Introdução: Em pacientes deprimidos, a mortalidade cardíaca devido à doença cardíaca coronária (CHD) é duas vezes maior do que em pacientes coronarianos nãodeprimidos, possivelmente devido à presença de disfunção endotelial. Pode haver uma associação entre a utilização de drogas antipsicóticas e doenças cardiovasculares, que são associadas à disfunção endotelial vasotônica. Os estudos relacionados aos efeitos das drogas antipsicóticas sobre a função endotelial vascular são praticamente inexistentes. Objetivo: Esse estudo foi delineado para avaliar os mecanismos envolvidos na reatividade vascular dependente do endotélio sob ação da droga antipsicótica atípica clozapina. Materiais e método: As curvas concentraçãoresposta em anéis pré-contraídos com PE (1 ?M), foram registradas na presença e na ausência de inibidores dos inibidores L-NAME e indometacina, os quais foram incubados, isolados e/ou em associação, por 30 minutos. Para estudar a participação dos canais para K+ foram realizados experimentos com os bloqueadores Tetraetilamonio e Cloreto de potássio (Kcl). Para finalizar o protocolo foram realizados experimentos com bloqueadores da guanilato ciclase (ODQ e azul de metileno) e os inibidores dos canais de K+ (apamina, paxilina e glibenclamida). Resultados: A clozapina causou relaxamento dependente do endotélio envolvendo as três vias de relaxamento (GMPc/NO, AMPc/PGI2 e hiperpolarização). Essa observação foi embasada na ineficiência dos bloqueadores, L-NAME, Indometacina e Tetraetilamônio (TEA), respectivamente. O relaxamento dependente do endotélio foi atenuado pela associação L-NAME/Indometacina e abolido em vasos contraídos por cloreto de potássio e incubados com glibenclamida. Conclusão: Estes resultados sugerem que há participação sinérgica, provavelmente através de um mecanismo de cross-talk, dos sistemas cAMP, cGMP e hiperpolarização. Portanto, mais estudos sobre possíveis funções endoteliais relacionadas aos efeitos colaterais dos antipsicóticos cardiovasculares seriam uma direção de pesquisa adequada. / Objective: In depressed patients, cardiac mortality due to coronary heart disease (CHD) is twice as high as in non-depressed coronary patients, possibly due to the presence of endothelial dysfunction. There may be an association between the use of antipsychotic drugs and cardiovascular diseases, which are associated with vasotonic endothelial dysfunction. Studies related to the effects of antipsychotic drugs on vascular endothelial function are practically non-existent. Aims: This study was designed to evaluate the mechanisms involved in endothelium-dependent vascular reactivity under the action of the atypical antipsychotic drug clozapine. Method: As concentration-response curves in pre-contracted rings with PE (1 ?M), they were recorded in the presence and absence of inhibitors of the L-NAME and indomethacin inhibitors, which were incubated, isolated and / or in association, for 30 minutes. To study the participation of the K + channels, experiments were performed with the tetraethylammonium and potassium chloride (Kcl) blockers. In order to obtain the protocol they were performed with guanylase cyclase blockers (ODQ and methylene blue) and K + channel inhibitors (apamina, paxilin and glibenclamide). Results: Clozapine caused endothelium-dependent relaxation as the three relaxation pathways (cGMP / NO, cAMP / PGI2 and hyperpolarization). This observation was based on the inefficiency of the blockers, L-NAME, Indomethacin and Tetraethylammonium (TEA), respectively. Endothelium-dependent relaxation was attenuated by the association LNAME / Indomethacin and abolished in vessels contracted by potassium chloride and incubated with glibenclamide. Conclusion: These results suggest that there is synergistic participation, probably through a cross-talk mechanism, of the cAMP, cGMP and hyperpolarization systems. Therefore, more studies of possible endothelium function related to cardiovascular antipsychotics side effects would be a proper research direction.

