Motivation: Cardiovascular dysfunction and hypertension are some of the leading causes of morbidity and mortality in the African population. According to the World Health Organisation the increases in these diseases are escalating in developing countries. Apart from the contributory role of genetics towards the incidence of hypertension, evidence regarding lifestyle as a determinant or marker of cardiovascular diseases in this group is not well known. The interaction of psychological and physiological mechanisms can contribute towards a broader scope of behavioural physiology in the higher prevalence of hypertension in Africans. Objectives: The main objective of the research in this thesis was to compare specific coping mechanisms of Africans with regard to cardiovascular dysfunction. Methodology: Manuscripts presented in Chapters 3, 4, and 5 made use of the cross-sectional comparative epidemiological "Transition and Health during Urbanisation in South Africa" (THUSA) project. The subjects included apparently healthy African men and women, which were recruited as a convenience sample from the North West Province, South Africa. Anthropometric measurements were taken and demographic questionnaires completed. An adapted Setswana COPE questionnaire was used to classify men and women as predominantly active (AC) or passive (PC) in coping style. Subjects were further subdivided into rural and urban groups (Manuscript Two), as well as younger (≤ 40) and older (≥ 45) age groups (Manuscript Three). The General Health Questionnaire (GHQ) was used to measure subjective perception of health in all three manuscripts. Blood pressure was recorded continuously before and during application of the handgrip test using the Finapres apparatus. Subjects were classified as normotensive and hypertensive after blood pressure measurement by the Finapres and the Riva-Rocci/Korotkoff method. The emphasis in this study was on the cardiovascular reactivity values. Fasting, resting serum renin activity, cortisol, prolactin, testosterone, high density lipoprotein, triglycerides, glucose and plasma fibrinogen values were correlated with cardiovascular and psychological variables. Significant differences between variables were determined by means of variance analyses (Manuscript One and Two adjusted for age; Manuscripts One, Two and Three adjusted for resting cardiovascular data). A logistic regression analysis was performed to determine the most significant determinants of urbanisation. All THUSA subjects and parents of under-aged adolescents gave informed consent and the study - was approved by the Ethics Committee of the Potchefstroom University for Christian Higher Education. The reader is referred to the abstracts at the beginning of each separate manuscript in Chapters 3 - 5 for a description of the subjects, study design and analytical methods used in each paper. Results and conclusions of the individual manuscripts: Results from the THUSA study showed that PC men and women reported more symptoms typical of an abnormal psychological and physiological profile than AC men and women. The PC men, compared to AC men, exhibited a larger vascular reactivity response as well as larger plasma renin activity. In contrast, the AC women showed a larger non-significant vascular reactivity response than PC women. All subjects though reacted with increased vascular reactivity on the stressor. Men with a PC strategy showed enhanced vascular reactivity, a perception of poorer health and larger stressor plasma renin activity. PC women reported more depressive symptoms and younger PC women indicated a higher prevalence of hypertension than younger AC women. As a follow-up on the first manuscript, the aim was focused mainly on including the environmental effect, namely urbanisation, as possible explanatory factor for the atypical physiological AC women’s' coping style. The rural AC subjects indicated more typical active coping central cardiac responses than rural PC subjects whereas urbanised AC and PC subjects indicated greater peripheral responses and hypertension prevalence rates. In addition, the urbanised AC men and women and PC women as opposed to their rural counterparts indicated symptoms more of a distress situation with increased values of prolactin and decreased values of testosterone. This was also accompanied by a perception of poorer health in women. Results of the AC style suggests that the typical physiological AC stimulation pattern of urbanised subjects and especially the women is dissociated from the "normal" physiological AC reaction and is now exhibited as a typical PC physiological stimulation pattern. The greater vascular reactivity, hypertension prevalence, perception of poorer health and endocrine distressed profile are associated with a PC and dissociated physiological AC style in an urban context in African men and women. No differences with regard to resting blood pressure or endocrine values were obtained when the AC and PC urbanised groups were compared. Africans develop cardiovascular dysfunction/hypertension during chronic stress or urbanisation. This implies a dissociation/habituation of physiological systems of African men and women despite having an active coping strategy. Active coping is, therefore, not necessarily "successful". Results of the first two manuscripts direct further investigation concerning the effects of ageing and urbanisation on the development of cardiovascular dysfunction and metabolic syndrome indicators in gender groups. The second manuscript showed that all rural AC subjects exhibit a more typical active coping central cardiac response and that rural PC and all urbanised subjects (AC and PC) exhibit enhanced peripheral vascular responses on the - handgrip test. Where peripheral vascular responses were more expected from older individuals in Manuscript Three, the occurrence of this pattern is strengthened in the younger subjects. The greater fibrinogen values in all younger urbanised women (AC and PC) compared to rural women further strengthen the risk for the development of cardiovascular disease. Increased vascular reactivity, abdominal obesity and increased levels of triglycerides as well as perception of poorer health were apparent in the urbanised AC women, PC men and women in comparison to their rural counterparts. The typical physiological AC stimulation pattern of urbanised women is dissociated from the "normal" physiological AC responses and is now exhibited as a typical PC physiological stimulation pattern. A typical PC style in older urbanised subjects is implicated in the greater hypertension prevalence. To conclude, it seems as if young urbanised Africans, and especially women, exhibit an AC style behaviourally with a dissociated physiological AC reaction pattern. Physiologically these women resemble a typical PC physiological cardiovascular and endocrine profile. This typical PC cardiovascular stimulation pattern is strengthened by a distressed endocrine profile, significant metabolic syndrome indicators and a 'perception of poorer health. Older PC style subjects also presented a greater hypertension prevalence. In this study it seems that cardiovascular changes that appear at a younger age might be influenced by other factors including urbanisation as a lifestyle factor as well as specific coping styles. Finally, a careful suggestion is made that specific coping mechanisms could be seen as a possible risk marker in the development of the metabolic syndrome. / Thesis (Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Motivation: