cGMP-dependent protein kinase in regulation of basal tone and in nitroglycerin- and nitric-oxide-induced relaxation in porcine coronary artery

Cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) may act as a critical enzyme for nitric-oxide-induced vasodilation. In this study, the role of PKG in regulation of basal tension and in relaxation induced by nitrovasodilators in coronary arteries was determined. Under basal conditions, Rp-8-Br-PET-cGMPS, a specific PKG inhibitor, evoked a significant contraction of isolated porcine coronary arteries, which was prevented by nitro-l-arginine or the removal of the endothelium. Relaxation to nitroglycerin and (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate) in vessels preconstricted with U46619 was largely abolished by 1H-[1,2,4]oxadiazolo[4,3]quinoxalin-1-one (ODQ) and inhibited by 48 to 79% by Rp-8-Br-PET-cGMPS. Relaxation of the vessels to 8-Br-cGMP was inhibited by 56% by Rp-8-Br-PET-cGMPS. The basal activity of PKG but not that of cyclic adenosine monophosphate-dependent protein kinase (PKA) was inhibited by nitro-l-arginine, ODQ, or Rp-8-Br-PET-cGMPS. The activity of PKG but not that of PKA was increased by nitroglycerin and DETA NONOate in intact vessels and increased by cGMP in the tissue homogenates. These effects were abolished by Rp-8-Br-PET-cGMPS but not by myristoylated PKI, a specific inhibitor of PKA. These results suggest that in porcine coronary arteries, PKG is involved in the regulation of basal tension and plays a primary role in relaxation induced by nitrovasodilators, whereas PKA may play a minor role.     

Attenuation of zinc-induced neuronal death by the interaction of growth inhibitory factor with Rab3A in rat hippocampal neurons

We examined whether early injury-associated activation of cyclic AMP response element binding protein (CREB) in the spinal dorsal horn was mediated by the cyclic AMP-dependent protein kinase A (PKA) pathway. Significant increases in the levels of phosphorylated CREB (pCREB), phosphorylated PKAII regulatory subunit (pPKA), and PKA catalytic subunit (PKA cat) were elicited 2 h after loose ligation of the sciatic nerve. These injury-elicited increases were significantly reduced by dorsal horn application of the cell-permeable PKA inhibitor Rp-8-Br-cAMPS. The cell-permeable PKA activator Sp-8-Br-cAMPS significantly increased the levels of pCREB, pPKA and PKA cat 2 h after application onto the dorsal horn of control, uninjured animals. Our data lent further support to the notion that activation of PKA may play an important role in the early stages of nerve injury-elicited plasticity in the dorsal horn.     

Ryanodine receptors contribute to cGMP-induced late-phase LTP and CREB phosphorylation in the hippocampus

We previously found that the nitric oxide (NO)-cGMP-cGMP-dependent protein kinase (PKG) signaling pathway acts in parallel with the cAMP-cAMP-dependent protein kinase (PKA) pathway to produce protein and RNA synthesis-dependent late-phase long-term potentiation (L-LTP) and cAMP response element-binding protein (CREB) phosphorylation in the CA1 region of mouse hippocampus. We have now investigated the possible involvement of a downstream target of PKG, ryanodine receptors. L-LTP can be induced by either multiple-train tetanization, NO or 8-Br-cGMP paired with one-train tetanization, or the cAMP activator forskolin, and all three types of potentiation are accompanied by an increase in phospho-CREB immunofluorescence in the CA1 cell body area. Both the potentiation and the increase in phospho-CREB immunofluorescence induced by multiple-train tetanization or 8-Br-cGMP paired with one-train tetanization are reduced by prolonged perfusion with ryanodine, which blocks Ca(2+) release from ryanodine-sensitive Ca(2+) stores. By contrast, neither the potentiation nor the increase in immunofluorescence induced by forskolin are reduced by depletion of ryanodine and inositol-1,4,5-triphosphate (IP3)-sensitive Ca(2+) stores. These results suggest that NO, cGMP, and PKG cause release of Ca(2+) from ryanodine-sensitive stores, which in turn causes phosphorylation of CREB in parallel with PKA during the induction of L-LTP.     

