Telmisartán y losartán: las marcadas diferencias entre sus propiedades químicas y farmacológicas pueden explicar la diferencia en la eficacia terapéutica en pacientes hospitalizados con covid-19 / Telmisartan and losartan: marked differences in chemical and pharmacological properties may explain differences in therapeutic efficacy in hospitalized patients with COVID-19

El objetivo de este artículo es comparar las propiedades químicas y farmacológicas del telmisartán y el losartán, y su metabolito activo EXP3174, con el fin de entender por qué el telmisartán es efectivo en pacientes hospitalizados con Covid-19 mientras que el losartán no lo es. Se llevó a cabo una revisión bibliográfica exhaustiva de las propiedades químicas, farmacocinéticas y farmacodinámicas de ambos fármacos y se destacaron las diferencias más importantes que podrían estar relacionadas con su efectividad en pacientes con Covid-19. Se concluyó que las propiedades farmacológicas del telmisartán, como su mayor afinidad por el receptor AT1, su duración de acción prolongada y su capacidad para modular la inflamación podrían explicar su efectividad en pacientes con Covid-19. Por otro lado, las propiedades farmacológicas del losartán, como su menor afinidad por el receptor AT1 y su rápido metabolismo, pueden limitar su efectividad en pacientes con Covid-19. Estos resultados resaltan la importancia de comprender las propiedades químicas y farmacológicas de los medicamentos para identificar posibles candidatos terapéuticos efectivos en el tratamiento de Covid-19. (AU)

The objective of this article is to compare the chemical and pharmacological properties of telmisartan and losartan and their active metabolite EXP3174 to understand why telmisartan is effective in hospitalized patients with COVID-19 while losartan is not. A comprehensive literature review of the chemical, pharmacokinetic and pharmacodynamic properties of both drugs was done to highlight the most important differences that may be related to their efficacy in patients with COVID-19. It was concluded that the pharmacological properties of telmisartan, such as its higher affinity for the AT1 receptor, its long duration of action and its ability to modulate inflammation, could explain its efficacy in patients with COVID-19. On the other hand, the pharmacological properties of losartan, such as its lower affinity for the AT1 receptor and its rapid metabolism, may limit its efficacy in patients with COVID-19. These results highlight the importance of understanding the chemical and pharmacological properties of drugs to identify potential effective therapeutic candidates for the treatment of COVID-19. (AU)

Renin-Angiotensin System Inhibitors in Patients With COVID-19: A Meta-Analysis of Randomized Controlled Trials Led by the International Society of Hypertension.

Background Published randomized controlled trials are underpowered for binary clinical end points to assess the safety and efficacy of renin-angiotensin system inhibitors (RASi) in adults with COVID-19. We therefore performed a meta-analysis to assess the safety and efficacy of RASi in adults with COVID-19. Methods and Results MEDLINE, EMBASE, ClinicalTrials.gov, and the Cochrane Controlled Trial Register were searched for randomized controlled trials that randomly assigned patients with COVID-19 to RASi continuation/commencement versus no RASi therapy. The primary outcome was all-cause mortality at ≤30 days. A total of 14 randomized controlled trials met the inclusion criteria and enrolled 1838 participants (aged 59 years, 58% men, mean follow-up 26 days). Of the trials, 11 contributed data. We found no effect of RASi versus control on all-cause mortality (7.2% versus 7.5%; relative risk [RR], 0.95; [95% CI, 0.69-1.30]) either overall or in subgroups defined by COVID-19 severity or trial type. Network meta-analysis identified no difference between angiotensin-converting enzyme inhibitors versus angiotensin II receptor blockers. RASi users had a nonsignificant reduction in acute myocardial infarction (2.1% versus 3.6%; RR, 0.59; [95% CI, 0.33-1.06]), but increased risk of acute kidney injury (7.0% versus 3.6%; RR, 1.82; [95% CI, 1.05-3.16]), in trials that initiated and continued RASi. There was no increase in need for dialysis or differences in congestive cardiac failure, cerebrovascular events, venous thromboembolism, hospitalization, intensive care admission, inotropes, or mechanical ventilation. Conclusions This meta-analysis of randomized controlled trials evaluating angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers versus control in patients with COVID-19 found no difference in all-cause mortality, a borderline decrease in myocardial infarction, and an increased risk of acute kidney injury with RASi. Our findings provide strong evidence that RASi can be used safely in patients with COVID-19.