Cardiovascular dysfunction and hypertension are some of the leading causes of morbidity and mortality in the African population. According to the World Health Organisation the increases in these diseases are escalating in developing countries. Apart from the contributory role of genetics towards the incidence of hypertension, evidence regarding lifestyle as a determinant or marker of cardiovascular diseases in this group is not well known. The interaction of psychological and physiological mechanisms can contribute towards a broader scope of behavioural physiology in the higher prevalence of hypertension in Africans. Objectives: The main objective of the research in this thesis was to compare specific coping mechanisms of Africans with regard to cardiovascular dysfunction. Methodology: Manuscripts presented in Chapters 3, 4, and 5 made use of the cross-sectional comparative epidemiological "Transition and Health during Urbanisation in South Africa" (THUSA) project. The subjects included apparently healthy African men and women, which were recruited as a convenience sample from the North West Province, South Africa. Anthropometric measurements were taken and demographic questionnaires completed. An adapted Setswana COPE questionnaire was used to classify men and women as predominantly active (AC) or passive (PC) in coping style. Subjects were further subdivided into rural and urban groups (Manuscript Two), as well as younger (≤ 40) and older (≥ 45) age groups (Manuscript Three). The General Health Questionnaire (GHQ) was used to measure subjective perception of health in all three manuscripts. Blood pressure was recorded continuously before and during application of the handgrip test using the Finapres apparatus. Subjects were classified as normotensive and hypertensive after blood pressure measurement by the Finapres and the Riva-Rocci/Korotkoff method. The emphasis in this study was on the cardiovascular reactivity values. Fasting, resting serum renin activity, cortisol, prolactin, testosterone, high density lipoprotein, triglycerides, glucose and plasma fibrinogen values were correlated with cardiovascular and psychological variables. Significant differences between variables were determined by means of variance analyses (Manuscript One and Two adjusted for age; Manuscripts One, Two and Three adjusted for resting cardiovascular data). A logistic regression analysis was performed to determine the most significant determinants of urbanisation. All THUSA subjects and parents of under-aged adolescents gave informed consent and the study - was approved by the Ethics Committee of the Potchefstroom University for Christian Higher Education. The reader is referred to the abstracts at the beginning of each separate manuscript in Chapters 3 - 5 for a description of the subjects, study design and analytical methods used in each paper. Results and conclusions of the individual manuscripts: Results from the THUSA study showed that PC men and women reported more symptoms typical of an abnormal psychological and physiological profile than AC men and women. The PC men, compared to AC men, exhibited a larger vascular reactivity response as well as larger plasma renin activity. In contrast, the AC women showed a larger non-significant vascular reactivity response than PC women. All subjects though reacted with increased vascular reactivity on the stressor. Men with a PC strategy showed enhanced vascular reactivity, a perception of poorer health and larger stressor plasma renin activity. PC women reported more depressive symptoms and younger PC women indicated a higher prevalence of hypertension than younger AC women. As a follow-up on the first manuscript, the aim was focused mainly on including the environmental effect, namely urbanisation, as possible explanatory factor for the atypical physiological AC women’s' coping style. The rural AC subjects indicated more typical active coping central cardiac responses than rural PC subjects whereas urbanised AC and PC subjects indicated greater peripheral responses and hypertension prevalence rates. In addition, the urbanised AC men and women and PC women as opposed to their rural counterparts indicated symptoms more of a distress situation with increased values of prolactin and decreased values of testosterone. This was also accompanied by a perception of poorer health in women. Results of the AC style suggests that the typical physiological AC stimulation pattern of urbanised subjects and especially the women is dissociated from the "normal" physiological AC reaction and is now exhibited as a typical PC physiological stimulation pattern. The greater vascular reactivity, hypertension prevalence, perception of poorer health and endocrine distressed profile are associated with a PC and dissociated physiological AC style in an urban context in African men and women. No differences with regard to resting blood pressure or endocrine values were obtained when the AC and PC urbanised groups were compared. Africans develop cardiovascular dysfunction/hypertension during chronic stress or urbanisation. This implies a dissociation/habituation of physiological systems of African men and women despite having an active coping strategy. Active coping is, therefore, not necessarily "successful". Results of the first two manuscripts direct further investigation concerning the effects of ageing and urbanisation on the development of cardiovascular dysfunction and metabolic syndrome indicators in gender groups. The second manuscript showed that all rural AC subjects exhibit a more typical active coping central cardiac response and that rural PC and all urbanised subjects (AC and PC) exhibit enhanced peripheral vascular responses on the - handgrip test. Where peripheral vascular responses were more expected from older individuals in Manuscript Three, the occurrence of this pattern is strengthened in the younger subjects. The greater fibrinogen values in all younger urbanised women (AC and PC) compared to rural women further strengthen the risk for the development of cardiovascular disease. Increased vascular reactivity, abdominal obesity and increased levels of triglycerides as well as perception of poorer health were apparent in the urbanised AC women, PC men and women in comparison to their rural counterparts. The typical physiological AC stimulation pattern of urbanised women is dissociated from the "normal" physiological AC responses and is now exhibited as a typical PC physiological stimulation pattern. A typical PC style in older urbanised subjects is implicated in the greater hypertension prevalence. To conclude, it seems as if young urbanised Africans, and especially women, exhibit an AC style behaviourally with a dissociated physiological AC reaction pattern. Physiologically these women resemble a typical PC physiological cardiovascular and endocrine profile. This typical PC cardiovascular stimulation pattern is strengthened by a distressed endocrine profile, significant metabolic syndrome indicators and a 'perception of poorer health. Older PC style subjects also presented a greater hypertension prevalence. In this study it seems that cardiovascular changes that appear at a younger age might be influenced by other factors including urbanisation as a lifestyle factor as well as specific coping styles. Finally, a careful suggestion is made that specific coping mechanisms could be seen as a possible risk marker in the development of the metabolic syndrome. / Thesis (Ph.D. (Physiology))--North-West University, Potchefstroom Campus, 2005.

