PKA) And C Research Articles

Oral treatment with Berberine reduces peripheral nociception: Possible interaction with different nociceptive pathways activated by different allogeneic substances

Ethnopharmacological relevanceBerberine was identified in extracts of Berberis ruscifolia Lam., a plant used in traditional medicine as an analgesic. Its presence may be involved in the reported pharmacological activity of this species. However, there is still a lack of scientific research concerning its analgesic activity in the peripheral nervous system. Aim of the studyTo investigate Berb-induced antinociception in the formalin test and to evaluate several pathways related to its pharmacological antinociceptive effects in chemical models of nociception in mice. Materials and methodsThe antinociceptive activity of Berb was assessed by inducing the paw licking in mice with different allodynic agents. In the formalin test, the antiedematous and antithermal effect of Berb was evaluated simultaneously in the same experiment. Other nociceptive behavior produced by endogenous [prostaglandin E2 (PGE2), histamine (His), glutamate (Glu) or bradykinin (BK)] or exogenous [capsaicin (Caps) and cinnamaldehyde (Cin)] chemical stimuli, and activators as protein kinase A (PKA) and C (PKC), were also evaluated.The in vivo doses for p.o. were 3 and 30 mg/kg. ResultsBerb, at 30 mg/kg p.o., showed a significant inhibition of the nociceptive action in formalin in both phases being stronger at the inflammatory phase (59 ± 9%) and more active than Asp (positive control) considering the doses evaluated. Moreover, Berb inhibited the edema (34 ± 10%), but not the temperature in the formalin test. Regarding the different nociceptive signaling pathways evaluated, the most relevant data were that the administration of p.o. of Berb, at 30 mg/kg, caused significant inhibition of nociception induced by endogenous [His (72 ± 11%), PGE2 (78 ± 4%), and BK (51 ± 7%)], exogenous [Cap (68 ± 4%) and Cinn (57 ± 5%)] compounds, and activators of the PKA [(FSK (86 ± 3%)] and PKC [(PMA(86 ± 6%)] signaling pathway. Berb did not inhibit the nociceptive effect produced by Glu. ConclusionThe present study demonstrated, for the first time, the potential of Berb in several nociceptive tests, with the compound present in B. ruscifolia contributing to the analgesic effect reported for this species.

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

Novelty promotes recognition memory persistence by D1 dopamine receptor and protein kinase A signalling in rat hippocampus.

Strategies for improving memory are increasingly studied, and exposure to a novel experience can be an efficient neuromodulator. Novelty effects on memory depend on D1-family dopamine receptors (D1Rs) activation. Here, we evaluated the novelty effect on memory persistence of Wistar rats and investigated the contribution of D1Rs and their signalling pathways by protein kinase A (PKA) and C (PKC). Animals with infusion cannulae inserted into the CA1 hippocampus area were trained on the novel object recognition (NOR) task, which involved exploring two different objects. After training, some rats received intrahippocampal infusions of vehicle or D1Rs agonist; others explored a novel environment for 5min and were infused with a variety of drugs targeting D1Rs and their signalling pathways. We demonstrated that pharmacological stimulation of D1Rs or novelty exposure promoted NOR memory persistence for 14 days and that the novelty effect depended on D1Rs activation. To determine if the D1 and D5 receptor subtypes were necessary for the impact of novelty exposure on memory, we blocked or stimulated PKA or PKC-protein kinases activated mainly by D1 and D5, respectively. Only PKA inhibition impaired the effect of novelty on memory persistence. After novelty and D1Rs blocking, PKA but not PKC stimulation maintained the memory persistence effect. Thus, we concluded that novelty promoted memory persistence by a mechanism-dependent on activating hippocampal D1Rs and PKA pathway.

Inhibitors of protein kinases A and C impair the motility of oncospheres of the model tapeworm Hymenolepis microstoma

The oncosphere larvae of tapeworms cyclically extend and retract their hooks during the penetration of the intestine of their intermediate hosts. The mechanisms regulating these movements are essentially unknown, in part due to the biohazardous nature of oncospheres from human pathogens. In this work, we standardized a method for the analysis of motility of hatched oncospheres (hexacanths) of the model tapeworm Hymenolepis microstoma. We used this assay to explore the relevance of protein kinases C (PKC) and A (PKA) in these processes. Pharmacological inhibition of the PKC pathway resulted in impaired larval motility. On the other hand, the PKA inhibitor H-89 potently blocked larval motility, as well as the motility of other life stages, although other inhibitors of the PKA pathway were not effective. This work represents the first study of the mechanisms that regulate the motility of oncospheres, and provides a path for further exploration.

