Urotensin-II (U-II), a peptide with multiple vascular effects, is detected in cholinergic neurons of the rat brainstem and spinal cord. Here, the effects of U-II on [Ca2+]i, was examined in dissociated rat spinal cord neurons by fura 2 microfluorimetry. The neurons investigated were choline acetyltransferase-positive and had morphological features of motoneurons. U-II induced [Ca2+]i, increases in these neurons with a threshold of 10-9 M, and a maximal effect at 10-6 M with an estimated EC50 of 6.2 × 10-9 M. The [Ca2+]i increase induced by U-II was mainly caused by Ca2+ influx from extracellular space, as the response was markedly attenuated in a Ca2+-free medium. Omega-conotoxin GVIA (10-7 M), a N-type Ca2+ channel blocker, largely inhibited these increases, whereas the P/Q Ca2+ channel blocker, omega-conotoxin GVIIC (10-7 M) and the L-type Ca2+ channel blocker, verapamil (10-5 M) had minimal effects. Down-regulation of protein kinase C by 4-α-phorbol 12-myristate 13-acetate (10-6 M) or enzyme inhibition using the specific inhibitor bisindolylmaleimide I (10-6 M) did not inhibit the observed effects. Similarly, inhibition of protein kinase G with KT5823 (10-6 M) or Rp-8-pCPT-cGMPS (3 × 10-5 M) did not modify U-II-induced [Ca2+]i increases. In contrast, protein kinase A inhibitors KT5720 (10-6 M) and Rp-cAMPS (3 × 10-5 M) reduced the response to 25 ± 3% and 42 ± 8%, respectively. Present results demonstrate that U-II modulates [Ca2+]i, in rat spinal cord neurons via protein kinase A cascade.

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.

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京都大学 / 0048 / 新制・課程博士 / 博士(人間・環境学) / 甲第19057号 / 人博第710号 / 新制||人||171(附属図書館) / 26||人博||710(吉田南総合図書館) / 32008 / 京都大学大学院人間・環境学研究科共生人間学専攻 / (主査)教授 林 達也, 教授 森谷 敏夫, 教授 石原 昭彦 / 学位規則第4条第1項該当 / Doctor of Human and Environmental Studies / Kyoto University / DGAM

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

Chlamydia are obligate intracellular bacterial pathogens that are responsible for infectious blindness, sexually transmitted infections, and acute respiratory disease in humans. These pathogens undergo an essential biphasic developmental cycle differentiating between two functionally distinct forms known as the infectious elementary body (EB) and the replicative reticulate body (RB). Identifying the signals and regulatory mechanisms that enable Chlamydia to establish infection, differentiate between the two developmental forms, and survive within the host cell is critical to understanding chlamydial pathogenesis and developing future therapeutic strategies. In pathogenic bacteria, serine, threonine, and tyrosine (Ser/Thr/Tyr) protein kinases and phosphatases are critical for development, metabolism, and virulence. Chlamydia encode two validated protein kinases (pkn1 and pknd), a putative protein phosphatase (ctl0511; CppA), and appear capable of global phosphorylation that differs between the developmental forms. While these findings support a role for protein phosphorylation in chlamydial pathogenesis, a validated cognate protein phosphatase for Pkn1 and PknD mediating reversible phosphorylation was lacking. We hypothesized that CppA is the partner phosphatase for the chlamydial protein kinases, and in this study we validated and characterized CppA as a broad specificity protein phosphatase type 2C. Using in vivo and in vitro approaches we demonstrated that CppA acts on P-Ser/Thr/Tyr residues and can dephosphorylate multiple chlamydial protein substrates including PknD and the FHA 2 domain of CdsD, a component of the type 3 secretion apparatus. The importance of CppA for chlamydial growth and development was determined using a chemical “knock-out” approach and study of CppA missense mutations identified in slow growing C. trachomatis L2 chemical mutants. Treatment of C. trachomatis L2, C. trachomatis D, and C. muridarum with CppA inhibitors significantly reduced progeny levels and inclusion size in a time dependent manner with more significant growth inhibition in the first 12 hours post infection. Collectively, our findings support that CppA works in conjunction with PknD, and likely Pkn1, to mediate reversible phosphorylation of multiple protein substrates leading to changes in chlamydial physiology that appear to be key for early steps in development.

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).

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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.