Purpose To investigate the impact of cigarrete smoking in a group of otherwise healthy young individuals on: 1) Retinal blood flow using Doppler based SD-OCT, 2) Retinal vascular reactivity using a gas sequencer to provoke hypercapnia via constant changes in PETCO2 (end-tidal partial pressure of CO2) and in PETO2 (end-tidal partial pressure of O2). Methods An automated gas flow controller was used to achieve normoxic hypercapnia in ten non-smokers (mean age 28.9 yrs, SD 4.58) and nine smokers (mean age 27.55 yrs, SD 4.77). Retinal blood flow measurements were obtained using Doppler OCT and cannon laser blood flowmeter (CLBF) during baseline, normoxic hypercapnia (15% increase in PETCO2 relative to homeostatic baseline) and post-hypercapnia in both the groups. Exhaled carbon monoxide level was measured in all subjects. Results In non-smokers, retinal arteriolar diameter, blood velocity and flow increased by +4.1% (SD 2.8, p<0.0001), +16.7% (SD 14.6, p=0.0004) and +29.6% (SD 12.5, p<0.0001) respectively, during normoxic hypercapnia; Similarly, the venous area, venous velocity and total retinal blood flow increased by 7% (SD 8.6, p=0.0418), 18.1% (SD 20.8, p=0.0068) and 26% (SD 22.9, p<0.0001) respectively. In smokers, normoxic hypercapnia resulted in a significant increase in velocity by 12.0% (SD 6.2, p=0.0019) and flow by 14.6% (SD 9.5, p=0.0029); though arteriolar diameter increased by 1.7% (SD 1.7, p=0.2616), the result was not statistically significant. Total retinal blood flow increased significantly by 19.3% (SD 18.4, p=0.002) in response to normoxic hypercapnia. However, there was no significant difference in venous area (p=0.3322) and venous velocity measurements (p=0.1185) during hypercapnia compared to baseline and recovery. Comparing smokers and non-smokers, only the percentage change in arteriolar diameter (p=0.0379) and flow (p=0.0101) was significantly different among the groups. Group mean PETCO2 was increased by 15.9% in the non-smoking group and by 15.7% in the smoking group, with a concomitant increase in PETO2 by approximately 1.5 to 2% in both groups. There was no significant difference in baseline PETCO2 level between smokers and non-smokers. Conclusions Retinal vascular reactivity in response to normoxic hypercapnia is significantly reduced in young healthy individuals who smoke compared to non-smokers. Further studies are needed to elucidate the exact reason behind the impaired retinal autoregulation to provocative stimuli in smokers.

