Distribution of group II phospholipase A2 protein and mRNA in rat tissues.

Group II phospholipase A2 (PLA2) is an acute-phase protein and an important component of the host defense against bacteria. In this study we investigated the distribution of PLA2 protein by immunohistochemistry and the distribution of mRNA of PLA2 by Northern blotting and in situ hybridization in rat tissues. PLA2 protein was localized in the Paneth cells of the intestinal mucosa, chondrocytes and the matrix of cartilage, and megakaryocytes in the spleen. By Northern blotting, mRNA of PLA2 was found in the gastrointestinal tract, lung, heart, and spleen. By in situ hybridization, PLA2 mRNA was localized in the Paneth cells of the small intestinal mucosa but in no other cell types. Our results show specific distribution of PLA2 in a limited number of cell types in rat tissues. The reagents developed in this study (the anti-rat PLA2 antibody and probes for Northern blotting and in situ hybridization of mRNA of rat PLA2) will provide useful tools for future studies concerning the role of PLA2 in various experimental disease models.     

Preferential distribution of group-II-like phospholipase A2 in mononuclear phagocytic cells in rat spleen and liver

The localization of calcium-dependent phospholipase A2, (PLA2) immunochemically closely related to the enzyme of the viperid and crotalid type (group II), in cells isolated from rat spleen and liver was examined using a polyclonal antibody directed against rat spleen group II, PLA2 (PLA2M). In isolated spleen cells, the monocyte/macrophage fraction had the highest PLA2 activity (1.28 +/- 0.35.min-1.10(6) cells-1) which was almost completely inhibited by the anti-PLA2M antibody. An immunoblot analysis confirmed the presence of the enzyme in this fraction. An immunocytochemical study revealed that the PLA2 was present in spleen macrophages. In the isolated liver cells, Kupffer cells (0.92 +/- 0.22 nmol.min-1.10(6) cells-1) contained higher anti-PLA2M-antibody-inhibitable PLA2 activity than parenchymal cells (0.26 +/- 0.06.min-1.10(6) cells-1). The immunocytochemical study showed that cells immunopositive with anti PLA2M antibody were Kupffer cells. These results suggest that the mononuclear phagocytic cells in rat spleen and liver have relatively high activity of group-II-like PLA2. Subcellular distribution patterns of the anti-PLA2M-antibody-inhibitable phospholipase A2 activity in different cell populations from spleen and liver were compared. A mode of the distribution of the enzyme in the spleen macrophages was essentially similar to that in the spleen lymphocytes. The distribution in Kupffer cells was similar to that in parenchymal cells.     

Bactericidal properties of group IIA and group V phospholipases A2

Group V phospholipase A(2) (PLA(2)) is a recently characterized 14-kDa secretory PLA(2) of mammalian heart and macrophage-derived cells. Group IIA PLA(2), which is structurally close to group V PLA(2), has been shown to kill Gram-positive bacteria in vitro and to prevent symptoms of Gram-positive infection in vivo. We studied the antibacterial properties of fully active recombinant rat group IIA and V PLA(2)s. Both group IIA and V PLA(2)s were highly bactericidal against Gram-positive bacteria, including methicillin-resistant staphylococci and vancomycin-resistant enterococci. Only high concentrations of group IIA PLA(2) showed some bactericidal effect against the Gram-negative bacterium Escherichia coli. Our results confirm that group IIA PLA(2) is a potent antibacterial enzyme against Gram-positive bacteria. Moreover, we show here that group V PLA(2) is a novel antibacterial mammalian protein, but is less potent than group IIA PLA(2). Both enzymes may be considered as future therapeutic agents against bacterial infections.     

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group IV cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2.

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2-4 h) of arachidonic acid (AA) and prostaglandin (PG) D2 in rat liver macrophages. Group IV cytosolic phospholipase A2 (cPLA2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA2 shows an enhanced in vitro activity. The Ca2+ dependence of cPLA2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA2 mRNA (after 2-4 h) and an enhanced expression of cPLA2 protein (after 8 h). The cellular cPLA2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA2 (sPLA2). LPS has no effect on the levels of Groups V and IIA sPLA2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA2 protein and sPLA2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA2.     

Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene

A novel P450 cDNA was isolated from a rat lung lambda gt11 library by hybridization with the rat P450 IIB1 cDNA probe. The cDNA-deduced amino acid sequence of this clone was 71% and 73% similar to rat IIA1 and IIA2 P450s; it was, therefore, designated IIA3 as the third member of the rat IIA subfamily. IIA3 demonstrates only 55% amino acid similarity with IIB1. Interestingly, this P450 also shared 85% and 94% amino acid similarities with human IIA3 and a mouse testosterone 15 alpha-hydroxylase P450, respectively, indicating that these P450s are orthologous counterparts to rat IIA3. Chromosome mapping, using mouse-hamster somatic cell hybrids, revealed that the IIA3 gene is localized on mouse chromosome 7. The IIA3 mRNA was detected in rat lung, and its level was induced 3-fold by treatment of rats with 3-methylcholanthrene. No IIA3 mRNA was seen in the liver, kidney, or intestine, even after long exposure of Northern blot filters to X-ray film. In contrast, the orthologous mouse and human IIA3 genes are expressed in liver.     

Gene conversion and differential regulation in the rat P-450 IIA gene subfamily. Purification, catalytic activity, cDNA and deduced amino acid sequence, and regulation of an adult male-specific hepatic testosterone 15 alpha-hydroxylase

Previous studies on regulation of the rat hepatic P-450 IIA1 cDNA have provided evidence for a second gene closely related to but regulated in a manner quite distinct from P-450 IIA1. Experiments were carried out to isolate the cDNA for this second P-450 gene, designated IIA2, in order to study more directly its regulation and relationship to IIA1. A full length cDNA to IIA2 was isolated from an adult male rat liver lambda gt11 library and sequenced completely. The IIA2 cDNA shared 93% nucleotide and 88% deduced amino acid similarities with the previously characterized IIA1 cDNA (Nagata, K., Matsunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 2787-2793). The protein, deduced from the cDNA, contained 492 amino acids and a calculated Mr of 56,352. Comparison of the IIA1 and IIA2 cDNAs revealed areas of low nucleotide similarity interspersed with areas of absolute identity, suggesting that gene conversions have played a role in the evolution of the IIA subfamily. Expression of IIA1 and IIA2 mRNAs in rat liver during development was studied with use of specific oligonucleotide probes. IIA1 mRNA was increased within 1 week after birth in both male and female rats; however, its postpubertal expression was decreased in males yet remained elevated in females. In contrast, IIA2 mRNA was markedly induced in male rat liver at puberty but was not detectable in females at any age examined. Furthermore, only IIA1 mRNA was induced by treatment of rats with 3-methylcholanthrene. Although IIA1 and IIA2 mRNAs were actively expressed in hepatic tissue, no evidence for their expression was found in lung, kidney, or intestine, suggesting that the IIA genes have tissue-specific promoters. Reconstituted enzyme assays on the purified protein products P-450 IIA1 and P-450 IIA2 showed that, although both enzymes share considerable sequence similarity, their positional specificities toward the prototype substrate testosterone are strikingly different.     

Functional role,cellular source,and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.     

Protection by group II phospholipase A2 against Staphylococcus aureus.

Group II phospholipase A2 (PLA2) is an enzyme that has marked antibacterial properties in vitro. To define the role of group II PLA2 in the defense against Staphylococcus aureus, we studied host responses in transgenic mice expressing human group II PLA2 and group II PLA2-deficient C57BL/6J mice in experimental S. aureus infection. After the administration of S. aureus, the transgenic mice showed increased expression of group II PLA2 mRNA in the liver and increased concentration of group II PLA2 in serum, whereas the PLA2-deficient mice completely lacked the PLA2 response. Expression of human group II PLA2 resulted in reduced mortality and improved the resistance of the mice by killing the bacteria as indicated by low numbers of live bacteria in their tissues. Human group II PLA2 was responsible for the bactericidal activity of transgenic mouse serum. These results suggest a possible role for group II PLA2 in the innate immunity against S. aureus infection.     

肠缺血/再灌注损伤后白介素1-β水平与磷脂酶A2激活的关系

为了探讨肠缺血/再灌注损伤后IL-1β基因表达和蛋白含量变化与磷脂酶A2抑制之间的关系,采用大鼠肠缺血/再灌注损伤模型,在对照组,损伤组和磷脂酶A2抑制剂处理组动物中收集血清,肺灌洗液,腹腔灌洗液及全身重要脏器组织样品,采用放射免疫法测定IL-1β含量,并且RT-PCR法测定肺组织中IL-1β和Ⅱ型PLA2基因表达,结果表明,损伤后6h血清中IL-1β含量明显高于对照组;损伤后1和3h,腹腔注保IL-1β也明显高于对照组;损伤后肝组织中IL-1β水平有明显增加,而肺,肾、肠组织中IL-1β没有明显变化。损伤后肺灌洗液中IL-1β也明显高于对照组水平,肺组织中IL-1βmRNA表达增加,而Ⅱ型PLA2mRNA在损伤后表达反而有所下降,采用磷脂酶A2抑制剂氯喹,环氧化物酶抑制剂消炎痛,血小板活化因子受体阻断剂SR27417后,IL-1β蛋白和基因表达有不同的改变,提示肠缺血/再灌注损伤后一定时间内,肝内IL-1βmRNA表达和血中IL-1β水平明显增高,但是否与磷脂酶A2激活或其代谢产物的释放有关尚需进一步证明。     

