Th The kinetics of exocytotic secretion depend not only on the spatial relationship between calcium channels and the exocytotic apparatus, but also on the total amount of Ca2+ influx through Ca2+ channels, the free Ca2+ around the release site and the filling state of the release-ready vesicles. These factors may differ between neurons and endocrine cells. Bovine chromaffin cells (BCCs) are neuroendocrine cells responsible for catecholamine release from the adrenal glands. Ca2+ imaging experiments have shown that localized zones of Ca2+ influx exist on BCC membranes, but how different Ca2+ channel subtypes are distributed, and the mechanisms by which they are targeted, remain to be elucidated. CASK (calcium, calmodulin associated serine kinase) and Mint1 (Munc-18-interacting protein 1), which are modular adaptor proteins involved in synaptic targeting, have recently been found to function in targeting of á1B Ca2+ channels in hippocampal neurons. These data led to the proposal that Ca2+ channels are clustered in BCCs and that CASK and Mint1 play important roles in targeting and/or anchoring channels to their proper location. p*Using RT-PCR and Western blotting, CASK is demonstrated present in isolated BCCs. Mint1 is shown to be present by Western blotting as well. Immunocytochemical experiments and experiments in which BCCs were transfected with plasmids expressing á1A, á1B, and á1C subunits labeled with green fluorescent protein, have shown that á1A and á1B subunits are clustered on the plasma membranes of BCCs, while the á1C subunit is distributed in diffuse patches. With immunoprecipitation, it was determined that CASK interacts biochemically with á1A and á1B Ca2+ channels. Transfection of BCCs with NC3-GFP, which codes for the sequence of the á1B Ca2+ channel that interacts with CASK and Mint1, results in a punctate pattern of fluorescence, which is consistent with the binding of GFP labeled peptide to complexes of CASK and Mint1 at sites of release. Furthermore, immunocytochemical analysis of cells transfected with NC3-GFP showed that á1B Ca2+ channels have a dispersed distribution suggesting that they have been displaced from the binding sites. These data suggest that CASK and Mint1 are important in clustering and targeting Ca2+ channels in the BCC plasma membrane. This study is the first to show the existence and function of CASK and Mint1 in BCCs, and may contribute to our understanding of the exocytotic process in neuroendocrine cells

Th The kinetics of exocytotic secretion depend not only on the spatial relationship between calcium channels and the exocytotic apparatus, but also on the total amount of Ca2+ influx through Ca2+ channels, the free Ca2+ around the release site and the filling state of the release-ready vesicles. These factors may differ between neurons and endocrine cells. Bovine chromaffin cells (BCCs) are neuroendocrine cells responsible for catecholamine release from the adrenal glands. Ca2+ imaging experiments have shown that localized zones of Ca2+ influx exist on BCC membranes, but how different Ca2+ channel subtypes are distributed, and the mechanisms by which they are targeted, remain to be elucidated. CASK (calcium, calmodulin associated serine kinase) and Mint1 (Munc-18-interacting protein 1), which are modular adaptor proteins involved in synaptic targeting, have recently been found to function in targeting of á1B Ca2+ channels in hippocampal neurons. These data led to the proposal that Ca2+ channels are clustered in BCCs and that CASK and Mint1 play important roles in targeting and/or anchoring channels to their proper location. p*Using RT-PCR and Western blotting, CASK is demonstrated present in isolated BCCs. Mint1 is shown to be present by Western blotting as well. Immunocytochemical experiments and experiments in which BCCs were transfected with plasmids expressing á1A, á1B, and á1C subunits labeled with green fluorescent protein, have shown that á1A and á1B subunits are clustered on the plasma membranes of BCCs, while the á1C subunit is distributed in diffuse patches. With immunoprecipitation, it was determined that CASK interacts biochemically with á1A and á1B Ca2+ channels. Transfection of BCCs with NC3-GFP, which codes for the sequence of the á1B Ca2+ channel that interacts with CASK and Mint1, results in a punctate pattern of fluorescence, which is consistent with the binding of GFP labeled peptide to complexes of CASK and Mint1 at sites of release. Furthermore, immunocytochemical analysis of cells transfected with NC3-GFP showed that á1B Ca2+ channels have a dispersed distribution suggesting that they have been displaced from the binding sites. These data suggest that CASK and Mint1 are important in clustering and targeting Ca2+ channels in the BCC plasma membrane. This study is the first to show the existence and function of CASK and Mint1 in BCCs, and may contribute to our understanding of the exocytotic process in neuroendocrine cells

No / Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal-containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+-sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T-type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

No / The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca2+ channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM−1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N′,N′-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter. / This work was supported by the British Heart Foundation, the Medical Research Council, and the Hebei Medical University

Made available in DSpace on 2015-05-14T12:59:34Z (GMT). No. of bitstreams: 1 arquivototal.PDF: 7069577 bytes, checksum: 994737d813443da6d7f127b17873faf9 (MD5) Previous issue date: 2012-03-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The flavonoid galetin 3,6 dimethyl ether (FGAL) was isolated from the aerial parts of Piptadenia stipulacea (Benth.) Ducke and was investigated its possible hemolytic activity in rat erythrocytes and spasmolytic activity in several isolated smooth muscles as rat uterus, guinea pig ileum and trachea, and rat aorta. FGAL showed no hemolytic effect on rat erythrocytes, which is suggestive of a low toxicity. In preliminary pharmacological screening performed in smooth muscle models, FGAL showed non-selective spasmolytic effect in the four organs tested, with a higher potency to relax the guinea pig trachea pre-contracted with carbachol and rat aorta pre-contracted with phenylephrine (FEN) in a functional epithelium and endothelium independent manner, respectively. The aim of this study was to investigate the action mechanism of FGAL relaxing effect in guinea pig trachea and rat aorta. The observation that FGAL was more potent in relaxing both pre-contracted organs with moderate increases in extracellular concentration of KCl than when contracted with larger increases of KCl is suggestive that FGAL modulates the channels positivety. This hypothesis was confirmed by decreasing of relaxant potency of FGAL in both organs in the presence of tetraethylammonium (TEA+) 10 mM, non-selective blocker of K+ channels. To determine the subtypes of K+ channel involved, were used selectives blockers: in trachea the effect of FGAL was not altered in the presence of TEA+ 1 mM, blocker of large conductance calcium-activated K+ channels (BKCa); glibenclamide, blocker of sensitive-ATP K+ channels (KATP); BaCl2, blocker of inward rectifier K+ channels (Kir) or 4-AP, blocker of voltage activated K+ channels (KV), but was reduced in the presence of apamin, blocker of small conductance calcium-activated K+ channels (SKCa). In aorta, the relaxant effect of FGAL was not altered in the presence of TEA+ 1 mM, but was reduced in the presence of apamin, glibenclamide, BaCl2 and 4-AP, suggesting the involvement of SKCa, KATP, Kir and KV in vasorelaxant action of flavonoid. The fact of FGAL rightward shifted, with Emax reduced the CaCl2-induced contractions in depolarizing medium, and CaCl2 in the presence of verapamil, a voltage activated calcium channel (CaV) blocker, and FEN, suggests the involvement of CaV and ROCs (receptor-operated calcium channel), respectively. Also in the aorta, FGAL inhibited FEN induced contractions in Ca2+-free medium, suggesting inhibition of Ca2+ release from the sarcoplasmic reticulum SR. We also evaluated the participation of the cyclic nucleotides pathway, and observed that the trachea and aorta relaxation induced by aminophylline, non selective inhibitor of phosphodiesterases (PDEs), was more potent in the presence of FGAL, suggesting the involvement of cAMP and/or cGMP. On the aorta was assessed FGAL effect on relaxation induced by selective inhibitors of PDE-3 (milrinone, cAMP selective) and PDE-5 (sildenafil, cGMP selective), and FGAL only potentiated the relaxation induced by sildenafil, suggesting the participation of cGMP. Since K+ channels are modulated negatively by PKC, we investigated a possible inhibition of PKC by FGAL and the flavonoid relaxed the aorta pre-contracted with a PKC activator (PMA), suggesting inhibition of this enzyme. In conclusion, the spasmolytic mechanism of FGAL in trachea