Compromised calcium homeostasis in the central nervous system (CNS) is implicated as a major contributor in the pathology of neurodegeneration. Dysregulation of Ca2+ homeostasis initiates downstream Ca2+–dependent events that lead to apoptotic and/or necrotic cell death. Increases in the intracellular free calcium concentration ([Ca2+]i) may be the result of Ca2+ influx from the extracellular environment or Ca2+ release from intracellular Ca2+ stores such as the endoplasmic reticulum (ER). Influx from the extracellular environment is controlled predominantly by voltage gated calcium channels (VGCC), such as L–type calcium channels (LTCC) and ionotropic glutamate receptors, such as the N–methyl–D–aspartate (NMDA) receptors. Ca2+ release from the ER occurs through the inositol–1,4,5–triphosphate receptors (IP3Rs) or ryanodine receptors (RyRs) via IP3–induced or Ca2+–induced mechanisms. Mitigation of Ca2+ overload through these Ca2+ channels offers an opportunity for pharmacological interventions that may protect against neuronal death. In the present study the ability of a novel series of polycyclic compounds, both the pentacycloundecylamines and triquinylamines, to regulate calcium influx through LTCC was evaluated in PC12 cells using calcium imaging with Fura–2/AM in a fluorescence microplate reader. We were also able for the first time to determine IC50 values for these compounds as LTCC blockers. In addition, selected compounds were evaluated for their ability to offer protection in apoptosis–identifying assays such as the lactate dehydrogenase release assay (LDH–assay), trypan blue staining assay and immunohistochemistry utilizing the Annexin V–FITC stain for apoptosis. We were also able to obtain single crystal structures for the tricyclo[6.3.0.02,6]undecane–4,9–dien–3,11–dione (9) and tricyclo[6.3.0.02,6]undecane–3,11–dione (10) scaffolds as well as a derivative, N–(3–methoxybenzyl)–3,11–azatricyclo[6.3.0.02,6]undecane (14f). We also evaluated the possibility that the polycyclic compounds might be able to modulate Ca2+ flux through intracellular Ca2+ channels. Computational methods were utilized to accurately predicted IC50 values and develop a QSAR model with marginal error. The linear regression model delivered r2 = 0.83, which indicated a favorable correlation between the predicted and experimental IC50 values. This model could thus serve as valuable predictor for future structural design and optimization efforts. Data obtained from the crystallographic analysis confirmed the NMR–data based structural assignments done for these compounds in previous studies. Obtaining structural information gave valuable insight into the differences in size and geometric constrains, which are key features for the LTCC activity of these compounds. vii In conclusion, we found that all of the compounds evaluated were able to attenuate Ca2+ influx through the LTCC, with some compounds having IC50 values comparable with known LTCC blockers such as nimodipine. Representative compounds were evaluated for their ability to afford protection against apoptosis induced by 200 ?M H2O2. With the exception of compound 14c (the most potent LTCC blocker in the series, IC50 = 0.398 ?M), most compounds were able to afford protection at two or more concentrations evaluated. Compound 14c displayed inherent toxicity at the highest concentrations evaluated (100 ?M). We concluded that compounds representing both types of structures (pentacycloudecylamines and triquinylamines) have the ability to attenuate excessive Ca2+ influx through the LTCC. In general the aza–pentacycloundecylamines (8a–c) were the most potent LTCC blocker which also had the ability to offer protection in the cell viability assays. However, NGP1–01 (7a) had the most favorable pharmacological profile overall with good activity as an LTCC blocker (IC50 = 86 ?M) and the ability to significantly attenuate cell death in the cell viability assays, exhibiting no toxicity. In addition to their ability to modulate Ca2+ influx from the extracellular environment, these compounds also displayed the ability to modulate Ca2+ flux through intracellular Ca2+ channels. The mechanisms by which they act on intracellular Ca2+ channels still remains unclear, but from this preliminary study it would appear that these compounds are able to partially inhibiting Ca2+–ATPase activity whilst possibly simultaneously inhibiting the IP3R. In the absence of extracellular Ca2+ these compounds showed the ability in inhibit voltage–induced Ca2+ release (VICaR), possibly by modulating the gating charge of the voltage sensor being the dihydropyridine receptors. In future studies it might be worthwhile to do an expanded QSAR study and evaluate the aza–pentacycloundecylamines. To clarify the mechanisms by which the polycyclic compounds interact with intracellular Ca2+ channels we should examine the direct interaction with the individual Ca2+ channels independently. The polycyclic compounds evaluated in this study demonstrate potential as multifunctional drugs due to their ability to broadly regulate calcium homeostasis through multiple pathways of Ca2+ entry. This may prove to be more effective in diseases where perturbed Ca2+ homeostasis have devastating effects eventually leading to excitotoxicity and cell death. / Thesis (Ph.D. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Pain signaling involves transmission of nociceptive stimuli in the spinal cord where a critical balance between excitatory and inhibitory inputs determines the response to noxious stimuli. The neuropeptide, substance P (SP), mediates transmission of pain in part by binding to the tachykinin receptor (NK-1R) in the dorsal horn (DH) of the spinal cord. One of SP’s downstream effects is to modulate N-type Ca2+(N-) channels. While phospholipid breakdown is a part of the inflammatory process that accompanies tissue damage, the role of this metabolic pathway has not been completely described with respect to N-channel modulation during pain signaling. Despite the incomplete understanding of this modulation, pharmacological antagonists of both NK-1R and N-channels have been used to treat pain. In Chapter II, using whole-cell patch clamp recording techniques, the SP signaling cascade that mediates inhibition of recombinant N-channel activity was characterized. By adopting a pharmacological approach, I show that this pathway resembles the slow pathway that was earlier described for modulation of N-current by the M1 muscarinic receptor (M1R). M1R couples to Gq to stimulate phospholipid breakdown. Together with previous observations, the data presented in this chapter provide evidence for involvement of the extracellular receptor kinase (ERK1/2), phospholipase A2 and release of phospholipid metabolites in the modulation of N-current by SP. Overall, this chapter shows that phospholipid metabolism involved in modulation of N-currents is not specific to M1Rs but that other Gq-coupled receptors may also modulate N-currents via the same signal transduction pathway. In Chapter III, enhancement of N-current by SP was studied as part of a collaborative project to understand current enhancement that occurs when a palmitoylated accessory CaVβ2a subunit is co-expressed with the pore-forming subunit CaV2.2 and the accessory subunit α2δ-1. When CaVβ3 is present, SP inhibits N-current as described in Chapter II. However, when palmitoylated CaVβ2a is co-expressed with CaV2.2 (and α2δ-1), current enhancement is observed at negative test potentials, demonstrating that both M1Rs and NK-1Rs exhibit the same profile of N-current modulation. This change in modulation by muscarinic agonists is not observed in the presence of a depalmitoylated CaVβ2a. However a chimeric CaVβ2aβ1b subunit that contains the palmitoylated N-terminus from CaVβ2a confers enhancement. Normally expression of the β1b subunit resulted in current inhibition. These findings indicated that the palmitoylated CaVβ2a participates in enhancement of