Cyclic cytidine 3′,5′-monophosphate (cCMP) signals via cGMP kinase I

We analysed the function and intracellular signalling of the cyclic pyrimidinic nucleotide cCMP. The membrane-permeable cCMP analogue dibutyryl-cCMP mediated mouse aorta relaxation. cCMP activated purified cGMP-dependent protein kinase (cGK) Iα and Iβ and stimulated cGK in aorta lysates. cCMP-induced relaxation was abolished in cGKI-knockout tissue. Additionally, deletion of inositol-trisphosphate receptor associated cGKI substrate (IRAG) suppressed cCMP-mediated relaxation. Signalling of cCMP via cGKI/IRAG appears to be of broader physiological importance because cCMP-mediated inhibition of platelet aggregation was absent in cGKI- and IRAG-deficient platelets. These results demonstrate that cCMP acts as intracellular messenger molecule, most unexpectedly utilizing the cGMP signal transduction pathway.     

Differential modulation by N4, 2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate of neutrophil activation.

The cyclic pyrimidine nucleotide, cCMP, is an endogenous substance in mammalian cells but little is known on its functional role. We studied the effects of cCMP, its cell-permeant analogue, N4,2'-O-dibutyryl cytidine 3':5'-cyclic monophosphate (Bt2cCMP), and of butyrate on superoxide (O2-) formation and cytosolic Ca2+ [( Ca2+]i) in human neutrophils. Bt2cCMP inhibited O2- formation and the rise in [Ca2+]i induced by a chemotactic peptide at submaximally effective concentrations. O2- formation induced by platelet-activating factor was potentiated by Bt2cCMP, whereas the cyclic nucleotide had no effect on the rise in [Ca2+]i induced by this agonist. Bt2cCMP enhanced O2- formation induced by tau-hexachlorocyclohexane at a submaximally effective concentration. O2- formation stimulated by complement C5a, concanavalin A, NaF, A 23187, phorbol myristate acetate and arachidonic acid was not affected by Bt2cCMP. cCMP was less effective than Bt2cCMP to inhibit fMet-Leu-Phe-induced O2- formation, and butyrate was without effect on any of the functional parameters studied. Our data show that a cell-permeant analogue of cCMP differentially inhibits and potentiates activation of human neutrophils.     

Decreased activities of cyclic cytidine 3',5'-monophosphate phosphodiesterase in Morris hepatomas having varying growth rates

1. The activities of cyclic cytidine 3',5'-monophosphate (cCMP) phosphodiesterase in normal rat liver and host liver (bearing hepatoma 5123 t.c.(h)) were compared with those of three Morris hepatomas of varying growth rates. 2. The results show that the order of enzyme activity was as follows: normal liver = host liver greater than 7794A (slow growth rate) greater than 5123 t.c.(h) (intermediate growth rate) greater than 7800 (fast growth rate). 3. The enzyme had a pH optimal value of about 7.0 and an apparent Km for cCMP about 2.8 mM; its activity was slightly affected by the presence of calmodulin (100 micrograms/ml) and/or CaCl2 (100 microM), but showed variable responses to other cations (La3+, Mg2+, Mn2+, Zn2+, Fe2+, Na+ and K+).     

Inhibition of DNA synthesis of adult rat hepatocytes in primary culture by dibutyrylcytidine 3', 5'-cyclic monophosphate

The effect of dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) on DNA synthesis of adult rat hepatocytes in primary culture was examined. Bt2cCMP caused dose-dependent inhibition of the DNA syntheses stimulated by various growth factors including human hepatocyte growth factor (hHGF). Dibutyryladenosine 3',5'-cyclic monophosphate (Bt2cAMP) inhibited the DNA synthesis more effectively than Bt2cCMP, but dibutyrylguanosine 3',5'-cyclic monophosphate (Bt2cGMP) and n-butyrate had a slight or null inhibitory effect. When added at the onset of DNA synthesis, Bt2cAMP was much less effective, but Bt2cCMP was still effective. Thus Bt2cCMP is able to inhibit growth factor-stimulated hepatocyte proliferation.     

Radioimmunoassay of cytidine 3',5' monophosphate (cCMP). I. Development of the assay.