Pulmonary arterial hypertension is a rare, chronic disease characterized by progressive remodeling of the pulmonary vasculature. Historically, prognosis has been very poor with relatively low 3-year survival rates. Common symptoms include fatigue and shortness of breath upon exercise, chest pain, and syncope. Patients exhibit increased pressure and resistance in pulmonary arteries due to fibrosis, vessel narrowing, and elevated levels of vasoconstrictive agents; diagnosis is confirmed by right heart catheterization. Reduced blood flow through the pulmonary vasculature not only reduces the amount of oxygenated blood available for the systemic circulation, but increases afterload on the right ventricle and, if left untreated, ultimately causes right ventricular heart failure. In the past, few medications were available to pulmonary arterial hypertension patients. However, recent advancements in our molecular understanding of the disease have led to the development of new therapeutic options that show promise of slowing, or in some cases reversing, disease progression. Currently available treatments have been shown to significantly improve 3-year survival rates and help promote a better quality of life for patients. While an exact molecular or genetic mechanism of disease progression is not yet known, several studies have noted the presence of dysfunctional endothelial cells and an imbalance in molecular modulators of the pulmonary vasculature. Specifically, patients exhibit chronically low levels of vasodilating agents such as prostacyclin and nitric oxide. In addition, there is a heightened vasoconstrictive effect due to elevated endothelin-1 and thromboxane A2. Drugs have been developed to target these signaling pathways and show considerable promise and efficacy for managing pulmonary hypertension in patients. Although these therapeutics have been shown to significantly improve survival rates and symptoms, many have complex and inconvenient administration protocols and a host of adverse side effects. Moreover, many require monitoring or frequent follow up visits due to their off-target effects. Recent innovative advancements in pulmonary arterial hypertension pharmaceuticals hope to deliver safe, efficacious treatment options to patients debilitated by this chronic disease.

Introdução: Em pacientes deprimidos, a mortalidade cardíaca devido à doença cardíaca coronária (CHD) é duas vezes maior do que em pacientes coronarianos nãodeprimidos, possivelmente devido à presença de disfunção endotelial. Pode haver uma associação entre a utilização de drogas antipsicóticas e doenças cardiovasculares, que são associadas à disfunção endotelial vasotônica. Os estudos relacionados aos efeitos das drogas antipsicóticas sobre a função endotelial vascular são praticamente inexistentes. Objetivo: Esse estudo foi delineado para avaliar os mecanismos envolvidos na reatividade vascular dependente do endotélio sob ação da droga antipsicótica atípica clozapina. Materiais e método: As curvas concentraçãoresposta em anéis pré-contraídos com PE (1 ?M), foram registradas na presença e na ausência de inibidores dos inibidores L-NAME e indometacina, os quais foram incubados, isolados e/ou em associação, por 30 minutos. Para estudar a participação dos canais para K+ foram realizados experimentos com os bloqueadores Tetraetilamonio e Cloreto de potássio (Kcl). Para finalizar o protocolo foram realizados experimentos com bloqueadores da guanilato ciclase (ODQ e azul de metileno) e os inibidores dos canais de K+ (apamina, paxilina e glibenclamida). Resultados: A clozapina causou relaxamento dependente do endotélio envolvendo as três vias de relaxamento (GMPc/NO, AMPc/PGI2 e hiperpolarização). Essa observação foi embasada na ineficiência dos bloqueadores, L-NAME, Indometacina e Tetraetilamônio (TEA), respectivamente. O relaxamento dependente do endotélio foi atenuado pela associação L-NAME/Indometacina e abolido em vasos contraídos por cloreto de potássio e incubados com glibenclamida. Conclusão: Estes resultados sugerem que há participação sinérgica, provavelmente através de um mecanismo de cross-talk, dos sistemas cAMP, cGMP e hiperpolarização. Portanto, mais estudos sobre possíveis funções endoteliais relacionadas aos efeitos colaterais dos antipsicóticos cardiovasculares seriam uma direção de pesquisa adequada. / Objective: In depressed patients, cardiac mortality due to coronary heart disease (CHD) is twice as high as in non-depressed coronary patients, possibly due to the presence of endothelial dysfunction. There may be an association between the use of antipsychotic drugs and cardiovascular diseases, which are associated with vasotonic endothelial dysfunction. Studies related to the effects of antipsychotic drugs on vascular endothelial function are practically non-existent. Aims: This study was designed to evaluate the mechanisms involved in endothelium-dependent vascular reactivity under the action of the atypical antipsychotic drug clozapine. Method: As concentration-response curves in pre-contracted rings with PE (1 ?M), they were recorded in the presence and absence of inhibitors of the L-NAME and indomethacin inhibitors, which were incubated, isolated and / or in association, for 30 minutes. To study the participation of the K + channels, experiments were performed with the tetraethylammonium and potassium chloride (Kcl) blockers. In order to obtain the protocol they were performed with guanylase cyclase blockers (ODQ and methylene blue) and K + channel inhibitors (apamina, paxilin and glibenclamide). Results: Clozapine caused endothelium-dependent relaxation as the three relaxation pathways (cGMP / NO, cAMP / PGI2 and hyperpolarization). This observation was based on the inefficiency of the blockers, L-NAME, Indomethacin and Tetraethylammonium (TEA), respectively. Endothelium-dependent relaxation was attenuated by the association LNAME / Indomethacin and abolished in vessels contracted by potassium chloride and incubated with glibenclamide. Conclusion: These results suggest that there is synergistic participation, probably through a cross-talk mechanism, of the cAMP, cGMP and hyperpolarization systems. Therefore, more studies of possible endothelium function related to cardiovascular antipsychotics side effects would be a proper research direction.

