Involvement of cyclic nucleotide-dependent protein kinases in cyclic AMP-mediated vasorelaxation

1. The involvement of cyclic AMP-dependent protein kinase (PKA) and cyclic GMP-dependent protein kinase (PKG) in the effects of cyclic AMP-elevating agents on vascular smooth muscle relaxation, cyclic nucleotide dependent-protein kinase activities and ATP-induced calcium signalling ([Ca²⁺]_i) was studied in rat aorta. Cyclic AMP-elevating agents used were a β-adrenoceptor agonist (isoprenaline), a phosphodiesterase 3 (PDE3) inhibitor (SK&F 94120) and a PDE4 inhibitor (rolipram).
2. In rat intact aorta, the relaxant effect induced by isoprenaline (0.01–0.3 μM) was decreased by a specific inhibitor of PKA, H-89, whereas a specific inhibitor of PKG, Rp-8-Br-cyclic GMPS, was without effect. No significant difference in PKA and PKG activity ratios was detected in aortic rings when isoprenaline 10 μM was used. At the same concentration, isoprenaline did not modify ATP-induced changes in [Ca²⁺]_i in smooth muscle cells. Neither H-89 nor Rp-8-Br-cyclic GMPS modified this response. These findings suggest that PKA is only involved in the relaxant effect induced by low concentrations of isoprenaline (0.01–0.3 μM), whereas for higher concentrations, other mechanisms independent of PKA and PKG are involved.
3. The relaxant effects induced by SK&F 94120 and rolipram were inhibited by Rp-8-Br-cyclic GMPS with no significant effect of H-89. Neither SK&F 94120, nor rolipram at 30 μM significantly modified the activity ratios of PKA and PKG. Rolipram inhibited the ATP-induced transient increase in [Ca²⁺]_i. This decrease was abolished by Rp-8-Br-cyclic GMPS whereas H-89 had no significant effect. These results suggest that PKG is involved in the vascular effects induced by the inhibitors of PDE3 and PDE4. Moreover, since it was previously shown that PDE3 and PDE4 inhibitors only increased cyclic AMP levels with no change in cyclic GMP level, these data also suggest a cross-activation of PKG by cyclic AMP in rat aorta.
4. The combination of 5 μM SK&F 94120 with rolipram markedly potentiated the relaxant effect of rolipram. This relaxation was decreased by H-89 and not significantly modified by Rp-8-Br-cyclic GMPS. Moreover, the association of the two PDE inhibitors significantly increased the activity ratio of PKA without changing the PKG ratio. The present findings show that PKA rather than PKG is involved in this type of vasorelaxation. The differences in the participation of PKA vs PKG observed when inhibitors of PDE3 and PDE4 were used alone or together could be due to differences in the degree of accumulation of cyclic AMP, resulting in the activation of PKA or PKG which are differently localized in the cell.
5. These findings support a role for both PKA and PKG in cyclic AMP-mediated relaxation in rat aorta. Their involvement depends on the cellular pathway used to increase the cyclic AMP level.

     

Potentiation by 2'-deoxycoformycin of the inhibitory effect of xylosyladenine on nuclear RNA synthesis in L1210 cells in vitro

The effect of the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF), on the inhibitory effect of 9-β-d-xylofuranosyladenine (XA) on nuclear RNA synthesis was examined in L1210 cells in vitro. Pretreatment of cells for 15 min with a 100 per cent inhibitory dose (1 × 10^?6 M) of dCF resulted in approximately a 3- to 8-fold reduction in the 50 per cent inhibitory dose (id₅₀) of XA for [³H]uridine and [³H]thymidine incorporation into RNA and DNA respectively. The id₅₀ for XA for RNA synthesis vs DNA synthesis was 5-fold lower in the absence of dCF and 20-fold lower in the presence of dCF, indicating the greater sensitivity of RNA synthesis to this inhibitor. Fractionation of nuclear RNA into rRNA, non-poly(A) heterogeneous RNA and poly(A)heterogeneous RNA revealed the latter species of RNA to be less sensitive to XA in the absence of dCF; however, in the presence of dCF, all three species of nuclear RNA showed similar sensitivities. Nuclear polyadenylic acid synthesis was among the most sensitive RNA fractions to XA, and was also inhibited to a greater degree by pretreatment of cells with dCF. These results indicate that XA is potentiated markedly by inhibition of adenosine deaminase, and that deamination serves as a major catabolic route for this drug.     