current. Our data support a model where inhibition dominates over enhancement; when inhibition is blocked, enhancement may be observed. Lastly, we show that N-current inhibition by SP is minimized when exogenous palmitic acid is applied to cells co-expressing CaVβ3 subunits with N-channels. These results indicate that the presence of palmitic acid can prevent N-current inhibition when SP is applied most likely by interacting with CaV2.2. We propose a model where palmitic acid occupies the inhibitory site and serves to antagonize inhibition by a lipid metabolite, which is most likely arachidonic acid. The CaVβ2a protein seems to have a role in positioning the palmitoyl groups near CaV2.2. This chapter provides a new role for protein palmitoylation where the palmitoyl groups of CaVβ2a are both necessary and sufficient to block inhibition of another protein: CaV2.2. In Chapter IV, I probe the role of the relative orientation of CaVβ2a and the pore-forming subunit of the N-channel in N-current modulation. Evidence is presented that shows that not just the presence of a palmitoylated CaVβ2a is necessary, but the relative orientation of CaVβ2a to CaV2.2 is critical for blocking inhibition. Using N-channel mutants that cause a change in the orientation of CaVβ2a relative to CaV2.2, I show that the block of inhibition is disrupted; inhibition by the slow pathway is rescued. These findings further support my model that the palmitoyl groups of CaVβ2a normally reside in a specific location that overlaps with the slow pathway inhibitory site on CaV2.2. Lastly I present data showing that the enhancement of N-current, observed when palmitoylated CaVβ2a is present, occurs via the slow pathway. In Chapter V the effect of CaVβ’s orientation on N-channel modulation by the dopamine D2 receptor is tested. In this form of modulation, inhibition is rapid and voltage-dependent. The signaling pathway is membrane-delimited since Gβγ, released after receptor stimulation, directly interacts with the N-channel at a site that overlaps with a high affinity binding site for CaVβs. While N-currents are modulated by this pathway, the deletion mutants show aberrant membrane-delimited modulation. The findings in this chapter further underscore the importance of proper positioning of CaVβ to CaV2.2 for eliciting proper N-current modulation after GPCR stimulation. Overall, the data presented in this dissertation provides a mechanistic approach into examining modulation of N-current by different GPCRs via two different signaling pathways as well as the role CaVβ subunits serve in each modulatory pathway.

Vasorrelaxant effects of R(+)-pulegone were tested in normotensive rats using two different methodological approaches. In vivo, increasing doses (1, 3, 10, 20 and 30 mg / kg) were administered in the animals, i.v. bolus and selected randomly, then the parameters for mean arterial pressure (MAP) and heart rate (HR) were evaluated. In this situation the substance triggered a hypotensive effect and bradycardia. In the ex vivo approach, we used the thoracic aorta of these animals and isometric tension experiments, evaluated the vasorelaxant activity of the substance. The administration of R (+)-pulegone triggered vasorelaxant effect concentration-dependent in both rings with intact endothelium and with this removed, but the substance had the lowest pD2 value in the presence of the endothelium (-3.64 ± 0.06, n = -3.17 vs 5 ± 0.034, n = 6, respectively), no change in peak effect (98.2 ± 1.2%, n = 5 vs. 106.0 ± 8.1%, n = 6) indicating that the substance acts triggering vasodilation in aortic dependent manner and independent of the vascular endothelium. In rings with intact endothelium, the vasorelaxant activity of R(+)-pulegone was not altered in the presence of diclofenac and atropine, but was modified by L-NAME (-3.00 ± 0.016; n=5), HDX (-3.07 ± 0.021; n=5), ODQ (-3.17 ± 0.03; n = 5) and the red ruthenium (-3.14 ± 0.04; n=5) vs control: -3.64 ± 0.06; n = 5. These results suggest that the substance is probably stimulating NO production via the activation of TRP channels. In smooth muscle vascular, R(+)-pulegone inhibited curve calcium concentration-dependent manner (Emax: 10-4 M: 68 9 ± 3.81%; 3x10-4 M: 40.97 ± 8.05%; 10-3 M: 24.79 ± 5.04% and 3x10-3 M: 0.29 ± 0.33%) via calcium channels type L, as in the presence of nifedipine, there was a reduction of the maximum effect (Emax: 93.3 ± 1.7% ; n = 6 vs Emax control: 106.8 ± 8.1%; n = 6 ). Additionally, it was surveyed the participation of for potassium channels, using 4-aminiopiridine, it was seen that the substance has inhibited response by blocking the potassium channels sensitive to voltage (-2.93 ± 0.012 - n = 5 vs control - 3.17 ± 0.034 - n = 6) as well as sensitive to ATP, since, in the presence of glybenclamide, the relaxant response to R(+)-pulegone was also inhibited (-2.94 vs. -3.17 ± 0.012 ± 0.03). Thus, to cause vasorelaxation in normotensive rat thoracic aorta, R(+)-pulegone, stimulates the production of NO in endothelial cells, probably by activating calcium influx via TRP channels. The effect independent of the endothelium is mediated by inhibition of calcium influx, likely through the CaV and for opening potassium channels (KATP and Kv). / O efeito vasorrelaxante de R(+)-pulegona foi estudado em ratos normotensos, utilizando duas abordagens metodológicas. Na avaliação in vivo, doses crescentes (1, 3, 10, 20 e 30 mg/Kg), foram administradas nos animais, via i.v. em bolus de maneira aleatória, posteriormente os parâmetros de pressão arterial média (PAM) e frequência cardíaca (FC) foram avaliados. Nesta situação a substância desencadeou um efeito hipotensor e bradicárdico dependente de dose. Na abordagem ex vivo, utilizou-se a aorta torácica destes animais e avaliou-se a atividade vasorrelaxante da substância. A administração de R(+)-pulegona desencadeou efeito vasorrelaxante, concentração dependente tanto em anéis com endotélio intacto quanto com este removido, porém a substância apresentou o valor do pD2 menor na presença do endotélio (-3,64 ± 0,06, n=5 vs -3,17 ± 0,034, n=6, respectivamente), sem nenhuma alteração no efeito máximo (98,2 ± 1,2%, n=5 vs 106,0 ± 8,1%, n=6), indicando que a substância atua desencadeando vasodilatação na aorta de maneira dependente e independente do endotélio vascular. Em anéis com endotélio intacto, a atividade vasorrelaxante de R(+)-pulegona não foi alterada na presença de diclofenaco e atropina, porém foi modificada por L-NAME (-3,00 ± 0,016, n=5), HDX (-3,07 ± 0,021, n=5), ODQ (-3,17 ± 0,03, n=5) e o vermelho de rutênio (-3,14 ± 0,04, n=5) todos vs controle: -3,64 ± 0,06, n=5. Estes resultados sugerem que a substância provavelmente está estimulando a produção de óxido nítrico (NO), via ativação dos canais TRP na célula endotelial. O efeito dependente do músculo liso vascular de R(+)-pulegona dá-se através da inibição da curva de cálcio de maneira dependente de concentração (Emáx: 10-4 M: 68,9 ± 3,81%; 3x10-4 M: 40,97 ± 8,05%; 10-3 M: 24,79 ± 5,04% e 3x10-3 M: 0,29 ± 0,33%), via canais para cálcio tipo L, pois na presença de NIF, ocorreu redução do efeito máximo (Emáx: 93,3 ± 1,7%, n= 6 vs controle Emáx: 106,8 ± 8,1%, n=6). Adicionalmente, pesquisou-se a participação dos canais para potássio e na presença de 4-AP ocorreu redução da resposta relaxante da substância indicando a participação dos canais para potássio sensíveis a voltagem (-2,93 ± 0,012, n=5 vs controle -3,17 ±0,034, n=6) e também sensíveis ao ATP, uma vez que, na presença de glibenclamida, a resposta relaxante para R(+)-pulegona também foi reduzida (-2,94 ± 0,012, n=5 vs -3,17 ± 0,03, n=6). Assim, para causar vasorrelaxamento em aorta torácica de ratos normotensos, R(+)-pulegona, estimula a produção de NO na célula endotelial, provavelmente por ativar o influxo de cálcio via canais TRP e o efeito independente do endotélio, é mediado pela inibição do influxo de cálcio, provavelmente através dos CaV e abertura dos canais para potássio (Kv e KATP).