A method is presented for the production of reagents for a radioimmunoassay for cCMP. cCMP was succinylated at the 2′0 position with [1,4 ¹⁴C] succinic anhydride, and the monosuccinyl cCMP coupled to Keyhole limpet hemocyanin and injected into rabbits. Antibodies to cCMP were produced that showed minimal crossreactivity with other cyclic nucleotides. Monosuccinyl cCMP was coupled to tyrosine methyl ester, then labeled with ¹²⁵I, and used as the radiolabeled ligand in the immunoassay of cCMP. By use of this assay, the concentration of cCMP in various tissues of rat and guinea pig have been determined.     

Interaction of cCMP with the cGK,cAK and MAPK Kinases in Murine Tissues

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-³²P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.     

Regulation of Hyperpolarization-activated Cyclic Nucleotide-gated (HCN) Channel Activity by cCMP

Activation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels is facilitated in vivo by direct binding of the second messenger cAMP. This process plays a fundamental role in the fine-tuning of HCN channel activity and is critical for the modulation of cardiac and neuronal rhythmicity. Here, we identify the pyrimidine cyclic nucleotide cCMP as another regulator of HCN channels. We demonstrate that cCMP shifts the activation curves of two members of the HCN channel family, HCN2 and HCN4, to more depolarized voltages. Moreover, cCMP speeds up activation and slows down deactivation kinetics of these channels. The two other members of the HCN channel family, HCN1 and HCN3, are not sensitive to cCMP. The modulatory effect of cCMP is reversible and requires the presence of a functional cyclic nucleotide-binding domain. We determined an EC(50) value of ～30 μm for cCMP compared with 1 μm for cAMP. Notably, cCMP is a partial agonist of HCN channels, displaying an efficacy of ～0.6. cCMP increases the frequency of pacemaker potentials from isolated sinoatrial pacemaker cells in the presence of endogenous cAMP concentrations. Electrophysiological recordings indicated that this increase is caused by a depolarizing shift in the activation curve of the native HCN current, which in turn leads to an enhancement of the slope of the diastolic depolarization of sinoatrial node cells. In conclusion, our findings establish cCMP as a gating regulator of HCN channels and indicate that this cyclic nucleotide has to be considered in HCN channel-regulated processes.     

Cytldlne Cyclic (3′, 5′) Monophosphate: Cyclic Nucleotide With a Non-Characteristic Ribose Ring Conformation

Abstract

Cytidine 3′,-5′-cyclic phosphate (cCMP) occurs in nature and has growth stimulatory activity on L-1210 cells. The initiation of cell growth by cCMP, under conditions where CAMP, cGMP and cUMP delay the onset of proliferation suggests that cCMP may play a regulatory role in the cell metabolism. It has been reported that in 3′,5′-cyclic nucleotides, the phosphate ring fused to the furanose ring resuicts the conformation of the furanose ring to the twist form C(3′) endo C(4′) exo (³T₄), in contrast to the C(2′) endo C(3′) endo (²T₃) and C(3′) endo C(2′) exo (³T₂) twist forms normally found in nucleotides and nucleosides. We have carried out an accurate crystal structure of cCMP and found that the furanose ring in cCMP has the C(3′) endo C(2′) exo conformation (³T₂), with a pseudo rotation amplitude (P) of 44° and phase angle τ_m of 12°. cCMP is in low anti conformation (X_CN = 15.4°) and O(5′) has the fixed g conformation. The phosphate ring is constrained to the chair conformation, as in other cyclic nucleotides. The two exocyclic P-O bond distances are short (1.489, 1.476Å) and the ring angle at N(3) is large (125.2°) suggesting that the molecule in the solid state is a zwitterion with a plus charge on N(3). The crystals are hydrated and highly unstable. The three water molecules are highly disordered in ten locations. The crystals of cCMP 3H₂O are hexagonal, a = 16.294(3), b = c = 11.099(4)Å, space group P6₁, final R value is 0.067 for 1620 reflections 230.     

Binding of regulatory subunits of cyclic AMP-dependent protein kinase to cyclic CMP agarose

The bacterial adenylyl cyclase toxins CyaA from Bordetella pertussis and edema factor from Bacillus anthracis as well as soluble guanylyl cyclase α(1)β(1) synthesize the cyclic pyrimidine nucleotide cCMP. These data raise the question to which effector proteins cCMP binds. Recently, we reported that cCMP activates the regulatory subunits RIα and RIIα of cAMP-dependent protein kinase. In this study, we used two cCMP agarose matrices as novel tools in combination with immunoblotting and mass spectrometry to identify cCMP-binding proteins. In agreement with our functional data, RIα and RIIα were identified as cCMP-binding proteins. These data corroborate the notion that cAMP-dependent protein kinase may serve as a cCMP target.     

