High blood pressure or hypertension is a major public health issue that affects more than 50% of adults in the United States. Hypertension is the leading risk factor for multiple cardiovascular events including stroke and myocardial infarction. In general, hypertension is considered a disease of the aged population as 2/3rds of adults over 65 are hypertensive. Critically, a sex dependent component exists as females under age 50 are less likely to develop hypertension than males. Dietary sodium intake significantly influences blood pressure regulation and its importance is underscored by the salt-sensitivity of blood pressure, which is characterized by acute increases in blood pressure in response to dietary salt intake. The salt-sensitivity of blood pressure is prevalent in 25% of normotensive individuals and 50% of hypertensive individuals. Coupled with statistics that show Americans on average consume 2 g of sodium in excess of the recommended daily allowance, the risk for developing salt-sensitive hypertension is drastically higher in salt-sensitive individuals. Moreover with age, there is an increase in the prevalence and severity of salt-sensitivity. Taken together, these findings underscore the need for novel therapeutics to combat hypertension. The pathophysiology of the salt-sensitivity of blood pressure and age dependent hypertension has been attributed in part to excessive sympathetic outflow that can drive increases in sodium reabsorption. Excessive sympathetic outflow via the release of norepinephrine has been linked to increased activity of a key renal sodium transporter, the sodium chloride cotransporter (NCC). This thesis investigates the adrenergic signaling pathway by which excessive sympathetic outflow drives NCC activity and sodium reabsorption as well investigates the mechanisms underlying sex differences in age dependent hypertension. Our findings demonstrate that 1) norepinephrine mediates its influence on NCC activity via an α1-adrenoceptor gated pathway involving WNK/SPAK/OxSR1 kinase signaling, 2) α1-adrenoceptor antagonism can prevent and attenuate the development and maintenance of salt-sensitive hypertension, 3) β-adrencoptor antagonism has no effect on NCC activity, 4) in male rats age dependent salt-sensitivity of blood pressure and hypertension is associated with age dependent- increases in NCC activity and impairments in renal sodium handling, and 5) female rats do not develop age dependent hypertension or salt-sensitivity of blood pressure. Collectively, these results support a sympathetic model of NCC regulation that plays a key role in salt-sensitive hypertension and age dependent hypertension. / 2021-05-26T00:00:00Z

We tested the hypothesis that oxytocin-induced labor augmented IL-1β-induced/-stimulated lung fluid absorption in preterm guinea pig fetuses. IL-1β was administered subcutaneously daily to timed-pregnant guinea pigs for 3 days with and without simultaneous cortisol synthesis inhibition by metyrapone. At day 3, oxytocin was administered, and fetuses were delivered by abdominal hysterotomy at 61 and by oxytocin-induced birth at 68 days gestation. Delivered fetuses were instilled with isosmolar 5% albumin into the lungs, and lung fluid movement was measured over 1 h by mass balance. Lung fluid absorption was induced in 61-day and stimulated in 68-day gestation lungs by IL-1β. Labor induction by oxytocin augmented IL-1β-induced/- stimulated lung fluid absorption. Metyrapone pretreatment did not affect oxytocin-induced/-stimulated lung fluid absorption, while completely blocking IL-1β-induced/-stimulated fluid absorption. Fetal lung fluid absorption, when present, was always propranolol and amiloride sensitive, suggesting that β-adrenoceptor stimulation and amiloride-sensitive sodium channels were critical for fluid absorption. Epithelial sodium channel and Na-K-ATPase subunit expressions were both increased by IL-1β, but not further by oxytocin. Our results indicate that IL-1β release into the maternal blood circulation positively affects lung maturation due to the IL-1β-induced release of cortisol and thus prepares the lungs for the epinephrine surge associated with labor.

