but not the PKC activator 4,-phorbol 12-myristate, 13-acetate (PMA). In L929 fibroblasts, the proliferative effect of neokyotorphin was suppressed by the Ca2+L -type channel inhibitors verapamil or nifedipine, the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N,,N, - tetraacetic acid acetoxymethyl ester, kinase inhibitors H-89 (PKA), KN-62 (Ca2+/calmodulin-dependent kinase II) and PD98059 (mitogen-activated protein kinase). The proliferative effect of 8-Br-cAMP was also suppressed by KN-62 and PD98059. PKC suppression (downregulation with PMA or inhibition with bisindolylmaleimide XI) did not affect neokyotorphin action. The results obtained point to a cAMP-like action for neokyotorphin. [source] Glucose sensing in the intestinal epitheliumFEBS JOURNAL, Issue 16 2003Jane Dyer Dietary sugars regulate expression of the intestinal Na+/glucose cotransporter, SGLT1, in many species. Using sheep intestine as a model, we showed that lumenal monosaccharides, both metabolisable and nonmetabolisable, regulate SGLT1 expression. This regulation occurs not only at the level of transcription, but also at the post-transcriptional level. Introduction of d -glucose and some d -glucose analogues into ruminant sheep intestine resulted in >,50-fold enhancement of SGLT1 expression. We aimed to determine if transport of sugar into the enterocytes is required for SGLT1 induction, and delineate the signal-transduction pathways involved. A membrane impermeable d -glucose analogue, di(glucos-6-yl)poly(ethylene glycol) 600, was synthesized and infused into the intestines of ruminant sheep. SGLT1 expression was determined using transport studies, Northern and Western blotting, and immunohistochemistry. An intestinal cell line, STC-1, was used to investigate the signalling pathways. Intestinal infusion with di(glucos-6-yl)poly(ethylene glycol) 600 led to induction of functional SGLT1, but the compound did not inhibit Na+/glucose transport into intestinal brush-border membrane vesicles. Studies using cells showed that increased medium glucose up-regulated SGLT1 abundance and SGLT1 promoter activity, and increased intracellular cAMP levels. Glucose-induced activation of the SGLT1 promoter was mimicked by the protein kinase A (PKA) agonist, 8Br-cAMP, and was inhibited by H-89, a PKA inhibitor. Pertussis toxin, a G-protein (Gi)-specific inhibitor, enhanced SGLT1 protein abundance to levels observed in response to glucose or 8Br-cAMP. We conclude that lumenal glucose is sensed by a glucose sensor, distinct from SGLT1, residing on the external face of the lumenal membrane. The glucose sensor initiates a signalling pathway, involving a G-protein-coupled receptor linked to a cAMP,PKA pathway resulting in enhancement of SGLT1 expression. [source] Cross-talk between olfactory second messenger pathwaysFEBS JOURNAL, Issue 14 2000Alexander Vogl The second messengers 3,-5,-cyclic-monophosphate (cAMP) and inositol 1,4,5-trisphosphate (InsP3) have been implicated in olfactory signal transduction in various species. The results of the present study provide evidence that the two olfactory second messenger pathways in rat olfactory neurons do not work independently but rather show a functional antagonism: whereas inhibition of phospholipase C (PLC) in isolated olfactory cilia by U-73122 led to an augmentation of odor-induced cAMP signaling, activation of the phosphoinositol pathway resulted in attenuation of odor-induced cAMP formation. Furthermore, this study indicates that elevated cAMP levels cause suppression of odor-induced InsP3 signaling, whereas inhibition of adenylate cyclase (AC) by cisN -(2-phenylcyclopentyl)azacylotridec-1-en-2-amine (MDL-12,330 A) results in potentiation of odor-induced InsP3 formation. Concerning the molecular mechanism involved in cross-interaction, the experimental data indicate that the observed antagonism of elevated cAMP is based on inhibition of PLC activation rather than on stimulation of InsP3 degradation. As blockage of the endogenous protein kinase A (PKA) prevented the inhibitory effect of cAMP, the suppression of odor-induced InsP3 signaling by cAMP may be mediated by a PKA-controlled reaction. [source] Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activitiesFEBS JOURNAL, Issue 9 2000Jackie D. Corbin In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem.265, 14971,14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50,70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2,0.5 ,m PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 ,m. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed. [source] Rab11 and its effector Rip11 participate in regulation of insulin granule exocytosisGENES TO CELLS, Issue 4 2009Kenji Sugawara Rab GTPases and their effectors play important roles in membrane trafficking between cellular compartments in eukaryotic cells. In the present study, we examined the roles of Rab11B and its effectors in insulin secretion in pancreatic ,-cells. In the mouse insulin-secreting cell line MIN6, Rab11 was co-localized with insulin-containing granules, and over-expression of the GTP- or the GDP-bound form of Rab11B significantly inhibited regulated secretion, indicating involvement of Rab11B in regulated insulin secretion. To determine the downstream signal of Rab11-mediated insulin secretion, we examined the effects of various Rab11-interacting proteins on insulin secretion, and found that Rip11 is involved in cAMP-potentiated insulin secretion but not in glucose-induced insulin secretion. Analyses by immunocytochemistry and subcellular fractionation revealed Rip11 to be co-localized with insulin granules. The inhibitory effect of the Rip11 mutant was not altered in MIN6 cells lacking Epac2, which mediates protein kinase A (PKA)-independent potentiation of insulin secretion, compared with wild-type MIN6 cells. In addition, Rip11 was found to be phosphorylated by PKA in MIN6 cells. The present study shows that both Rab11 and its effector Rip11 participate in insulin granule exocytosis and that Rip11, as a substrate of PKA, regulates the potentiation of exocytosis by cAMP in pancreatic ,-cells. [source] Beta-amyloid peptide stimulates endozepine release in cultured rat astrocytes through activation of N -formyl peptide receptorsGLIA, Issue 13 2008Tursonjan Tokay Abstract Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam-binding inhibitor (DBI). The authors have recently shown that ,-amyloid peptide (A,) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of A, in cultured rat astrocytes. A,25,35 and the N -formyl peptide receptor (FPR) agonist N -formyl-Met-Leu-Phe (fMLF) increased the secretion of endozepines in a dose-dependent manner with EC50 value of ,2 ,M. The stimulatory effects of A,25,35 and the FPR agonists fMLF and N -formyl-Met-Met-Met (fMMM) on endozepine release were abrogated by the FPR antagonist N - t -Boc-Phe-Leu-Phe-Leu-Phe. In contrast, A,25,35 increased DBI mRNA expression through a FPR-independent mechanism. A,25,35 induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of A,25,35 on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that A,25,35 stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC. © 2008 Wiley-Liss, Inc. [source] Involvement of ,1 integrin in microglial chemotaxis and proliferation on fibronectin: Different regulations by ADP through PKAGLIA, Issue 2 2005Kaoru Nasu-Tada Abstract Microglia are immune cells in the brain; their activation, migration, and proliferation have pivotal roles in brain injuries and diseases. Microglia are known to attach firmly to fibronectin, the upregulation of which is associated with several pathological conditions in the CNS, through ,1 integrin and become activated. Extracellular nucleotides can serve as potent signaling molecules. Recently, ATP and ADP were revealed to possess chemoattractive properties to microglia via Gi-coupled P2Y receptors. In the present study, we report that the ADP-induced chemotaxis of microglia is mediated by P2Y12/13 receptors and is ,1 integrin-dependent in the presence of fibronectin. Signals from P2Y12/13 receptors also cause ,1 integrin translocation to the membrane ruffle regions, but this redistribution was lost when the intracellular cyclic AMP (cAMP) was increased by forskolin or dibutyryl cAMP. This inhibitory effect of cAMP-elevating agents did not appear when microglia were co-incubated with a protein kinase A (PKA) inhibitor, KT-5720, suggesting that PKA is a negative regulator of the ,1 integrin translocation. We also show that the engagement of ,1 integrin enhanced microglial proliferation. Signals from P2Y12/13 receptors attenuated the proliferation, whereas ADP itself had no effect on microglial growth. Furthermore, ,1 integrin-induced proliferation is positively regulated by the cAMP-dependent PKA. Together, these results indicate the involvement of ,1 integrin in microglial proliferation and chemotaxis, both of which have clinical importance. The data also suggest that PKA is inversely involved in these two cellular functions. © 2005 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||