肼酞嗪对蛋白激酶A和蛋白激酶G的体外抑制作用(英文)

目的:研究血管扩张药肼酞嗪和KRN2391对蛋白激酶的直接作用以探讨其作用机制.方法:采用层析纯化的蛋白激酶进行体外活性测定.结果:肼酞嗪可以抑制PKA和PKG的活性(0.01-10 mmol·L~(-1)),其IC_(50)分别为1.2和2.5 mmol·L~(-1),而对PKC作用很小.KRN2391(1—1000μmol·L~(-1))对PKA,PKG和PKC活性均无明显影响.结论:肼酞嗪对PKA和PKG的直接作用可能是其扩张血管的机制之一.     

磷酸二酯酶同工酶Ⅲ和Ⅳ调节气管平滑肌环腺苷酸的区域化分布

磷酸二酯酶同工酶Ⅳ（ＰＤＥⅣ）抑制剂咯利普兰（Ｒｏｌ，３０μｍｏｌ·Ｌ－１）对３０μｍｏｌ·Ｌ－１异丙肾上腺素所致的犬气管平滑肌环腺苷酸（ｃＡＭＰ）积聚的增强作用比ＰＤＥⅢ抑制剂氰胍哒嗪（ＳＫＦ９４８３６，３０μｍｏｌ·Ｌ－１）要强，但对异丙肾上腺素所致的犬气管平滑肌松弛的增强作用比ＳＫＦ９４８３６要弱．３０μｍｏｌ·Ｌ－１Ｒｏｌ或ＳＫＦ９４８３６对３０μｍｏｌ·Ｌ－１福斯科林所致的犬气管平滑肌松弛与ｃＡＭＰ积聚的增强作用程度几乎相等．异丙肾上腺素，福斯科林单独或与ＳＫＦ９４８３６，Ｒｏｌ合用都仅引起犬气管平滑肌的可溶部ｃＡＭＰ积聚，对颗粒部ｃＡＭＰ含量无明显影响．上述结果提示犬气管平滑肌存在着ｃＡＭＰ的区域化分布，它受ＰＤＥⅢ，ＰＤＥⅣ的调节，而与平滑肌细胞的亚细胞结构无关     

Effect of modification of the 1-, 2-, and 6-positions of 9-beta-D-ribofuranosylpurine cyclic 3',5'-phosphate on the cyclic nucleotide specificity of adenosine cyclic 3',5'-phosphate- and guanosine cyclic 3',5'-phosphate-dependent protein kinases

A group of analogs of adenosine cyclic 3′,5′-phosphate (cAMP) and guanosine cyclic 3′,5′-phosphate (cGMP) with modifications in the 1-, 2- (or N²), and 6 (or N⁶)-positions of the purine ring were compared as activators of a cAMP-dependent protein kinase [PK(cAMP)] from bovine brain and of cGMP-dependent protein kinase [PK(cGMP)] from lobster tail muscle. The results suggest that the 6-amino group of cAMP is not required for the activation of PK(cAMP) by cAMP and that the 6-oxygen and 2-amino moieties of cGMP are required for the activation of PK(cAMP) by cAMP aIn the case of PK(cGMP) activation by cGMP, the 6-oxygen apparently accepts a proton from the enzyme and the 2-amino group apparently donates a proton to the enzyme.     

