Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot. Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein. Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells. Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.

Ambient mass spectrometry is one of the mainstream techniques in modem mass spectrometry development. The developing purpose of ambient mass spectrometry is improving the analysis efficiency and simplifying operation process. So that, there are many techniques about ambient mass spectrometry had been published and commercialized. And direct electrospray probe mass spectrometry (DEP/MS) is one of ambient mass spectrometry techniques which is emphasized its rapid analysis and high sampling space resolution. In this research we improved the DEP system for more suitable in rapid analysis. We rule of the use of Syringe pump and its tube. Replace it by using solution tank so that we can save the time cost by disassemble syringe pump and tube when sampling. We also change the electric field by different high voltage applied mode for safety concern. In this study DEP/MS were used in plants analysis applications: capsaicin in peppers, systematic pesticide absorption in tomato and toxic compounds distribution in potato surface.

Vagal sensory afferent innervation corresponds to regulation of neurogenic inflammation in the airways. Capsaicin is mostly used for stimulation of sensory nerves that induce pain and inflammatory responses. It can specifically stimulate sensory afferent nerves, inducing neurogenic inflammation in the airways. According the past studies, we have found the right thoracic vagus nerve (RTVN) and right recurrent laryngeal nerve (RRLN); branches of right thoracic vagus trunk (RTVT) mediate different degree of neurogenic inflammation by intraenous injection of capsaicin (300 nmol/ml/kg). In order to investigate the innervation from the RTVN and RRLN of rat tracheobronchi and their involvement in plasma exudation, we injected 3 £gl of capsaicin (10 mg/ml) into RTVT and denervated the RRLN or RTVN and used India ink as tracer dye to label the leaky microvessels. Our observation indicated that injection of capsaicin into the RTVT coud induce obvious plasma exudation in trachea (area density of leaky blood vessels was about 22%), but plasma exudation was significantly decreased after denervation of RRLN. The left upper side of trachea was decreased by 77.6% and the right upper side decreased by 84.5%. This phenomenon was not caused by denervation of RTVN. The results suggest that vagal nerve innervation of upper trachea mostly came from the RLN. Otherwise, capsaicin injection into the RTVT also induced neurogenic inflammation in the larynx. Experimental denervation of both superior and recurrent laryngeal nerves resulted in a decrease of plasma extravasation by 84.98%. Denervation of either RTVN or RRLN also decreased the plasma extravasation in the larynx. The evidence suggest that sensory fibers in the superior laryngeal nerve, recurrent laryngeal nerve, and thoracic vagus nerve might come from the same population of vagal ganglion sensory neurons.

<p>Background and aim: The calcium ion (Ca2+) is an important ion involved in intracellular signalling. An increase in the free intracellular calcium concentration ([Ca2+]i) is essential for triggering insulin secretion from pancreatic beta cells. Beta cell death or disturbed insulin secretion are key factors in the pathogenesis of type 1 and type 2 diabetes respectively. A number of Ca2+ channels located on the plasma membrane or on the endoplasmic reticulum (ER) mediate Ca2+ increase in beta cells. Among the plasma membrane Ca2+ channels, members of the Transient Receptor Potential (TRP) family are currently of great interest. Transient Receptor Potential Vanilloid subtype 1 (TRPV1) is one of the 28 members of the TRP family. This ion channel is activated by heat and pungent chemicals like capsaicin. The main aim of this study was to investigate if functional TRPV1 channels are present in insulin secreting cells. Further more we examined if TRP channels could be studied by using microfluorometry in single cells. A third objective was to investigate if members of the TRP family could be identified by western blot.</p><p>Methods: We used S5 cells, a highly differentiated rat insulinoma cell line, as a model of beta cells. A ratiometric fluorescence technique was used for measurement of [Ca2+]i concentration from single Fura-2 loaded cells. [Ca2+]i was measured continuously using microscope based fluorometry with the time resolution of 1 Hz. For western blot we used proteins extracted from S5 cells and human islets. The blots were probed with antibodies directed against both the N-terminal and the C-terminal end of the protein.