involves positive modulation of SKCa and cyclic nucleotides, and in the aorta involves the positive modulation of KATP, SKCa, Kir, Kv and inhibition of CaV, ROCs, Ca2+ release of SR, PDE-5 and PKC. / O flavonoide 3,6-dimetil éter galetina (FGAL) foi isolado das partes aéreas de Piptadenia stipulacea (Benth.) Ducke e foi investigada sua possível atividade hemolítica em eritrócitos de rato e espasmolítica em vários músculos lisos isolados como útero de rata, íleo e traqueia de cobaia, e aorta de rato. FGAL não causou efeito hemolítico em eritrócitos de ratos, o que é sugestivo de baixa toxicidade. Na triagem farmacológica preliminar realizada em músculos lisos, FGAL apresentou efeito espasmolítico não seletivo nos 4 órgãos testados, apresentando uma maior potência em relaxar a traqueia de cobaia pré-contraída com carbacol e a aorta de rato pré-contraída com fenilefrina (FEN), de maneira independente de epitélio e endotélio funcional, respectivamente. Assim, o objetivo deste trabalho foi investigar o mecanismo de ação relaxante de FGAL em traqueia de cobaia e aorta de rato. A observação de que FGAL foi mais potente em relaxar ambos os órgãos pré-contraídos com aumentos moderados na concentração extracelular de KCl do que quando contraídos com aumentos maiores de KCl é sugestivo de que FGAL está agindo por modular positivamente os canais de K+, hipótese esta confirmada pela diminuição da potência relaxante de FGAL em ambos os órgãos na presença de tetraetilamônio (TEA+) 10 mM, bloqueador não seletivo dos canais de K+. Para verificar os subtipos de canais de K+, usou-se bloqueadores seletivos: em traqueia o efeito de FGAL não foi alterado na presença de TEA+ 1 mM, bloqueador dos canais K+ de grande condutância ativados pelo Ca2+ (BKCa), glibenclamida, bloqueador dos canais de K+ sensíveis ao ATP (KATP), BaCl2, bloqueador dos canais de K+ retificadores de entrada (Kir) ou de 4-AP, bloqueador dos canais de K+ sensíveis à voltagem (KV), porém foi reduzido na presença de apamina, bloqueador dos canais de K+ de pequena condutância ativados pelo Ca2+ (SKCa). Em aorta, o efeito relaxante de FGAL não foi alterado na presença de TEA+ 1 mM, por outro lado foi reduzido na presença de apamina, glibenclamida, BaCl2 e 4-AP, sugerindo a participação dos SKCa, KATP, Kir e KV na ação vosorrelaxante do flavonoide. O fato de FGAL deslocar para direita com redução do Emax as contrações induzidas por CaCl2 em meio despolarizante, e por CaCl2 na presença de FEN e verapamil, bloqueador de CaV, sugere o envolvimento dos CaV e dos ROCs, respectivamente. Ainda em aorta, FGAL inibiu as contrações induzidas por FEN em meio livre de Ca2+, sugerindo inibição da liberação de Ca2+ do retículo sarcoplasmático (RS). Avaliou-se ainda a participação dos nucleotídios cíclicos, e observou-se que o relaxamento induzido pela aminofilina, inibidor não seletivo de fosfodiesterases (PDEs) em traqueia e aorta foi potencializado com FGAL, sugerindo a participação de AMPc e/ou GMPc. Em aorta foi avaliado o efeito de FGAL sobre o relaxamento induzido com inibidores seletivos de PDE-3 (milrinona, seletiva para AMPc) e PDE-5 (sildenafila, seletiva para GMPc), sendo que FGAL só potencializou o relaxamento induzido por sildenafila, sugerindo a participação do GMPc. Como os canais de K+ são modulados negativamente pela PKC, investigou-se uma possível inibição da PKC por FGAL, que relaxou a aorta pré-contraída com o ativador de PKC (PMA), sugerindo inibição dessa enzima. Em conclusão, o mecanismo de ação espasmolítica de FGAL em traqueia envolve modulação positiva dos SKCa e dos nucleotídios cíclicos, e em aorta modulação positiva dos KATP, SKCa, Kir, KV; inibição dos CaV e dos ROCs, da liberação de Ca2+ do RS, da PDE-5 e PKC.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19376号 / 工博第4121号 / 新制||工||1635(附属図書館) / 32390 / 新制||工||1635 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 森 泰生, 教授 梅田 眞郷, 教授 濵地 格 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Protein-protein interactions play a crucial role in a wide range of biological processes. Research on the design and synthesis of small molecules to modulate these proteinprotein interactions can lead to new targets and drugs to modulate their function. In Chapter one, we discuss the design and synthesis of small molecules to probe a proteinprotein interaction in a voltage-gated Ca2+ channel. Virtual screening identified a compound (BTT-3) that contained a 3,4-dihydro-3,4’-pyrazole core. This compound had modest biological activity when tested in a fluorescence polarization (FP) assay. The synthetic route to