Telmisartan for treatment of Covid-19 patients: An open multicenter randomized clinical trial.

Background: Angiotensin receptor blockers (ARBs), such as telmisartan, have been postulated to treat Covid-19-induced lung inflammation. Methods: This is a parallel-group, randomized, two-arm, open-label, adaptive, multicenter superiority trial with 1:1 allocation ratio. Participants included patients from 18 years of age hospitalized with Covid-19 with 4 or fewer days since symptom onset enrolled at a university and a community hospital in Buenos Aires, Argentina. Exclusion criteria included prior intensive care unit (ICU) admission and use of ARBs/angiotensin converting enzyme inhibitors at randomization. Control arm received standard care alone and treatment arm telmisartan 80 mg twice daily for 14 days. Primary outcomes were C-reactive protein (CRP) plasma levels at day 5 and 8 after randomization. Secondary outcomes included time to discharge within 15 days, admission to ICU and death at 15- and 30-days. NCT04355936 (Completed). Findings: A pragmatic decision to end the study before the third interim analysis was made on Oct. 30th due to sharp reduction in recruitment. A total of 162 patients were randomized. 158 patients enrolled between May 14 and October 30 2020, were included in the analysis, 80 in the standard care and 78 in the telmisartan added to standard care group. Baseline absolute CRP serum levels were 5.53 ± 6.19 mg/dL (95% CI 6.91 to 4.15, n = 80) and 9.04 ± 7.69 (95% CI 9.04 to 10.82, n = 74) in the standard care and telmisartan added to standard care groups, respectively. Day 5 control-group CRP levels were 6.06 ± 6.95 mg/dL (95% CI 7.79-4.35, n = 66) while telmisartan group were 3.83 ± 5.08 mg/dL (95% CI 5.08-2.59, n = 66, p = 0.038). Day 8 CRP levels were 6.30 ± 8.19 mg/dL (95% CI 8.79-3.81, n = 44) and 2.37 ± 3.47 mg/dL (95% CI 3.44-1.30, n = 43, p = 0.0098) in the control and telmisartan groups, respectively (all values expressed as mean ± SD). Kaplan-Meier analysis showed that telmisartan-treated patients had a lower median time-to-discharge (control=15 days; telmisartan=9 days). Death by day 30 was reduced in the telmisartan-treated group (control 22.54%, 16/71; telmisartan 4.29%, 3/70 participants; p = 0.0023). Composite ICU, mechanical ventilation or death was reduced by telmisartan treatment at days 15 and 30. No adverse events were reported. Interpretation: Our study suggests that the ARB telmisartan, a widely used antihypertensive drug, is safe and could reduce morbidity and mortality in hospitalized patients infected with SARS -CoV-2 by anti-inflammatory effects. Further studies employing telmisartan are needed for confirmation of our results and to define its true therapeutic value as a tool against Covid-19.

Angiotensin Receptor Blockers for COVID-19: Pathophysiological and Pharmacological Considerations About Ongoing and Future Prospective Clinical Trials.

COVID-19 pandemic demands a swift response to find therapeutic tools that effectively reduce morbidity and mortality. Despite initial fears, evidence from retrospective observational studies supports the inhibition of the renin-angiotensin system as an emerging pathway to delay or moderate angiotensin II-driven lung inflammation. This has triggered several prospective clinical trials. In this commentary we provide an overview and analysis of current ongoing clinical trials aimed at evaluating the therapeutic efficacy of angiotensin receptor blocker (ARB) use in COVID-19. The relevance of the results of these trials will have to be interpreted depending on the stage and severity of the disease and in light of the start time of their prescription related to the time of diagnosis of COVID-19 as well as the administered doses.