</p><p>Results: Capsaicin, an activator of TRPV1, increased [Ca2+]i in a dose-dependent manner with a half maximal effective concentration (EC50) ~ 100 nM. In nominally Ca2+ free buffer the capsaicin-induced [Ca2+]i increase was completely lost, while the intracellular depots of Ca2+ were not emptied as shown by administration of carbachol. The capsaicin-induced [Ca2+]i increase was completely blocked by capsazepine, an antagonist of TRPV1. An increase in temperature in the range of 43 – 49 °C increased [Ca2+]i, whereas temperatures < 42 °C did not. In nominally Ca2+ free medium the response to heat was reduced. Subsequent administration of carbachol showed that intracellular depots of Ca2+ were not emptied. Ruthenium red, an antagonist of TRPV1, also reduced the heat induced [Ca2+]i response. Another heat-sensitive, Ca2+ permeable protein Transient Receptor Potential Melastatin-like subtype 2 (TRPM2) was detected in S5 cells and human islets by western blot. The 171 kDa band represents the full length TRPM2 and is clearly visible in human islets, while the 95 KDa band represents the truncated form of TRPM2 and is more prominent in S5 cells.</p><p>Interpretation and conclusions: Microscope based fluorometry is a powerful method for studying ion channels of the TRP family in single living cells. We found that pancreatic beta cells express functional TRPV1 channels that were activated by capsaicin and heat. TRPV1 channels of beta cells are located on the plasma membrane and not on the ER. TRP channel proteins can also be detected by the western blot technique. The ease of studying TRP channels by microfluorometry and our demonstration of functionalTRPV1 channels in beta cells paves the way for studying the role of these channels in insulin secretion and in the pathogenesis of diabetes.</p>

The underlying nociceptive mechanisms in temporomandibular joint (TMJ) and masticatory muscles in many pain conditions are still unclear, largely due to the limited study of peripheral and central neural mechanisms affecting craniofacial musculoskeletal tissues. This study provided evidence in support of Hypothesis 1: Peripheral glutamatergic and capsaicin-sensitive mechanisms modulate the properties of primary afferents and brainstem neurons processing deep craniofacial nociceptive information. Effects of glutamate and capsaicin injected into the receptive field of deep craniofacial nociceptive afferents or TMJ of TMJ-responsive nociceptive neurons in trigeminal subnucleus caudalis/upper cervical cord (Vc/UCC) were studied in halothane-anesthetized rats. When injected alone, glutamate and capsaicin activated and induced peripheral sensitization in many afferents. Following glutamate injection, capsaicin-evoked activity was greater than that evoked by capsaicin alone, whereas following capsaicin injection, glutamate-evoked responses were similar to those of glutamate alone. When injected alone, glutamate and capsaicin also activated and induced central sensitization in most Vc/UCC neurons. Following glutamate injection, capsaicin evoked greater activity and less sensitization compared with capsaicin alone, whereas following capsaicin, glutamate was less effective in activating and sensitizing most Vc/UCC neurons. This apparent desensitizing effect of capsaicin on glutamate-evoked excitability of Vc/UCC neurons contrasts with the lack of capsaicin-induced modulation of glutamate-evoked afferent excitability, suggesting that peripheral and central sensitization may be differentially involved in the nociceptive effects of glutamate and capsaicin applied to deep craniofacial tissues. Further evidence of glutamate-capsaicin interactions was documented in the attenuation by TMJ pre-injection of glutamate receptor antagonists of jaw muscle activity reflexly evoked by TMJ injection of capsaicin. Moreover, additional findings support Hypothesis 2: Surgical cutaneous incision modulates the properties of brainstem neurons processing deep craniofacial nociceptive information. TMJ-responsive nociceptive Vc/UCC neurons could be activated by surgical incision of the skin overlying the TMJ and this incision-induced afferent barrage caused nociceptive neurons to be temporarily refractory to further capsaicin-induced central sensitization. These novel findings suggest that peripheral glutamate and capsaicin receptor mechanisms as well as surgical cutaneous incision may be involved in the nociceptive processing of deep craniofacial afferent inputs and may interact to modulate both activation as well as sensitization evoked from these tissues.