BTT-3 consisted of six steps. In addition, analogs of BTT-3 were made for a structure-activity study to establish the importance of a carboxylate moiety. We also synthesized a biotinylated benzophenone photo-affinity probe and linked it to BTT-3 to identify additional protein targets of the compound. In Chapter two, small-molecule antagonists targeting uPA-uPAR protein-protein interaction are presented. A total of 500 commercially-available compounds were previously identified by virtual screening and tested by a FP assay. Three classes of compounds were found with biological activity. The first class of compounds contains pyrrolidone core structures represented by IPR- 1110, the second class has a novel pyrrolo[3,4-c]pyrazole ring system, represented by xv IPR-1283 and the last series had compounds with a 1,2-disubstituted 1,2- dihydropyrrolo[3,4-b]indol-3(4H)-one core structure, represented by IPR-540. Each of these three compounds were synthesized and assessed by FP and ELISA assays. A binding mode of IPR-1110 with uPA was subsequently proposed. Based on this binding mode, another 61 IPR-1110 derivatives were synthesized by us to illustrate the SAR activity. Analogs of the other two series were also synthesized.

<p>Oscillations in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca²⁺]_i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca²⁺]_i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca²⁺]_i in isolated mouse β-cells into sustained elevation. Increased Ca²⁺ entry promoted the reappearance of the slow [Ca²⁺]_i oscillations. The [Ca²⁺]_i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca²⁺]_i oscillations due to periodic entry of Ca²⁺ as well as with transients evoked by mobilization of intracellular stores. The [Ca²⁺]_i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca²⁺]_i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca²⁺]_i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca²⁺ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca²⁺]_i oscillations. Nevertheless, there was an excessive firing of [Ca²⁺]_i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca²⁺]_i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.</p>

Oscillations in cytoplasmic Ca2+ concentration ([Ca2+]i) is the key signal in glucose-stimulated β-cells governing pulsatile insulin release. The glucose response of mouse β-cells is often manifested as slow oscillations and rapid transients of [Ca2+] i. In the present study, microfluorometric technique was used to evaluate the role of amino acids, glucagon, ryanodine and caffeine on the generation and maintenance of [Ca2+] i oscillations and transients in individual murine β-cells and isolated mouse pancreatic islets. The amino acids glycine, alanine and arginine, at around their physiological concentrations, transformed the glucose-induced slow oscillations of [Ca2+] i in isolated mouse β-cells into sustained elevation. Increased Ca2+ entry promoted the reappearance of the slow [Ca2+] i oscillations. The [Ca2+] i oscillations were more resistant to amino acid transformation in intact islets, supporting the idea that cellular interactions are important for maintaining the oscillatory activity. Individual rat β-cells responded to glucose stimulation with slow [Ca2+] i oscillations due to periodic entry of Ca2+ as well as with transients evoked by mobilization of intracellular stores. The [Ca2+] i oscillations in rat β-cells had a slightly lower frequency than those in mouse β-cells and were more easily transformed into sustained elevation in the presence of glucagon or caffeine. The transients of [Ca2+] i were more common in rat than in mouse β-cells and often appeared in synchrony also in cells lacking physical contact. Depolarization enhanced the generation of [Ca2+] i transients. In accordance with the idea that β-cells have functionally active ryanodine receptors, it was found that ryanodine sometimes restored oscillatory activity abolished by caffeine. However, the IP3 receptors are the major Ca2+ release channels both in β-cells from rats and mice. Single β-cells from ob/ob mice did not differ from those of lean controls with regard to frequency, amplitudes and half-widths of the slow [Ca2+] i oscillations. Nevertheless, there was an excessive firing of [Ca2+] i transients in the β-cells from the ob/ob mice, which was suppressed by leptin at close to physiological concentrations. The enhanced firing of [Ca2+] i transients in ob/ob mouse β-cells may be due to the absence of leptin and mediated by activation of the phospholipase C signaling pathway.