Analysis of free nucleotide pools of mouse liver tissue by high-pressure liquid chromatography (HPLC)

In this paper, analysis of free nucleotides from mouseliver tissue during different day times has been described. Perchloric acid extract of mouse liver tissue was neutralized with tri-N-octylamine in trichlorotriflouroethane and after removal of ClO4(-), subjected to preliminary purification on a Cu(2+)-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure used in the study gave a good resolution of free nucleotides on a single column.     

Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: identification of a rare cCMP cofactor preference

Proteomics efforts have created a need for better strategies to functionally categorize newly discovered proteins. To this end, we have employed saturation transfer difference NMR with pools of closely related cofactors, to determine cofactor preferences. This approach works well for dehydrogenases and has also been applied to cyclic nucleotide-binding proteins. In the latter application, a protein (radial spoke protein-2, RSP2) that plays a central role in forming the radial spoke of Chlamydomonas reinhardtii flagella was shown to bind cCMP. cCMP-binding proteins are rare, although previous reports of their presence in sperm and flagella suggest that cCMP may have a more general role in flagellar function. 31P NMR was used to monitor the preferential hydrolysis of ATP versus GTP, suggesting that RSP2 is a kinase.     

Human cyclic nucleotide phosphodiesterases possess a much broader substrate-specificity than previously appreciated

Phosphodiesterases (PDEs) capable of degrading cAMP and cGMP are indispensable for the regulation of cyclic nucleotide-mediated signals. The existence of other cyclic nucleotides such as cCMP and cUMP has been discussed controversially in the literature. Despite publications on PDEs hydrolyzing cCMP or cUMP, the molecular identity of such enzymes remained elusive. Recently, we have provided evidence for a role of cCMP as second messenger in vascular relaxation and inhibition of platelet aggregation. Using an HPLC-MS based assay, here, we show that human PDEs belonging to various families hydrolyze not only cAMP and cGMP but also other cyclic nucleotides.     

多维色谱-串联质谱联用技术分离和鉴定小鼠肝脏质膜蛋白质

采用自动在线纳流多维液相色谱 串联质谱联用的方法分离和鉴定蔗糖密度梯度离心法分离和富集的小鼠肝脏质膜蛋白质 .以强阳离子交换柱为第一相 ,反相柱为第二相 ,在两相之间连接一预柱脱盐和浓缩肽段 .用含去污剂的溶剂提取细胞质膜中的蛋白质 ,获得的质膜蛋白质经酶解和适当的酸化后通过离子交换柱吸附 ,分别用 10个不同浓度的乙酸铵盐溶液进行分段洗脱 .洗脱物经预柱脱盐和浓缩后进入毛细管反相柱进行反相分离 ,分离后的肽段直接进入质谱仪离子源进行一级和二级质谱分析 .质谱仪采得的数据经计算机处理后用Mascot软件进行蛋白质数据库搜寻 ,共鉴定出 12 6种蛋白质 ,其中 4 1种为膜蛋白 ,包括与膜相关的蛋白质和具有多个跨膜区的整合膜蛋白 ,为建立质膜蛋白质组学研究的适宜方法和质膜蛋白质数据库提供了有价值的基础性研究资料 .     

PDE7A1 hydrolyzes cCMP

The degradation and biological role of the cyclic pyrimidine nucleotide cCMP is largely elusive. We investigated nucleoside 3′,5′-cyclic monophosphate (cNMP) specificity of six different recombinant phosphodiesterases (PDEs) by using a highly-sensitive HPLC–MS/MS detection method. PDE7A1 was the only enzyme that hydrolyzed significant amounts of cCMP. Enzyme kinetic studies using purified GST-tagged truncated PDE7A1 revealed a cCMP K_M value of 135 ± 19 μM. The V_max for cCMP hydrolysis reached 745 ± 27 nmol/(min mg), which is about 6-fold higher than the corresponding velocity for adenosine 3′,5′-cyclic monophosphate (cAMP) degradation. In summary, PDE7A is a high-speed and low-affinity PDE for cCMP.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α₁β₁ has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn²⁺ but not Mg²⁺, the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α₁β₁ and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn²⁺ is a physiological sGC cofactor.     

