RECEPTORES b-ADRENÉRGICOS NO SISTEMA CARDIOVASCULAR

b-adrenoceptors (b-AR) are classified as classic b-AR (b1, b2) and atypical b-AR (b3,b4). b-AR are coupled to G protein and enzymes such as adenylyl-cyclase and protein kinase-A(PKA) that is activated by 3´-5´adenosine-monophosphate (cAMP). This b-AR-enzymes complex can directly bind to ionic channels in the membrane, but the main mechanism of action induced by cAMP is the phosphorylation of several proteins via the action of PKA. The activation of b-AR in the endothelial cells increases the Ca+2 cytosolic levels that facilitate Ca+2 binding to calmodulin activating endothelial nitric oxide synthase and the production of nitric oxide. The main mechanism of cAMP in inducing vascular smooth muscle relaxation is due to the phosphorylation via PKA that decreases the cytosolic Ca+2 concentration by several mechanisms that lowers its affinity for calmodulin. Major b-AR agonists are classified as nonselective b-AR (isoprenaline), selective b1-AR (xamoterol) and b2-AR (terbutaline, salbutamol). Several b-AR agonists have been used in clinical situations but they play their main role in the treatment of the patients with asthma or as cardiac stimulant. The development of novel compounds b3-AR agonists as BRL-37344, CGP-12177 and CL-316243 open new pespectives for the treatment of hypertension. Considerable interest has focused on the development of b-AR antagonists due to their efficacy on the treatment of hypertension, cardiac arrhytmias and angina. Efforts for more selective b-AR antagonists have resulted in the synthesis of several compounds as atenolol, bisoprolol, betaxolol, practolol and CFP20712A (b1-AR), ICI 118551 (b2-AR), SR59230A (b3-AR), bupranolol and CGP20712A (b4-AR).Os receptores b-adrenérgicos (b-AR) integram um sistema proteico ternário: b-AR, proteína G de acoplamento e enzimas como a adenilato-ciclase (AC) que produz o 3´-5´monofosfato de adenosina (AMPc). O principal mecanismo de ação do AMPc é a ativação da proteína quinase A (PKA), capaz de fosforilar inúmeros substratos. Em células endoteliais, a ativação dos b-AR promove o aumento dos níveis citoplasmáticos de Ca+2 favorecendo a ligação do Ca+2 com a calmodulina e deste complexo com a enzima óxido nítrico sintase endotelial (eNOS), resultando na produção de NO. A ativaçãob -AR no músculo liso vascular e conseqüente ativação da PKA reduz a concentração citoplasmática de Ca+2 e a sua afinidade pela calmodulina, resultando no relaxamento vascular. Os receptores b-AR podem ser agrupados em b-AR clássicos (b1 e b2) e b-AR atípicos (b3 e b4). Os agonistas b-AR são classificados em não seletivos isoprenalina), seletivos b1 (xamoterol) e seletivos b2 (terbutalina, salbutamol) e podem ser usados em várias situações clínicas, como broncodilatadores ou como estimulantes cardíacos. O desenvolvimento de agonistas b3-AR como BRL-37344, CGP 12177 e CL 316243, bem como de antagonistas b -AR têm merecido especial atenção devido à sua eficácia no tratamento da hipertensão arterial, certas arritmias cardíacas e isquemia cardíaca. A busca por antagonistas seletivos resultou na síntese de vários compostos como: atenolol, bisoprolol, betaxolol, practolol e CFP20712A (antagonistas b1-AR seletivos), ICI 118551 (antagonista b2-AR seletivo), SR59230A (antagonista b3-AR seletivo), bupranolol e CGP20712A (antagonistas b4-AR seletivos). 

Reduced caveolae density in arteries of SHR contributes to endothelial dysfunction and ROS production