Submitted by Clebson Anjos (clebson.leandro54@gmail.com) on 2016-03-29T17:44:52Z No. of bitstreams: 1 arquivototal.pdf: 1922192 bytes, checksum: 6f50e098ac1e02adb9b434bdbc12154e (MD5) / Made available in DSpace on 2016-03-29T17:44:52Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1922192 bytes, checksum: 6f50e098ac1e02adb9b434bdbc12154e (MD5) Previous issue date: 2014-08-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / TRP channels have been extensively studied in many physiological and pathological processes involved in blood pressure regulation. Carvacrol is well known to act on TRP channels in the vasculature, however there are no studies of its effects in hypertensive rats. Our aim was to evaluate the contribution of TRP channels in hypertension and evaluate the effects of carvacrol on TRP channels of SHR. In an electrophysiological approach, carvacrol (300 μM) inhibited the barium current, suggesting a reduction of calcium influx through L-type voltage-operated Ca2+ channels. We found that the mRNA expression of the following TRP channels: TRPV1 (p=0.0007), TRPV4 (p=0.0002), TRPM7 (p=0.0091) and TRPM8 (p=0.0008) are decreased and TRPC1 (p=0,02) are increased in SHR compared to control. In aortic rings preparations precontracted with 1 μM of phenylephrine, carvacrol (10-8 - 3x10-4 M) induced vasorelaxation in WKY (pD2 = 4.88  0.09, Emax = 100.73  2.24%, n = 6) and SHR (pD2 = 4.93  0.08, Emax= 110.06  2.07%, n = 6) in the presence of functional endothelium and that effect was not altered after endothelium removal in WKY (pD2 = 5.09  0.08, Emax = 99.60  0.88%, n = 6) and SHR (pD2 = 5.00  0.08, Emax = 101.23  1.96%, n = 6), proposing an endotheliumindependent mechanism. To assess the role of TRP channels, aortic rings were incubated with ruthenium red. In this assay, the vasorelaxant response was not changed in the WKY. On the other hand both potency (p<0.001) and efficacy (p<0.001) were reduced in SHR, suggesting that carvacrol could activate the subtypes TRPV in hypertensive animals. When using magnesium, equally potency (p<0.001) and pharmacological efficacy (p<0.01) were attenuated in both WKY and SHR, suggesting the involvement of TRPM7. In preparations with 2-APB, CPZ and BCTC, the vasorelaxant effect was potentiated (p<0.01) in both WKY and SHR, suggesting the participation of TRPV1, TRPM8 and TRPM7 channels in the vasorelaxant effect induced by carvacrol. Nevertheless, in the presence of capsaicin, the vasodilator effect was attenuated (p<0.001) in both WKY and SHR endorsing a possible action of carvacrol on TRPV1 and TRPV4 channel. In addition, in vivo studies showed that carvacrol produced hypotension and bradycardia in unanesthetized WKY and SHR. In order to address the cardiovascular responses in vivo, we performed experiments using ruthenium red and capsaicin to evaluate the contribution of TRP channels in this effect. Our results suggested an action of carvacrol on TRPV1 and TRPV4, confirming the in vitro assays. In conclusion, these results suggest that the expression of TRPV1, TRPV4, TRPM7 and TRPM8 was reduced and