The Interplay between S-Glutathionylation and Phosphorylation of Cardiac Troponin I and Myosin Binding Protein C in End-Stage Human Failing Hearts.

Oxidative stress is defined as an imbalance between the antioxidant defense system and the production of reactive oxygen species (ROS). At low levels, ROS are involved in the regulation of redox signaling for cell protection. However, upon chronical increase in oxidative stress, cell damage occurs, due to protein, DNA and lipid oxidation. Here, we investigated the oxidative modifications of myofilament proteins, and their role in modulating cardiomyocyte function in end-stage human failing hearts. We found altered maximum Ca2+-activated tension and Ca2+ sensitivity of force production of skinned single cardiomyocytes in end-stage human failing hearts compared to non-failing hearts, which was corrected upon treatment with reduced glutathione enzyme. This was accompanied by the increased oxidation of troponin I and myosin binding protein C, and decreased levels of protein kinases A (PKA)- and C (PKC)-mediated phosphorylation of both proteins. The Ca2+ sensitivity and maximal tension correlated strongly with the myofilament oxidation levels, hypo-phosphorylation, and oxidative stress parameters that were measured in all the samples. Furthermore, we detected elevated titin-based myocardial stiffness in HF myocytes, which was reversed by PKA and reduced glutathione enzyme treatment. Finally, many oxidative stress and inflammation parameters were significantly elevated in failing hearts compared to non-failing hearts, and corrected upon treatment with the anti-oxidant GSH enzyme. Here, we provide evidence that the altered mechanical properties of failing human cardiomyocytes are partially due to phosphorylation, S-glutathionylation, and the interplay between the two post-translational modifications, which contribute to the development of heart failure.

One single physical exercise session improves memory persistence by hippocampal activation of D1 dopamine receptors and PKA signaling in rats

Previously, we demonstrated that one single physical exercise session could positively modulate recognition memory persistence by D1/D5 activation. Here, we aim to investigate whether the effect of physical exercise on memory occurs due to the activation of both receptors, D1 and D5, or only one of them. Adult male Wistar rats were habituated on a treadmill one week before experiments. After learning session in the object recognition task, some animals received intrahippocampal infusions of the vehicle or a D1/D5 agonist (SKF 38393, 12.5 μg/μL/side), whereas others performed a single session of physical exercise on a treadmill (30 min at an intensity of 60–70% of indirect VO2 max.). Immediately after physical exercise, some animals received intrahippocampal infusions of vehicle or D1/D5 antagonist (SCH 23390, 1 μg/μL/side). Signaling pathways of D1 and D5 receptors in the hippocampus were evaluated by pharmacological activation or inactivation of protein kinases A (PKA) and C (PKC), respectively. According to previous findings, D1/D5 agonist and a single physical exercise session after learning promoted memory persistence, and D1/D5 block impaired physical exercise effect. Importantly, here we demonstrated for the first time that PKA inhibition, but not PKC, impairs the effect of acute physical exercise on memory persistence. Besides, PKA stimulation can promote its effects on memory. Therefore, we provide evidence that corroborates the idea that D1-like dopaminergic receptors, by activation of the PKA pathway, are involved in the effects of acute physical exercise on memory.