Interaction with chlorethylclonidine has been used to subdivide populations of α1-adrenoceptors in some tissues. WB 4101 can distinguish high and low affinity states of the receptor. The present study was carried out to determine if different populations or affinity states of α1-adrenoceptors distinguished by either of these compounds, could explain the variation in α1-adrenoceptor agonist affinity found amongst rabbit arteries. Five arteries were studied whose affinity for noradrenaline vary between 4.8 and 6.4. These were the thoracic aorta, renal, superior mesenteric, ear and ovarian arteries. WB 4101 was found to be equally effective in antagonizing noradrenaline on all arteries. Chlorethylclonidine caused a 20-fold rightward shift of the noradrenaline dose-contraction curve in the thoracic aorta; but had little or no effect on the other vessels. Thus, the combination of different proportionsof subsets of α1-adrenoceptors distinguished by WB 4101 or chlorethylclonidine does not explain the variation in α1-adrenoceptor affinity found in these rabbit arteries.

The ability of derivatives of 2-aminotetralins (2AT), cis- or trans-isomers of octahydrobenzo[f]quinolines (BfQ) and clonidine to modulate acetylcholine release was studied using field-stimulated guinea pig ilea (GPI). Antihistaminic and antiacetylcholine activities were also determined using isolated superfused segments of GPI. Hydroxylated 2AT, BfQ and clonidine inhibited field stimulation-induced contractions through α-adrenoceptor mechanisms which were antagonized by phentolamine. In contrast, the inhibition produced by nonhydroxylated 2AT was not attenuated by α-adrenoceptor antagonism. 2AT, trans-7,8-dihydro-BfQ and cis-8,9-dihydroxy-BfQ inhibited contractions induced by nicotine bitartrate using superfused GPI. Clonidine was inactive as an antinicotinic agent and there was no correlation between a compound's ability to inhibit contractions induced by field stimulation and its antinicotinic activity. Various 2AT derivatives demonstrated weak antimuscarinic and/or antihistaminic activities on superfused ileal segments. These data demonstrate that these agents posses a spectrum of pharmacological activity.

Thesis (MScMedSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: It is well-established that transient activation of the β-adrenergic signalling pathway with ligands such as isoproterenol, formoterol and dobutamine, elicits cardioprotection against subsequent long periods of ischaemia. Initially the focus was on the β1- and β2-adrenergic receptors (β1-AR, β2-AR), but recently the β3-AR also emerged as a potential target in the treatment of heart disease. In heart failure, β1- and β2-AR are typically known to be down-regulated while β3-ARs, on the other hand, are up-regulated (Moniotte et al., 2001). Thus, it has become important to examine the significance of the β3-AR and its downstream signalling under similar states of stress. It has been shown that β3-AR stimulation is resistant to short term agonist-promoted desensitization in vitro and in vivo (Liggett et al., 1993) and after being activated, this receptor is able to convey continual intracellular signals (Lafontan et al., 1994). Thus, it could be an ideal target for therapeutic intervention, also in ischaemic heart disease. We hypothesized that selective β3-AR stimulation during ischaemia / reperfusion may be cardioprotective, whereas selective inhibition of this receptor may prove useful in the end stages of sustained ischaemia and early reperfusion. Methods: The isolated working rat heart, subjected to 35 min of regional ischaemia (RI) and 60 min reperfusion was used as model. The β3-AR agonist (BRL37344) (1 μM) or antagonist (SR59230A) (0.1 μM) were applied as follows: (i) before 35 min RI (PT), (ii) during the last 10 min of RI (PerT) and /or (iii) at the onset of reperfusion (PostT) and (iv) administration of BRL37344 during the last 10 min of RI BRL37344 (PerT) was followed by SR59230A during first 10 min of reperfusion SR59230A (Post). The contribution of nitric oxide synthase (NOS) in β3-AR was assessed, using the non-specific NOS inhibitor, L-NAME (50 μM). Endpoints were functional recovery and infarct size. In another set of experiments