Extracts from Tithonia diversifolia (TD) have been widely employed in Central and South America, Asia and Africa to help in the treatment of malaria, a disease to which millions of people are subjected in tropical countries. In spite of this, we were not able to find any scientific report dealing with their effects on the heart muscle functioning or describing their toxic effects. Such gaps are barriers for the TD derivatives to become a phytotherapic agent. Therefore, we have planned this study aiming to clarify the inotropic and toxicological effects of TD derivatives. Their effects on the muscle inotropism were investigated in isolated guinea pig atrium mounted in an organ chamber containing Tyrode solution oxygenated by carbogen mixture (5% CO2, 95% O2). The atria were stretched (1 gf) and stimulated at 2 Hz with suprathreshold electrical pulses. Their contractile forces were captured isommetrically and stored in computer to be analysed off line. The effect of AqF was determined after this fraction be added to the organ bath. The evaluation concerned to its effect on the L-type calcium current (ICa,L) was carried out in ventricular cardiomyocytes isolated according to the steps described by Shioya (2007). The intracellular calcium transients were also studied in these cells previously loaded with FLUO4-AM and monitored by confocal microscopy. The acute (ATx) and subacute (STx) toxicities of the crude extract of TD (CE) were evaluated in rats according to the steps proposed by OECD (2001a,b). Briefly: animals were separated by gender to create one control group and two treated groups. For the ATx study, the animals pertaining to the control group received orally only distilled water, whereas rats pertaining to the treated groups received, respectively, 2.5 g/kg and 5.0 g/kg of CE administered in a unique dose. The animals involved in the study were monitored for abnormal signals during 14 days. In the STx evaluation, the animals of the two treated groups received, respectively, 0.125 g/kg/day CE and 0.250 g/kg/day CE during 30 days. Animals were carefully observed for signals representing physiological and/or behavioral abnormalities. At the end of each toxicological protocol, the animals were anaesthetized for collecting blood and after that they were euthanized for the removal of organs. Our results showed that AqF depresses the atrial contractility in a dose-dependent fashion. However, concentrations greater than 2 g/l cause mechanical alternans , indicating that AqF can disturb the dynamics of the calcium release channels, which are present in the sarcoplasmic reticulum membrane. The effect of AqF on the L-type calcium current (ICa,L) was studied by recording how this fraction changes the inotropic effect produced by increasing concentrations of CaCl2. Our data showed that 2 g/l AqF increased the CaCl2 EC50 from 1.207 ± 0.139 to 2.846 ± 0.035 mM (n = 4, p < 0.05), suggesting that AqF is able to reduce ICa,L. Such conclusion was reinforced by testing the Bowditch phenomenon in atria incubated with 1.3 g/l AqF. In these experiments, AqF completely abolished the force overshoot , which is normally seen in the positive staircase Bowditch phenomenon. The depressant effect of AqF on the ICa,L was also supported by the decrease of intracellular fluorescence related to the subcellular calcium from 6.9 ± 0.4 (n = 53) to 5.3 ± 0.3 u.a. (n = 51; p < 0.05). In fact, direct measurements of ICa,L performed in patch clamped cardiomyocytes, confirmed the depressant effect of AqF on ICa,L. In these experiments, 80 g/ml of AqF reduced the density of ICa,L from 10.00 ± 1.22 to 7.80 ± 1.19 A/F (n = 4, p < 0.05). An expected consequence for the ICa,L reduction is the increase of the time required for the myocardial electrical wave to cross the atrioventricular node. Electrocardiograms from isolated guinea pig hearts confirmed the reduction of ICa,L because in the presence of AqF the heart presented a complete atrioventricular block (AVB). There was no involvement of potassium channels, neither the muscarinic nor the opioids receptors in the AqF myocardial depressant effect. We can conclude that the depressant effects of AqF in the myocardium contractility are due to a reduction of ICa,L. Studies concerned to the ATx and STx showed that the CE does not promote any signal of pain, abnormal behavior or mortality in the rats (DL50>5.0 g/kg). In STx, CE does not change water or food intakes, but in the animals treated with 0.250 g/kg/day a gain of weight occurred, suggesting water retention in the animal s body. Neutropenia, eosinopenia, lymphocytosis, and monocytosis were observed in treated animals during the ATx study. In animals pertaining to the STx study it was seen leucopenia, neutropenia, and monocytopenia. In ATx evaluation, the biochemical profile of the blood showed a reduction in the plasmatic urea but the aspartate and alanine aminotransferases, alkaline phosphatase, total and direct bilirubin, total protein, globulin, sodium, and uric acid increased. Similar results were found in the STx study, except for the urea that remained unchanged and for the glucose, creatinin, albumin, and potassium that presented an increase in their serum levels. These findings advise for precaution when TD derivatives were proposed as a therapeutic agent. We conclude that AqF reduces ICa,L leading to a negative inotropism and an AVB. The CE interferes with the hematopoietic system and promotes injuries for the liver and kidneys leading to a non-inflammatory tissue reaction. / Extratos das folhas da Tithonia diversifolia (TD) têm sido empregados nas Américas do Sul e Central, Ásia e África como agentes antimaláricos entre outros usos. Seus possíveis efeitos sobre o coração e sua toxicidade, contudo, não estão relatados. Estas lacunas comprometem a expectativa de os derivados da planta virem a interessar à indústria de fitoterápicos e por esta razão, o presente trabalho visou complementar tais lacunas, trazendo para o conhecimento as propriedades inotrópicas e toxicológicas de derivados das folhas desta planta. Os estudos inotrópicos foram realizados com a fração aquosa (FAq) e em átrio esquerdo isolado de cobaia montado em cuba para órgão. Os átrios foram estirados a 1 gf e estimulados com pulsos de corrente supralimiares. A força atrial foi captada isometricamente e armazenada em computador. A ação da FAq foi avaliada adicionando-a ao banho. Para estudos com célula isolada, foram obtidos cardiomiócitos pela técnica de dissociação proposta por Shioya (2007). O efeito da FAq sobre a corrente de cálcio tipo L (ICa,L) foi estudado em cardiomiócitos ventriculares de rato e os de camundongo foram usados para estudar os transientes de cálcio subcelular, empregando-se, para isso, a sonda fluorescente Fluo4-AM. Os registros foram feitos com microscopia confocal de varredura a laser. A toxicidade aguda (TxA) e subaguda (TxS) do extrato bruto da TD (EB) foi avaliada em ratos de acordo com as regras da OECD. Resumidamente: os animais foram separados por gênero em grupos controle e tratados. Nos estudos de TxA, os animais do grupo controle receberam água destilada, enquanto que os dos grupos tratados receberam, p.o., 2,5 g/kg e 5,0 g/kg de EB. Os efeitos foram monitorados por 14 dias. Na avaliação referente à TxS, os grupos tratados receberam, p.o., durante 30 dias, 0,125 g/kg/dia ou 0,250 g/kg/dia de EB. Os animais foram observados para sinais de alterações fisiológicas e/ou comportamentais. Em seguida, foram anestesiados, o sangue coletado e depois eutanasiados para a histopatologia dos órgãos. Os resultados mostraram que a FAq deprime a contratilidade atrial e que concentrações acima de 2 g/l induzem o alternância mecânica, sugerindo