Object: The aim of the study was to evaluate the early changes in pituitary hormone levels after severe traumatic brain injury (sTBI) and compare hormone levels to basic neuro-intensive care data, a systematic scoring of the CT-findings and to evaluate whether hormone changes are related to outcome. Methods: Prospective study, including consecutive patients, 15-70 years, with sTBI, Glasgow Coma Scale (GCS) score <= 8, initial cerebral perfusion pressure > 10 mm Hg, and arrival to our level one trauma university hospital within 24 hours after head trauma (n = 48). Serum samples were collected in the morning (08-10 am) day 1 and day 4 after sTBI for analysis of cortisol, growth hormone (GH), prolactin, insulin-like growth factor 1 (IGF-1), thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), follicular stimulating hormone (FSH), luteinizing hormone (LH), testosterone and sex hormone-binding globulin (SHBG) (men). Serum for cortisol and GH was also obtained in the evening (17-19 pm) at day 1 and day 4. The first CT of the brain was classified according to Marshall. Independent staff evaluated outcome at 3 months using GOS-E. Results: Profound changes were found for most pituitary-dependent hormones in the acute phase after sTBI, i.e. low levels of thyroid hormones, strong suppression of the pituitary-gonadal axis and increased levels of prolactin. The main findings of this study were: 1) A large proportion (54% day 1 and 70% day 4) of the patients showed morning s-cortisol levels below the proposed cut-off levels for critical illness related corticosteroid insufficiency (CIRCI), i.e. < 276 nmol/L (= 10 ug/dL), 2) Low s-cortisol was not associated with higher mortality or worse outcome at 3 months, 3) There was a significant association between early (day 1) and strong suppression of the pituitary-gonadal axis and improved survival and favorable functional outcome 3 months after sTBI, 4) Significantly lower levels of fT3 and TSH at day 4 in patients with a poor outcome at 3 months. 5) A higher Marshall CT score was associated with higher day 1 LH/FSH-and lower day 4 TSH levels 6) In general no significant correlation between GCS, ICP or CPP and hormone levels were detected. Only ICPmax and LH day 1 in men was significantly correlated. Conclusion: Profound dynamic changes in hormone levels are found in the acute phase of sTBI. This is consistent with previous findings in different groups of critically ill patients, most of which are likely to be attributed to physiological adaptation to acute illness. Low cortisol levels were a common finding, and not associated with unfavorable outcome. A retained ability to a dynamic hormonal response, i.e. fast and strong suppression of the pituitary-gonadal axis (day 1) and ability to restore activity in the pituitary-thyroid axis (day 4) was associated with less severe injury according to CT-findings and favorable outcome.

No / There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.

Neste estudo foram avaliados os efeitos das serinoproteinases PA-BJ e giroxina, isoladas dos venenos das serpentes Bothrops jararaca e Crotalus durissus terríficus, respectivamente, sobre células endoteliais (CEs) em cultura. Os resultados obtidos demonstraram que essas toxinas, nas concentrações utilizadas, não afetaram a viabilidade e a integridade das CEs. Por outro lado, induziram a liberação de PGI2, que foi significativamente reduzida por inibidores não seletivos e seletivos das ciclooxigenases -1 e -2 (COX-1 e -2), mas não afetaram a expressão protéica constitutiva das mesmas. Adicionalmente, foi demonstrado que o antagonista de receptores PAR-1, o SCH 79797, não alterou a liberação de PGI2, induzida pelas toxinas. Em conclusão, essas toxinas, em concentrações não citotóxicas, induziram a liberação de PGI2 a partir de CEs, de modo dependente da ativação das COX-1 e -2. Por outro lado, o receptor PAR-1 não parece ser importante para este efeito, nessas células. / In this study, the effects of PA-BJ and gyroxin, isolated from Bothrops jararaca and Crotalus durissus terrificus snake venoms, respectively, on endothelial cells in culture were investigated. Results showed that neither PA-BJ nor gyroxin affected the integrity of monolayers nor modified ECs viability in the periods of incubation tested. In contrast, these serine proteinases increased the release of prostacyclin from ECs. This effect was inhibited by both non-selective and selective COX-1 and COX-2 inhibitors, but these toxins did not affect the protein expression of COX-1 and -2. Inhibition of the catalytic activity of PA-BJ and gyroxin or pre-incubation of ECs with PAR-1 antagonist did not abrogate the ability of these toxins to induce PGI2 release. These findings demonstrate that these serine proteinases are able to stimulate production of prostacyclin by ECs by a mechanism dependent on stimulation of COX-1 and COX-2 enzyme activity. Moreover, neither enzyme activity of both serine proteinases nor the receptor PAR-1 contribute for this effect on endothelium.