Effects of type I protein kinase A modulation on the T cell distal pole complex

The distal pole complex (DPC) assembles signalling proteins at the T cell pole opposite the immunological synapse (IS) and is thought to facilitate T cell activation by sequestering negative regulatory molecules away from the T cell receptor-proximal signalling machinery. Here, we report the translocation of type I protein kinase A (PKA) to the DPC in a fraction of T cells following activation and the localization of type I PKA with known components of the DPC. We propose that sequestration of type I PKA and concomitant loss of cAMP-mediated negative regulation at the IS may be necessary to allow full T cell activation. Moreover, composition of the DPC appears to be modulated by type I PKA activity, as the antagonist Rp-8-Br-cAMPS inhibited translocation of type I PKA and other DPC proteins.     

cAMP protects neutrophils against TNF-alpha-induced apoptosis by activation of cAMP-dependent protein kinase, independently of exchange protein directly activated by cAMP (Epac)

It is unclear by which receptor cyclic adenosine monophosphate (cAMP) acts to promote neutrophil survival. We found that 8-(4-chlorophenylthio)-2'-O-methyl-cAMP, a specific activator of the recently discovered cAMP receptor, cAMP-regulated guanosine 5'-triphosphate exchange protein directly activated by cAMP, failed to protect human neutrophils from cell death. In contrast, specific activators of cAMP-dependent protein kinase type I (cA-PKI) could protect against death receptor [tumor necrosis factor receptor 1 (TNFR-1), Fas]-mediated apoptosis as well as cycloheximide-accelerated "spontaneous" apoptosis. A novel "caged" cA-PK-activating analog, 8-bromo (8-Br)-acetoxymethyl-cAMP, was more than 20-fold more potent than 8-Br-cAMP to protect neutrophils challenged with TNF-alpha against apoptosis. This analog acted more rapidly than forskolin (which increases the endogenous cAMP production) and allowed us to demonstrate that cA-PK must be activated during the first 10 min after TNF-alpha challenge to protect against apoptosis. The protective effect was mediated solely through cA-PK activation, as it was abolished by the cA-PKI-directed inhibitor Rp-8-Br-cAMPS and the general cA-PK inhibitor H-89. Neutrophils not stimulated by cAMP-elevating agents showed increased apoptosis when exposed to the cA-PK inhibitors Rp-8-Br-cAMPS and H-89, suggesting that even moderate activation of cA-PK is sufficient to enhance neutrophil longevity and thereby contribute to neutrophil accumulation in chronic inflammation.     

Acute hypoxia induces vasodilation and increases coronary blood flow by activating inward rectifier K+ channels

We examined the effects of acute hypoxia on vascular tone and coronary blood flow (CBF) in rabbit coronary arteries. In the pressurized arterial preparation of small arteries (<100 μm) and the Langendorff-perfused rabbit hearts, hypoxia induced coronary vasodilation and increased CBF in the presence of glibenclamide (K_ATP channel blocker), Rp-8-Br-PET-cGMPs [cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor, Rp-cGMPs], and methionyl transfer RNA synthetase (MRS) 1334 (adenosine A₃ receptor inhibitor); these increases were inhibited by the inward rectifier K⁺ (Kir) channel inhibitor, Ba²⁺. These effects were blocked by the adenylyl cyclase inhibitor SQ 22536 and by the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) inhibitors Rp-8-CPT-cAMPs (Rp-cAMPs) and KT 5720. However, cGMP-dependent protein kinase was not involved in the hypoxia-induced increases of the vascular diameter and CBF. In summary, our results suggest that acute hypoxia can induce the opening of Kir channels in coronary artery that has small diameter (<100 μm) by activating the cAMP and PKA signalling pathway, which could contribute to vasodilation and, therefore, increased CBF.     