Modulation of a GIRK1 Active Mutant Subunit by Protein Kinase C Isoforms

G-protein gated inwardly rectifying potassium channels (GIRK)are activated by the Gᵦᵧ dimer of Gi/o-protein coupled receptors to inhibit excitability. There are four distinct subunits, GIRK1-GIRK4, which exist as heterotetramers or homotetramers throughout the body. Unlike GIRK1 and GIRK3 subunits that do not form functional homotetramers, GIRK2 and GIRK4 can each form homotetramers but can also assemble with the other subunits to form heterotetramers. These channels are activated by Na+, ethanol and PIP2, and are modulated by various factors like Protein kinase A and C (PKC). Phosphorylation of the channels by PKC has been widely reported to inhibit channel activity, however the effects of the various PKC isoforms on channel activity has yet to be fully defined. The 14 different isoforms of PKC are categorized into conventional, novel and atypical PKC's. In this study, we aimed to understand the various effects of two isoforms of PKC, the conventional PKCα and novel PKCε on GIRK1 through GIRK1(F137S), an active mutant of GIRK1 that can form functional homotetrameric channels. PKCε augmented GIRK1(F137S)activity, while PKCα inhibited GIRK1(F137S)activity. Due to the differential effect of these PKC isoforms on GIRK1, we sought to identify distinct phosphorylation sites on this subunit. Phosphorylation sites for both PKC isoforms on GIRK1(F137S) were predicted using the Group-based Prediction 3.0 software, and a site-directed mutagenesis approach is being employed to study alanine mutants of the channel in two-electrode voltage clamp experiments. Additionally, PIP2 dependence of PKC-mediated GIRK1 channel activity will be determined by depleting PIP2. This work aims to inform the elucidation of PKC modulation of GIRK1/2 and GIRK1/4 heterotetramers and the relevance of these effects on phosphorylation-mediated channelopathies.

Sensitization of small-diameter sensory neurons is controlled by TRPV1 and TRPA1 association.

Unique features of sensory neuron subtypes are manifest by their distinct physiological and pathophysiological functions. Using patch-clamp electrophysiology, Ca2+ imaging, calcitonin gene-related peptide release assay from tissues, protein biochemistry approaches, and behavioral physiology on pain models, this study demonstrates the diversity of sensory neuron pathophysiology is due in part to subtype-dependent sensitization of TRPV1 and TRPA1. Differential sensitization is influenced by distinct expression of inflammatory mediators, such as prostaglandin E2 (PGE2), bradykinin (BK), and nerve growth factor (NGF) as well as multiple kinases, including protein kinase A (PKA) and C (PKC). However, the co-expression and interaction of TRPA1 with TRPV1 proved to be the most critical for differential sensitization of sensory neurons. We identified N- and C-terminal domains on TRPV1 responsible for TRPA1-TRPV1 (A1-V1) complex formation. Ablation of A1-V1 complex with dominant-negative peptides against these domains substantially reduced the sensitization of TRPA1, as well as BK- and CFA-induced hypersensitivity. These data indicate that often occurring TRP channel complexes regulate diversity in neuronal sensitization and may provide a therapeutic target for many neuroinflammatory pain conditions.

Molecular Mechanisms of Short-Term Plasticity: Role of Synapsin Phosphorylation in Augmentation and Potentiation of Spontaneous Glutamate Release.

We used genetic and pharmacological approaches to identify the signaling pathways involved in augmentation and potentiation, two forms of activity dependent, short-term synaptic plasticity that enhance neurotransmitter release. Trains of presynaptic action potentials produced a robust increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs). Following the end of the stimulus, mEPSC frequency followed a bi-exponential decay back to basal levels. The time constants of decay identified these two exponential components as the decay of augmentation and potentiation, respectively. Augmentation increased mEPSC frequency by 9.3-fold, while potentiation increased mEPSC frequency by 2.4-fold. In synapsin triple-knockout (TKO) neurons, augmentation was reduced by 83% and potentiation was reduced by 74%, suggesting that synapsins are key signaling elements in both forms of plasticity. To examine the synapsin isoforms involved, we expressed individual synapsin isoforms in TKO neurons. While synapsin IIIa rescued both augmentation and potentiation, none of the other synapsin isoforms produced statistically significant amounts of rescue. To determine the involvement of protein kinases in these two forms of short-term plasticity, we examined the effects of inhibitors of protein kinases A (PKA) and C (PKC). While inhibition of PKC had little effect, PKA inhibition reduced augmentation by 76% and potentiation by 60%. Further, elevation of intracellular cAMP concentration, by either forskolin or IBMX, greatly increased mEPSC frequency and occluded the amount of augmentation and potentiation evoked by electrical stimulation. Finally, mutating a PKA phosphorylation site to non-phosphorylatable alanine largely abolished the ability of synapsin IIIa to rescue both augmentation and potentiation. Together, these results indicate that PKA activation is required for both augmentation and potentiation of spontaneous neurotransmitter release and that PKA-mediated phosphorylation of synapsin IIIa underlies both forms of presynaptic short-term plasticity.

