Delta-opioid receptor-induced late preconditioning is mediated by cyclooxygenase-2 in conscious rabbits

Although activation of delta-opioid receptors is known to induce both early and late preconditioning (PC) against myocardial infarction, the mechanisms for this salubrious effect are unclear. Furthermore, it is unknown whether delta-opioid receptors can also induce late PC against myocardial stunning. By using conscious rabbits (n = 120) in this study, we found that the delta-opioid receptor agonist (+/-)-4-[(alpha-R*)-alpha-[(2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl]-3-hydroxybenzyl]-N,N-diethylbenzamide (BW-373U86) induced late PC against myocardial stunning 24 h after treatment and that this effect was abolished by the selective cyclooxygenase-2 (COX-2) inhibitors N-[2-(cyclohexyloxy)4-nitrophenyl]methanesulfonamide (NS-398) and celecoxib. This protective effect was also abrogated by the selective delta(1)-opioid receptor antagonist 7-benzylidenenaltrexone, indicating that the delta(1)-opioid receptor is necessary for BW-373U86-induced late PC. BW-373U86 did not induce early PC against stunning. In addition, BW-373U86 induced late PC against infarction, which was blocked by NS-398. At 24 h after BW-373U86 administration, myocardial COX-2 protein expression and PGE(2) and 6-keto-PGF(1alpha) levels were significantly increased. These results demonstrate that activation of delta-opioid receptors induces late PC against both stunning and infarction via a COX-2-dependent mechanism.     

Role of Src protein tyrosine kinases in late preconditioning against myocardial infarction

Although Src protein tyrosine kinases (PTKs) have been shown to be essential in late preconditioning (PC) against myocardial stunning, their role in triggering versus mediating late PC against myocardial infarction remains unclear. Four groups of conscious rabbits were subjected to a 30-min coronary occlusion on day 2, with or without PC ischemia on day 1. Administration of the Src PTK inhibitor lavendustin A (LD-A; 1 mg/kg iv) before the PC ischemia on day 1 (group III, n = 7) failed to block the delayed protective effect against myocardial infarction 24 h later. Late PC against infarction, however, was completely abrogated when LD-A was given 24 h after the PC ischemia, prior to the 30-min occlusion on day 2 (group IV, n = 8). We conclude that, in conscious rabbits, Src PTK activity is necessary for the mediation of late PC protection against myocardial infarction on day 2, but not for the initiation of this phenomenon on day 1. Taken together with previous studies in the setting of stunning, these findings reveal heretofore unrecognized differences in the roles of Src PTKs in late PC against stunning versus late PC against infarction.     

Non-destructive monitoring of rpoS promoter activity as stress marker for evaluating cellular physiological status

To monitor the extent of cellular physiological stress, the activity of the rpoS promoter was evaluated as a marker of the stress pathway. A reporter plasmid was constructed by inserting the GFPuv gene under the rpoS promoter and used to transform Escherichia coli cells. The fluorescence of the GFPuv protein was measured in intact cells in a non-destructive manner. The physiological status of the cells could be conveniently monitored using the rpoS-GFPuv reporter gene with respect to the cellular growth phase and to elevated ethanol and NaCl concentrations as two examples of environmental stress factors. Comparison of the response of different E. coli strains demonstrated an essential role of the relA gene in the induction of the rpoS-GFPuv reporter gene.     

Possible involvement of the vascular endothelial growth factor-Flt-1-focal adhesion kinase pathway in chemotaxis and the cell proliferation of osteoclast precursor cells in arthritic joints

Vascular endothelial growth factor (VEGF) plays a crucial role in the pathogenesis of inflammatory joint disease, including angiogenesis and synovitis. Rheumatoid arthritis is a chronic inflammatory disease characterized by progressive synovitis and subsequent bone destruction mediated by osteoclasts (OCs). In this study, we investigate the effects of VEGF on OC precursor cells (pOCs) using Raw cells and adjuvant-induced arthritis in rats. OCs and pOCs in the arthritic joints express VEGF and VEGF receptor type I (Flt-1). Raw cells also express Flt-1, and VEGF treatment stimulated chemotaxis, cell proliferation, the association of Flt-1 with focal adhesion kinase (FAK), and the tyrosine phosphorylation of FAK in Raw cells. The tyrosine phosphorylation of FAK was also observed in pOCs in the arthritic joints of adjuvant-induced arthritis. Adenovirus-mediated expression of FAK-related nonkinase in Raw cells inhibited the effects of VEGF in a dominant negative manner. Furthermore, intra-articular injection of the FAK-related nonkinase virus suppressed the recruitment of pOCs and bone destruction. Our results suggest the possible involvement of the VEGF-Flt-1-FAK pathway in inflammatory disease-induced joint destruction.     

Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.     

Rat macrophage activation after treatment with the bleomycin group of antitumour antibiotics in vivo

Summary We have previously reported that bleomycin and its derivative peplomycin enhance the release of cytokines by rat spleen cells during mitogen-stimulated cell culture in vitro, but liblomycin, another derivative of bleomycin, decreases cytokine release to below untreated control levels. Cytokine release correlated well with the inhibition of subcutaneous tumour growth after treatment with equivalent doses of the three analogues. In contrast, ascites tumour growth is completely inhibited by liblomycin and appears to be at least partly macrophage-mediated because the antitumour effect can be significantly inhibited by carageenan. This study shows that bleomycin and its analogues activate rat peritoneal macrophages and increase interleukin-6 release, O₂ ^– production, cell spreading, phagocytosis and random migration of macrophages, but only bleomycin enhances peritoneal macrophage invasion into a monolayer of rat lung endothelial cells in vitro. This study also shows that although liblomycin decreases spleen cell cytokine production and is less effective than bleomycin against subcutaneous tumour, as we have previously reported, the antitumour drug activates peritoneal macrophages and, compared to bleomycin, has a remarkable therapeutic effect on rat ascites tumour.     

Enzymatic Formation of Sphingenine from Ketosphinganine in Rat Liver Particulates

Mutants of Klebsiella aerogenes that synthesized tyramine oxidase and arylsulfatase constitutively were isolated. The properties of four of seven constitutive mutants isolated were unstable, segregating spontaneously to the parental type at high frequency. Some of these segregants also lost arylsulfatase (AtsA^?) or tyramine oxidase (TynA^?) spontaneously. These unstable constitutive mutations were shown by genetic analysis to involve mutations of tynP and tynR, and transductants receiving the tynP gene were also unstable. These results showed that the instability was due to unstable tynP gene, which may be the promoter region for tyramine oxidase.     

The construction of endothelial cellular biosensing system for the control of blood pressure drugs

In order to assess blood pressure control drugs, the endothelial cellular biosensing system for assessing blood pressure control drugs was constructed. This system consists of human umbilical vein endothelial cells (HUVEC) on a polyion-coated gold electrode, a platinum counter electrode and an Ag/AgCl reference electrode. Nitric oxide (NO) as an indicator of blood vessel relaxation was detected with a polyion-coated electrode in the system. The NO detection limit of this electrode was 8.4 nM by differential pulse voltammetry (DPV). The drugs of blood pressure control (acetylcholine chloride (AcChCl), NOC 7 and NG-monomethyl-L-arginine (L-NMMA)) were assessed with this endothelial cellular biosensing system. One milli molar of AcChCl make NO released from HUVEC stimulated by activating endothelial nitric oxide synthase (eNOS) in HUVEC. In the case of 5 mM of L-NMMA, NO releasing was inhibited by inhibiting eNOS activation by 1 mM of AcChCl. NOC 7 immediately released NO regardless of eNOS activation in endothelial cells.