Heterogeneity in relaxation of different sized porcine coronary arteries to nitrovasodilators: role of PKG and MYPT1

The present study was to determine the role of the type I isoform of cGMP-dependent protein kinase (PKG I) and its downstream effector myosin phosphatase target subunit 1 (MYPT1) in the responses of different sized coronary arteries to nitrovasodilators. Relaxations of isolated porcine coronary arteries were determined by isometric tension recording technique. Protein levels of PKG I and its effectors were analyzed by Western blotting. The activities of PKG I and MYPT1 were studied by analyzing phosphorylation of vasodilator-stimulated phosphoprotein (VASP) and MYPT1, respectively. Nitroglycerin, DETA NONOate, and 8-Br-cGMP caused greater relaxations in large than in small coronary arteries. Relaxations were attenuated to a greater extent by Rp-8-Br-PET-cGMPS (a PKG inhibitor) in large vs. small arteries. The expressions of PKG I and MYPT1 in large arteries were more abundant than in small arteries. DETA NONOate stimulated phosphorylation of VASP at Ser239 and inhibited phosphorylation of MYPT1 at Thr853 to a greater extent in large than in small arteries. A suppressed phosphorylation of MYPT1 at Thr853 was caused by 8-Br-cGMP in large but not small arteries, which was inhibited by Rp-8-Br-PET-cGMPS. These results suggest that the greater responsiveness of large coronary arteries to nitrovasodilators result in part from greater activities of PKG I and MYPT1. Dysfunction in nitric oxide signaling is implicated in the vulnerability of large coronary arteries to certain disorders such as atherosclerosis and spasm. Augmentation of PKG I–MYPT1 signaling may be of therapeutic benefit for combating these events.     

Cyclic AMP-dependent protein kinase mediates ocular dominance shifts in cat visual cortex

Visual experience during a critical period early in postnatal development can change connections within mammalian visual cortex. In a kitten at the peak of the critical period (approximately P28-42), brief monocular deprivation can lead to complete dominance by the open eye, an ocular dominance shift. This process is driven by activity from the eyes, and depends on N-methyl-D-aspartate (NMDA) receptor activation. The components of the intracellular signaling cascade underlying these changes have not all been identified. Here we show that inhibition of protein kinase A (PKA) by Rp-8-Cl-cAMPS blocks ocular dominance shifts that occur following monocular deprivation early in the critical period. Inhibition of protein kinase G by Rp-8-Br-PET-cGMPS had no effect, indicating a specificity for the PKA pathway. Enhancement of PKA activity late in the critical period with Sp-8-Cl-cAMPS did not increase plasticity. PKA is a necessary component of the pathway leading to cortical plasticity during the critical period.     

Inhibition of cGMP-dependent protein kinase reduces the response to sonic hedgehog in neuralized embryoid bodies

Previously, we have shown that increasing the intracellular cGMP concentration enhances the sonic hedgehog (Shh) response in neural plate cells. The use of two mouse embryonic stem (ES) cell lines allowed a highly sensitive and reproducible quantification of the Shh response in neuralized embryoid bodies. Here we demonstrate that the specific, membrane-permeable cGMP-dependent protein kinase G-Ialpha (PKG-Ialpha) inhibitor DT-2 prevents an efficient Shh response, indicating that the effects of cGMP on the Shh response are mediated via PKG. We also demonstrate that the PKG acts upon the Shh response upstream of the Ptc1 promoter, which is up-regulated invariably and early in response to Shh, significantly limiting the targets for PKG phosphorylation to molecules involved in the early steps of the Shh response. These effects of cGMP and PKG are antagonistic to those of cAMP and PKA, and thus provide a mechanism by which the sensitivity of cells to the effects of Shh can be regulated, by modulating the intracellular cyclic nucleotide concentration.     

Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells

Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (> or =100 micromol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).     