BRL37344 and SR59230A were applied according to the same protocols, but the left ventricle was dissected from the heart and freeze clamped at 10 min reperfusion for Western blot analysis of extracellular signal-regulated kinase (ERK p44/p42), protein kinase B (PKB/Akt), glycogen synthase kinase-3β (GSK-3β), and endothelial nitric oxide synthase (eNOS). Data were analyzed with one or two-way analysis of variance (ANOVA). Results: Administration of the selective β3-AR agonist (BRL37344) (1μM) before 35 min RI (BRL37344 (PT), significantly reduced infarct size when compared to the non-pretreatment group (NPT) (21.43±2.52 vs 43.17±1.20, p < 0.001). BRL37344 had similar effects on infarct size when applied during the last 10 min of regional ischaemia BRL37344 (PerT) (14.94±2.34, vs NPT, p < 0.001) or at the onset of reperfusion BRL37344 (PostT) (19.06±1.81, vs NPT, p < 0.001). When BRL37344 was applied as a (PerT+PostT) strategy, infarct size was once again significantly reduced (20.55±2.01 vs 43.17±1.20, p <0.001). In contrast, administration of the β3-antagonist SR59230A according to the same protocol did not reduce infarct size and values similar to those of untreated hearts (NPT) were obtained. Surprisingly, when BRL37344 was applied during the last 10 min of regional ischaemia followed by the administration of the β3-AR antagonist (SR59230A) at the onset of reperfusion, [BRL37344 (PerT) & SR59230A (PostT)], infarct size was significantly reduced to 20.78±3.02 (p <0.001 vs NPT and SR59230A (PerT + PostT). Involvement of nitric oxide (NO) was shown since the reduction in infarct size elicited by BRL37344 was totally abolished by, L-NAME, when administered in combination with BRL37344 for 10 minutes prior to RI or at the onset of reperfusion for 10 minutes (% infarct size: 41.48±3.18 and 35.75±3.54, p <0.001 vs BRL37344 (PT) and BRL37344 (PostT), respectively. Western blot results show that PKB/Akt is activated by BRL37344 regardless of the time of administration. The intervention BRL37344 (PerT+PostT), exhibited the most significant phosphorylation of PKB/Akt (fold increase: 14.2±3.71, p<0.01 vs NPT and p<0.05 vs BRL37344 (PostT). In addition, BRL37344 (PT), (PerT), (PostT) and [BRL37344 (PerT) +SR59230A (PostT)] showed significant activation of this kinase (2.92±0.22, 5.54±0.43, 4.73±0.47, and 6.60±0.78, respectively). ERKp44/p42 however, was not significantly activated by any of the treatments. Phosphorylation of eNOS and GSK-3β was significant only in the BRL37344 (PerT+PostT) and [BRL37344 (PerT) + SR59230A (PostT)] groups. The activation of eNOS-S-1177 in the BRL37344 (PerT+PostT) group was (2.82±0.46, p<0.01 and 0.05 vs NPT and BRL37344 (PostT), respectively) and in the [BRL37344 (PerT) + SR59230A (PostT)] group was (2.26±0.48, p<0.05 vs NPT). A very significant increased phosphorylation of GSK-3β was seen in the same two groups (68.8±7.73, p<0.001 vs NPT and 25.5±5.42 vs NPT, p<0.05, respectively). Conclusion: β3-AR has potent cardioprotective effects when administered either before, during and after ischaemia during early reperfusion as indicated by the reduction in infarct size as well as activation of PKB, GSK-3β and eNOS. These beneficial effects can be linked to NO production through activation of eNOS. / AFRIKAANSE OPSOMMING: Dit is bekend dat verbygaande aktivering van die β-adrenerge seinpad, met ligande soos isoproterenol, formoterol en dobutamien, die hart teen daaropvolgende lang periodes van iskemie beskerm. Aanvanklik was die fokus op die β1- en β2-adrenerge reseptore (β1-AR, β2-AR); maar onlangs is ook die β3-AR as 'n potensiële teiken in die behandeling van hartsiektes ge-eien. In hartversaking, is dit bekend dat β1- en β2-AR afreguleer word, terwyl β3-ARs, aan die ander kant, opreguleer word (Moniotte et al., 2001). Dit het dus belangrik geword om die belang van die β3-AR en sy stroomaf seinpad onder soortgelyke strestoestande te ondersoek. Dit is bewys dat β3-AR stimulasie teen korttermyn agonis geïnduseerde desensitisering in vitro en in vivo bestand is (Liggett et al., 1993) en wanneer geaktiveer, is hierdie reseptor in staat om intrasellulêre seine voortdurend oor te dra (Granneman, 1995). Dit kan dus ‘n ideale teiken vir terapeutiese intervensie