que ela leva à disfunção dos canais liberadores de cálcio existentes no retículo sarcoplasmático. Para avaliar a ação da FAq sobre a ICa,L, foi estudada a sua ação sobre a curva concentração-efeito para o CaCl2. Neste estudo, a FAq produziu aumento da CE50 de 1,207 ± 0,139 mM para 2,846 ± 0,035 mM (n = 4, p < 0,05). A incubação do átrio com 1,3 g/l de FAq aboliu completamente o overshoot de força do fenômeno de Bowditch (n = 4, p < 0,05), indicando que ela reduz ICa,L. Esta hipótese foi igualmente suportada no fato de que a fração diminuiu a fluorescência para o cálcio subcelular de 6,9 ± 0,4 u.a. (n = 53) para 5,3 ± 0,3 u.a. (n = 51; p < 0,01) e também porque, quando empregada a 80 g/ml, ela reduziu a densidade da ICa,L de 10,00 ± 1,22 A/F para 7,80 ± 1,19 A/F (n = 4, p < 0,05). A FAq produziu bloqueio de condução no nódulo atrioventricular. O mecanismo de ação da FAq não envolveu receptores muscarínicos, opióides, nem canais para potássio, mas está baseado na diminuição da ICa,L. A avaliação da TxA e TxS mostrou que o EB é de baixa toxicidade pois sua DL50 foi maior do que 5,0 g/kg. O EB não alterou o consumo de água ou de comida, mas nos animais tratados com 0,250 g/kg/dia houve ganho de peso, sugerindo retenção hídrica, pois o peso dos órgãos não se modificou. Neutropenia, eosinofilopenia, linfocitose e monocitose foram observadas na TxA, mas na TxS ocorreu leucopenia, neutropenia e monocitopenia. A análise bioquímica na TxA mostrou redução da ureia plasmática, porém aumento da aspartato e da alanina aminotransferases, fosfatase alcalina, bilirrubinas total e direta, proteína total, globulina, sódio e ácido úrico. Resultados semelhantes foram encontrados nos estudos sobre a TxS, exceto para ureia, que se manteve constante e para glicose, creatinina, albumina e potássio, cujos níveis sanguíneos aumentaram. A histopatologia mostrou sinais de injúria hepática e renal. Concluimos que a FAq reduz a ICa,L como consequência ela é inotrópica negativa e induz BAV. Ela interfere com o sistema hematopoiético e promove lesão hepática e renal sem causar mortalidade e induz reação inflamatória tissular.

Receptores purinérgicos e canais de cálcio voltagem-dependentes estão envolvidos em diversos processos biológicos como na gastrulação, durante o desenvolvimento embrionário, e na diferenciação neural. Quando ativados, canais de cálcio voltagem-dependentes e receptores purinérgicos do tipo P2, ativados por nucleotídeos, desencadeiam transientes de cálcio intracelulares controlando diversos processos biológicos. Neste trabalho, nós estudamos a participação de canais de cálcio voltagem-dependentes e receptores do tipo P2 na geração de transientes de cálcio espontâneos e sua regulação na expressão de fatores de transcrição relacionados com a neurogênese utilizando como modelo células tronco (CTE) induzidas à diferenciação em células tronco neurais (NSC) com ácido retinóico. Descrevemos que CTE indiferenciadas podem ter a proliferação acelerada pela ativação de receptores P2X7, enquanto que a expressão e a atividade desse receptor precisam ser inibidas para o progresso da diferenciação em neuroblasto. Além disso, ao longo da diferenciação neural, por análise em tempo real dos níveis de cálcio intracelular livre identificamos 3 padrões de oscilações espontâneas de cálcio (onda, pico e unique), e mostramos que ondas e picos tiveram a frequência e amplitude aumentadas conforme o andamento da diferenciação. Células tratadas com o inibidor do receptor de inositol 1,4,5-trifosfato (IP3R), Xestospongin C, apresentaram picos mas não ondas, indicando que ondas dependem exclusivamente de cálcio oriundo do retículo endoplasmático pela ativação de IP3R. NSC de telencéfalo de embrião de camundongos transgênicos ou pré-diferenciadas de CTE tratadas com Bz-ATP, o agonista do receptor P2X7, e com 2SUTP, agonista de P2Y2 e P2Y4, aumentaram a frequência e a amplitude das oscilações espontâneas de cálcio do tipo pico. Dados, obtidos por microscopia de luminescência, da expressão em tempo real de gene repórter luciferase fusionado à Mash1 e Ngn2 revelou que a ativação dos receptores P2Y2/P2Y4 aumentou a expressão estável de Mash1 enquanto que ativação do receptor P2X7 levou ao aumento de Ngn2. Além disso, células na presença do quelante de cálcio extracelular (EGTA) ou do depletor dos estoques intracelulares de cálcio do retículo endoplasmático (thapsigargin) apresentaram redução na expressão de Mash1 e Ngn2, indicando que ambos são regulados pela sinalização de cálcio. A investigação dos canais de cálcio voltagem-dependentes demonstrou que o influxo de cálcio gerado por despolarização da membrana de NSC diferenciadas de CTE é decorrente da ativação de canais de cálcio voltagem-dependentes do tipo L. Além disso, esse influxo pode controlar o destino celular por estabilizar expressão de Mash1 e induzir a diferenciação neuronal por fosforilação e translocação do fator de transcrição CREB. Esses dados sugerem que os receptores P2X7, P2Y2, P2Y4 e canais de cálcio voltagem-dependentes do tipo L podem modular as oscilações espontâneas de cálcio durante a diferenciação neural e consequentemente alteram o padrão de expressão de Mash1 e Ngn2 favorecendo a decisão do destino celular neuronal. / Purinergic receptors and voltage gated Ca2+ channels have been attributed with developmental functions including gastrulation and neural differentiation. Upon activation, nucleotide-activated P2 purinergic receptor and voltage-gated Ca2+ channel subtypes trigger intracellular calcium transients controlling cellular processes. Here, we studied the participation of voltage-gated calcium channels and P2 receptor activity in spontaneous calcium transients and consequent regulation expression of transcription factors related to retinoic acid-induced neurogenesis of mouse neural stem and embryonic stem cells (ESC). In embryonic pluripotent stem cells, proliferation is accelerated by P2X7 receptor activation, while receptor expression / activity needs to be down-regulated for the progress of neuroblast differentiation. Moreover, along neural differentiation time lapse imaging with means of a cytosolic calcium-sensitive fluorescent probe provided different patterns of spontaneous calcium transients (waves and spikes) showing that both, frequency and amplitude increased along differentiation. Cells treated with the inositol 1,4,5-trisphosphate receptor (IP3R) inhibitor Xestospongin C showed spikes but not waves, indicating that waves exclusively depended on calcium release from endoplasmic reticulum by IP3R activation. Cells treated with the P2X7 receptor subtype agonist Bz-ATP and the P2Y2 and P2Y4 receptor 2-S-UTP increased frequency and amplitudes of calcium transients, mainly spikes, in embryonic telencephalon neural stem cells (NSC) and NSC pre-differentiated from ESC. Data obtained by luminescence time lapse imaging of stable transfected cells with Mash1 or Ngn2 promoter-protein fusion to luciferase reporter construct revealed increased Mash1 expression due to activation of P2Y2/P2Y4 receptor subtypes, while increased expression of Ngn2 was observed following P2X7 receptor activation. In addition, cells imaged in presence of the extracellular calcium chelator EGTA or following endoplasmic reticulum calcium store depletion by thapsigargin showed a decrease in Mash1 and Ngn2 expression, indicating that both are regulated by calcium signaling. Investigation of the roles of voltage gated Ca2+ channels in neural differentiation showed that Ca2+ influx in NSC pre-differentiated from ESC is due to membrane depolarization and L-type voltage gated Ca2+ channel activation, thereby controlling cell fate decision, by stabilizing the expression of MASH1 and inducing differentiation, by phosphorylation of the transcription factor CREB. Altogether these data suggest that P2X7, P2Y2, P2Y4 receptors and L-type voltage gated Ca2+ channels can modulate spontaneous calcium oscillations during neural differentiation and consequently change the Mash1 and Ngn2 expression patterns, thus favoring the cell fate decision to the neuronal phenotype.