Cx40、43调节缺氧处理大鼠肠系膜上动脉收缩反应性的信号途径

目的： 研究连接蛋白（connexin,Cx)40或43对cAMP-PKA、cGMP-PKG和DG-PKC信号通路的影响及对缺氧处理大鼠肠系膜上动脉内膜依赖的血管收缩反应性的调节作用。方法: 以SD大鼠肠系膜上动脉（superior mesenteric artery,SMA）为研究对象,用Cx40或Cx43的反义寡脱氧核苷酸（antisense oligodeoxyribonucleotide,AODN）阻断SMA的Cx40或Cx43的表达,观察缺氧处理后血管的环一磷酸腺苷(cyclic adenosine monophosphate,cAMP)、环一磷酸鸟苷(cyclic guanosine monophosphate,cGMP)、二酰基甘油(diacylglycerol,DG)浓度和蛋白激酶A（protein kinase A,PKA)、蛋白激酶G（protein kinase G,PKG)、蛋白激酶C（protein kinase C,PKC)活性的变化,以及这些变化与内膜依赖的血管收缩反应性变化的关系。结果: Cx40AODN可以降低血管cAMP、cGMP的浓度和PKA、PKG的活性,增加DG的浓度、PKC的活性和血管内膜依赖的收缩反应性;Cx43AODN可以增加血管cAMP、cGMP的浓度和PKA、PKG的活性,降低DG的浓度、PKC的活性和血管内膜依赖的收缩反应性。结论: Cx40、Cx43通过cAMP-PKA、cGMP-PKG、DG-PKC信号通路参与了休克后内膜依赖的血管收缩反应性的调节。     

Both cGMP and peroxynitrite mediate chronic interleukin-6-induced negative inotropy in adult rat ventricular myocytes

We previously showed that chronic exposure to interleukin (IL)-6 decreases contractile and sarcoplasmic reticular (SR) function assessed by postrest potentiation (PRP) via a nitric oxide (NO)-dependent mechanism in adult rat ventricular myocytes (ARVM). Cyclic GMP (cGMP) has been associated with NO-associated negative inotropic effects of IL-6 during acute exposure; however, its role in chronic cardiac effects of IL-6 remains unclear. The present study examined the roles of cGMP and peroxynitrite (ONOO⁻) in chronic IL-6-induced negative inotropy in ARVM. After ARVM were exposed to IL-6 for 2–24 h, intracellular cGMP contents were time dependently increased; this was mimicked by a NO donor and abolished by 1 H -[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), or Rp-8-Br-cGMP, an inhibitor of cGMP-dependent protein kinase G (PKG). Meanwhile, the IL-6-induced decrease in PRP at 2 h was blocked by ODQ or Rp-8-Br-cGMP. By contrast, ODQ or Rp-8-Br-cGMP only attenuated the inhibition of PRP induced by IL-6 after 24 h exposure. Furthermore, IL-6 time dependently increased superoxide anion production and ONOO⁻ formation; the latter was abolished by 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron (III) (FeTPPS), an ONOO⁻ decomposition catalyst. Interestingly, FeTPPS had no effect on the IL-6-elicited decrease in PRP at 2 h, but attenuated it after 24 h exposure. Moreover, inhibition of sGC/cGMP/PKG, but not ONOO⁻ formation, abolished the IL-6-induced inhibition of kinetics of myocyte contraction during 24 h exposure. We conclude that while the sGC/cGMP/PKG pathway was the primary mechanism for chronic IL-6-induced negative inotropy at 2 h, both sGC/cGMP/PKG and ONOO⁻, at least in part, mediate the IL-6-induced inhibition of SR function after 24 h exposure.     