The effect of thiol-combining agents on polypeptide synthesis in cell-free system from Escherichia coli

Among the thiol-combining reagents studied 2,3-dicyano-1,4-dithiaanthraquinone, quajazulene, p-bromobenzylisothiocyanate, ethylester of 2-isothiocyano-3-methyl-butanoic acid, N-trichloromethylthiotetrahydrophthalimide and N,N-dimethyl-N′-phenyl-(N′-fluorodichloromethylthio) sulfamide exhibited a marked inhibitory effect on poly(U)-lysyl polyphenylalanine synthesis. These compounds inactivated the S-100 postribosomal supernatant fraction. A distinct inhibitory effect on ribosomes was found with two isothiocyanates, namely, ethylester of 2-isothiocyano-3-methylbutanoic acid and in particular p-bromobenzylisothiocyanate. The latter, however, did not influence the ribosomal peptidyl transferase activity measured in the presence of puromycin. All the compounds studied acted as inhibitors of the elongation factor EF-G. Particularly effective in this respect was 2,3-dicyano-1,4-dithiaanthraquinone. Several of the compounds investigated represent new thiol-combining reagents differing by their chemical reactivity with R-SH compounds as well as by other physico-chemical properties.     

Quercetin impairs learning and memory in normal mice via suppression of hippocampal phosphorylated cyclic AMP response element-binding protein expression

Quercetin is a naturally occurring dietary flavonol and several reports have shown that quercetin substantially affects cognitive function in disease models, which suggests that quercetin might be a useful agent for treatment of memory dysfunction. However, only one report has examined the effects of quercetin on normal cognitive function. In the present study, we investigated the potential deleterious effects of quercetin on normal cognitive function using Western blot assays and the following behavioral tasks: passive avoidance, Y-maze, and Morris water maze. In the passive avoidance task, pre-acquisition administration of quercetin (10, 20, or 40 mg/kg, p.o.) caused significant cognitive impairments in mice (P < 0.05 or P < 0.01). Quercetin-treated groups (10, 20, or 40 mg/kg, p.o.) also showed significant memory impairments compared with the control group in the Y-maze task (P < 0.05). In the Morris water maze task, there were no significant differences among the groups during training trial sessions, but at the probe trial session, the quercetin-treated group (40 mg/kg, p.o.) spent significantly less time in the target quadrant than did the control group (P < 0.05). In Western blot assays of hippocampal tissue, we found that quercetin-treated groups showed decreased expression of phosphorylated Akt (pAkt), phosphorylated calcium-calmodulin kinase II (pCaMKII), and phosphorylated cyclic AMP response element-binding protein (pCREB). These results suggest that acute administration of quercetin impairs cognitive function by suppression of pAkt and pCaMKII, which, in turn, decreases pCREB expression in the hippocampus.     

Involvement of mitogen-activated protein kinases and protein kinase C in cadmium-induced prostaglandin E2 production in primary mouse osteoblastic cells

We previously reported that cadmium (Cd) induced prostaglandin E₂ (PGE₂) biosynthesis through the activation of cytosolic phospholipase A₂ (cPLA₂) and induction of cyclooxygenase 2 (COX-2) in primary mouse osteoblastic cells. In the present study, we further investigated the mechanism of PGE₂ production by Cd focusing on the main mitogen-activated protein kinase (MAPK) subfamilies that mediate prostaglandin synthesis, extracellular signal-regulated kinase (ERK1/2 MAPK), c-jun-amino-terminal kinase (JNK MAPK) and p38 MAPK, and protein kinase C (PKC) which is activated by Cd in several kinds of cells. Cd at 2 μM and above stimulated PGE₂ production in osteoblastic cells and its production was inhibited by the kinase-specific inhibitors PD98059, SB203580, curcumin, and calphostin C. Calphostin C also inhibited the production of PGE₂ by phorbol 12-myristate 13-acetate (PMA), which is a potent activator of PKC. PD98059 inhibited PGE₂ production stimulated by PMA as well as Cd, indicating that activation of PKC by ERK1/2 MAPK was necessary for Cd-stimulated PGE₂ production. Moreover, Cd stimulated the phosphorylation of these three MAPKs, and inhibition of the phosphorylation of ERK1/2 MAPK by calphostin C was also observed. On the other hand, Cd was found to phosphorylate cPLA₂ and the phosphorylation was inhibited by PD98059, indicating that cPLA₂ was activated by Cd through ERK1/2 MAPK and released arachidonic acid (AA), a substrate of COX-2, from membranous phospholipids. From these results, it was suggested that activation of each of the ERK1/2, p38, and JNK MAPK cascades in addition to that of PKC and cPLA₂ played an important role in the Cd-stimulated biosynthesis of PGE₂ in mouse osteoblastic cells.     