wees, ook in iskemiese hartsiekte. Ons hipotetiseer dat selektiewe β3-AR stimulasie tydens iskemie / reperfusie kardiobeskermende mag wees, terwyl selektiewe inhibisie van hierdie reseptor effektief kan wees in die eindstadia van volgehoue iskemie en vroeë herperfusie. Metodes: Die geïsoleerde werkende rothart, onderwerp aan 35 min van streeksiskemie (SI) en 60 min herperfusie, is as model gebruik. Die β3-AR agonis (BRL37344) (1μM) of antagonis (SR59230A) (0.1 μM), is as volg toegedien: (i) voor 35 min SI (PT), (ii) gedurende die laaste 10 min van SI (PerT) en / of (iii) tydens die aanvang van herperfusie (PostT) en (iv) gedurende die laaste 10 min van SI is BRL toediening BRL37344 (PerT) gevolg deur SR59230A tydens die eerste 10 min van herperfusie SR59230A (Post). Die rol van stikstofoksiedsintase (NOS) in β3-AR is met behulp van die nie-spesifieke NOS inhibitor, L-NAME (50 μM) ondersoek. Eindpunte was funksionele herstel tydens herperfusie en infarktgrootte. In 'n ander reeks eksperimente is BRL37344 en SR59230A volgens dieselfde protokolle toegedien, maar die linker ventrikel is uit die hart gedissekteer na 10 min herperfusie en gevriesklamp vir Western klad analise van ekstrasellulêre-sein gereguleerde kinase (ERK p44/p42), proteïen kinase B (PKB/Akt), glikogeen sintase kinase-3β (GSK-3β), en endoteel stikstofoksied- sintase (eNOS). Data is met een of twee-rigting variansie analise (ANOVA) ontleed. Resultate: Administrasie van die selektiewe β3-AR agonis (BRL37344) (1μM) voor 35 min SI BRL37344 (PT), het die infarktgrootte beduidend verminder vergeleke met die nie-behandelde groep (NPT) (21.43±2.52 vs 43.17±1.20, p<0.001). BRL37344 het ‘n soortgelyke effek op infarktgrootte wanneer dit gedurende die laaste 10 min van streeksiskemie BRL37344 (PerT) (14.94±2.34, vs NPT, p<0.001) of by die aanvang van herperfusie (BRL37344 (PostT) (19.06±1.81, vs NPT, p<0.001) toegedien word. Wanneer BRL37344 as 'n (PerT+PostT) strategie toegedien is, was infarktgrootte weereens beduidend verlaag (20.55±2.01 vs 43.17±1.20, p<0.001). In teenstelling hiermee, het administrasie van die β3-antagonis SR59230A volgens dieselfde protokol, nie infarktgrootte verminder nie en waardes soortgelyk aan dié van onbehandelde harte (NPT) is verkry. Interessant, wanneer BRL37344 gedurende die laaste 10 min van streeksiskemie toegedien is, gevolg deur die administrasie van die β3-AR antagonis (SR59230A) by die aanvang van herperfusie, [BRL37344(PerT) & SR59230A(PostT)], was infarktgrootte aansienlik verminder tot 20.78±3.02 (p<0.001 vs NPT en SR59230A (PerT+PostT). Die betrokkenheid van stikstofoksied (NO) is waargeneem deurdat die vermindering in infarktgrootte ontlok deur BRL37344, heeltemal deur L-NAME opgehef is, wanneer dit in kombinasie met BRL37344 vir 10 minute voor SI of by die aanvang van herperfusie vir 10 minute toegedien is (% infarktgrootte: 41.48±3.18 en 35.75±3.54, p<0.001 vs BRL37344 (PT) en BRL37344 (PostT) onderskeidelik). Western kladresultate toon dat PKB/Akt deur BRL37344 geaktiveer word ongeag die tyd van die administrasie. Die intervensie BRL37344 (PerT+PostT), toon die mees beduidende fosforilering van PKB/Akt (voudige toename: 14.2±3.71, p<0.01 vs NPT en p<0.05 vs BRL37344 (PostT). Daarbenewens het BRL37344 (PT), (PerT), (PostT) en [BRL37344 (PerT) + SR59230A (PostT)] ook beduidende aktivering van hierdie kinase tot gevolg gehad (2.92±0.22, 5.54±0.43, 4.73±0.47 en 6.60±0.78, onderskeidelik). ERKp44/p42 is egter nie deur enige van die behandelings geaktiveer nie. Fosforilering van eNOS en GSK-3β was net beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. Die aktivering van eNOS-S-1177 was beduidend in die BRL37344 (PerT+PostT) en [BRL37344 (PerT) + SR59230A (PostT)] groepe. 'n Baie beduidende toename in fosforilering van GSK-3β is in dieselfde twee groepe (68.8±7.73, p<0.001 en 25.5±5.42, p<0.05 vs NPT onderskeidelik) waargeneem. Gevolgtrekking: β3-AR het kragtige kardiobeskermende effekte wanneer dit, hetsy voor, tydens en na iskemie gedurende vroeë herperfusie toegedien word, soos deur die vermindering in infarktgrootte sowel as die aktivering van PKB, GSK-3β en eNOS aangedui is. Hierdie voordelige effekte kan aan NO produksie deur aktivering van eNOS gekoppel word.