Expression level of adenosine kinase in rat tissues. Lack of phosphate effect on the enzyme activity

In this report we describe cloning and expression of rat adenosine kinase (AK) in Esccherichaia coli cells as a fusion protein with 6xHis. The recombinant protein was purified and polyclonal antibodies to AK were generated in rabbits. Immunoblot analysis of extracts obtained from various rat tissues revealed two protein bands reactive with anti-AK IgG. The apparent molecular mass of these bands was 48 and 38 kDa in rat kidney, liver, spleen, brain, and lung. In heart and muscle the proteins that react with AK antibodies have the molecular masses of 48 and 40.5 kDa. In order to assess the relative AK mRNA level in rat tissues we used the multiplex PCR technique with beta-actin mRNA as a reference. We found the highest level of AK mRNA in the liver, which decreased in the order kidney > spleen > lung > heart > brain > muscle. Measurement of AK activity in cytosolic fractions of rat tissues showed the highest activity in the liver (0.58 U/g), which decreased in the order kidney > spleen > lung > brain > heart > skeletal muscle. Kinetic studies on recombinant AK as well as on AK in the cytosolic fraction of various rat tissues showed that this enzyme is not affected by phosphate ions. The data presented indicate that in the rat tissues investigated at least two isoforms of adenosine kinase are expressed, and that the expression of the AK gene appears to have some degree of tissue specificity.     

Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells.

Two potent inflammatory mediators, interleukin 1 (IL-1) and tumor necrosis factor (TNF) as well as lipopolysaccharide (LPS) increased group II phospholipase A2 (PLA2) mRNA levels, which resulted in enhanced secretion of the PLA2 enzyme from rat smooth muscle cells. cAMP-elevating agents also stimulated the release of PLA2 and increased the mRNA, but IL-1, TNF and LPS did not affect cAMP levels. Furthermore, the effects of TNF and cAMP-elevating agents were not additive but synergistic. Therefore, we concluded that the level of rat group II PLA2 mRNA is controlled at least by two distinct mechanisms, one involves cAMP and the other is mediated by TNF, IL-1 and LPS. This study also suggests important roles of group II PLA2 in pathogenesis of vascular inflammation.     

Rat ATP citrate-lyase. Molecular cloning and sequence analysis of a full-length cDNA and mRNA abundance as a function of diet, organ, and age

ATP citrate-lyase is the primary enzyme responsible for the synthesis of cytosolic acetyl-CoA in many tissues. We have isolated a full-length cDNA copy of 4.3 kilobase pairs encoding the ATP-citrate lyase mRNA by screening rat liver cDNA library using oligonucleotide probes designed from peptide sequences obtained from the purified rat enzyme. Expression of this cDNA in bacteria, followed by immunoblotting with antibody directed against the ATP citrate-lyase, further demonstrated the identity of this clone. Nucleic acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1100 amino acids in length with a calculated molecular weight of 121,293. RNA blot analysis indicated an mRNA species of about 4.3 kilobase pairs in livers of chow-fed rats. Rats maintained on low fat, high carbohydrate diets exhibited a striking increase (50-fold) in the level of liver ATP citrate-lyase mRNA as compared with the control animals maintained on a normal diet. The tissue distribution of this mRNA in chow-fed animals revealed a relatively high abundance of the message in liver and adrenal, moderate levels were found in lung, brain, and large intestine with only trace amounts of the message in small intestine, stomach, testis, spleen, pancreas, kidney, and heart. During rat development, the ATP citrate-lyase mRNA was relatively high in the liver at parturition, followed by a reduction in its level during suckling. Higher amounts of the mRNA were detected again in adult animals. The isolation and characterization of the mRNA for ATP citrate-lyase will allow further studies on the reaction mechanism and metabolic regulation of this key enzyme in lipogenesis and cholesterogenesis.     

Light microscopic immunohistochemical analysis of the distribution of group II phospholipase A2 in human digestive organs.