Inhibition of calmodulin-dependent protein kinase II by cyclic and linear peptide alkaloids fromZizyphus species

The effects of sedative peptide alkaloids from Zizyphus species on calmodulin- dependent protein kinase II were investigated. Protein kinase II activity was assayed on the basis of its ability to activate tryptophan 5-monooxygenase as its substrate in the presence of calmodulin. All thirteen alkaloids tested were stronger inhibitors than chlorpromazine (IC50, 98 microM) on calmodulin-dependent protein kinase II. Among them, the most potent inhibitor was daechuine S27 (IC50 2.95 microM), which was stronger than pimozide (IC50, 15.0 microM).     

ATP敏感性钾电流与蛋白激酶关系的研究

目的观察蛋白激酶A(PKA)及蛋白激酶C(PKC)抑制剂和蛋白脱磷酸化物质对大鼠心室肌细胞ATP敏感性钾电流 (IKATP)的影响 ,探讨克罗卡林 (cromakalim)开放ATP敏感性钾通道的作用机制。方法采用全细胞膜片钳技术记录心室肌细胞IKATP。结果在37℃时克罗卡林 (1μmol·L-1)可阻断ATP的抑制作用 ,诱导出IKATP。PKA抑制剂PKI(6～22)amide (1μmol·L -1),可模拟克罗卡林的作用 ,激活IKATP;而PKC抑制剂calphosticC无此作用。同时还观察到蛋白脱磷酸化物质butanedioemonoxime(BDM,5mmol·L -1)也可诱发出IKATP。结论克罗卡林开放IKATP 机制与抑制PKA的活性有关 ,而与PKC无关。     

Beneficial effects of cordycepin on metabolic profiles of liver and plasma from hyperlipidemic hamsters

In this study, ¹H NMR-based metabonomics was applied to evaluate the beneficial effects of cordycepin (3′-deoxyadenosine), a natural monomer compound, on endogenous metabolic profiles of liver and plasma from hyperlipidemic Syrian golden hamsters. Hyperlipidemia was successfully established in hamsters fed by a high-fat diet for 2 weeks. The hyperlipidemic hamsters were treated with an oral administration of simvastatin (2 mg kg^? 1) or cordycepin (140 mg kg^? 1) for consecutive 4 weeks. The metabolic profiles of plasma and intact liver tissues were established using ¹H NMR spectroscopy. The results showed higher contents of lipids (triglyceride and cholesterol), lactate, acetate, alanine, glutamine together with lower contents of choline-containing compounds (e.g. phosphocholine, phosphatidylcholine, and glycerophosphocholine), glucose, and glycogen in plasma and liver samples from hyperlipidemic hamsters than those in controls. Cordycepin afforded a little lipid-regulating activity on plasma but more beneficial effects on liver, implicating that cordycepin might have a protective effect on liver under fatty liver condition.     