Differential activation of cAMP- and cGMP-dependent protein kinases by cyclic purine and pyrimidine nucleotides

The cyclic purine nucleotides cAMP and cGMP are well-characterized second messengers and activators of PKA and PKG, respectively. In contrast, the functions of the cyclic pyrimidine nucleotides cCMP and cUMP are poorly understood. cCMP induces relaxation of smooth muscle via PKGI, and phosphodiesterases differentially hydrolyze cNMPs. Here, we report that cNMPs differentially activate PKA isoforms and PKGIα. The combination of cCMP with cAMP reduced the EC₅₀ of cAMP for PKA. PKGIα exhibited higher specificity for the cognate cNMP than PKA. Our data support a role of cCMP and cUMP as second messengers.     

Hepatic ornithine decarboxylase from the frog, Rana negromaculata: dietary induction, purification and some properties

1. In the liver of the frog, Rana negromaculata, the activity of ornithine decarboxylase (ODC) was induced by dietary stimuli and was rapidly lost upon intraperitoneal injection of cycloheximide or putrescine. 2. Frog liver ODC, purified by DEAE-Cellulofine and immunoaffinity column chromatographies, was used in a comparative study with mouse kidney ODC, also purified by the same method. 3. The purified frog ODC showed three bands on SDS-polyacrylamide gel electrophoretic analysis, as confirmed by [3H]alpha-difluoromethylornithine binding. 4. Frog ODC was found to be similar to mouse enzyme in some properties, for example molecular weight, immunoreactivity and inhibition by rat antizyme, except for a slightly higher Km value for ornithine.     

Purification,molecular cloning,and functional expression of inducible liver acylcarnitine hydrolase in C57BL/6 mouse,belonging to the carboxylesterase multigene family

To identify the peroxisome proliferator-inducible acylcarnitine hydrolase in C57BL/6 mice, acylcarnitine hydrolase was purified to homogeneity using column chromatography. The purified enzyme, named ACH M1, had a subunit molecular weight of 60kDa. ACH M1 could hydrolyze classical carboxylesterase (CES) substrates as well as palmitoyl-dl-carnitine and these activities were inhibited by anti-rat CES antibodies. The peptide fragments of ACH M1 were identical to those of the deduced amino acid sequence of mouse CES2 isozyme. These findings suggested that ACH M1 was a member of the CES2 family. The mouse CES2 cDNA, designated mCES2, was cloned from mouse liver. The recombinant mCES2 expressing in Sf9 cells showed high level of catalytic activity toward acylcarnitines. Furthermore, the biological characteristics of the expressed protein were identical with those of ACH M1 in many cases, suggesting that mCES2 encodes mouse liver ACH M1.     

Multiplicity of affinity modification of RNAse during its alkylation with a reactive analog of 5-deoxyribonucleotide

The interaction of pancreatic RNase with 5'-deoxyribodinucleotide alkylating derivative, 4-(N-2-chloroethyl-N-methylamino)benzylamide of d(pTpA) d[(ClRCH2NH)pTpA], was studied. The unreactive oxyanalogue d[(HORCH2NH)pTpA] was shown to act as competitive inhibitor of cCMP hydrolysis by RNase. d[(ClRCH2NH)pTpA] irreversibly inactivated RNase. A protective effect was exerted by d(pTpA) and d[(HORCH2NH)pTpA]. The modification, although having an affinity character, was not accompanied by total inactivation of the enzyme. It was supposed that covalent bonding between the reagent and enzyme induced the dinucleotide displacement from the recognition site. The formation of four RNase monolabeled forms retaining the activity in the hydrolysis of cCMP and poly(U) was demonstrated.     

CEREBROSIDE MAY BE FALSELY IDENTIFIED AS A SOLUBLE ''BRAIN SPECIFIC PROTEIN''

—Injection of a soluble protein fraction from mouse brain into rabbits gave rise to an antibody which was specific for galactocerebroside. The antigen had the following characteristics: (1) it was present in the soluble fraction of a mouse brain homogenate but absent from the soluble fraction of homogenates of mouse liver, spleen, kidney and testis; (2) it was non-dialysable; (3) it voided from a Sephadex G200 column; (4) on immunodiffusion with antibody directed against it, it gave a sharp single precipitin band; (5) it bound to DEAE cellulose column and was eluted with high salt. Given these characteristics the antigen might have been identified as a ‘brain specific protein’. However, the lipid nature of the antigen was revealed when it was found that it was not destroyed by Pronase digestion and could be quantitatively extracted with chloroform-methanol. The antigen has been identified as a galaetocerebroside and is 100 times more abundant in the myelin fraction than in the soluble fraction of the mouse brain homogenates. The antigen could have been falsely identified as a ‘brain specific protein’ if the antigenicity and macromolecular behaviour of lipids was overlooked.