柚皮素通过环核苷酸通路拮抗血小板聚集的体外研究

目的:探讨柚皮素拮抗二磷酸腺苷(ADP)诱导血小板聚集的作用机制。方法:采用ELISA检测柚皮素对ADP诱导的大鼠血小板内环磷酸腺苷(cAMP)、环磷酸鸟苷(cGMP)水平的影响。采用高效液相法检测柚皮素对血小板磷酸二酯酶(PDE)活性影响。采用Western blot检测柚皮素对ADP刺激的血小板内血管扩张刺激磷蛋白(VASP)磷酸化形式p-VASP(Ser157)、p-VASP(Ser239)的蛋白水平的影响。分别给予蛋白激酶A(PKA)抑制剂H89、蛋白激酶G(PKG)抑制剂Rp-8-Br-PET-cGMPS和蛋白激酶C(PKC)抑制剂GF109203X预先孵育血小板后,再用柚皮素处理,然后给予ADP刺激,Western blot检测p-VASP(Ser239)蛋白水平。采用血小板聚集仪进一步观察PKA抑制剂、PKG抑制剂预先孵育血小板是否影响柚皮素对ADP诱导血小板聚集的抑制作用。结果:柚皮素剂量依赖性地升高ADP抑制的血小板内cGMP水平,而并不改变cAMP水平。柚皮素还能显著升高血小板PDE活性。Western blot结果显示,柚皮素可明显升高由ADP抑制的p-VASP(Ser239)水平,但不影响p-VASP(Ser157)的蛋白水平,预先孵育PKG或PKC抑制剂并不影响柚皮素对p-VASP(Ser239)蛋白表达的作用,而预先孵育PKA抑制剂后,则能抑制柚皮素对p-VASP(Ser239)蛋白表达的作用。PKA抑制剂能阻断柚皮素对血小板聚集的拮抗作用,而PKG抑制剂并不影响其作用。结论:柚皮素可能通过升高血小板内cGMP水平和激活PKA依赖的信号通路来介导VASP的磷酸化,从而发挥抗血小板聚集的作用。     

Regulation of epithelial sodium channels by cGMP/PKGII

Airway and alveolar fluid clearance is mainly governed by vectorial salt movement via apically located rate-limiting Na⁺ channels (ENaC) and basolateral Na⁺/K⁺-ATPases. ENaC is regulated by a spectrum of protein kinases, i.e. protein kinase A (PKA), C (PKC), and G (PKG). However, the molecular mechanisms for the regulation of ENaC by cGMP/PKG remain to be elucidated. In the present study, we studied the pharmacological responses of native epithelial Na⁺ channels in human Clara cells and human αβγδ ENaCs expressed in oocytes to cGMP. 8-pCPT-cGMP increased amiloride-sensitive short-circuit current ( I _sc) across H441 monolayers and heterologously expressed αβγδ ENaC activity in a dose-dependent manner. Similarly, 8-pCPT-cGMP (a PKGII activator) but not 8-Br-cGMP (a PKGI activator) increased amiloride-sensitive whole cell currents in H441 cells in the presence of CFTRinh-172 and diltiazem. In all cases, the cGMP-activated Na⁺ channel activity was inhibited by Rp-8-pCPT-cGMP, a specific PKGII inhibitor. This was substantiated by the evidence that PKGII was the sole isoform expressed in H441 cells at the protein level. Importantly, intratracheal instillation of 8-pCPT-cGMP in BALB/c mice increased amiloride-sensitive alveolar fluid clearance by ∼30%, consistent with the in vitro results. We therefore conclude that PKGII is an activator of lung epithelial Na⁺ channels, which may expedite the resolution of oedematous fluid in alveolar sacs.     

PKA regulates HMGB1 through activation of IGFBP-3 and SIRT1 in human retinal endothelial cells cultured in high glucose

Objective and Design

Inflammation is a key component of a number of diseases, including diabetic retinopathy. We investigated the cellular pathway by which protein kinase A (PKA) inhibited high mobility group box 1 (HMGB1).

Methods

Primary human retinal endothelial cells (REC) were grown in normal glucose (5 mM) or high glucose (25 mM). Cells in high glucose were treated with exchange protein for cAMP 1 (Epac1) and IGFBP-3 siRNA. Additional cells in high glucose were treated with forskolin, a PKA agonist, and Epac1 siRNA. Some cells were treated with a plasmid for insulin-like growth factor binding protein 3 (IGFBP-3) that does not bind IGF-1. Finally, some REC received Ex527, a sirtuin 1 (SIRT1) antagonist, prior to forskolin treatment. Protein analyses were done for HMGB1, Epac1, IGFBP-3, SIRT1, and PKA.

Results

PKA inhibited cytoplasmic HMGB1, independent of Epac1 actions. PKA activated IGFBP-3 and SIRT1 to inhibit cytoplasmic HMGB1. High glucose inhibited SIRT1 levels and increased cytoplasmic HMGB1 in REC.

Conclusions

PKA requires active IGFBP-3 and SIRT1 to inhibit HMGB1 inflammatory actions in the retina vasculature. Activation of these pathways may offer new targets for therapy development.