Charles university in Prague, Pharmaceutical faculty in Hradci Králové, Department of biological and medical sciences Rheinische Friedrich-Wilhelms-University Bonn, Institute of Pharmacology and Toxicology Candidate: Michaela Čajková Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: Dr. Linda Sarah Hoffmann Title of diploma thesis: Role of Gq-coupled receptors in brown adipocytes In my diploma thesis, we focused on four Gq-coupled receptors (F2R, LPHN1, α1DAR, TSHR) in brown adipocytes (BAs), which were identified in the screen as the highest expressed in immature and mature BAs. Our goal was to validate suggestion, that Thyroid stimulating hormone receptor (TSHR) plays a key role in differentiation of BAs and that F2R, LPHN1, α1D-AR might be important for BAs. In our study, we investigated gene expression of these four receptors in BAs, using analytical methodsquantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blot. Results from analysis revealed, that expression of TSHR was increased in mature BAs, it means, that TSHR induce differentiation of BAs. The BAs transduced with short hairpin RNA (sh-RNA) against TSHR were less differentiated, this we proved also with Oil Red-O staining. Expression of adipocyte Protein 2 (aP2), peroxisome proliferator-activated...

Phospholipase Cβ (PLCβ) cleaves phosphatidylinositol-4,5-bisphosphate (PIP₂) into the second messengers inositol-1,4,5-triphosphate (IP₃) and diacylglycerol (DAG). IP₃ increases intracellular Ca²⁺, while DAG remains in the membrane, and together with increased Ca²⁺, activates protein kinase C (PKC). PLCβ has low basal activity but is activated following stimulation of G_i- and G_q-coupled receptors through direct interactions with Gα_q and Gβγ. PLCβ is essential for normal cardiomyocyte and vascular smooth muscle function and regulates cell proliferation, survival, migration, and differentiation. However, increased PLCβ activity and expression results in arrhythmias, hypertrophy, and heart failure. PLCβ must interact with the cell membrane for its activity. While heterotrimeric G proteins stimulate PLCβ, they are insufficient for full activation, suggesting the membrane itself contributes to increased lipid hydrolysis, potentially via interfacial activation. However, how the composition of the membrane and its resulting properties, such as surface charge, contribute to adsorption and interfacial activation is not well-established. Furthermore, whether or how interfacial activation also impacts other regulatory elements in PLCβ and Gα_q-dependent activation is unknown. Using an innovative combination of atomic force microscopy on compressed lipid monolayers and biochemical assays, we are beginning to understand how the membrane itself, PLCβ autoinhibitory elements and Gα_q regulate PLCβ activation. These studies provide the first structure-based approach to understanding how the cell membrane regulates the activity of this essential effector enzyme.

cAMP-elevating agents such as adenosine and epinephrine (after binding to beta-adrenergic receptor) contribute to local vascular dilation and some of these dilations are endothelium-dependent. Previous intracellular Ca 2+ imaging studies in mouse microvessel endothelial cells reported that addition of adenosine or epinephrine induced a Ca2+ influx which is blocked by CNG channel blockers such as L-cis-diltiazem or LY83583. Inside-out patch clamp studies confirmed the existence of a cAMP-activated current in endothelial cells, strongly suggesting a functional role of CNG, in particular CNGA2, channels in endothelial cells. The current study went further to show that similar Ca2+ influx in response to adenosine or epinephrine occurred in endothelial cells in freshly isolated mouse aortic strips and was again blocked by L-cis-diltiazem. By measuring the isometric force developed in mouse aortic strips, we showed that CNGA2 channel-mediated Ca2+ influx in endothelial cells contributed to the endothelium-dependent vascular dilatation in response to adenosine and epinephrine. / In conclusion, cyclic nucleotides playa vital role in the regulation of intracellular Ca2+ concentration in vascular cells and Jurket T cells. / In Jurkat T cells, cyclic nucleotides regulated Ca2+ mobilization in a different way. Fluorescence-imaging studies showed that cGMP inhibited store-operated Ca2+ influx and histamine-induced Ca 2+ rise in Jurkat T cells through activation of PKG. / Thromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can partly be attributed to TxA2-induced Ca2+ influx, which activates the Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells. Application of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relied on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by a L-type Ca2+ channel inhibitor nifedipine (0.5-1 muM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner, Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+, which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence Of CNGA2 protein in vascular smooth muscle cells, These data suggest a functional role of CNG channels in U-46619-induced Ca 2+ influx and contraction of smooth muscle cells. / Leung, Yuk Ki. / "August 2010." / Adviser: Yao Xiaoxiang. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 116-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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Indiana University-Purdue University Indianapolis (IUPUI) / Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that has proven to be an important signaling molecule both as an extracellular primary messenger and as an intracellular second messenger. Extracellular S1P acts through a family of five S1P receptors, S1PR1-5, all of which are G protein-coupled receptors associated with different G proteins. Previous work from our laboratory shows that externally applied S1P increases the excitability of small-diameter sensory neurons by enhancing the action potential firing. The increased neuronal excitability is mediated primarily, but not exclusively, through S1PR1. This raises the question as to which other S1PRs mediate the enhanced excitability in sensory neurons. To address this question, the expression of different S1PR subtypes in small-diameter sensory neurons was examined by single-cell quantitative PCR. The results show that sensory neurons express the mRNAs for all five S1PRs, with S1PR1 mRNA level significantly greater than the other subtypes. To investigate the functional contribution of other S1PRs in augmenting excitability, sensory neurons were treated with a pool of three individual siRNAs targeted to S1PR1, R2 and R3. This treatment prevented S1P from augmenting excitability, indicating that S1PR1, R2 and/or R3 are essential in mediating S1P-induced sensitization. To study the role of S1PR2 in S1P-induced sensitization, JTE-013, a selective antagonist at S1PR2, was used. Surprisingly, JTE-013 by itself enhanced neuronal excitability. Alternatively, sensory neurons were pretreated with FTY720, which is an agonist at S1PR1/R3/R4/R5 and presumably downregulates these receptors. FTY720 pretreatment prevented S1P from increasing neuronal excitability, suggesting that S1PR2 does not mediate the S1P-induced sensitization. To test the hypothesis that S1PR1 and R3 mediate S1P-induced sensitization, sensory neurons were pretreated with specific antagonists for S1PR1 and R3, or with siRNAs targeted to S1PR1 and R3. Both treatments blocked the capacity of S1P to enhance neuronal excitability. Therefore my results demonstrate that the enhanced excitability produced by S1P is mediated by S1PR1 and/or S1PR3. Additionally, my results indicate that S1P/S1PR1 elevates neuronal excitability through the activation of mitogen-activated protein kinase kinase. The data from antagonism at S1PR1 to regulate neuronal excitability provides insight into the importance of S1P/S1PR1 axis in modulating pain signal transduction.