Calcium imaging has been used as a promising technique to monitor the dynamic activity of neuronal populations. However, the calcium trace is temporally smeared which restricts the extraction of quantities of interest such as spike trains of individual neurons. To address this issue, spike reconstruction algorithms have been introduced. One limitation of such reconstructions is that the underlying models are not informed about the biophysics of spike and burst generations. Such existing prior knowledge might be useful for constraining the possible solutions of spikes. Here we describe, in a novel Bayesian approach, how principled knowledge about neuronal dynamics can be employed to infer biophysical variables and parameters from fluorescence traces. By using both synthetic and in vitro recorded fluorescence traces, we demonstrate that the new approach is able to reconstruct different repetitive spiking and/or bursting patterns with accurate single spike resolution. Furthermore, we show that the high inference precision of the new approach is preserved even if the fluorescence trace is rather noisy or if the fluorescence transients show slow rise kinetics lasting several hundred milliseconds, and inhomogeneous rise and decay times. In addition, we discuss the use of the new approach for inferring parameter changes, e.g. due to a pharmacological intervention, as well as for inferring complex characteristics of immature neuronal circuits.

La douleur chronique occupe une place centrale dans les préoccupations de santé publique. En France, elle touche environ 20% de la population et a un impact négatif sur la qualité de vie des patients. Les traitements actuels sont généralement inefficaces ou associés à d’importants effets indésirables. Par conséquent, de nouvelles approches thérapeutiques sont nécessaires. Parmi les cibles potentielles, les canaux calciques voltage-dépendants de type T, en particulier l’isoforme Cav3.2, constituent des candidats d’intérêt. Aussi, l’objectif de cette thèse est de caractériser leur implication fonctionnelle dans deux types de douleur chronique : viscérale et somatique. Concernant la douleur viscérale, nous avons développé un modèle murin d’hypersensibilité colique associée à une inflammation à bas bruit, deux caractéristiques séméiologiques proches de la symptomatologie rencontrée chez la plupart des patients souffrant du syndrome de l’intestin irritable (SII) ou de maladies inflammatoires chroniques intestinales (MICI) durant les périodes de rémission. En ce qui concerne la douleur somatique, nous avons utilisé deux modèles murins de douleur inflammatoire, l’un présentant une inflammation subaiguë et l’autre persistante. Dans ces différents modèles, une inhibition pharmacologique via l’administration d’un antagoniste des canaux Cav3.2, le TTA-A2, ou génétique grâce à des souris présentant un Knock out (KO) de Cav3.2 a induit un puissant effet antalgique, démontrant une implication fonctionnelle des canaux Cav3.2 dans le développement et le maintien de ces types de douleur. De plus, l’utilisation de souris présentant un KO conditionnel de Cav3.2, spécifiquement dans les fibres C des ganglions de la racine dorsale (DRG), ainsi que l’emploi de l’ABT-639, un agent pharmacologique bloqueur des canaux de type T à action périphérique, nous ont permis de préciser la localisation de cette implication. Ainsi, une action majoritairement spinale présynaptique des canaux Cav3.2 a été mise en évidence pour la douleur viscérale alors qu’une action plus complexe de ces canaux est mise en jeu pour la douleur somatique inflammatoire. En effet, pour cette dernière, le canal Cav3.2 présente une implication à la fois spinale et périphérique. De plus, nous avons montré un rôle des canaux Cav3.2 dans le processus inflammatoire, s’effectuant au travers d’une implication de ces derniers dans les cellules immunitaires. Enfin, dans une démarche de recherche translationnelle, nous avons évalué l’effet de l’éthosuximide (ETX), un bloqueur des canaux de type T, utilisé en clinique dans le traitement de l’épilepsie. Nous avons décrit un effet antalgique de ce dernier dans chacun des modèles étudiés ainsi qu’une action anti-inflammatoire, apportant ainsi une preuve de concept préclinique pour une évaluation d’efficacité clinique de l’ETX dans un contexte de douleurs viscérales ou somatiques inflammatoires. L’ensemble de ces résultats apporte de nouvelles connaissances concernant l’implication des canaux Cav3.2 dans la douleur chronique et permet de proposer ces canaux comme des cibles d’intérêt pour le développement de stratégies thérapeutiques innovantes visant à soulager les patients. / Chronic pain is a central concerns to public health. In France, it affects about 20% of the population and has a negative impact on the patients’ quality of life. Current treatments are generally ineffective or associated with strong adverse effects. Therefore, new therapeutic approaches are needed. Among the potential targets, the T-type voltage-dependent calcium channels, in particular the Cav3.2 isoform, constitute candidates of interest. Thus, the objective of this thesis is to characterize their functional implication in two types of chronic pain: visceral and somatic. We have developed a murine model of colonic hypersensitivity associated with low grade inflammation, two symptomatic features close to the symptomatology found in most patients suffering from irritable bowel syndrome (IBS) or with diseases inflammatory bowel disease (IBD) during remission periods. Concerning the somatic pain, we used two murine models of inflammatory pain, one with subacute inflammation and another with persistent inflammation. In these different models, a pharmacological inhibition with the administration of a Cav3.2 channel antagonist, TTA-A2, or a genetic approach using Cav3.2 knockout (KO) mice induced a robust analgesic effect demonstrating a functional implication of Cav3.2 channels in the development and maintenance of these types of pain. Moreover, the use of mice with a Cav3.2 conditional KO, specifically in the C-dorsal root ganglia (DRG) fibers, and the use of ABT-639, a peripherally acting pharmacological blocker of type T channels, allowed us to specify the localization of this implication. Thus, a pre-synaptic spinal action of the Cav3.2 channels has been demonstrated for visceral pain whereas a more complex action of these channels is involved for inflammatory somatic pain. Indeed, for the latter, Cav3.2 channels present a spinal and peripheral implication. In addition, we have shown the role of Cav3.2 channels in the inflammatory process, with an involvement located in the immune cells. Finally, with a translational research approach, we evaluated the effect of ethosuximide (ETX), a T-channel blocker, clinically used in the treatment of epilepsy. We have described an analgesic effect of the latter in both studied models as well as an anti-inflammatory action. These results constitute a pre-clinical proof of concept for a clinical efficacy evaluation of ETX in a context of visceral pain or somatic inflammatory diseases. Altogether these results provide new insight about the involvement of Cav3.2 channels in chronic pain and allow us to propose these channels as targets of interest for the development of new therapeutic strategies to relieve patients.