The light microscopic and immunohistochemical distribution of human Group II phospholipase A2 (M-PLA2) in digestive organs of both human fetus and adult, with a new monoclonal antibody (MAb) against M-PLA2, was investigated semiquantitatively. The immunoreactivity was distributed similarly in the adult and fetal epithelium of the esophagus, duodenum, and small intestine, and in the acinar, islet, and duct cells of the pancreas. The epithelium of adult gallbladder was immunoreactive. Paneth cells, especially the secretory apparatus, were strongly immunoreactive. Hepatic Küpffer cells and macrophages of the adult spleen were also immunoreactive. These results suggest that these cells contain secretory-type Group II PLA2, which may be involved in host defensive mechanisms, such as phagocytosis in human digestive organs. In the adult colon, the immunoreactivity was observed only in the ascending colon and was not found in the transverse, descending, sigmoid, or rectal colon. The immunoreactivity was not found in fetal colon. Similarly, immunoreactivity was found in hepatocytes and Küpffer cells of adult liver but not in fetal liver. By contrast, strong immunoreactivity was observed in the epithelium of the fetal stomach but not in adult stomach except in gastric neck cells. This suggests that the expression of M-PLA2 may be related to cell differentiation in particular organs.     

Reevaluation of erythropoietin production by the nephron

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in β-intercalated or non α/non β-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.     

Akt as a mediator of secretory phospholipase A2 receptor-involved inducible nitric oxide synthase expression

The induction of inducible NO synthase (iNOS) by group IIA phospholipase A(2) (PLA(2)) involves the stimulation of a novel signaling cascade. In this study, we demonstrate that group IIA PLA(2) up-regulates the expression of iNOS through a novel pathway that includes M-type secretory PLA(2) receptor (sPLA(2)R), phosphatidylinositol 3-kinase (PI3K), and Akt. Group IIA PLA(2) stimulated iNOS expression and promoted nitrite production in a dose- and time-dependent manner in Raw264.7 cells. Upon treating with group IIA PLA(2), Akt is phosphorylated in a PI3K-dependent manner. Pretreatment with LY294002, a PI3K inhibitor, strongly suppressed group IIA PLA(2)-induced iNOS expression and PI3K/Akt activation. The promoter activity of iNOS was stimulated by group IIA PLA(2), and this was suppressed by LY294002. Transfection with Akt cDNA resulted in Akt protein overexpression in Raw264.7 cells and effectively enhanced the group IIA PLA(2)-induced reporter activity of the iNOS promoter. M-type sPLA(2)R was highly expressed in Raw264.7 cells. Overexpression of M-type sPLA(2)R enhanced group IIA PLA(2)-induced promoter activity and iNOS protein expression, and these effects were abolished by LY294002. However, site-directed mutation in residue responsible for PLA(2) catalytic activity markedly reduced their ability to production of nitrites and expression of iNOS. These results suggest that group IIA PLA(2) induces nitrite production by involving of M-type sPLA(2)R, which then mediates signal transduction events that lead to PI3K/Akt activation.     

Cellular retinol-binding protein messenger RNA levels in normal and retinoid-deficient rats

A study was conducted to determine the levels of cellular retinol-binding protein (CRBP) mRNA and protein in various tissues of the rat, to explore relationship between CRBP mRNA and protein levels in different tissues, and to examine the effects of changes in retinol nutritional status on the tissue distribution and levels of CRBP mRNA. Previous studies have shown that tissue CRBP protein levels are reduced in totally retinoid-deficient rats, but are otherwise minimally affected by changes in retinoid status. Three groups of male rats were compared: normal controls, retinoid-deficient, and retinol-repleted deficient rats. CRBP mRNA levels were measured by RNase protection assay and CRBP protein levels by radioimmunoassay in seven tissues. High levels of both CRBP mRNA and CRBP protein were found in the proximal epididymis, kidney, and liver; lower levels were seen in lung, testis, spleen, and small intestine. Tissue CRBP mRNA and protein levels were highly correlated (P less than 0.01) with each other. Retinoid deficiency did not alter the levels of CRBP mRNA found in the proximal epididymis, kidney, and liver. In contrast, CRBP mRNA levels in the lung, testis, spleen, and small intestine were reduced substantially in retinoid-deficient rats, to values that were only 23% to 50% of the corresponding values in the tissues of control rats. After oral repletion with retinol (4-18 h earlier), CRBP mRNA levels for these latter four tissues were found to have risen to control or near-control levels. The suggestion is raised that retinol repletion may have directly induced the expression of the CRBP gene in these particular tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

大白鼠DNA拓扑异构酶Ⅰ生物学性质的研究

对大白鼠组织作DNA拓扑弄构酶Ⅰ(拓扑酶Ⅰ)活力测定,见酶活力出现在胚胎早期,在胚胎发育过程及出生后不同年龄期,酶活力基本稳定;几种成年大鼠组织的酶活力彼此无显著差异;肝细胞再生及癌变,酶活力亦无显著变化。