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

The 1-adrenoceptors (1-ARs) serve as an interface between the sympathetic nervous system and the cardiovascular system where they are mediators of systemic arterial blood pressure, initiators of positive inotropy, and regulators of cellular growth responses. There are three subtypes: 1A-, 1B-, and 1D-ARs. This dissertation research investigated the trafficking properties of the 1-ARs at the cellular level as well as physiological relevance of the 1-ARs at the tissue level. In vitro studies using transiently transfected 1-AR/GFP subtypes revealed distinct basal localization patterns and different agonist-mediated activation and desensitization properties. The 1A- and the 1B-AR/GFP subtypes displayed agonist-mediated receptor redistribution, in which rate and degree of redistribution differed. Additionally, redistribution of either of these two receptor subtypes required arrestin-1, a protein often associated with receptor internalization. In contrast, the 1D-AR/GFP did not require arrestin-1 for maintaining the basal receptor orientation pattern. Although these data increase our knowledge of trafficking properties of the 1-AR subtypes, it is of equal importance to determine the role(s) that each subtype contributes to cardiovascular function. The lack of subtype-selective 1-AR pharmacological agents prompted the use of genetically manipulated mouse models with a systemic overexpression of a constitutively active 1B-AR. Echocardiographic analysis of transgenic hearts indicated both an enlarged left ventricular chamber in the absence of hypertrophy and a depressed cardiac function. From isolated transgenic hearts, experimental results suggested a role for the 1B-AR in attenuating the inotropic responses. However, experiments using isolated thoracic aortae from transgenic animals suggested that the 1B-AR does not participate in vascular smooth muscle contractile responses. Additional studies investigated the role of 1D-AR in cardiovascular function by using animals systemically lacking the 1D-AR subtype. Experimental data suggested an 1D-AR participation in vascular smooth muscle function since the deficiency of the 1D-AR subtype affected vasoconstriction in the coronary arteries but not inotropy in the heart. The data presented in this dissertation research suggest subtype specific differences of 1-ARs in cellular localization, signal regulation, and trafficking. Additionally, the data provide an investigation into the physiologic significance of both the 1B- and the 1D-ARs in cardiovascular tissue.