Orientador: Luiz Claudio Di Stasi / Banca: Marcelo Fabio Gouveia Nogueira / Banca: Alessandra Gambero / Resumo: A Doença Inflamatória Intestinal (DII) engloba, fundamentalmente, duas doenças distintas: a Doença de Crohn (DC) e a Retocolite Ulcerativa (CU), ambas caracterizadas por uma inflamação crônica do intestino, com períodos de exacerbação seguidos de intervalos prolongados de remissão dos sintomas. Apesar da DII ser objeto de pesquisa há várias décadas, a sua etiologia ainda é desconhecida e a principal limitação no entendimento dos mecanismos fisiopatológicos desta doença é a disponibilidade de modelos experimentais adequados que mimetizem o caráter crônico e de recidiva da DII em humanos e que possam ser de baixo custo, reprodutível, fácil de induzir e que apresente características clínicas e histopatológicas, respostas terapêuticas e mediadores inflamatórios similares ao que ocorre com a doença em humanos. Dentre os vários modelos experimentais disponíveis, o modelo de colite induzida por ácido trinitrobenzenosulfônico (TNBS) em ratos tem sido considerado o mais adequado para a avaliação de novos fármacos, assim como aquele que melhor mimetiza esta doença em humanos. Assim sendo, a caracterização do papel de diferentes mediadores do processo inflamatório intestinal neste modelo permitiria a determinação de novos alvos terapêuticos, assim como geraria informações importantes da fisiopatologia desta doença. Neste sentido, o presente projeto teve como objetivo determinar a participação da HSP70, Heparanase e HPRT, mediadores do processo inflamatório intestinal em humanos, na fase aguda do processo inflamatório intestinal induzido TNBS em ratos, assim como estudar os efeitos de fármacos das três principais classes farmacológicas usadas no tratamento da DII em humanos, os aminossalicilatos (sulfassalazina), os glicocorticóides (prednisolona) e os imunomoduladores (azatioprina) sobre esses mediadores. Este estudo demonstrou que HSP70, Heparanase... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The idiopatic inflammatory bowel diseases comprise two types of chronic intestinal disorders: Crohn's disease and ulcerative colitis that are characterized by a chronic inflammation of the intestine, with periods of remission and reactivation of the inflammatory process. Although IBD is the subject of research for several decades, its etiology remains unknown, and the major limitation to understanding the IBD pathophysiology is the availability of experimental models that mimic the chronic and relapse of human IBD. Is still important that experimental models can be inexpensive, reproducible, easy to induce and present clinical and histopathological features, therapeutic responses and inflammatory mediators similar to what occurs in humans. Among experimental models available, the model of colitis induced by trinitrobenzenesulphonic acid (TNBS) in rats has been considered the most suitable for the evaluation of new drugs, as well as the one that best mimics the disease in humans. Therefore, the involvement of different IBD mediators in this experimental model would allow the determination of new therapeutic targets, as well as generate important information on the pathophysiology of this disease. In light of this, the aim of present study was to determine the participation of HSP70, Heparanase and HPRT, mediators of intestinal inflammatory process in humans, in acute phase of inflammatory process induced by TNBS in rats, as well as, to study the effects of drugs of the three main classes used in the treatment of human IBD, i.e., aminosalicylates (sulphasalazine), glucocorticoids (prednisolone) and immunomodulators (azathioprine) on these mediators. This study showed that HSP70, Heparanase and HPRT participate as mediators of intestinal inflammation induced by TNBS since these mediators are increased in colitic animals when compared to healthy animals... (Complete abstract click electronic access below) / Mestre

Systemic hypoxia frequently occurs in patients with cardiopulmonary diseases. Maintenance of vascular reactivity and endothelial viability is essential to preserving oxygen delivery in these patients. The role of matrix metalloproteinase-2 (MMP-2) and heme oxygenase-2 (HO-2) in the vascular response to hypoxia were investigated. In the first part of the thesis, the role of MMP-2 in regulating systemic arterial contraction after prolonged hypoxia was investigated. MMP-2 inhibition with cyclic peptide CTTHWGFTLC (CTT) reduced phenylephrine (PE)-induced contraction in aortae and mesenteric arteries harvested from rats exposed to hypoxia for 7 d. Responses to PE were reduced in MMP-2-/- mice exposed to hypoxia for 7 d compared to wild-type controls. CTT reduced contraction induced by big endothelin-1 (big ET-1) in aortae harvested from rats exposed to hypoxia. Increased contraction to big ET-1 after hypoxia was observed in wild-type controls, but not MMP-2-/- mice. Rat aortic MMP-2 and MT1-MMP protein levels and MMP activity were increased after 7 d of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. These results suggest that hypoxic induction of MMP-2 activity potentiates contraction in systemic conduit and resistance arteries through proteolytic activation of big ET-1. The second part of the thesis investigated oxygen regulation of HO-2 protein and whether it plays a role in preserving endothelial cell viability during hypoxia. HO-2, but not HO-1, protein level was maintained during hypoxia in human endothelial cells through enhanced translation of HO-2 transcripts. Inhibition of HO-2 expression increased the production of reactive oxygen species, decreased mitochondrial membrane potential, and enhanced apoptotic cell death and activated caspases during hypoxia, but not during normoxia. These data indicate that HO-2 is translationally regulated and important in maintaining endothelial viability and function during hypoxia. In summary, the thesis demonstrates the importance of MMP-2 and HO-2 in preserving vascular function during prolonged systemic hypoxia. These enzymatic pathways may, therefore, represent novel therapeutic targets that may be exploited to ameliorate the effects of hypoxia in patients with cardiopulmonary disease.