The underlying nociceptive mechanisms in temporomandibular joint (TMJ) and masticatory muscles in many pain conditions are still unclear, largely due to the limited study of peripheral and central neural mechanisms affecting craniofacial musculoskeletal tissues. This study provided evidence in support of Hypothesis 1: Peripheral glutamatergic and capsaicin-sensitive mechanisms modulate the properties of primary afferents and brainstem neurons processing deep craniofacial nociceptive information. Effects of glutamate and capsaicin injected into the receptive field of deep craniofacial nociceptive afferents or TMJ of TMJ-responsive nociceptive neurons in trigeminal subnucleus caudalis/upper cervical cord (Vc/UCC) were studied in halothane-anesthetized rats. When injected alone, glutamate and capsaicin activated and induced peripheral sensitization in many afferents. Following glutamate injection, capsaicin-evoked activity was greater than that evoked by capsaicin alone, whereas following capsaicin injection, glutamate-evoked responses were similar to those of glutamate alone. When injected alone, glutamate and capsaicin also activated and induced central sensitization in most Vc/UCC neurons. Following glutamate injection, capsaicin evoked greater activity and less sensitization compared with capsaicin alone, whereas following capsaicin, glutamate was less effective in activating and sensitizing most Vc/UCC neurons. This apparent desensitizing effect of capsaicin on glutamate-evoked excitability of Vc/UCC neurons contrasts with the lack of capsaicin-induced modulation of glutamate-evoked afferent excitability, suggesting that peripheral and central sensitization may be differentially involved in the nociceptive effects of glutamate and capsaicin applied to deep craniofacial tissues. Further evidence of glutamate-capsaicin interactions was documented in the attenuation by TMJ pre-injection of glutamate receptor antagonists of jaw muscle activity reflexly evoked by TMJ injection of capsaicin. Moreover, additional findings support Hypothesis 2: Surgical cutaneous incision modulates the properties of brainstem neurons processing deep craniofacial nociceptive information. TMJ-responsive nociceptive Vc/UCC neurons could be activated by surgical incision of the skin overlying the TMJ and this incision-induced afferent barrage caused nociceptive neurons to be temporarily refractory to further capsaicin-induced central sensitization. These novel findings suggest that peripheral glutamate and capsaicin receptor mechanisms as well as surgical cutaneous incision may be involved in the nociceptive processing of deep craniofacial afferent inputs and may interact to modulate both activation as well as sensitization evoked from these tissues.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (M.S.)--Missouri State University, 2008. / "May 2008." Includes bibliographical references (leaves 56-63). Also available online.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The transient receptor potential vanilloid 1 (TRPV1) is relevant to the perception of noxious information and has been studied as a therapeutic target for the development of new analgesics. The goal of this study was to perform in vivo and in vitro screens to identify novel, efficacious, and safe TRPV1 antagonists isolated from leaves of the medicinal plant Vernonia tweedieana Baker. All of the fractions and the hydroalcoholic extract produced antinociception in mice during the capsaicin test, but the dichloromethane fraction (Dcm) also had antioedematogenic effect. Among the compounds isolated from the Dcm fraction, only α-spinasterol reduced the nociception and oedema induced by capsaicin injection. Moreover, α-spinasterol demonstrated good oral absorption and high penetration into the brain and spinal cord of mice. Besides, α-spinasterol was able to displace [3H]-resiniferatoxin (RTX) binding and diminish calcium (Ca2+) influx mediated by capsaicin. Orally administration of the Dcm fraction and α-spinasterol also produced antinociceptive effect in the noxious heat-induced nociception test; however, they did not change the mechanical threshold of naive mice. The treatment with α-spinasterol did not produce antinociceptive effect in mice systemically pre-treated with RTX. In addition, α- spinasterol and the Dcm fraction also reduced the oedema, mechanical and heat hyperalgesia elicited by complete Freund s adjuvant (CFA) paw injection. The Dcm fraction and α-spinasterol did not affect body temperature or locomotor activity. In conclusion, α-spinasterol is an efficacious and safe antagonist of the TRPV1 receptor with antinociceptive effect. / O receptor de potencial transitório vanilóide 1 (TRPV1) é relevante para a percepção de estímulos nocivos e tem sido estudado como um alvo terapêutico para o desenvolvimento de novos analgésicos. O objetivo deste estudo foi desenvolver uma triagem in vivo e in vitro para caracterizar novos antagonistas do receptor TRPV1 isolados das folhas de Vernonia Tweedieana Baker, uma planta medicinal, com atividade antinociceptiva em camundongos. Todas as