La grossesse s’accompagne d’importantes modifications hormonales et hémodynamiques. Parmi celles-ci, le système rénine-angiotensine-aldostérone (SRAA) est activé très tôt durant la grossesse. De plus, cette augmentation du SRAA est accompagnée d’élévations du débit cardiaque et du volume plasmatique ainsi que des baisses paradoxales de la pression artérielle et de la résistance vasculaire périphérique. Ceci suggère que la grossesse induit un remaniement des réponses physiologiques normales au SRAA. Une résistance vasculaire à l’action des vasopresseurs est également observée durant la gestation. Ce phénomène serait causé par la modification de la fonction des canaux calciques et potassiques. De plus, il serait possiblement dû à la participation de la Na+/K+-ATPase, par son influence sur le potentiel membranaire des cellules des muscles lisses vasculaires (VSMC). La présence des récepteurs minéralocorticoïdes (MR) dans les VSMC laisse croire que l’aldostérone peut influencer le tonus vasculaire par des effets génomiques et non-génomiques. Compte tenu des connaissances actuelles, nous avons émis l’hypothèse que l’augmentation des taux sériques d’aldostérone durant la grossesse est responsable des changements hémodynamiques observés et que ces effets sont causés par l’activation des MR. Des rates gestantes ont été traitées avec du canrénoate de potassium (20 mg/kg•jr), un antagoniste des MR, durant la dernière semaine de gestation (sur 3). Sur des anneaux aortiques dénudés de leur endothélium, nous avons mesuré les réponses contractiles à la phényléphrine (PhE) et au KCl en présence d’un bloqueur des canaux calciques dépendants du voltage (VDCC), la nifédipine, et d’activateurs des canaux potassiques à large conductance (BKCa) et ceux dépendants de l’ATP (KATP), respectivement le NS-1619 et la cromakalim. Les réponses à la PhE et au KCl sont réduites à partir du 17e jour de gestation et le traitement au canrénoate augmente ces réponses dans tous les groupes. Les modulateurs de canaux ioniques atténuent les réponses à la PhE et au KCl. Cependant, le canrénoate modifie aussi les effets des modulateurs sur les aortes. Aucun effet ou une baisse des réponses est observable chez les rates non gestantes, tandis qu’une hausse de leur effet inhibiteur est notée chez les rates gestantes. Ces effets du canrénoate font croire que l’aldostérone participe à l’adaptation de la réactivité vasculaire durant la grossesse. Par ailleurs, le potentiel membranaire des VSMC pourrait être affecté dans la gestation. Pour vérifier cette hypothèse, nous avons évalué l’activité de la Na+/K+-ATPase, impliquée dans le contrôle du potentiel membranaire. Nos résultats démontrent que l’activité de la pompe est inhibée à partir du 19e jour de gestation. Cet effet est renversé par le canrénoate. Toutefois, comme le renversement de l’inhibition de la pompe est également présent chez les rates gestantes traitées avec du PST 2238, un antagoniste de l’ouabaïne sur la Na+/K+-ATPase, et que le canrénoate agit également comme agoniste partiel de la pompe, nous croyons que la diminution d’activité associée à la gestation est liée à une inhibition de la Na+/K+-ATPase par des stéroïdes cardiotoniques plutôt qu’à un effet des minéralocorticoïdes. L’augmention d’activité de la pompe liée au canrénoate s’accompagne d’une diminution de l’expression de la sous-unité α1, suggérant que la sous-unité α2 est responsable des variations de contractilité de l’aorte, puisque son expression n’est pas modifiée par le canrénoate. Les effets de la diminution de l’expression de la sous-unité α1, influencée par la signalisation du MR, restent à être déterminés. Néanmoins, nos résultats montrent que les modifications d’activité de la Na+/K+-ATPase influencent l’activité des canaux potassiques et que la pompe pourraient être un des éléments primordiaux dans le contrôle de la réactivité vasculaire durant la grossesse. Comme le canrénoate modifie la réactivité vasculaire, nous voulions déterminer ses impacts sur la pression artérielle. Des rates gestantes ont été traitées avec du canrénoate (20 ou 60 mg/kg•jr) et les paramètres hémodynamiques ont été évalués par radiotélémétrie. Aucune modification de la pression artérielle, du rythme cardiaque et de la pression pulsée ne sont mesurées chez les rates recevant le traitement. Toutefois, des augmentations de l’osmolalité, des taux sériques d’aldostérone et de corticostérone ainsi que de l’activité rénine plasmatique sont observées chez les animaux recevant 60 mg/kg•jr. Le canrénoate bloque donc le rétrocontrôle du SRAA. Par contre, les MR ne sont pas les principaux responsables du contrôle de la pression artérielle durant la grossesse. En conclusion, nous avons démontré que le traitement des rates au canrénoate influence la réactivité vasculaire de l’aorte durant la gestation. Cet effet est causé par la modification de l’activité de certains canaux ioniques (VDCC, BKCa et KATP). De plus, le canrénoate renverse l’inhibition de la Na+/K+-ATPase observée durant la gestation. Finalement, les actions locales de cet antagoniste des MR sur les vaisseaux sanguins ne se répercutent pas sur l’effet systémique global et aucune modification de la pression artérielle n’est observée. D’autres études seront toutefois nécessaires pour déterminer les voies de signalisation par lesquelles l’aldostérone module les réponses des canaux ioniques dans les VSMC. / Pregnancy is accompanied by important hormonal and hemodynamic modifications. Among them, the renin-angiotensin-aldosterone system (RAAS) is activated early during pregnancy. Furthermore, this increase of RAAS is accompanied by raises of cardiac output and blood volume as well as paradoxal decreases in blood pressure and peripheral vascular resistance. These suggest that pregnancy induces reorganization of the normal physiological responses to RAAS. In addition, a decreased vascular reactivity to vasoconstrictive agents is observed during pregnancy. Modifications of calcium and potassium channels function would be implicated in this phenomenon. Furthermore, an implication of the Na+/K+-ATPase is suspected, through its influence on the membrane potential of vascular smooth muscle cells (VSMC). Aldosterone, through the presence of mineralocorticoid receptors (MR) in VSMC, could control vascular tone by its genomic and non-genomic effects. With our knowledge at this time, we submit the hypothesis that the increased serum aldosterone levels of pregnancy are responsible for the hemodynamic changes associated with pregnancy and that these effects are caused by the activation of MR. Pregnant rats were treated with potassium canrenoate (20 mg/kg•d), a MR antagonist, during the last week of pregnancy (out of 3 weeks). Vascular reactivity of endothelium-denuded aortic rings was measured. Contractile responses to phenylephrine (PhE) and KCl were studied in