Avaliou-se a participação do óxido nítrico (NO), do fator hiperpolarizante derivado do endotélio (EDHF), dos produtos da ciclooxigenase (COX), das espécies reativas de oxigênio (EROs) e o efeito do tratamento com metformina (Met) nas alterações vasculares em ratos com obesidade induzida por glutamato monossódico (MSG). O tratamento com Met corrigiu alterações metabólicas em ratos MSG, reduzindo o acúmulo de gordura visceral, corrigindo a resistência à insulina, a hiperinsulinemia e a dislipidemia. Ratos MSG apresentaram aumentada resposta contrátil e diminuída sensibilidade à Ach, associadas a alterações na via do NO, do EDHF, dos produtos da COX e das EROs. Ratos MSG com 16 semamas apresentaram hiperresponsividade ao nitroprussiato de sódio, que foi mantida nos ratos tratados com Met. A Met corrige as alterações da resposta vascular atuando sobre o NO e o EDHF, reduzindo a geração de EROs e interferindo na resposta do músculo liso vascular, mantendo a hiperresponsividade ao NO. / The role of the nitric oxide (NO), the endothelium derived hyperpolarizing factor (EDHF), the ciclooxygenase (COX) products and the reactive oxygen species (ROS), as well as the effect of metformin (Met) treatment on the vascular alterations in rat model of obesity induced by monosodium glutamate (MSG) were evaluated. Met treatment corrected metabolic alterations in MSG, reducing fat accumulation, correcting dyslipidemia, insulin resistance and hyperinsulinemia. MSG rats had an increased response to norepinephrine and decreased sensitivity to acetilcholine, which were associated with alterations in NO, COX products and ROS. Sixteen-week-old MSG rats presented hyperresponsiveness to sodium nitroprusside, which was preserved in Met-treated group. Met corrects the alterations of the vascular reactivity acting on NO and EDHF, and decreasing the ROS generation, besides its effect on the vascular smooth muscle response, preserving the hyperresponsiveness to NO.

A pesquisa e o uso de produtos naturais como agentes terapêuticos tem crescido muito nos últimos anos. Os diterpenos são os principais constituintes de extratos de plantas que são usadas na medicina popular no tratamento da hipertensão arterial. Diterpenos da classe dos labdanos exercem atividade inibitória sobre a contração vascular e induzem relaxamento desses tecidos. Nesse sentido, o presente estudo foi delineado de forma a investigar os mecanismos envolvidos no efeito cardiovascular do labdano ácido ent-3-acetóxi-labda-8(17),13-dieno-15-óico (labda-15-óico) em ratos. Os resultados mostraram que o labdano exerce seu efeito inibitório máximo sobre a contração vascular induzida pelo KCl após 30 min de incubação. O efeito inibitório do labda-15-óico sobre a contração induzida por KCl foi totalmente revertido 60 e 120 minutos após a remoção do diterpeno das preparações em anéis sem endotélio (E-) e com endotélio (E+), respectivamente. Os valores de Emax para as curvas de fenilefrina e serotonina foram reduzidos na presença do labda-15-óico em anéis de aorta de rato E+ e E-. O labda-15-óico reduziu a contração induzida pelo CaCl2 em anéis E- nas concentrações de 10, 50 e 100 mol/L. O labda-15-óico não alterou a mobilização do Ca2+ intracelular induzida por fenilefrina ou cafeína. O labdano induziu relaxamento em artérias aorta E+ ou E- pré-contraídas com fenilefrina ou KCl. Em anéis de aorta E+ pré-contraídos com fenilefrina, os valores de Emax para o relaxamento foram reduzidos na presença de L-NAME, ODQ, hemoglobina, 7-nitroindazol, Rp-8-Br-Pet, tapsigargina e tetraetilamônio. Por outro lado, indometacina, wortmanin, LY294002, H-89, SQ22,536, atropina e propranolol não afetaram o relaxamento induzido pelo labdano. O labdano aumentou os níveis de nitrato e GMPc em anéis E+, mas não alterou os níveis de AMPc. O composto em estudo elevou ainda, a intensidade de fluorescência emitida por amostras de células endoteliais marcadas com DAF-2DA, indicando um aumento dos níveis de NO citosólico. Além disso, o labda-15-óico (3 mg/Kg) induziu hipotensão em ratos não anesteziados. O L-NAME reduziu a resposta hipotensora induzida pelo labdano. Concluímos que o labdano exerce um efeito vasorelaxante in vitro e hipotensor in vivo. O labda-15-óico age no músculo liso vascular, onde bloqueia canais para Ca2+ sensíveis a voltagem e operados por receptores, levando à redução do influxo de Ca2+ extracelular. A resposta de relaxamento é parcialmente dependente do endotélio onde o labda-15-óico ativa a via de sinalização do NO-GMPc e promove abertura de canais para K+ no músculo liso vascular. Os estudos in vivo confirmam a participação do NO na resposta cardiovascular induzida pelo labda-15-óico. / The research, development and use of natural compounds as therapeutic agents have been increasing in recent years. Diterpenoids are the main constituents of plant extracts that are used in folk medicine for the treatment of hypertension. Labdane-type diterpenes are described to exert antispasmodic and relaxant action in vascular tissues. The present investigation aimed to evaluate the mechanisms (in vitro and in vivo) underlying the cardiovascular effects displayed by the labdane ent-3-acetoxy-labda-8(17),13-dien-15-oic acid (labda-15-oic) in rats. Our findings show that labda-15-oic achieved its maximal inhibitory action on KCl-induced contraction at 30 min. The inhibitory effect on the the contraction induced KCl elicided by labda-15-oic was totally abolished 60 and 120 min after the removal of the labdane from the medium bath in endothelium-denuded (E-) rings and endothelium-intact (E+) rings. The Emax values for phenylephrine and serotonin in E+ and E- rings were reduced in the presence of labda-15-oic. The labda-15-oic inhibited the contraction induced CaCl2 in E- rings at 10, 50 and 100 mol/L. The labdane did not alter the intracellular Ca2+ mobilization induced by phenylephrine or caffeine. The labdane induced relaxation in E+ and E- rings pre-contracted with phenylephrine or KCl. In E+ rings pre-contracted with phenylephrine, labda-15-oic-induced relaxation was reduced in the presence of L-NAME, ODQ, haemoglobin and RP-8-Br-Pet. On the other hand, indomethacin, wortmannin, LY294002, H-89, SQ22,536, atropine, propranolol did not have a significant effect on the relaxation induced by the labdane. The labdane increased the levels of cGMP and nitrate but not cAMP in E+ rings. The compound studied also increased the intensity of fluorescence emitted by samples of endothelial cells labeled with DAF-2DA indicating an increase in the cytosolic levels of NO. Furthermore, labda-15-oic (3 mg/Kg) induced hypotension in unanesthezided rats and this effect was attenuated by L-NAME. Taken together, our results demonstrate that the labdane exerts a vasorelaxant effect in vitro and hypotensive effect in vivo. The labda-15-oic acts on vascular smooth muscle where it blocks Ca2+ influx through interference with both voltage and receptor-operated channels. The relaxant action of the labdane is also partly mediated by the activation of endothelial NO-cGMP pathway and the opening of K+ channels present in vascular smooth muscle. The studies in vivo confirm the role of NO in the cardiovascular response induced labda-15-oic acid.

