AC－cAMP－PKA途径对淋巴细胞DNA合成的调节作用

以ＣＤ３ＭｃＡｂ为激动剂，以淋巴细胞体外ＤＮＡ合成为研究手段，探讨了ＡＣ－ｃＡＭＰ－ＰＫＡ信号途径在ＣＤ３ＭｃＡｂ诱导的淋巴细胞活化中的意义。研究结果表明ＡＣ、ｃＡＭＰ和ＰＫＡ在决定细胞对外界刺激的反应中起着重要作用。在淋巴细胞活化早期细胞内ｃＡＭＰ出现一过性升高，随着细胞活化增殖，ｃＡＭＰ降至正常水平以下。活化ＡＣ、升高细胞内ｃＡＭＰ水平可显著降低ＣＤ３ＭｃＡｂ诱导的淋巴细胞ＤＮＡ合成，而ＰＫＩ却能在一定程度上促进淋巴细胞活化增殖。     

Adenosine suppresses protein kinase A- and C-induced enhancement of glutamate release in the hippocampus

Cultured hippocampal neurons from neonatal rats were used to investigate the effect of adenosine on the release of glutamate. Spontaneous tetrodotoxin-resistant miniature excitatory postsynaptic currents (mEPSCs) through AMPA receptor channels were recorded by means of the whole-cell patch-clamp technique. Adenosine (50 microM) reversibly reduced the frequency of mEPSCs by approximately 50-60%, but did not change their amplitudes. The protein kinase A inhibitor Rp-cyclic adenosine monophosphate (100-150 microM) did not block the adenosine-dependent reduction of the mEPSC frequency, showing that adenosine is not depressing synaptic transmission via a protein kinase A (PKA)-dependent mechanism. The D1 dopamine agonist SKF-38393 (250 microM), forskolin (5 microM) and 8Br-cAMP (2 mM), known to activate the cAMP/PKA-dependent signalling pathway, all enhanced the mEPSC frequency. A subsequent application of adenosine (50 microM) strongly reduced the potentiation produced by any one of these three drugs. It also reversed protein kinase C (PKC)-dependent stimulation of glutamate release induced by phorbol myristate acetate (100 nM). Taken together, adenosine not only inhibits the spontaneous release of glutamate independently of protein kinases A and C but also reverses the enhancement of exocytosis produced by protein kinases A and C activators.     

Abnormalities of cAMP signaling in affective disorders: implication for pathophysiology and treatment

Objective: During the last decade, much attention has been given to the role of signal transduction pathways in affective disorders. This review describes the possible role of the cAMP signaling in such disorders.

Methods: Among the components of cAMP signaling, this review focuses on the cAMP-dependent phosphorylation system. We analyzed the basic components of the cAMP-dependent phosphorylation system and the preclinical evidence supporting their involvement in the biochemical action of antidepressants and mood stabilizers. The clinical data available until now, concerning the possible link between the cAMP-dependent phosphorylation system and the pathophysiology of affective disorders, are also reviewed.

Results: The studies herein presented demonstrated that the levels and the activity of cAMP-dependent protein kinase are altered by antidepressants and mood stabilizers. Furthermore, these medications are able to modify the phosphorylation state, as well as the levels of some of the cAMP-dependent protein kinase substrates. More recently, clinical studies have reported abnormalities in the cAMP-dependent phosphorylation system in both peripheral cells and the postmortem brain of patients with affective disorders.

Conclusions: Overall, these studies support an involvement of cAMP signaling in affective disorders. The precise knowledge of the findings has the potential to improve the understanding of pharmacotherapy and to provide directions for the development of novel biochemical and genetic research strategies on the pathogenesis of affective disorders.     

In vitro ex vivo assessment of Morinda citrifolia on drug metabolizing enzymes in spontaneously hypertensive rats

Morinda citrifolia Linn. (Rubiaceae), common name noni, has been used as a herbal medicine for over 2000 years. The consumption of noni, and especially the fruit, stresses the importance, urgency, and possibility of the examination of drug interaction when concomitantly administered with a drug. The objectives of this study were to determine the effects of noni juice (NJ) on aminopyrine N-demethylase (APND), uridine diphosphoglucuronosyl-transferase (UGT), and cytosolic glutathione S-transferase (GST) drug metabolizing enzymes and the molecular mechanism elucidation of NJ on APND using different inhibitors and stimulators. The in vitro results for APND showed that different concentrations of NJ significantly increased the activity in isolated hepatocytes at 1.0?ng/mL, 10?ng/mL, 10?μg/mL, 20?μg/mL, 50?μg/mL, and 100?μg/mL. The ex vivo results demonstrated that NJ (210?mg/kg) produced a statistically significant increase in APND activity following 1 day of NJ treatment. The results for UGT and GST showed a decrease in the activity of UGT at a dose of 21?mg/kg following 1 day of treatment, and at 2.1 and 21?mg/kg following 14 days of treatment. GST enzyme demonstrated an increase in activity by 100% for all doses following 1 day of treatment. Molecular mechanism elucidation of the ex vivo effect of NJ on phase I APND showed that KT5720 significantly reduced the activity as compared to control. A change in activity of APND, UGT, and GST following 1 day and 14 days of treatment suggests that all three metabolic pathways may play a role in herb–drug interaction by modulation of metabolic enzymes.     