Caveolae are plasma membrane invaginations enriched with high cholesterol and sphingolipid content; they also contain caveolin proteins in their structure. Endothelial nitric oxide synthase (eNOS), an enzyme that synthesizes nitric oxide (NO) by converting L-arginine to L-citrulline, is highly concentrated in plasma membrane caveolae. Hypertension is associated with decreased NO production and impaired endothelium-dependent relaxation. Understanding the molecular mechanisms that follow hypertension is important. For this study, we hypothesized that spontaneously hypertensive rat (SHR) vessels should have a smaller number of caveolae, and that the caveolae structure should be disrupted in these vessels. This should impair the eNOS function and diminish NO bioavailability. Therefore, we aimed to investigate caveolae integrity and density in SHR aortas and mesenteric arteries and the role played by caveolae in endothelium-dependent relaxation. We have been able to show the presence of caveolae-like structures in SHR aortas and mesenteric arteries. Increased phenylephrine-induced contractile response after treatment with dextrin was related to lower NO release. In addition, impaired acetylcholine-induced endothelium-dependent relaxation could be related to decreased caveolae density in SHR vessels. The most important finding of this study was that cholesterol depletion with dextrin induced eNOS phosphorylation at Serine1177 (Ser1177) and boosted reactive oxygen species (ROS) production in normotensive rat and SHR vessels, which suggested eNOS uncoupling. Dextrin plus L-NAME or BH4 decreased ROS production in aorta and mesenteric arteries supernatant's of both SHR and normotensive groups. Human umbilical vein endothelial cells (HUVECs) treated with dextrin confirmed eNOS uncoupling, as verified by the reduced eNOS dimer/monomer ratio. BH4, L-arginine, or BH4 plus L-arginine inhibited eNOS monomerization. All these results showed that caveolae structure and integrity are essential for endothelium-dependent relaxation. Additionally, a smaller number of caveolae is associated with hypertension. Finally, caveolae disruption promotes eNOS uncoupling in normotensive and hypertensive rat vessels and in HUVECs.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2016/22180-9, 2016/21239-0, 2017/14797-9]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [400.164/2014-0, 304.137/2014-6]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Biodegradable nanoparticles containing benzopsoralens : an attractive strategy for modifying vascular function in pathological skin disorders

Psoralens are often used to treat skin disorders such as psoriasis, vitiligo and others. The toxicity and fast degradation of these drugs can be diminished by encapsulation in drug delivery systems (DDS). Nanoparticles (NPs) containing the benzopsoralen (BP) (3-ethoxy carbonyl-2H-benzofuro[3,2-e]-1-benzopiran-2-one) were prepared by the solvent evaporation technique, and parameters such as particle size, zeta potential, drug encapsulation efficiency, and external morphology were evaluated. The analysis revealed that the NPs are spherical and possessed a smooth external surface with diameter of 815 ± 80 nm, they present low tendency toward aggregation, as confirmed by their zeta potential (+17.3±2.9 mV) and the encapsulation efficiency obtained was 74%. The intracellular distribution of NPs as well as their uptake by tissues was monitored by using laser confocal microscopy and transmission electron microscopy. The use of benzopsoralen in association with ultraviolet light (360 nm) revealed morphological characteristics of cell damage such as cytosolic vesiculation, mitochondria condensation, and swelling of both the granular endoplasmic reticulum and the nuclear membrane. The primary target of DDS and drugs in vascular system are endothelial cells and an attractive strategy for modifying vascular function in various pathological states of skin disorders, cancer and inflammation. The result presented in this work indicates that PLGA NP could be a promising delivery system for benzopsoralen in connection with ultraviolet irradiation therapy (PUVA) for further application in different therapies.CNPq , CAPES, FAPESP (Braisl

Antiproteinuric and Hyperkalemic Mechanisms Activated by Dual Versus Single Blockade of the RAS in Renovascular Hypertensive Rats

This study aimed to investigate the antiproteinuric and hyperkalemic mechanisms activated by dual renin-angiotensin system (RAS) blockade in renovascular hypertensive rats (2-kidney 1-clip model [2K-1C]). Six weeks after clipping the left renal artery or sham operation (2K), rats were treated with losartan, enalapril, or both drugs for two weeks. We found that 2K-1C rats displayed higher tail-cuff blood pressure (BP), increased non-clipped kidney Ang II concentration, and more pronounced urinary albumin excretion than 2K. BP was decreased by the treatment with either enalapril or losartan, and the combination of both drugs promoted an additional antihypertensive effect in 2K-1C rats. Renal Ang II content and albuminuria were reduced by either enalapril or losartan in monotherapy and restored to control levels by dual RAS blockade. Albuminuria in 2K-1C rats was accompanied by downregulation of the glomerular slit protein podocin, reduction of the endocytic receptors megalin and cubilin, and a marked decrease in the expression of the ClC-5 chloride channel, compared to 2K animals. Treatment with losartan and enalapril in monotherapy or combination increased the expression of podocin, cubilin, and ClC-5. However, only the combined therapy normalized podocin, cubilin, and ClC-5 protein abundance in the non-clipped kidney of 2K-1C rats. Renovascular hypertensive 2K-1C rats had a lower concentration of plasma potassium compared to 2K rats. Single RAS blockade normalized potassium plasma concentration, whereas 2K-1C rats treated with dual RAS blockade exhibited hyperkalemia. Hypokalemia in 2K-1C rats was accompanied by an increase in the cleaved activated forms of α-ENaC and γ-ENaC and the expression of β-ENaC. Combined RAS blockade but not monotherapy significantly reduced the expression of these ENaC subunits in 2K-1C rats. Indeed, double RAS blockade reduced the abundance of cleaved-α-ENaC to levels lower than those of 2K rats. Collectively, these results demonstrate that the antiproteinuric effect of dual RAS blockade in 2K-1C rats is associated with the restored abundance of podocin and cubilin, and ClC-5. Moreover, double RAS blockade-induced hyperkalemia may be due, at least partially, to an exaggerated downregulation of cleaved α-ENaC in the non-clipped kidney of renovascular hypertensive rats