Telmisartan as tentative angiotensin receptor blocker therapeutic for COVID-19.

In late 2019, a new coronavirus emerged in Wuhan Province, China, causing lung complications similar to those produced by the SARS coronavirus in the 2002-2003 epidemic. This new disease was named COVID-19 and the causative virus SARS-CoV-2. The SARS-CoV-2 virus enters the airway and binds, by means of the S protein on its surface to the membrane protein ACE2 in type 2 alveolar cells. The S protein-ACE2 complex is internalized by endocytosis leading to a partial decrease or total loss of the enzymatic function ACE2 in the alveolar cells and in turn increasing the tissue concentration of pro-inflammatory angiotensin II by decreasing its degradation and reducing the concentration of its physiological antagonist angiotensin 1-7. High levels of angiotensin II on the lung interstitium can promote apoptosis initiating an inflammatory process with release of proinflammatory cytokines, establishing a self-powered cascade, leading eventually to ARDS. Recently, Gurwitz proposed the tentative use of agents such as losartan and telmisartan as alternative options for treating COVID-19 patients prior to development of ARDS. In this commentary article, the authors make the case for the election of telmisartan as such alternative on the basis of its pharmacokinetic and pharmacodynamic properties and present an open-label randomized phase II clinical trial for the evaluation of telmisartan in COVID-19 patients (NCT04355936).

Macrophage function in tissue repair and remodeling requires IL-4 or IL-13 with apoptotic cells.

Tissue repair is a subset of a broad repertoire of interleukin-4 (IL-4)- and IL-13-dependent host responses during helminth infection. Here we show that IL-4 or IL-13 alone was not sufficient, but IL-4 or IL-13 together with apoptotic cells induced the tissue repair program in macrophages. Genetic ablation of sensors of apoptotic cells impaired the proliferation of tissue-resident macrophages and the induction of anti-inflammatory and tissue repair genes in the lungs after helminth infection or in the gut after induction of colitis. By contrast, the recognition of apoptotic cells was dispensable for cytokine-dependent induction of pattern recognition receptor, cell adhesion, or chemotaxis genes in macrophages. Detection of apoptotic cells can therefore spatially compartmentalize or prevent premature or ectopic activity of pleiotropic, soluble cytokines such as IL-4 or IL-13.

Gene Expression Architecture of Mouse Dorsal and Tail Skin Reveals Functional Differences in Inflammation and Cancer.

Inherited germline polymorphisms can cause gene expression levels in normal tissues to differ substantially between individuals. We present an analysis of the genetic architecture of normal adult skin from 470 genetically unique mice, demonstrating the effect of germline variants, skin tissue location, and perturbation by exogenous inflammation or tumorigenesis on gene signaling pathways. Gene networks related to specific cell types and signaling pathways, including sonic hedgehog (Shh), Wnt, Lgr family stem cell markers, and keratins, differed at these tissue sites, suggesting mechanisms for the differential susceptibility of dorsal and tail skin to development of skin diseases and tumorigenesis. The Pten tumor suppressor gene network is rewired in premalignant tumors compared to normal tissue, but this response to perturbation is lost during malignant progression. We present a software package for expression quantitative trait loci (eQTL) network analysis and demonstrate how network analysis of whole tissues provides insights into interactions between cell compartments and signaling molecules.

Involvement of Extracellular Signal-Regulated Kinase 5 in Kinin B1 Receptor Upregulation in Isolated Human Umbilical Veins.

