Angiotensin II (All) was found to upregulate tissue inhibitor of metalloproteineses-1 (TIMP-1) gene expression in rat heart endothelial cells in a dose and time-dependent manner. The maximal stimulation of TIMP-1 mRNA was achieved by 2 h after the addition of All. This effect was blocked by losartan, an AT1 receptor antagonist and by calphostin C, a protein kinase C inhibitor. Addition of cycloheximide superinduced and actinomycin D abolished the induction. These results suggest that All stimulates TIMP-1 production by a protein kinase C dependent pathway which is dependent upon de novo RNA synthesis. Immunoprecipitation experiment showed an enhanced band of 28 kDa from the conditioned medium of All-treated cultures. Immunoblot analysis revealed that TIMP-1 was detectable in the conditioned medium 4 h after All stimulation. Since endothelial cells line the blood vessels and sense the rise in All associated with hypertension, the TIMP-1 released by these cells may provide an initial trigger leading to cardiac fibrosis in angiotensin-renin dependent hypertension.

Protein kinase C (PKC) has been linked to functional and morphological changes in endothelial cells involved in increased microvessel permeability. Bradykinin and TNF are potent inflammatory mediators which translocate PKC from the cytosol to the membrane of various cell types, including endothelial cells. The PKC isoforms α, ε and ζ have been demonstrated as the most prominent in human umbilical vein endothelial cells (HUVEC). We propose that bradykinin and TNF cause increased microvascular permeability via a PKC-dependent endothelial cell signalling pathway. HUVEC were incubated at 37°C and 5% CO2 for 1 min, 15 min and 3 h with either bradykinin (1 μM) or TNF (100 U/ml). PMA incubation served as a positive control (100 nM, 15 min). Cytosolic and membrane-bound extracts were obtained by incubation in digitonin (0.5%) and Triton X100 (1%). PKC isoforms were assayed by Western blot and membrane fractions calculated. These experiments revealed that: HUVEC clearly displayed a non-uniform basal membrane fraction distribution of PKC isoforms, with ζ (35.4%) greater than ε (30.6%) and both much greater than α (8.6%); Bradykinin caused significant translocation of PKC α with 15 min and 3 h of treatment but not 1 min; TNF caused dramatic translocation of PKC α at 1 min treatment which subsided at 15 min and 3 h but remained significantly elevated; and PMA caused dramatic translocation of α and ε but not ζ. Treatments of bradykinin and TNF that translocated PKC also showed cytoskeletal rearrangement of rhodamine-phalloidin stained actin, causing it to become more prevalent near cell membranes and concentrated at focal points between cells. These results suggest that PKC α may contribute to long term low grade increases in microvessel permeability in response to bradykinin, and that PKC α could be involved in both transient and sustained microvessel permeability changes induced by TNF. Also, cytoskeletal actin organization appears to be a downstream pathway in the activation process, possibly leading to alteration in endothelial cell shape and contact points.

Chronic surgical denervation of the rat vas deferens leads to an enhanced contractile response of the tissue to norepinephrine in vitro. Norepinephrine produces a higher rate of protein kinase C translocation to the particulate fraction of denervated tissues as compared with the paired, control vas deferens. Diacylglycerol generation in response to norepinephrine and contractile responses to phorbol diacetate were not altered by chronic denervation of the vas deferens. However, the contractile response to norepinephrine in these tissues was less susceptible to the inhibitory effects of the calcium channel blocker nifedipine. A potential role of protein kinase C in sensitizing the contractile apparatus to mobilized calcium in denervation supersensitivity is discussed.

Transforming growth factor-β1 (TGF-β1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-β1 is not clearly understood. In this study, we found that the addition of TGF-β1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-β1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-κB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-β1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-β1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-β1 may be attributed to the production of VEGF.

Dopamine (DA) is central to movement, reward, learning, sleep, and anxiety. The dopamine transporter (DAT) spatially and temporally controls extracellular dopamine levels by taking DA back up into the presynaptic neuron. Multiple lines of evidence from studies using pharmacological DAT blockade or genetic DAT deletion demonstrate that DAT availability at the plasma membrane is required for maintenance of homeostatic DA levels and DA tone. Therefore, intrinsic mechanisms that regulate the transporter’s availability at the plasma membrane may directly impact downstream DA signaling cascades and DA-dependent behavior. Acute, regulated DAT internalization in response to protein kinase C (PKC) activation has been well documented, however the physiological importance of this mechanism remains untested. Due to DAT’s critical role in regulating DA levels, It is essential to understand mechanisms that acutely regulate DAT function and surface expression, and further, how these mechanisms contribute to DA related behaviors. DAT has intracellular amino and carboxy termini, which contain domains for transporter phosphorylation, recruitment to and from the plasma membrane, and sites for protein-protein interactions. To test whether these domains work synergistically for DAT function and regulated endocytosis I made DAT/SERT chimeras, in which I switched DAT’s amino, carboxy, or both termini with that of SERT, a homologous transporter with highly divergent intracellular domains. I demonstrated that DAT’s amino and carboxy termini synergistically contribute to substrate and select competitive inhibitor affinities. Additionally, I demonstrated that the amino terminus is required for PKC-stimulated DAT endocytosis, and that both N- and C-termini are required for downstream Ack1-dependent regulation of DAT endocytosis. To test the physiological importance of PKC-stimulated DAT endocytosis in vivo, I knocked down Rin, a GTPase required for PKC-stimulated DAT trafficking, in mouse DA neurons. This study was the first to achieve AAV-mediated, conditional, and inducible gene silencing in neurons. Using this AAV approach, I demonstrated a critical role for Rin GTPase signaling and DAT trafficking in both anxiety and locomotor response to cocaine. Taken together, this thesis 1) adds to the understanding of DAT functional and endocytic mechanisms and 2) is the first to report the physiological impact of Rin signaling and DAT endocytosis in DA behavior.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21153号 / 農博第2279号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5127(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C.

Objective: The aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during 'metabolic' preconditioning with glucose deprivation or adenosine agonists. Methods: Isolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U·ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling. Results: Myocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 μM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 μM), while the A1/A3 antagonist BW 1433U (1 μM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX. Conclusions: Ischaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.