Preparation and Characterization of pH-Sensitive Anionic Hydrogel Microparticles for Oral Protein-Delivery Applications

pH-sensitive P(MAA-g-EG) anionic hydrogel microparticles having an average diameter of approx. 4 μm were prepared by suspension photopolymerization. The pH-sensitive swelling and release behaviors of the P(MAA-g-EG) hydrogel microparticles were investigated as a biological on–off switch for the design of an oral protein delivery system triggered by external pH changes in the human GI tract. There was a drastic change of the equilibrium weight swelling ratio of P(MAA-g-EG) particles at a pH of around 5, which is the pK _a of PMAA. At pH < 5, the particles were in a relatively collapsed state, while at a pH > 5 the particles swelled to a high degree. When the concentration of the cross-linker of the hydrogel increased, the swelling ratio of the P(MAA-g-EG) hydrogel microparticles decreased at a pH higher than 5 and the pK _a of all the microparticles was in the pH range 4.0–6.0. In release experiments using Rhodamine B (Rh-B) as a model solute, the P(MAA-g-EG) hydrogel microparticles showed a pH-responsive release behavior. At low pH (pH 4.0) only a small amount of Rh-B was released while at high pH (pH 6.0) a relatively large amount of Rh-B was released from the hydrogel particles.     

Cocaine facilitates protein synthesis-dependent LTP: The role of metabotropic glutamate receptors

Cocaine addiction alters synaptic plasticity in many brain areas involved in learning and memory processes, including the hippocampus. Long-term potentiation (LTP) is one of the best studied examples of hippocampal synaptic plasticity and it is considered as one of the molecular basis of learning and memory. We previously demonstrated that in the presence of cocaine, a long lasting form of hippocampal LTP is induced by a single pulse of high frequency stimulation, which in normal conditions evokes only an early form of LTP. In this study, we further explore the molecular basis of this modulation of synaptic plasticity by cocaine. By performing pharmacological experiments on hippocampal slices, we were able to show that cocaine converts early LTP to a form of LTP dependent on protein synthesis, probably through the cAMP-dependent protein kinase and extracellular signal-regulated kinase signaling cascades. We also found that metabotropic glutamate receptors are involved in this phenomenon. These studies further clarify the molecular machinery used by cocaine to alter synaptic plasticity and modulate learning and memory processes.     

Hypothalamic insulin and glucagon-like peptide-1 levels in an animal model of depression and their effect on corticotropin-releasing hormone promoter gene activity in a hypothalamic cell line

Background

In depression, excessive glucocorticoid action may cause maladaptive brain changes, including in the pathways controlling energy metabolism. Insulin and glucagon-like peptide-1 (GLP-1), besides regulation of glucose homeostasis, also possess neurotrophic properties. Current study was aimed at investigating the influence of prenatal stress (PS) on insulin, GLP-1 and their receptor (IR and GLP-1R) levels in the hypothalamus. GLP-1 and GLP-1R were assayed also in the hippocampus and frontal cortex – brain regions mainly affected in depression. The second objective was to determine the influence of exendin-4 and insulin on CRH promoter gene activity in in vitro conditions.

The novel distribution of phosphodiesterase-4 subtypes within the rat retina

Phosphodiesterases (PDEs) are important regulators of signal transduction processes. While much is known about the function of cyclic GMP-specific PDEs in the retina, much less is known about the closely related, cyclic AMP-specific PDEs. The purpose of the present study is to characterize and localize PDE4 within the adult rat retina. We have used Western blotting, RT-PCR, and immunohistochemistry together with retrograde labeling to determine the presence and location of each PDE4 subtype. Western blot analysis revealed that multiple isoforms of PDE4A, B, and D subtypes are present within the retina, whereas the PDE4C subtype was absent. These data were confirmed by RT-PCR. Using immunohistochemistry we show that all three PDE4s are abundantly expressed within the retina where they all colocalize with retrograde-labeled retinal ganglion cells, as well as bipolar cells, horizontal cells, and cholinergic amacrine cells, whereas Müller cells lack PDE4 expression. Uniquely, PDE4B was expressed by the inner and outer segments of rod photoreceptors as well as their terminals within the outer plexiform layer. Collectively, our results demonstrate that PDE4s are abundantly expressed throughout the rodent retina and this study provides the framework for further functional studies.     