Embora seja na obesidade onde se observa hipertrofia e hiperplasia dos adipócitos e aumento da síntese e liberação de adipocinas, condição associada com resistência à insulina e disfunção endotelial, é de suma importância entender os efeitos biológicos de adipocinas, mais especificamente da adipocina chemerin, em condições não patológicas. Os mecanismos pelos quais as citocinas liberadas pelo tecido adiposo podem interferir na função vascular ainda não estão totalmente esclarecidos. Além disso, praticamente não se conhecem os efeitos da citocina/adipocina chemerin sobre a função vascular. Levando-se em consideração que o receptor para chemerin está presente no músculo liso vascular e no endotélio, este trabalho avaliou a atividade biológica e celular desta adipocina sobre a vasculatura de animais não obesos. Investigou-se os efeitos produzidos por esta citocina na reatividade vascular, bem como os mecanismos pelos quais ela modifica a função vascular em animais não obesos. A hipótese deste trabalho é que chemerin aumenta a reatividade vascular a estímulos constritores de endotelina-1 (ET-1) e fenilefrina (PhE) e diminui a vasodilatação induzida pela acetilcolina (ACh) e nitroprussitao de sódio (NPS). Nossos objetivos específicos incluíram determinar: 1) se chemerin promove alterações na reatividade vascular; 2) se as alterações de reatividade vascular promovidas por chemerin são mediadas por modificações da função das células endoteliais ou células de músculo liso vascular; 3) quais vias de sinalização (foco na via das MAPKs) estão sendo modificadas por chemerin e como elas contribuem para as alterações de reatividade vascular produzidas por esta citocina. Nosso estudo demonstrou que a adipocina chemerin possui atividade biológica e celular em aortas de ratos não obesos. Chemerin aumentou respostas vasculares a estímulos contráteis (ET-1 e PhE), atuando tanto no endotélio quanto diretamente em células do músculo liso vascular. O aumento da resposta a estímulos contráteis à ET-1 e PhE foi mediado pela via MEK-ERK1/2, COX-1 e COX-2 e aumento da expressão dos receptores para ET-1, ETA e ETB. Além disso, esta adipocina diminuiu a vasodilatação induzida pela ACh, por meio do desacoplamento da eNOS e aparente envolvimento de estresse oxidativo, e pelo NPS, através de ação sobre a guanilato ciclase. Nossos estudos poderão contribuir para um melhor entendimento sobre o papel dos fatores liberados pelo tecido adiposo visceral sobre a função vascular e, consequentemente, sobre as alterações vasculares presentes na obesidade e patologias associadas. / Although hypertrophy and hyperplasia of adipocytes as well as increased synthesis and release of adipokines are commonly observed in obesity, a condition associated with insulin resistance and endothelial dysfunction, it is extremely important to understand the biological effects of adipokines, or more specifically of the adipokine chemerin, in non-pathological conditions,. The mechanisms by which cytokines released by the adipose tissue may interfere with vascular function are not yet fully understood. Furthermore, the effects of the cytokine/adipokine chemerin on vascular function are not known. Considering that the chemerin receptor is expressed by vascular smooth muscle and endothelial cells, this study investigated the effects produced by this cytokine in vascular reactivity, as well as the mechanisms by which it modifies vascular function in non-obese animals. Our working hypothesis is that chemerin enhances vascular reactivity to constrictor stimuli, such as endothelin-1(ET-1) and phenylephrine (Phe), and decreases the vasodilation induced by acetylcholine (ACh) and sodium nitroprussiate (SNP). Our specific aims were to determine: 1) whether chemerin induces changes in vascular reactivity, 2) if the alterations of vascular reactivity induced by chemerin are mediated by changes in the function of endothelial cells or vascular smooth muscle cells, 3) which signaling pathways (focus on the MAPKs pathway) are being modified by chemerin and how they contribute to changes in vascular reactivity produced by this cytokine. Our study showed that the adipokine chemerin has biological and cellular activity in aortas from non-obese rats. Chemerin increased vascular responses to contractile stimuli (ET-1 and PhE), producing effects both in the endothelial and vascular smooth muscle cells. The increased contractile responses to ET-1 and PhE were mediated via activation of MEK-ERK1/2, COX-1 and COX-2 and increased expression of the ETA and ETB receptors. Furthermore, this adipokine reduced the vasodilation induced by ACh via eNOS uncoupling and oxidative stress, and by SNP, via effects in the enzyme guanylate cyclase. Our studies may contribute to a better understanding of the role of factors released by the visceral adipose tissue on vascular function and, consequently, on the vascular lesions in obesity and obesity-associated diseases.