Involvement of protein kinase C in the UTP-mediated potentiation of cyclic AMP accumulation in mouse J774 macrophages

1. We have investigated the effects of nucleotide analogues on cyclic AMP formation in mouse J774 macrophages and the mechanisms involved.
2. UTP, in the concentration range 0.1–100 μM, induced concentration-dependent potentiation of prostaglandin E₁ (PGE₁)-induced cyclic AMP formation, but had no effect on basal cyclic AMP formation. UDP showed an equal potency, while 2-methylthio ATP, α,β-methylene ATP and β,γ-methylene ATP gave either a slight increase or had no effect at concentrations up to 100 μM. ATP, although 100 fold less effective than UTP, also caused cyclic AMP potentiation, but had no effect on agonist-stimulated or basal cyclic AMP levels.
3. The cyclic AMP potentiation effect of UTP correlated with increased [Ca²⁺]_i and inositol phosphate (IP) formation over the same concentration range.
4. Ionomycin, which evokes an increase in [Ca²⁺]_i without affecting IP formation, did not cause an increase in cyclic AMP content, indicating that UTP-induced cyclic AMP regulation is not due to activation of Ca²⁺-sensitive adenylyl cyclase isoforms.
5. Although reduced, UTP potentiation was seen in cells incubated in a Ca²⁺-free and/or BAPTA-containing medium. Under these conditions, the UTP-increased IP accumulation was similarly reduced.
6. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) also increased PGE₁ stimulation of cyclic AMP levels, and the UTP-induced potentiation of cyclic AMP formation was inhibited by either staurosporine or Ro 31-8220. Pretreatment of cells with PMA for 4–24 h resulted in marked attenuation of UTP-stimulated cyclic AMP potentiation.
7. Pretreatment with pertussis toxin (24 h, 100 ng ml⁻¹) did not significantly affect UTP-induced cyclic AMP potentiation and IP formation, although it increased the cyclic AMP response to PGE₁.
8. Analysis of J774 cells by Western blotting with antibodies specific for different protein kinase C (PKC) isoforms shows the presence of the βI, βII, δ, ε, ζ, μ, λ and ζ isoforms. Moreover, UTP significantly increased the level of PKC βI, βII, δ, ε, μ, λ and ζ immunoreactivity in the membrane fraction and decreased the cytosolic reactivity of PKC βII, δ, ε and ζ.
9. Immunoblot studies also indicate the presence of type II adenylyl cyclase.
10. These results indicate that PKC is required for the potentiation of adenylyl cyclase activity by macrophage pyrimidinoceptors, which exhibit a higher specificity for UTP and UDP than for ATP.

     

Inhibitory effect of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy

The myocardial protective effects of trilinolein, isolated from the Chinese herb Sanchi (Panax notoginseng), may be related to its antioxidant effects. In the present study, we investigated the effects of trilinolein on angiotensin II-induced cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were stimulated with angiotensin II, [3H]leucine incorporation and the beta-myosin heavy chain promoter activity were examined. We also examined the effects of trilinolein on angiotensin II-induced intracellular reactive oxygen species generation. Trilinolein significantly inhibited angiotensin II-increased protein synthesis, beta-myosin heavy chain promoter activity, and intracellular reactive oxygen species generation. Antioxidant N-acetylcysteine also decreased angiotensin II-increased protein synthesis and beta-myosin heavy chain promoter activity. Furthermore, trilinolein and N-acetylcysteine decreased angiotensin II- or hydrogen peroxide (H2O2)-activated mitogen-activated protein kinases (MAPKs) phosphorylation, and activator protein-1 (AP-1)- [or nuclear factor-kappaB (NF-kappaB)]-reporter activities. These data indicate that trilinolein inhibits angiotensin II-induced cardiomyocyte hypertrophy and beta-myosin heavy chain promoter activity via attenuation of reactive oxygen species generation.     