Aortas isolated from sinoaortic-denervated rats exhibit rhythmic contractions that are regulated by pharmacologically distinct calcium sources

Sinoaortic denervation is characterized by arterial pressure lability, without sustained hypertension. Aortas isolated from rats with sinoaortic denervation present rhythmic contractions. We studied the participation of distinct Ca2+ sources in the maintenance of the oscillations. Three days after the surgeries, aortic rings were placed in an organ chamber, and the incidence of aortas presenting rhythmic contractions was measured. Specific drugs were employed to analyse the participation of the Ca2+ released from the sarcoplasmic reticulum [2-APB (diphenylborinic acid 2-aminoethyl ester), thapsigargin and ryanodine] and external Ca2+ entry [Bay K 8644, verapamil and DMB (dimethylbenzyl amiloride)] on the rhythmic contractions. Additionally, we verified the effects of chloride channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)benzoic acid] on the maintenance of the rhythmic contractions. Under phenylephrine stimulus, sinoaortic-denervated rat aortas exhibited rhythmic contractions in the frequency of 4.5 +/- 0.50 cycles/min. and an amplitude of 0.465 +/- 0.05 g. 2-APB, thapsigargin and ryanodine inhibited the rhythmic contractions. Bay K 8644 increased the oscillations, reaching maximum values with a concentration of 50 nM (18.5 +/- 2.5 cycles/min.). The rhythmic contractions were inhibiting by verapamil and Ca2+-free solution. DMB and NPPB did not alter the oscillations. In conclusion, we observed that aorta isolated from sinoaortic-denervated rats present rhythmic contractions. Moreover, drugs that impaired intracellular Ca2+ release from sarcoplasmic reticulum interrupted the oscillations. The oscillations also depend on the extracellular Ca2+ entry through L-type Ca2+

Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.Fundacao de Amparo A Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Ruthenium-nitrite complex as pro-drug releases NO in a tissue and enzyme-dependent way

Nitric oxide (NO) plays an important role in the control of the vascular tone and the most often employed NO donors have limitations due to their harmful side-effects. In this context, new NO donors have been prepared, in order to minimize such undesirable effects. cis-[Ru(bpy)(2)(py)NO(2)](PF(6)) (RuBPY) is a new nitrite complex synthesized in our laboratory that releases NO in the presence of the vascular tissue only. In this work the vasorelaxation induced by this NO donor has been studied and compared to that obtained with the well known NO donor SNP. The relaxation induced by RuBPY is concentration-dependent in denuded rat aortas pre-contracted with phenylephrine (EC(50)). This new compound induced relaxation with efficacy similar to that of SNP, although its potency is lower. The time elapsed until maximum relaxation is achieved (E(max) = 240 s) is similar to measured for SNP (210 s). Vascular reactivity experiments demonstrated that aortic relaxation by RuBPY is inhibited by the soluble guanylyl-cyclase inhibitor 1H-[1,2,4] oxadiozolo[4,3-a]quinoxaline-1-one (ODQ 1 mu M). In a similar way, 1 mu M ODQ also reduces NO release from the complex as measured with DAF-2 DA by confocal microscopy. These findings suggest that this new complex RuBPY that has nitrite in its structure releases NO inside the vascular smooth muscle cell. This ruthenium complex releases significant amounts of NO only in the presence of the aortic tissue. Reduction of nitrite to NO is most probably dependent on the soluble guanylyl-cyclase enzyme, since NO release is inhibited by ODQ. (C) 2011 Elsevier Inc. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)US National Science Foundation (NSF