frações e o extrato hidroalcólico apresentaram efeito antinociceptivo no teste da capsaicina, sendo que a fração diclorometano (Dcm) também mostrou efeito antiedematogênico. Entre os compostos isolados da fração Dcm, apenas o α-espinasterol reduziu a nocicepção e o edema induzidos pela injeção intraplantar de capsaicina. Além disso, o α- espinasterol foi capaz de deslocar o radioligante [3H]-resiniferatoxina, e também de diminuir o influxo de cálcio estimulado pela capsaicina. A fração Dcm e o composto α-espinasterol apresentaram efeito anti-hiperalgésico na nocicepção induzida por estímulo térmico, mas não induzida por estímulo mecânico em animais sem injúria. Porém, o composto α-espinasterol não apresentou atividade antinociceptiva em animais pré-tratados sistemicamente com resiniferatoxina. Este composto e a fração Dcm foram capazes de reduzir a hiperalgesia mecânica e térmica, e também o edema induzidos por adjuvante completo de Freund. A fração Dcm e o α- espinasterol não foram capazes de induzir alteração na temperatura corporal ou atividade locomotora. Também, o α-espinasterol mostrou boa absorção por via oral, e alta penetração no cérebro e na medula espinhal de camundongos. Assim, o α- espinasterol, isolado da fração Dcm, age como um antagonista do receptor TRPV1 com eficaz efeito antinociceptivo, sem indução de efeitos adversos.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Cinnamaldehyde, a natural compound frequently present in cosmetic formulations, induces skin irritation when topically applied, but the mechanism by which cinnamaldehyde produces such skin reactions is unclear. Here, we showed that cinnamaldehyde induced ear edema in mice (1-6 μg/ear) with a maximum effect with 4 μg/ear (Emax of 0.18 ± 0.02 mm and an ED50 value of 2.0 (1.1- 3.4 μg/ear). Cinnamaldehyde can induce leukocyte infiltration detected by an increase in MPO activity and confirmed by histological analyses. The edema and cellular infiltration evoked by 4 μg/ear of cinnamaldehyde was prevented through topical application of ruthenium red, a non selective TRP antagonist or by camphor and HC030031, two TRPA1 receptor antagonists. In contrast, the edema and the leukocyte infiltration was unaffected by the TRPV1 receptor antagonist SB366791. Cinnamaldehydeinduced edema but not cellular infiltration was also prevented though topical application of the tachykinin NK1 antagonist aprepitant, indicating a neuropeptides release phenomenon in this process. Also, we observed that repeated topical applications of cinnamaldehyde (4 μg/ear) did not induced sensitization/desensitization alterations. Interestingly, the TRPV1 antagonist, capsaicin, repeated treatment abrogated its edematogenic response, confirming the desensitization process and decrease partially the cinnamaldehyde induced edema, suggesting the involvement of capsaicin-sensitive fibers and additional targets in cinnamaldehyde response. The present results demonstrated that cinnamaldehyde induces mouse skin inflammation through a mechanism involved the TRPA1 receptor activation and subsequent leukocyte infiltration. In addition, evidence supports the assumption that the tachykinin NK1 receptor is involved in these inflammatory responses. / O cinamaldeído é um composto natural frequentemente encontrado em formulações cosméticas, capaz de induzir irritação na pele quando aplicado topicamente, porém o mecanismo pelo qual o cinamaldeído produz estas reações ainda é desconhecido. Neste trabalho demonstramos que o cinamaldeído foi capaz de induzir edema de orelha em camundongos (1-6 μg/orelha) com um efeito máximo obtido com a dose de 4 μg/orelha (Emax de 0,18 ± 0,02 mm e um DE50 de 2,0 (1,1- 3,4) μg/orelha). O cinamaldeído foi capaz ainda de induzir infiltração leucocitária detectada por um aumento na atividade da MPO e confirmada por análise histológica. O edema e a infiltração leucocitária iniciados após aplicação tópica de 4 μg/orelha de cinamaldeído foi prevenido pela aplicação tópica de vermelho de rutênio, um antagonista TRP não seletivo, e por cânfora e HC030031, dois antagonistas seletivos TRPA1. Por outro lado, a aplicação de SB366791, um antagonista seletivo TRPV1, não alterou o edema nem a infiltração leucocitária. Ainda, o edema induzido pelo cinamaldeído foi prevenido pela aplicação tópica de aprepitant, um antagonista seletivo do receptor NK1 para taquicininas, sugerindo que a liberação de neuropeptídeos esteja envolvida neste processo. Também foi observado que a aplicação tópica repetida de cinamaldeído 4 μg/orelha não foi capaz de induzir processos de ensibilização/dessensibilização. No entanto, o tratamento repetidocom o antagonista TRPV1, capsaicina, aboliu o edema induzido pela própria capsaicina, confirmando a ocorrência de dessensibilização, e diminuiu parcialmente o edema induzido pelo cinamaldeído sugerindo o envolvimento de fibras sensíveis a capsaicina, além de outras vias, neste processo. Os resultados demonstram que o cinamaldeído induz um processo inflamatório na pele através de um mecanismo que envolve a ativação do receptor TRPA1 e consequente infiltração leucocitária.