Differential regulation of the Na+-Ca2+ exchanger 3 (NCX3) by protein kinase PKC and PKA

Isoform 3 of the Na+-Ca2+ exchanger (NCX3) participates in the Ca2+ fluxes across the plasma membrane. Among the NCX family, NCX3 carries out a peculiar role due to its specific functions in skeletal muscle and the immune system and to its neuroprotective effect under stress exposure. In this context, proper understanding of the regulation of NCX3 is primordial to consider its potential use as a drug target. In this study, we demonstrated the regulation of NCX3 by protein kinase A (PKA) and C (PKC). Disparity in regulation has been previously reported among the splice variants of NCX3 therefore the activity of Ca2+ uptake and extrusion of the two murine variants was measured using fura-2-based Ca2+ imaging and revealed that both variants are similarly regulated. PKC stimulation diminished the Ca2+ uptake performed by NCX3 in the reverse mode, triggered by a rise in [Ca2+]i or [Na+]i, whereas an opposite response was observed upon PKA stimulation, with a significant increase of the Ca2+ uptake after a rise in [Ca2+]i. The latter stimulation affected similarly the efflux capacity of NCX3 whereas Ca2+ extrusion capacity remained unaffected under activation of PKC. Next, using site-directed mutagenesis, the sensitivity of NCX3 to PKC was abolished by singly mutating its predicted phosphorylation sites T529 or S695. The sensitivity to PKC might be due to the influence of T529 phosphorylation on the Ca2+-binding domain 1. Additionally, we showed that stimulation of NCX3 by PKA occurred through residue S524. This effect may well participate in the fight-or-flight response in skeletal muscle and the long-term potentiation in hippocampus.

Catecholamines are produced by ascidian immune cells: The involvement of PKA and PKC in the adrenergic signaling pathway

The stress response is a complex mechanism, which includes changes in the immune system to enable organisms to maintain homeostasis. The neurohormones dopamine, noradrenaline (NA) and adrenalin are responsible for the physiological modulations that occur during acute stress. In the present study, we analyzed the effects of NA on the immune system specific to nitric-oxide (NO) production by subpopulations of immune cells (hemocytes) of the ascidian Phallusia nigra. We also investigated the capability of immune cells to produce catecholamine (CA). Finally, we tested the involvement of protein kinase A (PKA) and C (PKC) in the NA downstream signaling pathway. The results revealed that NA can reduce NO production by P. nigra hemocytes threefold, and that signet-ring cells, univacuolar refractile granulocytes and morula cells are the cell types most involved in this event. A challenge effected with Zymosan A induced CA production, and co-incubation with both inhibitors of the second messengers PKA and PKC revealed the involvement of these molecules in the adrenergic pathway of P. nigra hemocytes. Taken together, these results suggest that NO production can be down-regulated by NA through α- and β-adrenoceptors via the second messengers PKA and PKC.

Ionotropic Glutamate Receptors: Regulation by G-Protein-Coupled Receptors

The function of many ion channels is under dynamic control by coincident activation of G-protein-coupled receptors (GPCRs), particularly those coupled to the Gαs and Gαq family members. Such regulation is typically dependent on the subunit composition of the ionotropic receptor or channel as well as the GPCR subtype and the cell-specific panoply of signaling pathways available. Because GPCRs and ion channels are so highly represented among targets of U.S. Food and Drug Administration-approved drugs, functional cross-talk between these drug target classes is likely to underlie many therapeutic and adverse effects of marketed drugs. GPCRs engage a myriad of signaling pathways that involve protein kinases A and C (PKC) and, through PKC and interaction with β-arrestin, Src kinase, and hence the mitogen-activated-protein-kinase cascades. We focus here on the control of ionotropic glutamate receptor function by GPCR signaling because this form of regulation can influence the strength of synaptic plasticity. The amino acid residues phosphorylated by specific kinases have been securely identified in many ionotropic glutamate (iGlu) receptor subunits, but which of these sites are GPCR targets is less well known even when the kinase has been identified. N-methyl-d-aspartate, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and heteromeric kainate receptors are all downstream targets of GPCR signaling pathways. The details of GPCR-iGlu receptor cross-talk should inform a better understanding of how synaptic transmission is regulated and lead to new therapeutic strategies for neuropsychiatric disorders.

PTH1 Receptor Is Involved in Mediating Cellular Response to Long-Chain Polyunsaturated Fatty Acids

The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.