A insuficiência cardíaca (IC) é uma síndrome complexa que envolve múltiplos sistemas e mecanismos compensatórios neuro-hormonais, acompanhada de altos índices de morbidade e mortalidade, e caracterizada por sintomas clínicos como fadiga, dispneia, e intolerância aos esforços físicos. Embora a IC seja uma síndrome de origem cardíaca, observam-se alterações em outros tecidos, como na musculatura esquelética. As modificações do fenótipo muscular e a perda de massa muscular esquelética observadas na IC contribuem para o mau prognóstico e para o aumento da mortalidade dos pacientes. Considerando que os receptores 2-adrenérgicos medeiam a atividade do sistema nervoso simpático na musculatura esquelética, e que a hiperatividade simpática é um dos principais componentes envolvidos no desenvolvimento da miopatia esquelética observada IC, sugere-se que estes receptores estejam associados às alterações morfofuncionais da musculatura esquelética na síndrome. Na presente Dissertação, avaliamos a contribuição dos receptores 2-adrenérgicos nas alterações metabólicas e morfofuncionais da musculatura esquelética e na intolerância aos esforços físicos decorrentes da IC. Para isso, utilizamos camundongos da linhagem FVB controles e com inativação gênica dos receptores 2-adrenérgicos (2KO) que foram submetidos à cirurgia de infarto ou à cirurgia fictícia (sham). O infarto induziu disfunção e remodelamento cardíacos nos animais controles, acompanhados de aumento nos níveis de noradrenalina e adrenalina circulantes, intolerância ao esforço físico, mudança na tipagem de fibras musculares e rarefação capilar nos músculos sóleo e plantar. Essas mesmas respostas foram observadas nos animais com inativação gênica dos receptores 2-adrenérgicos após o infarto do miocárdio, no entanto, os animais 2KO infartados apresentaram uma maior redução da tolerância ao esforço físico e um quadro mais grave de miopatia esquelética. Acompanhando essa atrofia muscular esquelética presente nos animais 2KO infartados, foi observado aumento da expressão de proteínas relacionadas ao sistema proteolítico ubiquitina-proteassoma, aumento da atividade do 26S-proteassoma e tendência a diminuição na expressão proteica de p-Akt (ser473), p-4E-BP1 (thr37/46) e p-FoxO3 (ser253). Os resultados sugerem que a ausência dos receptores 2-adrenérgicos agrava e/ou antecipa as alterações musculoesqueléticas observadas na insuficiência cardíaca, principalmente a atrofia muscular, e que a ativação do sistema proteolítico ubiquitina proteassoma e a inibição da síntese proteica por meio da via Akt/4E-BP1 parecem colaborar para estas respostas / Heart failure (HF) is a complex syndrome involving multiple systems and neurohumoral compensatory mechanisms accompanied by high morbidity and mortality, and it is characterized by clinical signs like fatigue, dyspnea, and increased exercise intolerance. Although HF is a syndrome of cardiac origin, it promotes changes in other tissues, such as skeletal muscle, where modifications of muscle phenotype and the loss of skeletal muscle mass observed in HF contribute to poor prognosis and increased mortality of patients. Taking into consideration that 2-adrenoceptors mediate the activity of sympathetic nervous system in skeletal muscle and that sympathetic hyperactivity is one of the main components involved in developing skeletal muscle abnormalities in HF, it has been suggested that 2-adrenoceptors would be associated with morphofunctional alterations due to chronic sympathetic hyperactivity in skeletal muscles in HF. In the present study, we evaluated the contribution of 2-adrenoceptors on metabolic and morphofunctional alterations in skeletal muscle and also on exercise intolerance-induced by HF. For this, we used FVB mice and mice lacking 2-adrenoceptors (2KO) that were submitted to myocardial infarction or sham surgery. Myocardial infarction induced cardiac dysfunction and remodeling in FVB mice, which was accompanied by significantly increased plasma levels of norepinephrine and epinephrine, exercise intolerance, changes in muscle fiber type and vascular rarefaction in both soleus and plantaris muscles. These same responses were observed in mice lacking 2-adrenoceptors submitted to myocardial infarction. However, infarcted 2KO mice presented a higher decrease of exercise tolerance and more severe skeletal myopathy. Accompanying the skeletal muscle atrophy of infarcted 2KO mice, it was observed a significantly increased protein expression of proteins related with the ubiquitin-proteasome system, an increased 26S-proteassome activity and a trend toward decreased protein expression of p-Akt (ser473), p-4E-BP1 (thr37/46) and p- FoxO3a (ser253). These results suggest that the lack of 2-adrenoceptors worsens and/or anticipates the skeletal muscle alterations observed in HF, mainly muscular atrophy, and the activation of ubiquitin-proteassome system and inhibition of protein synthesis by Akt/4E-BP1 pathway seem to play an important role in skeletal muscle myopathy