槲皮素-7,4′-二硫酸酯钠对重组人CK2全酶的抑制作用及动力学分析

目的观察槲皮素-7,4′-二硫酸酯钠(sodium quercetin-7,4′-disulphate,SQDS)对重组人CK2全酶的直接作用及酶动力学机制。方法通过测定转移到CK2底物上的［γ-³²P］ATP的³²P放射活度检测不同条件下的重组人CK2全酶的活性。结果重组人CK2是一种Ca²⁺,cAMP和cGMP等第二信使非依赖性蛋白激酶,与天然CK2的性质一致。SQDS对重组人CK2全酶有很强的抑制作用,IC₅₀为4.4 μmol·L^-1,抑制作用大于已知CK2抑制剂DRB和A3。CK2的动力学研究表明:SQDS与ATP和酪蛋白分别呈竞争性和非竞争性抑制作用。结论SQDS是有效的CK2抑制剂,对于开发更有效的CK2抑制剂及将SQDS用于临床提供了一定的实验依据。     

三药组合抑制由α-溶血素导致的巨噬细胞炎性因子激活

为了考察一种新型三药组合(氯唑西林、硫利达嗪和四环素)对金葡菌α-溶血素分泌的影响,以及探讨该三药组合对由金葡菌α-溶血素引起巨噬细胞炎症发生的抑制作用。试验采用抗菌药物敏感实验、三维棋盘法、溶血实验、Western blot实验方法。结果表明：三药组合显示出协同杀菌作用;三药组合能够显著协同抑制α-溶血素的分泌;三药组合可显著抑制由金葡菌α-溶血素引起巨噬细胞raw264.7相关炎症通路MAPKs、NF-κB、NLRP3蛋白的表达。说明该新型三药组合通过抑制金葡菌α-溶血素的分泌,进而阻断金葡菌α-溶血素引起的巨噬细胞炎性因子的激活。     

Inhibitory effects of wogonin on the invasion of human breast carcinoma cells by downregulating the expression and activity of matrix metalloproteinase-9

Wogonin, a naturally occurring monoflavonoid extracted from Scutellariae radix, has been shown to possess tumor therapeutic potential in vitro and in vivo. However, the effects of wogonin on tumor cells invasion remains poorly understood. In this study, we performed in vitro experiments to investigate the anti-invasive and anti-metastatic activity of wogonin in MDA-MB-231 human breast carcinoma cells. Wogonin caused a concentration-dependent suppression of cell migration, adhesion and invasion. The mechanism revealed that wogonin significantly inhibited the expression and activity of both endogenous and phorbol-12-myristate-13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) potentially associating with the suppression of translocation of protein kinase C (PKC) δ and phosphorylation of extracellular signal-regulated kinase (ERK1/2). These results suggested that wogonin could inhibit the invasion of tumor cells by downregulating the expression and activity of MMP-9, the possible targets may be PKCδ and ERK1/2.     

Different roles of PKC and PKA in effect of interferon-y on proliferation and collagen synthesis of fibroblasts

AIM: To study the signal roles of protein kinase C (PKC) and protein kinase A (PKA) in the influence of interferon-γ(IFN-γ) on proliferation and collagen synthesis of flbroblasts derived from hypertrophic scar (HS-FB) and normal skin (NS-FB). METHODS: HS-FB and NS-FB were cultured and passaged in Dulbecco's modified Eagle's medium (DMEM). Activity of PKC and PKA were assayed by transferring phosphorus (32P) into substrate after treatment with IFN-γ1000 kU/L at 10, 30, 60, and 120 min. Cell proliferation was determined with MTT assay. The collagen synthesis was measured with [3H]proline incorporation and Type Ⅲ pre-collagen was determined with radioimmunoassay. RESULTS: After exposure to IFN-γ1000 kU/L for 30 min, PKC activity of HS-FB and NS-FB increased from 2.57±0.14 and 2.13±0.12 nmol·min-1·g-1 of control to 3.75±0.19 and 3.36±0.16 nmol·min-1·g-1 respectively (P<0.05). After exposure to IFN-y 1000 kU/L for 60 and 120 min, PKA activities of HS-FB increased gradually from 0.82±0.04 nmol·m