TRPC1 increased in SHR and carvacrol induced a vasorelaxant effect probably by acting on TRPV1, TRPV4, TRPC1, TRPM7 and TRPM8 in SHR. Furthermore, the in vivo effects induced by carvacrol exhibited a hypotensive and bradycardic activity and this effect, at least in part, is due to an activation of TRPV1 and TRPV4 channels in these responses. / Os canais TRP têm sido amplamente estudados, em diversos processos de regulação fisiológico e patológico no sistema cardiovascular. Carvacrol (5-isopropil-2metilfenol) é conhecido por agir na vasculatura ativando ou bloqueando canais TRP, entretanto não há relatos dos seus efeitos em ratos hipertensos. Nosso objetivo foi avaliar o envolvimento dos canais TRP na hipertensão e o papel do carvacrol nos efeitos cardiovasculares em ratos espontaneamente hipertensos. Em ensaios eletrofisiológicos carvacrol (300μM) promoveu inibição das correntes de bário, sugerindo uma inibição do influxo de cálcio por canais de Ca2+ tipo-L. Ao avaliar a expressão do RNAm dos canais TRP em SHR, observamos pela primeira vez que a expressão de TRPV1 (p=0,0007), TRPV4 (p=0,0002), TRPM7 (p=0,0091), TRPM8 (p=0,0008) foram diminuídas e TRPC1 (p=0,02) aumentada. Em anéis de aorta précontraídos com 1 μM de FEN, o carvacrol (10-8 - 3 ₓ 10-4 M) induziu vasorelaxamento em ratos wistar kyoto (WKY) (pD2 = 4,88  0,09, Emáx = 100,73  2,24%, n = 6; pD2 = 5,09 0,08, Emáx = 99,60  0,88%, n = 6) e em ratos espontaneamente hipertensos (SHR) (pD2 = 4,93  0,08, Emáx = 110,06  2,07%, n = 6) na presença e na ausência do endotélio funcional, respectivamente. Para avaliar a participação dos canais TRP, na ausência do endotélio funcional as preparações foram incubadas com vermelho de rutênio, em WKY não houve alteração da resposta, mas em animais SHR tanto sua potência (p<0,001) como sua eficácia (p<0,001) foram diminuídas, sugerindo que carvacrol pode estar agindo em TRPV nos SHR. Ao utilizar magnésio, em WKY e SHR tanto sua potência (p<0,01) quanto sua eficácia (p<0,001) farmacológica foram atenuadas, sugerindo ação sobre o canal TRPM7. Nas preparações com 2-APB, CPZ e BCTC os seus efeitos foram potencializados (p<0,01), sugerindo ação sobre os canais TRPV1, TRPC1, TRPM7 e TRPM8. Já com capsaicina, um ativador de TRPV1, esse efeito foi atenuado (p<0,001) confirmando uma possível ação do carvacrol sobre TRPV1. Nos estudos in vivo, com WKY e SHR não anestesiados, carvacrol produziu hipotensão e bradicardia, onde ao avaliar a ação dos canais TRP em ensaios com vermelho de rutênio e capsaicina pode-se sugerir uma possível ação de carvacrol sobre TRPV1 e TRPV4, diminuindo a pressão arterial, corroborando com os ensaios in vitro. Em conclusão, esses resultados sugerem que os canais TRPV1, TRPV4, TRPM8 e TRPM7 têm sua expressão diminuída e TRPC1 a expressão aumentada em animais SHR e carvacrol induz efeito vasorelaxante provavelmente agindo em TRPV1, TRPV4, TRPC1, TRPM7 e TRPM8 em SHR. Além disso, os efeitos induzidos por carvacrol in vivo mostraram uma atividade hipotensora e bradicárdica e uma possível influencia dos canais TRPV1 e TRPV4 nessas respostas.