Neste estudo investigou-se: 1) os subtipos de receptores b-adrenérgicos (b-AR) envolvidos nas alterações de reatividade vascular induzida pela hiperativação dos receptores b-AR com do uso de camundongos nocaute para os receptores b1- ou b2-AR e selvagens tratados por 7 dias com isoproterenol; 2) a expressão gênica e protéica de mediadores pró-inflamatórios na aorta de ratos tratados por 7 dias com isoproterenol. Os dados demonstram que: em aorta de camundongos selvagens o receptor b1-AR é mais importante em mediar vasodilatação, enquanto o b2AR modula negativamente a contração vascular de maneira dependente do endotélio. A hiperativação dos receptores b-AR aumenta a vasoconstrição à fenilefrina via desequilíbrio NO/ ânion superóxido em aorta de camundongos e o b2-AR parece estar envolvido no desencadeamento destes efeitos. Sugere-se que a ativação crônica dos receptores b-AR aumenta a síntese de fatores pró-inflamatórios e induz maior ativação do NF-kB no tecido vascular, efeitos que parecem estar associados ao aumento da reatividade vascular à fenilefrina. / In this study, it was investigated: 1) the b-adrenoceptor (AR) subtypes involved in the altered vascular reactivity induced by overactivation of b-AR using b1- or b2-adrenoceptor knock-out mice and respective wild-types treated for 7 days with isoproterenol or with vehicle; 2) gene and protein expression of vascular inflammatory mediators in aorta from 7-day isoproterenol-treated rats. In conclusion, the data suggest that b1-AR is more important in the modulation of vasodilator mechanisms while b2 down regulates mice vascular contraction in an endothelium-dependent manner. Overactivation of b-AR increased vasoconstrictor response to phenylephrine, by NO/ superoxide anion unbalance in mice aortic rings, and b2-AR seems to be involved in the triggering of these chronic isoproterenol effects. The results also suggest that chronic activation of b-AR enhances the synthesis of proinflammatory factors and induces higher NF-kB activation on vascular tissue, and these effects seem to be related to hiperreactivity to phenylephrine induced by chronic isoproterenol.

Os efeitos benéficos do exercício físico aeróbico (EFA) frente à prevenção e ao tratamento de doenças ocorrem por meio de adaptações agudas e crônicas em diversos tecidos e sistemas orgânicos, dentre os quais o tecido muscular esquelético assume papel de destaque. Considerando que a musculatura esquelética é rica em mitocôndrias, não surpreende o fato de que o aumento da capacidade e potência aeróbicas induzido pelo EFA ocorra principalmente em função das adaptações mitocondriais. Contudo, os mecanismos moleculares envolvidos nessas adaptações mitocondriais induzidas pelo EFA não são completamente compreendidos. Considerando-se que a atividade nervosa simpática aumenta consideravelmente durante a realização de uma sessão de EFA, parte das respostas mitocondriais ao EFA poderia ser mediada pela ativação dos receptores \'beta\'-adrenérgicos (\'beta\'-AR) na musculatura esquelética, representados predominantemente pelo subtipo \'beta\'2 (~ 90%). Com isso, na primeira parte do presente estudo, testou-se a hipótese de que a sinalização \'beta\'2-AR contribuiria para as respostas de função e dinâmica mitocondriais induzidas pela ativação adrenérgica aguda na musculatura esquelética. Para isso, o agonista \'beta\'-AR não seletivo isoproterenol foi administrado na presença ou ausência do antagonista \'beta\'2-AR específico, ICI 118,551, tanto em miotúbulos diferenciados a partir da linhagem de células C2C12 como em camundongos da cepa FVB. Os dados demonstraram que a ativação \'beta\'-AR aumentou significativamente a respiração mitocondrial em miotúbulos e em fibras musculares isoladas do músculo tibial anterior de camundongos, e que tal resposta foi dependente da ativação dos receptores \'beta\'2-AR via eixo proteína G&alpha;s-AMPc-PKA. Em relação à dinâmica mitocondrial, os dados obtidos demonstraram que a ativação dos receptores \'beta\'2-AR aumentou a fusão mitocondrial na musculatura esquelética, e que esta resposta estava associada a um aumento da proteína de fusão Mfn1. Na