Severe Traumatic Brain Injury (sTBI) is a major cause of mortality and morbidity. At the Department of Neurosurgery Umeå University Hospital subjects with sTBI are treated with an intracranial pressure (ICP) guided therapy based on physiological principles, aiming to optimise the microcirculation of the brain so avoiding secondary brain injuries. The investigations in this thesis are unique in the sense that all patients with sTBI were treated according to the guidelines of an ICP targeted therapy based on the “Lund concept”. As the treatment is based on normalisation of the ICP, the accuracy and reliability of the measuring device is of outmost importance. Therefore the accuracy, drift, and complications related to the measuring device was prospectively studied (n=128). The drift was 0,9 ± 0,2 mmHg during a mean of 7,2 ± 0,4 days and the accuracy high. No clinical significant complications were noted. In 1997 uni- or bilateral decompressive hemi-craniectomy (DC) was introduced into the treatment guidelines. The effect of DC on the ICP and outcome was retrospectively analysed for subjects with sTBI treated 1998-2001. In the subjects who underwent DC the ICP was 36,4 mmHg immediately before and 12,6 mmHg immediately after the DC. The ICP then levelled out at just above 20 mmHg. The ICP was significant lower during the 72 hours following DC. The outcome did not differ between subjects who had undergone DC or not. Subclinical electroencephalographic seizures and status epilepticus have been reported to be common in subjects treated for traumatic brain injury (TBI). This can negatively influence the outcome giving rise to secondary brain injuries. The occurrence of seizures in subjects treated for TBI using continuous EEG monitoring was therefore prospectively studied. During 7334 hours of EEG recording in 47 patients no electroencephalographic seizures were observed. Theoretically, and based on animal studies, prostacyclin (PGI2) can improve the microcirculation of the brain, decreasing the risk for secondary ischaemic brain injury. PGI2 was introduced to the treatment in a prospective randomised double blinded study (epoprostenol 0,5 ng/kg/min). The effect of PGI1 pkt was analysed using the lactate/pyruvate ratio (L/P) measured by cerebral microdialysis in order to study the energy metabolism in the brain. The outcome was measured as Glasgow Outcome Scale (GOS) at 3 months follow-up. Forty-eight subjects were included. The L/P was pathological high during the first day, thereafter decreasing. There was no significant difference in L/P or outcome between the treated and non-treated group. At 3 months the mortality was 12,5% (95,8% was discharged alive from the ICU), and favourable outcome (GOS 4-5) was 52%. In the same study the brain injury biomarkers S-100B and NSE were followed twice a day for five days to evaluate brain injury and investigate the possible use of these biomarkers for outcome prediction. Initially the biomarkers were elevated to pathological levels which decreased over time. The biomarkers were significant elevated in subjects with Glasgow Coma Scale 3 (GCS) and GOS 1 compared with subjects with GCS 4-8 and GOS 2–5, respectively. A correlation to outcome was found but this correlation could not be used to predict clinical outcome. It is concluded that the ICP measurements are valid and the treatment protocol is a safe and solid protocol, yielding among the best reported results in the world, in regard to favourable outcome as well as in regard to mortality. Epoprostenol in the given dose was not shown to have any effects on the microdialysis parameters nor the clinical outcome. In sTBI L/P and brain injury biomarkers can not be used to predict the final outcome.