Vários mecanismos controlam os processos de contração e relaxamento das células musculares lisas vasculares (CMLVs). Receptores e moléculas sinalizadoras intracelulares, os quais controlam os processos de contração e relaxamento das CMLVs, são alvo de modificações pós-traducionais, como a O-GlcNAcilação que modula respostas vasculares. O aumento de proteínas modificadas por O-GlcNAc apresenta efeito ambíguo sobre as CMLVs, sendo protetor em situações de aumento agudo, mas lesivo quando mantido cronicamente. O aumento crônico de O-GlcNAc em animais está associado a repostas contráteis mais intensas e redução do relaxamento vascular, assim como o aumento da produção de espécies reativas de oxigênio (EROs), denominado estresse oxidativo, alterações constantemente descritas em doenças crônicas como diabetes e hipertensão arterial. Considerando que algumas proteínas que controlam a contratilidade vascular são modificadas por O-GlcNAc e que pouco se sabe a respeito da via de Biossíntese das Hexosaminas (VBH) e sua relação com o sistema vascular em humanos, nós investigamos a hipótese de que modificações de proteínas por O-GlcNAc estão relacionadas a alterações vasculares na hipertensão arterial em humanos. Durante a realização de nossos experimentos, demonstramos que os principais componentes da VBH estão presentes em CMLVs humanas. O tratamento com Thiamet G (TMG) por 24 h aumentou os níveis de proteínas modificadas por O-GlcNAc nas CMLVs pela redução da atividade de OGA, assim como induziu efeito compensatório de aumento da expressão dessa enzima. TMG reduziu a atividade de OGA em CMLVs no grupo Controle, mas não promoveu alteração na geração de EROs. Após tratamento com TMG, artérias de grupo Controle apresentaram maior sensibilidade à noradrenalina (NA) e maior relaxamento ao nitroprussiato de sódio (NPS); enquanto o grupo Hipertenso não apresentou alterações na contratilidade ou no relaxamento arterial. Artérias do grupo Hipertenso apresentaram maior sensibilidade à NA que o grupo Controle antes de qualquer tratamento, além de deficiência no relaxamento, com menor sensibilidade e menor resposta máxima ao NPS em comparação ao grupo Controle. O grupo Hipertenso apresentou aumento da pressão arterial média de internação (PAMi), assim como da idade, quando comparado com o grupo Controle. Em conclusão, a VBH está presente nas CMLVs humanas. A inibição da atividade da OGA por TMG aumenta os níveis de proteínas modificadas por O-GlcNAc, a expressão de OGA e modula a reatividade vascular no grupo Controle, mas não no grupo Hipertenso. Os resultados demonstram que pacientes hipertensos apresentam respostas vasculares a drogas vasoativas diferentes daquelas observadas em pacientes controle, antes e após o aumento dos níveis de proteínas modificadas por O-GlcNAc nas CMLVs. Novos estudos serão necessários para determinar se as alterações observadas são decorrentes da hipertensão arterial e/ou do tratamento farmacológico aos quais os pacientes estão submetidos. / Several mechanisms control the contraction and relaxation processes in the vascular smooth muscles cells (VSMC). Intracellular receptors and signaling molecules involved in contraction and relaxation mechanisms are targets of post-translational modifications (PTM), such as O-GlcNAcylation, which modulates vascular responses. Augmented levels of O-GlcNAc-modified proteins show a dual effect in VSMC, being protective during acute stressful events and deleterious when O-GlcNAc is chronically augmented. In animals, chronic increases in O-GlcNAc-modified proteins are linked to increased vascular responses to constrictor agents, to reduced vascular relaxation in response to dilator drugs, and to increased production of reactive oxygen species (ROS), named oxidative stress. All these changes are frequently described in chronic diseases such as diabetes and arterial hypertension. Since proteins involved in vascular contractility are modified by O-GlcNAc, and our knowledge on the influence of the hexosamine biosynthesis pathway (HBP) in the human vascular system is limited, we tested the hypothesis that proteins modified by O-GlcNAc contribute to vascular changes in hypertensive patients. Our data show that human VSMC express the main components of the HBP; the treatment of human VSMC with Thiamet G (TMG) for 24 h augmented O-GlcNAc levels, decreased OGA activity and induced a compensatory increase in OGA\'s expression. TMG reduced OGA activity, increased levels of O-GlcNAc-modified proteins but did not change ROS generation in human arteries from the Control group. After treatment with TMG, arteries from the Control group exhibited increased sensitivity to norepinephrine (NE) and to sodium nitroprusside (SNP), as well as increased maximum relaxation to SNP. Augmented O-GlcNAc levels produced no changes in contractile or relaxation responses in the Hypertensive group. Arteries from the Hypertensive group exhibited an increased sensitivity to NE as well as decreased sensitivity and maximum relaxation to SNP when compared to arteries from the Control group. Mean arterial blood pressure (hMABP) and the average age were increased in patients from the Hypertensive group. In conclusion, the HBP is present in human VSMC and the inhibition of OGA activity with TMG increases O-GlcNAc levels, increases OGA expression and modifies vascular responses to constrictor and dilator stimuli in human arteries from the Control group, but not from the Hypertensive group. These results indicate that hypertensive patients have altered vascular responses to vasoactive drugs both in the absence and in the presence of augmented O-GlcNAc levels. Further research is needed to explain whether these differences are due to the hypertensive disease and/or to the patient\'s medical treatment.