segunda parte do presente estudo, testou-se a hipótese de que a ativação \'beta\'2-AR contribuiria para as respostas mitocondriais agudas induzidas pelo EFA na musculatura esquelética. Para isso, camundongos FVB foram submetidos a uma sessão de EFA em esteira rolante com e sem o bloqueio dos receptores \'beta\'2-AR com o antagonista ICI 118,551, e parâmetros de função e dinâmica mitocondriais foram avaliados. O EFA foi capaz de aumentar a respiração máxima mitocondrial em fibras musculares isoladas, e este efeito foi parcialmente prevenido pelo bloqueio dos receptores \'beta\'2-AR. Além disso, assim como observado com o tratamento com isoproterenol, o EFA promoveu aumento da fusão mitocondrial na musculatura esquelética, sendo que a ativação \'beta\'2-AR estava associada a essa resposta, porém em uma menor magnitude em relação aos efeitos observados com o estímulo farmacológico. Assim como observado no tratamento com isoproterenol, a expressão de Mfn1 foi aumentada pela sessão de EFA, a qual não foi atenuada pelo bloqueio dos receptores \'beta\'2-AR com ICI 118,551. Dessa forma, a partir dos resultados obtidos no presente estudo, pode-se concluir que os receptores \'beta\'2-AR têm papel importante no metabolismo muscular esquelético, modulando a função e a dinâmica mitocondriais. Essa resposta é evidente mediante ativação direta via agonistas \'beta\'-AR no músculo esquelético, no entanto, os receptores \'beta\'2-AR modulam parcialmente as respostas mitocondriais em resposta à uma sessão de EFA / The beneficial effects of aerobic exercise (AE) for prevention and treatment of diseases are due to acute and chronic adaptations in several organ systems, including skeletal muscle, one of the main tissues involved in these adaptations. Since skeletal muscle contains high amounts of mitochondria, the increased aerobic capacity induced by AE occurs mainly due to mitochondrial adaptations. However, the molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by AE are not fully understood. One of the mechanisms by which these mitochondrial adaptations may occur is sympathetic activation mediated by \'beta\'2-adrenergic receptors (\'beta\'-AR) in skeletal muscle, predominantly represented by \'beta\'2 subtype (~90%). Considering that sympathetic activity increases during AE, \'beta\'2-AR signaling might be involved in skeletal muscle mitochondrial adaptations in response to AE. Thus, in the first part of this study, we tested the hypothesis that acute \'beta\'2-AR activation contributes to mitochondrial function and dynamics adaptations in skeletal muscle. For this, both differentiated C2C12 myotubes and FVB mice were treated with the nonselective \'beta\'-AR agonist isoproterenol in the presence or absence of the specific \'beta\'2-AR antagonist, ICI 118,551. The data showed that &beta;-AR activation significantly increased mitochondrial respiration in myotubes and muscle fiber bundles isolated from mice tibialis anterior, and that this response was dependent on \'beta\'2-AR receptors activation via protein G\' alpha\'s-cAMP-PKA signaling cascade. Regarding mitochondrial dynamics, the data showed that \'beta\'2-AR receptor activation increased mitochondrial fusion in skeletal muscle and that this response was associated to an increase in Mfn1. In the second part of this study, we tested the hypothesis that \'beta\'2-AR activation would contribute to acute mitochondrial responses induced by AE in skeletal muscle. For this, FVB mice underwent one session of AE on a treadmill with and without \'beta\'2-AR receptor blockade with ICI 118,551, and parameters of mitochondrial function and dynamics were evaluated. AE was able to increase maximal mitochondrial respiration in isolated muscle fibers, and this effect was partially prevented by blocking \'beta\'2-AR receptors. Furthermore, as observed with isoproterenol treatment, AE increased mitochondrial fusion in skeletal muscle. \'beta\'2-AR activation was associated with this response, but in a smaller magnitude compared to the effects observed with pharmacological stimulation. As observed with the isoproterenol treatment, Mfn1 expression was increased by AE, which was not attenuated by blocking \'beta\'2-AR receptors with ICI 118,551. Therefore, one can conclude that \'beta\'2-AR receptors play an important role in skeletal muscle metabolism, modulating mitochondrial function and dynamics. This response is evident by direct activation through \'beta\'-AR agonists in skeletal muscle, however, \'beta\'2-AR receptors partially modulate mitochondrial responses induced by AE