Defective protein kinase A and C pathways are common causes of disordered zona pellucida (ZP)–induced acrosome reaction in normozoospermic infertile men with normal sperm-ZP binding

To determine association between defective protein kinases C (PKC) and A (PKA) and disordered zona pellucida (ZP)-induced acrosome reaction (DZPIAR) in normozoospermic infertile men with normal sperm-ZP binding. Sperm from DZPIAR infertile men were treated without (control) or with (test) phorbol myristate acetate (PMA, PKC activator) or dibutyryl cyclic AMP (dbcAMP, PKA activator) under in vitro standard culture condition. The ZP-induced AR was assessed and compared between control and test. Public and private hospital-based clinical assisted reproduction technology (ART) centers. A total of 51 DZPIAR infertile men were involved in this study. None. Sperm-ZP binding and the ZP-induced IAR. Both PMA and dbcAMP enhanced ZP-induced AR up to a normal level (≥25%) in some subjects with DZPIAR: 29 (57%) with PMA and 27 (53%) with dbcAMP. Overall 35 (69%) had the ZP-induced AR enhanced to normal by PMA or dbcAMP but 16 (31%) had little or no response to either agent. Fourteen men responded to the two activators differently: 8 effective only with PMA and 6 effective only with dbcAMP. Defective upstream of PKC and PKA pathways are highly associated with disordered ZPIAR in normozoospermic infertile men with normal sperm-ZP binding.

Identification of a cAMP-response Element in the Regulator of G-protein Signaling-2 (RGS2) Promoter as a Key Cis-regulatory Element for RGS2 Transcriptional Regulation by Angiotensin II in Cultured Vascular Smooth Muscles

Mice deficient in regulator of G-protein signaling-2 (RGS2) have severe hypertension, and RGS2 genetic variations occur in hypertensive humans. A potentially important negative feedback loop in blood pressure homeostasis is that angiotensin II (Ang II) increases vascular smooth muscle cell (VSMC) RGS2 expression. We reported that Group VIA phospholipase A(2) (iPLA(2)β) is required for this response (Xie, Z., Gong, M. C., Su, W., Turk, J., and Guo, Z. (2007) J. Biol. Chem. 282, 25278-25289), but the specific molecular causes and consequences of iPLA(2)β activation are not known. Here we demonstrate that both protein kinases C (PKC) and A (PKA) participate in Ang II-induced VSMC RGS2 mRNA up-regulation, and that actions of PKC and PKA precede and follow iPLA(2)β activation, respectively. Moreover, we identified a conserved cAMP-response element (CRE) in the murine RGS2 promoter that is critical for cAMP-response element-binding protein (CREB) binding and RGS2 promoter activation. Forskolin-stimulated RGS2 mRNA up-regulation is inhibited by CREB sequestration or specific disruption of the CREB-RGS2 promoter interaction, and Ang II-induced CREB phosphorylation and nuclear localization are blocked by iPLA(2)β pharmacologic inhibition or genetic ablation. Ang II-induced intracellular cyclic AMP accumulation precedes CREB phosphorylation and is diminished by inhibiting iPLA(2), cyclooxygenase, or lipoxygenase. Moreover, three single nucleotide polymorphisms identified in hypertensive patients are located in the human RGS2 promoter CREB binding site. Point mutations corresponding to these single nucleotide polymorphisms interfere with stimulation of human RGS2 promoter activity by forskolin. Our studies thus delineate a negative feedback loop to attenuate Ang II signaling in VSMC with potential importance in blood pressure homeostasis and the pathogenesis of human essential hypertension.

Activation of β‐adrenergic receptors increases the in vitro migration of malignant hepatocytes

Activation of adrenergic receptors (AR) has been reported to enhance the growth and invasion of various malignancies. The effects of AR agonists on malignant hepatocyte proliferation and migration have yet to be determined. PLC/PRF/5 (PLC) and Huh-7 cells were exposed to a wide range of concentrations of the AR agonists noradrenaline (NA) and isoprenaline. Cell proliferation, migration, intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) and C (PKC), matrix metalloproteinases (MMP)-2, -3, -7 and -9, and α(1) -, β(1) - and β(2) -AR expression were documented in both cell lines. Cell proliferative activity was unaltered following exposure to physiological and stress-related concentrations of AR agonists but migration was accelerated, an effect that was inhibited by the nonselective β-AR antagonist labetalol. cAMP, PKA, PKC or MMP expression remained unchanged. Although α(1) - and β(1) -AR expressions were abundant, β(2) -AR expression was limited in both cell lines. Unlike other malignancies studied to date, in this study, the proliferative activity of malignant hepatocytes was not increased by exposure to AR agonists, a finding that could be explained by downregulation of β(2) -AR expression. The increase in malignant hepatocyte migration observed remains unexplained but does not appear to involve adenyl cyclase or MMP signaling pathways.