the presence of a voltage-dependent calcium channel (VDCC) blocker, nifedipine, as well as calcium-actived (BKCa) and ATP-dependent (KATP) potassium channels activators, NS-1619 and cromakalim respectively. Vascular responses to PhE and KCl were reduced as of the 17th day of gestation. Canrenoate increased the responses to both agonists in the aortas of all the groups. Ionic channel modulators reduced the contractile responses to PhE and KCl. However, canrenoate also modified the responses to the modulators. In the aortas of non pregnant rats, no effect or a decrease of their inhibitory effect were observed while with the pregnant rats we noticed an increased effect. These results suggest that aldosterone could be implicated in the adaptation of vascular responses to pregnancy. On the other hand, VSMC membrane potential could be affected during pregnancy. To verify this hypothesis, Na+/K+-ATPase activity was evaluated since it is implicated in its control. Our results demonstrated that the pump is inhibited as of the 19th day of pregnancy. This effect was reversed by canrenoate. However, since the reversal of the inhibition of the pump was also present in pregnant rats treated with PST 2238, an ouabain antagonist on the Na+/K+-ATPase, and because canrenoate can also act as a partial agonist of the pump, we believe that the decreased activity associated with pregnancy is linked to the inhibition of the Na+/K+-ATPase by cardiotonic steroids rather than the effect of mineralocorticoids. The increased activity of the pump by canrenoate is linked with a decreased of the α1 subunit expression. This suggests that the α2 subunit of the pump would be responsible for the variations of aortic contractility since its expression is not modified by canrénoate. The effects of the diminished expression of the α1 subunit, influenced by the MR signalization, still need to be determined. Nevertheless, our results showed that modifications of Na+/K+-ATPase activity had an impact on the activity of potassium channels and that the pump could be one of the principal elements implicated in the control of vascular reactivity during pregnancy. Since canrenoate modified vascular reactivity, we wanted to determine its impact on blood pressure. Pregnant rats were treated with canrenoate (20 or 60 mg/kg•d) and hemodynamic functions were determined by radiotelemetry. No modification was observed in blood pressure, cardiac output and pulse pressure among the treated rats. However, increases in osmolality, aldosterone and corticosterone levels as well as plasma renin activity were observed in the animal receiving the 60 mg/kg•d canrénoate. Thus, canrenoate blocks the negative feedback of the RAAS. However, MR are not the principal element responsible for the control of blood pressure during pregnancy. In conclusion, we have demonstrated that canrenoate affects aortic vascular reactivity during pregnancy. This effect is perpetuated by modifying the activity of certain ionic channels (VDCC, BKCa and KATP). Furthermore, canrenoate is able to reverse the inhibition of the Na+/K+-ATPase observed during pregnancy. Finally, the local actions of this MR antagonist on blood vessels are not reflected by the global systemic effect and no modification of the blood pressure was observed. Other studies will be necessary to determine the signaling pathways by which aldosterone modulate ionic channel responses in VSMC.

La grossesse s’accompagne d’importantes modifications hormonales et hémodynamiques. Parmi celles-ci, le système rénine-angiotensine-aldostérone (SRAA) est activé très tôt durant la grossesse. De plus, cette augmentation du SRAA est accompagnée d’élévations du débit cardiaque et du volume plasmatique ainsi que des baisses paradoxales de la pression artérielle et de la résistance vasculaire périphérique. Ceci suggère que la grossesse induit un remaniement des réponses physiologiques normales au SRAA. Une résistance vasculaire à l’action des vasopresseurs est également observée durant la gestation. Ce phénomène serait causé par la modification de la fonction des canaux calciques et potassiques. De plus, il serait possiblement dû à la participation de la Na+/K+-ATPase, par son influence sur le potentiel membranaire des cellules des muscles lisses vasculaires (VSMC). La présence des récepteurs minéralocorticoïdes (MR) dans les VSMC laisse croire que l’aldostérone peut influencer le tonus vasculaire par des effets génomiques et non-génomiques. Compte tenu des connaissances actuelles, nous avons émis l’hypothèse que l’augmentation des taux sériques d’aldostérone durant la grossesse est responsable des changements hémodynamiques observés et que ces effets sont causés par l’activation des MR. Des rates gestantes ont été traitées avec du canrénoate de potassium (20 mg/kg•jr), un antagoniste des MR, durant la dernière semaine de gestation (sur 3). Sur des anneaux aortiques dénudés de leur endothélium, nous avons mesuré les réponses contractiles à la phényléphrine (PhE) et au KCl en présence d’un bloqueur des canaux calciques dépendants du voltage (VDCC), la nifédipine, et d’activateurs des canaux potassiques à large conductance (BKCa) et ceux dépendants de l’ATP (KATP), respectivement le NS-1619 et la cromakalim. Les réponses à la PhE et au KCl sont réduites à partir du 17e jour de gestation et le traitement au canrénoate augmente ces réponses dans tous les groupes. Les modulateurs de canaux ioniques atténuent les réponses à la PhE et au KCl. Cependant, le canrénoate modifie aussi les effets des modulateurs sur les aortes. Aucun effet ou une baisse des réponses est observable chez les rates non gestantes, tandis qu’une hausse de leur effet inhibiteur est notée chez les rates gestantes. Ces effets du canrénoate font croire que l’aldostérone participe à l’adaptation de la réactivité vasculaire durant la grossesse. Par ailleurs, le potentiel membranaire des VSMC pourrait être affecté dans la gestation. Pour vérifier cette hypothèse, nous avons évalué l’activité de la Na+/K+-ATPase, impliquée dans le contrôle du potentiel membranaire. Nos résultats démontrent que l’activité de la pompe est inhibée à partir du 19e jour de gestation. Cet effet est renversé par le canrénoate. Toutefois, comme le renversement de l’inhibition de la pompe est également présent chez les rates gestantes traitées avec du PST 2238, un antagoniste de l’ouabaïne sur la Na+/K+-ATPase, et que le canrénoate agit également comme agoniste partiel de la pompe, nous croyons que la diminution d’activité associée à la gestation est liée à une inhibition de la Na+/K+-ATPase par des stéroïdes cardiotoniques plutôt qu’à un effet des minéralocorticoïdes. L’augmention d’activité de la pompe liée au canrénoate s’accompagne d’une diminution de l’expression de la sous-unité α1, suggérant que la sous-unité α2 est responsable des variations de contractilité