Introdução: Os distúrbios ácido-básicos são comuns na prática médica e podem variar desde uma acidose ou alcalose simples até um distúrbio misto complexo e potencialmente fatal. A acidose metabólica ocorre por aumento na quantidade de ácidos não-voláteis sob condições como: insuficiência renal, sepse, diabetes grave, diarréia e outras. Há mais de um século tem-se conhecimento de que mudanças no pH promovem alterações no tônus vascular, o que afeta a circulação e controle da pressão sanguínea. Objetivos: 1) estabelecer um modelo eficiente de acidose metabólica crônica (AMC) em ratos; 2) avaliar os parâmetros ventilatórios durante a indução da AMC; 3) investigar os efeitos da AMC sobre a reatividade da aorta de ratos, bem como os mecanismos envolvidos nesta resposta. Metodologia: A AMC foi induzida com cloreto de amônio ad libitum 0,5 M + 0,02M por gavagem, durante 10 dias. Os parâmetros ventilatórios avaliados foram frequência respiratória (fR), volume corrente (Vc) e ventilação pulmonar (VE). No estudo de reatividade vascular foram realizadas curvas dose-resposta para acetilcolina (ACh), fenilefrina (Phe), endotelina 1 (ET-1) e angiotensina II (Ang II), em aorta com e sem endotélio, na ausência e presença do L-NAME. Resultados: A AMC induzida por cloreto de amônio (NH4Cl) reduziu o pH para 7.17 (controle 7.39), com níveis de bicarbonato (HCO3-) próximos a 9.8 mmol/L (controle 21.9 mmol/L). Quanto aos parâmetros ventilatórios, houve um aumento do Vc no segundo dia de tratamento, levando a um aumento na VE. Nos estudos de reatividade vascular, a AMC não alterou a resposta para a ACh e ET-1, entretanto reduziu a vasoconstrição induzida pela Ang II e Phe, sendo a resposta para Phe restaurada na presença de L-NAME. Conclusões: 1) a partir do protocolo empregado foi possível obter um modelo de AMC reprodutível; 2) houve alterações ventilatórias apenas no início do tratamento, associada à redução de pressão parcial de dióxido de carbono (pCO2), embora não significativa, mostrando uma resposta compensatória transitória; 3) o efeito da AMC sobre a reatividade vascular, parece ser agonista-seletiva e o óxido nítrico (NO) está envolvido nessa resposta. / Introduction: The acid-base disorders are common in the medical practice and can vary from an acidosis or simple alkalosis to a mixed, complex and potentially fatal disorder. The metabolic acidosis occurs because of the increase in the quantity of nonvolatile acids under conditions such as kidney disease, sepsis, serious diabetes, diarrhea, and so on. More than one century there is some knowledge that the extracellular pH has promoted changes in the vascular tonus, which affects the circulation and the blood pressure control. Objectives: 1) Develop an efficient model of chronic metabolic acidosis (CMA) in rats; 2) Evaluate the ventilatory parameters during the induction of the CMA; 3) to investigate the effects of the CMA on rat aorta reactivity as well as the mechanisms involved in this response. Methodology: The CMA was induced by NH4CI 0.5M ad libitum + 0.02M by gavage during 10 days. The assessed ventilatory parameters were respiratory frequency (fR), tidal volume (Vt) and pulmonary ventilation (VE). The studies of the vascular reactivity were carried out by dose-response curve to acetylcholine (ACh), phenylephrine (Phe), endotelina1 (ET-1) and angiotensin II (Ang II), in aorta with and without endothelium, in absence and presence of the L-NAME. Results: The acidosis induced by ammonium chloride (NH4Cl) reduced the pH to 7.17 (7.39 control), with levels of bicarbonate (HCO3-) about 9.8 mmol/L (21.9 control mmol/L). As for the ventilatory parameters, there was an increase of the Vt in the second day of the treatment, which lead to an increase in the VE. In the studies of the vascular reactivity, the CMA have not changed the response to the Ach and ET-1, however the vasoconstriction induced by Ang II and Phe were reduced after CMA, and this was restored by L-NAME. Conclusions: 1) From the used protocol, it was possible to obtain a model of reproducible CMA. 2) There were ventilatory alterations only in the beginning of the treatment, associated to the reduction of pCO2, although not significant, which showed a transitory compensatory response. 3) The effect of the CMA on the vascular reactivity seems to be agonist-selective and the nitric oxide (NO) is involved in this response.