Epigallocatechin gallate stimulates the neuroreactive salivary secretomotor system in autoimmune sialadenitis of MRL-Faslpr mice via activation of cAMP-dependent protein kinase A and inactivation of nuclear factor κB

Abstract

The water channel aquaporin 5 (AQP5) plays a crucial role in regulating salivary flow rates. Xerostomia is often observed in patients with Sjögren's syndrome, and this is attributed to reduced AQP5 expression in the salivary glands. Recently, anti-type 3 muscarinic cholinergic receptors (M₃R) autoantibodies and nuclear factor κB (NF-κB) have been found to be negative regulators of AQP5 expression in the salivary gland. Anti-M₃R autoantibodies desensitize M₃R to salivary secretagogues in Sjögren's syndrome, while activated NF-κB translocates to nuclei and binds to the AQP5 gene promoter, resulting in the suppression of AQP5 expression. We previously documented that epigallocatechin gallate (EGCG), which is a robust antioxidant contained in green tea, ameliorates oxidative stress-induced tissue damage to the salivary glands of MRL/MpJ-lpr/lpr (MRL-Fas^lpr) mice, which are widely used as a model of Sjögren's syndrome. Reactive oxygen species (ROS) can activate NF-κB and inactivate protein kinase A (PKA), which is a key driver of AQP5 expression. In this study, we examined the effects of administering EGCG to MRL-Fas^lpr mice with autoimmune sialadenitis on the levels of AQP5, activated NF-κB p65 subunit, activated PKA, activated c-Jun N-terminal kinase (JNK) (an activator of NF-κB), inhibitor κB (IκB) and histone deacetylase 1 (HDAC1) (an inhibitor of NF-κB). In EGCG-treated mice, intense aster-like immunostaining for AQP5 was observed on the apical plasma membranes (APMs) of submandibular gland acinar cells. Likewise, PKA, IκB and HDAC1 were highly expressed in salivary gland tissues, whereas the expression of JNK and NF-κB p65 was negligible. Rank correlation and partial correlation analyses revealed that treatment with EGCG upregulated AQP5 expression on the APM of acinar cells through activation of PKA and inactivation of NF-κB, while IκB and HDAC1 played a pivotal role in the induction of AQP5 expression by PKA. Our study indicates that EGCG may have therapeutic potential for Sjögren's syndrome patients.     

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein–membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

Myristoylation is a major type of posttranslational modification that occurs at the N terminus of a myriad of proteins (1–4). Depending on the target, myristoylation can contribute to the structure, stability, protein–protein interactions, and subcellular localization of the modified proteins (2–4). In particular, myristoylation often facilitates protein association with the membrane. However, it is thought that, with an acyl chain of only 14 carbons, myristate confers insufficient energy for stable association of a protein with the membrane (5, 6). Subsequent studies have shown that a second membrane-affinity motif, such as a stretch of basic residues or a second lipid modification, is required for the membrane association of several myristoylated proteins (reviewed in refs. 2–4). When the second membrane-affinity motif is removed or neutralized, either physiologically or via mutagenesis, the membrane localization of the protein is disrupted. Thus, the canonical view is that myristoylation alone is not sufficient to provide a functionally significant association of a protein with the plasma membrane, even though myristoylation has been observed to be associated with reconstituted lipid bilayers (7).Myristoylation was first discovered in the catalytic subunit of protein kinase A (PKA) (1, 8), which is a primary mediator of the second messenger cAMP that plays diverse essential roles in nearly all organisms, from bacteria to humans. At rest, PKA is a tetrameric protein that consists of two regulatory subunits (PKA-Rs) and two catalytic subunits (PKA-Cs). PKA holoenzymes are anchored to specific subcellular locations via the binding of PKA-R with A-Kinase anchoring proteins (9–12). In the presence of cAMP, PKA-C is released from PKA-R and becomes an active kinase (8, 13–16).Despite being myristoylated, PKA-C is thought to function as a cytosolic protein because of its high solubility (14, 15). Consistently, a PKA-C mutant with disrupted myristoylation has been shown to support the phosphorylation of certain substrates and to maintain several PKA functions in heterologous cells (17). Structural studies found that the PKA-C myristoylation is folded into a hydrophobic pocket, and it was proposed that this myristoylation serves a structural role (18–20). This view has started to shift based on recent reports showing that activated PKA-C can associate with the membrane in a myristoylation-dependent manner (16, 21, 22), including in neurons. However, the extent to which PKA-C associates with the plasma membrane in living cells and its functional significance are not known. Furthermore, as discussed above, myristolyation-mediated membrane association is thought to require a second-membrane motif. The identity of this second membrane-affinity motif has not been determined. Therefore, we set out to address these questions.