Myosin Binding Protein-C Slow is a Novel Substrate for Protein Kinase A (PKA) and C (PKC) in Skeletal Muscle

Myosin Binding Protein-C slow (MyBP-C slow), a family of thick filament-associated proteins, consists of four alternatively spliced forms, namely variants 1-4. Variants 1-4 share common structures and sequences; however, they differ in three regions: variants 1 and 2 contain a novel 25-residue long insertion at the extreme NH(2)-terminus, variant 3 carries an 18-amino acid long segment within immunoglobulin (Ig) domain C7, and variant 1 contains a unique COOH-terminus consisting of 26-amino acids, while variant 4 does not possess any of these insertions. Variants 1-4 are expressed in variable amounts among skeletal muscles, exhibiting different topographies and potentially distinct functions. To date, the regulatory mechanisms that modulate the activities of MyBP-C slow are unknown. Using an array of proteomic approaches, we show that MyBP-C slow comprises a family of phosphoproteins. Ser-59 and Ser-62 are substrates for PKA, while Ser-83 and Thr-84 are substrates for PKC. Moreover, Ser-204 is a substrate for both PKA and PKC. Importantly, the levels of phosphorylated skeletal MyBP-C proteins (i.e., slow and fast) are notably increased in mouse dystrophic muscles, even though their overall amounts are significantly decreased. In brief, our studies are the first to show that the MyBP-C slow subfamily undergoes phosphorylation, which may regulate its activities in normalcy and disease.

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.

Basic FGF Promotes Proliferation of Ovarian Granulosa Cells in the Laying Chickens Via FGFR1 and PKC Pathway

The proliferating effect of basic fibroblast growth factor (bFGF) on granulosa cells from ovarian pre-hierarchical follicles was evaluated in the laying chickens. Expression of bFGF receptor 1 (FGFR1) from small yellow follicles was determined by immunohistochemistry and RT-PCR. The FGFR1 protein and mRNA were intensively expressed in the granulosa layer. After 8- to 24-h treatment with bFGF (0.1-100 ng/ml), the proliferation of cultured granulosa cells was remarkably enhanced in a dose- and time-dependent manner. The FGFR1 antagonist SU5402 inhibited bFGF-induced cell proliferation. This stimulating effect was further confirmed by 5-bromo-2-deoxyuridine incorporation and terminal transferase dUTP nick end-labelling assay. Immunocytochemistry of protein kinases A (PKA) and C (PKC) showed that the pro-proliferation action of bFGF predominantly activated PKC expression. Meanwhile, the bFGF-induced cell proliferation was significantly promoted by PKC activator PMA and inhibited by PKC inhibitor H(7) (p < 0.05). In addition, the bFGF-elicited cell proliferation was accompanied with increased mRNA expression of the cell cycle-regulating genes including cyclins D1 and E1, cyclin-dependent kinases 2 and 6. In conclusion, bFGF promoted the proliferation of ovarian granulosa cells through binding with FGFR1 and involving PKC pathway in the pre-hierarchical follicles of the laying chickens.

Follicle-stimulating hormone promotes proliferation of cultured chicken ovarian germ cells through protein kinases A and C activation

The study was conducted to investigate the effects of follicle-stimulating hormone (FSH) on embryonic chicken ovarian germ cell proliferation and its possible involvements of protein kinases A (PKA) and C (PKC) pathways. Ovarian cells were treated with FSH alone or in the presence of forskolin (FRSK), PKA inhibitor (H(89)), PKC activator (PMA) or inhibitor (H(7)). The germ cell number was counted from micropictures. The immunocytochemistry of proliferating cell nuclear antigen (PCNA) was applied to identify the proliferating cells. The germ cell labeling index (LI) was determined for cell proliferation. The FSH treatment increased the germ cell number, and this stimulating effect was enhanced by FRSK or PMA, but inhibited by H(89) or H(7) in a dose-dependent manner. Moreover, the PCNA-LI showed parallel changes with germ cell numbers. This study suggests that FSH may stimulate proliferation of cultured chicken ovarian germ cells by activation of both the PKA and PKC signaling pathways.