Cell membrane receptors help to mediate communication between the cell and its environment. The largest group of these membrane receptors belong to the family of G protein-coupled receptors (GPCRs). GPCRs contain seven transmembrane (TM) helices and signal predominantly through heterotrimeric G proteins in response to diverse extracellular stimuli. Previously, three levels of amino acid conservation were proposed to understand the structure and function of a GPCR. This includes “signature” amino acids, “group –conserved” amino acids and amino acids conserved only within a specific subfamily. The group-conserved residues in class A GPCR family involve amino acid conservation of up to 99% when considered as a group of small and weakly polar residues (Ala, Gly, Ser, Cys and Thr). These group-conserved residues have been proposed as key determinants in helix-helix interactions. Therefore, I selected these residues for structure-function analysis in the amine and the prostanoid receptor sub-families of class A GPCRs. Molecular and biochemical assays clearly demonstrate the importance of group-conserved residues in β2-adrenergic receptor and thromboxane A2 receptor (TP) structure and function. These studies led to the identification of a non-synonymous single nucleotide polymorphic variant (nsSNP) A160T in TP to be a constitutively active mutant (CAM). Further, the TP-CAM was used as a pharmacological tool that enabled classification of well-known TP-blockers, into neutral antagonists and inverse agonists. The role of TP-A160T in prostanoid receptors, TP- Prostacyclin receptor (IP) heterodimerization and signaling was investigated. Activation of a GPCR ultimately leads to structural changes in its intracellular loops (ICLs), which in turn activates G-protein. TP activates its cognate G protein (Gαq), while IP mediates signaling, through Gαs. Using TP-IP chimeric receptors, molecular modelling, and site directed mutagenesis studies I determined the specific ICL regions required for G protein coupling in TP and IP. Significant challenges exist in expressing and purifying GPCR-CAMs in amounts required to pursue biophysical studies. Using tetracycline inducible HEK293S system, A160T was expressed at high-levels and CD spectropolarimetry studies were successfully pursued on the purified A160T. The CD spectra showed that the loss of thermal stability of the A160T mutant is due to the subtle changes in the secondary structure of the A160T protein. These studies involving molecular, biochemical and pharmacological approaches provide novel insights into the structure and function of prostanoid receptors TP and IP.

Neste estudo foram avaliados os efeitos das serinoproteinases PA-BJ e giroxina, isoladas dos venenos das serpentes Bothrops jararaca e Crotalus durissus terríficus, respectivamente, sobre células endoteliais (CEs) em cultura. Os resultados obtidos demonstraram que essas toxinas, nas concentrações utilizadas, não afetaram a viabilidade e a integridade das CEs. Por outro lado, induziram a liberação de PGI2, que foi significativamente reduzida por inibidores não seletivos e seletivos das ciclooxigenases -1 e -2 (COX-1 e -2), mas não afetaram a expressão protéica constitutiva das mesmas. Adicionalmente, foi demonstrado que o antagonista de receptores PAR-1, o SCH 79797, não alterou a liberação de PGI2, induzida pelas toxinas. Em conclusão, essas toxinas, em concentrações não citotóxicas, induziram a liberação de PGI2 a partir de CEs, de modo dependente da ativação das COX-1 e -2. Por outro lado, o receptor PAR-1 não parece ser importante para este efeito, nessas células. / In this study, the effects of PA-BJ and gyroxin, isolated from Bothrops jararaca and Crotalus durissus terrificus snake venoms, respectively, on endothelial cells in culture were investigated. Results showed that neither PA-BJ nor gyroxin affected the integrity of monolayers nor modified ECs viability in the periods of incubation tested. In contrast, these serine proteinases increased the release of prostacyclin from ECs. This effect was inhibited by both non-selective and selective COX-1 and COX-2 inhibitors, but these toxins did not affect the protein expression of COX-1 and -2. Inhibition of the catalytic activity of PA-BJ and gyroxin or pre-incubation of ECs with PAR-1 antagonist did not abrogate the ability of these toxins to induce PGI2 release. These findings demonstrate that these serine proteinases are able to stimulate production of prostacyclin by ECs by a mechanism dependent on stimulation of COX-1 and COX-2 enzyme activity. Moreover, neither enzyme activity of both serine proteinases nor the receptor PAR-1 contribute for this effect on endothelium.

No / Prostanoids modulate the activity of human pregnant myometrium and their functional role can be appreciated through characterisation of prostanoid receptors and tissue concentration of prostanoids. We have applied a lipidomic approach to elucidate the profile of prostanoids in human non-labouring and labouring myometrium. We have identified a total of nineteen prostanoids including prostacyclin, thromboxanes, prostaglandins and dihydro-prostaglandins. Prostacyclin was the predominant prostanoid in both non-labouring and labouring myometria, with PGD2 and PGF2¿ being the second most abundant. Although the total amount of prostanoids was increased in the labouring tissue, PGE2 and 13,14-dihydro-15-keto-PGE2 were the only prostanoids to increase significantly at early and late labour (p¿0.001). Our data suggest that PGF2¿ plays an important role in parturition, whilst the increase in PGE2 could occur to facilitate cervical dilation and relaxation of the lower myometrium during labour. Although the elevation in TXA2 was less marked than expected, in terms of translation to function even a relatively small increase in the level of this potent spasmogen may have significant effects.