de l’aorte, puisque son expression n’est pas modifiée par le canrénoate. Les effets de la diminution de l’expression de la sous-unité α1, influencée par la signalisation du MR, restent à être déterminés. Néanmoins, nos résultats montrent que les modifications d’activité de la Na+/K+-ATPase influencent l’activité des canaux potassiques et que la pompe pourraient être un des éléments primordiaux dans le contrôle de la réactivité vasculaire durant la grossesse. Comme le canrénoate modifie la réactivité vasculaire, nous voulions déterminer ses impacts sur la pression artérielle. Des rates gestantes ont été traitées avec du canrénoate (20 ou 60 mg/kg•jr) et les paramètres hémodynamiques ont été évalués par radiotélémétrie. Aucune modification de la pression artérielle, du rythme cardiaque et de la pression pulsée ne sont mesurées chez les rates recevant le traitement. Toutefois, des augmentations de l’osmolalité, des taux sériques d’aldostérone et de corticostérone ainsi que de l’activité rénine plasmatique sont observées chez les animaux recevant 60 mg/kg•jr. Le canrénoate bloque donc le rétrocontrôle du SRAA. Par contre, les MR ne sont pas les principaux responsables du contrôle de la pression artérielle durant la grossesse. En conclusion, nous avons démontré que le traitement des rates au canrénoate influence la réactivité vasculaire de l’aorte durant la gestation. Cet effet est causé par la modification de l’activité de certains canaux ioniques (VDCC, BKCa et KATP). De plus, le canrénoate renverse l’inhibition de la Na+/K+-ATPase observée durant la gestation. Finalement, les actions locales de cet antagoniste des MR sur les vaisseaux sanguins ne se répercutent pas sur l’effet systémique global et aucune modification de la pression artérielle n’est observée. D’autres études seront toutefois nécessaires pour déterminer les voies de signalisation par lesquelles l’aldostérone module les réponses des canaux ioniques dans les VSMC. / Pregnancy is accompanied by important hormonal and hemodynamic modifications. Among them, the renin-angiotensin-aldosterone system (RAAS) is activated early during pregnancy. Furthermore, this increase of RAAS is accompanied by raises of cardiac output and blood volume as well as paradoxal decreases in blood pressure and peripheral vascular resistance. These suggest that pregnancy induces reorganization of the normal physiological responses to RAAS. In addition, a decreased vascular reactivity to vasoconstrictive agents is observed during pregnancy. Modifications of calcium and potassium channels function would be implicated in this phenomenon. Furthermore, an implication of the Na+/K+-ATPase is suspected, through its influence on the membrane potential of vascular smooth muscle cells (VSMC). Aldosterone, through the presence of mineralocorticoid receptors (MR) in VSMC, could control vascular tone by its genomic and non-genomic effects. With our knowledge at this time, we submit the hypothesis that the increased serum aldosterone levels of pregnancy are responsible for the hemodynamic changes associated with pregnancy and that these effects are caused by the activation of MR. Pregnant rats were treated with potassium canrenoate (20 mg/kg•d), a MR antagonist, during the last week of pregnancy (out of 3 weeks). Vascular reactivity of endothelium-denuded aortic rings was measured. Contractile responses to phenylephrine (PhE) and KCl were studied in the presence of a voltage-dependent calcium channel (VDCC) blocker, nifedipine, as well as calcium-actived (BKCa) and ATP-dependent (KATP) potassium channels activators, NS-1619 and cromakalim respectively. Vascular responses to PhE and KCl were reduced as of the 17th day of gestation. Canrenoate increased the responses to both agonists in the aortas of all the groups. Ionic channel modulators reduced the contractile responses to PhE and KCl. However, canrenoate also modified the responses to the modulators. In the aortas of non pregnant rats, no effect or a decrease of their inhibitory effect were observed while with the pregnant rats we noticed an increased effect. These results suggest that aldosterone could be implicated in the adaptation of vascular responses to pregnancy. On the other hand, VSMC membrane potential could be affected during pregnancy. To verify this hypothesis, Na+/K+-ATPase activity was evaluated since it is implicated in its control. Our results demonstrated that the pump is inhibited as of the 19th day of pregnancy. This effect was reversed by canrenoate. However, since the reversal of the inhibition of the pump was also present in pregnant rats treated with PST 2238, an ouabain antagonist on the Na+/K+-ATPase, and because canrenoate can also act as a partial agonist of the pump, we believe that the decreased activity associated with pregnancy is linked to the inhibition of the Na+/K+-ATPase by cardiotonic steroids rather than the effect of mineralocorticoids. The increased activity of the pump by canrenoate is linked with a decreased of the α1 subunit expression. This suggests that the α2 subunit of the pump would be responsible for the variations of aortic contractility since its expression is not modified by canrénoate. The effects of the diminished expression of the α1 subunit, influenced by the MR signalization, still need to be determined. Nevertheless, our results showed that modifications of Na+/K+-ATPase activity had an impact on the activity of potassium channels and that the pump could be one of the principal elements implicated in the control of vascular reactivity during pregnancy. Since canrenoate modified vascular reactivity, we wanted to determine its impact on blood pressure. Pregnant rats were treated with canrenoate (20 or 60 mg/kg•d) and hemodynamic functions were determined by radiotelemetry. No modification was observed in blood pressure, cardiac output and pulse pressure among the treated rats. However, increases in osmolality, aldosterone and corticosterone levels as well as plasma renin activity were observed in the animal receiving the 60 mg/kg•d canrénoate. Thus, canrenoate blocks the negative feedback of the RAAS. However, MR are not the principal element responsible for the control of blood pressure during pregnancy. In conclusion, we have demonstrated that canrenoate affects aortic vascular reactivity during pregnancy. This effect is perpetuated by modifying the activity of certain ionic channels (VDCC, BKCa and KATP). Furthermore, canrenoate is able to reverse the inhibition of the Na+/K+-ATPase observed during pregnancy. Finally, the local actions of this MR antagonist on blood vessels are not reflected by the global systemic effect and no modification of the blood pressure was observed. Other studies will be necessary to determine the signaling pathways by which aldosterone modulate ionic channel responses in VSMC.

No description available.