The upregulated kinin B1 receptors exert a pivotal role in modulating inflammatory processes. In isolated human umbilical veins (HUVs), kinin B1 receptor is upregulated as a function of in vitro incubation time and proinflammatory stimuli. The aim of this study was to evaluate, using functional and biochemical methods, the involvement of extracellular signal-regulated kinase 5 (ERK5), p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2) on the kinin B1 receptor upregulation process in HUV. Real-time polymerase chain reaction analysis revealed for the first time that kinin B1 receptor mRNA expression closely parallels the functional sensitization to kinin B1 receptor selective agonist des-Arg(10)-kallidin (DAKD) in HUV. Moreover, the selective inhibition of ERK5, p38 MAPK, and JNK, but not ERK1/2, produced a dose-dependent rightward shift of the concentration-response curves to DAKD after 5-hour incubation and a reduction in kinin B1 receptor mRNA expression. Biochemical analyses showed that ERK5, p38 MAPK, and JNK phosphorylation is maximal during the first 2 hours postisolation, followed by a significant reduction in the last 3 hours. None of the treatments modified the responses to serotonin, an unrelated agonist, suggesting a specific effect on kinin B1 receptor upregulation. The present work provides for the first time pharmacologic evidence indicating that ERK5 plays a significant role on kinin B1 receptor upregulation. Furthermore, we confirm the relevance of p38 MAPK and JNK as well as the lack of effect of ERK1/2 in this process. This study may contribute to a better understanding of MAPK involvement in inflammatory and immunologic diseases.

Comparación de la estratificación de riesgo de recurrencia de cáncer de mama con score ihc4+c y score de recurrencia de 21 genes. Estudio retrospectivo de evaluación de impacto teórico en toma de decisión / Comparisson of distant recurrence risk stratification in breast cancer with IHC4+C and 21-gene Recurrence Score. A retrospective theoretical study on decision making

El incremento del rastreo del cáncer de mama ha producido un aumento en el diagnóstico de tumores en estadios tempranos. Mientras que los tumores que expresan receptores hormonales son tratados con hormonoterapia, la decisión acerca del uso de quimioterapia citotóxica es más compleja. En pacientes seleccionados, el test multigénico de 21 genes ha demostrado ser pronóstico y predictivo del beneficio del tratamiento con citotóxicos. En forma más reciente, se ha desarrollado un algoritmo clínico-patológico denominado ihc4+c que ha demostrado ser equivalente a la plataforma de 21 genes en los estudios iniciales, a un costo menor. Sin embargo, existe escasa experiencia internacional y nula experiencia local en este campo. Objetivo: El objetivo del presente estudio es evaluar retrospectivamente el riesgo de recurrencia a distancia a 10 años provisto por ihc4+c y Onwcotype dx. Material y método: Se evaluó en forma retrospectiva el score ihc4+c en 25 pacientes con tumores receptores hormonales positivos de las cuales se disponía del score provisto por Oncotype DX. Resultados: Se encontró una coincidencia en la estratificación de riesgo global del 76%. En un 20%, ihc4+c determinó una disminución, mientras que en un 4% resultó en un aumento de la categoría de riesgo. Discusión: El presente estudio, primero en su tipo en la Argentina, demostró una concordancia global alta entre ihc4+c y Oncotype dx comparable a otros estudios publicados...

Identification of Hipk2 as an essential regulator of white fat development.

Homeodomain-interacting protein kinase 2 (Hipk2) has previously been implicated in the control of several transcription factors involved in embryonic development, apoptosis, cell proliferation, and tumor development, but very little is understood about the exact mechanisms through which Hipk2 influences these processes. Analysis of gene expression in normal tissues from genetically heterogeneous mouse or human populations can reveal network motifs associated with the structural or functional components of the tissue, and may predict roles for genes of unknown function. Here we have applied this network strategy to uncover a role for the Hipk2 gene in the transcriptional system controlling adipogenesis. Both in vitro and in vivo models were used to show that knockdown or loss of Hipk2 specifically inhibits white adipose cell differentiation and tissue development. In addition, loss of Hipk2 leads to induction of pockets of multilocular brown fat-like cells in remaining white adipose depots, which express markers of brown and beige fat such as uncoupling protein 1 and transmembrane protein 26. These changes are accompanied by increased insulin sensitivity in Hipk2 knockout mice and reduced high-fat diet-induced weight gain, highlighting a potential role for this kinase in diseases such as diabetes and obesity. Our study underscores the versatility and power of a readily available tissue, such as skin, for network modeling of systemic transcriptional programs involved in multiple pathways, including lipid metabolism and adipogenesis.

Expression and functional evidence of the prostaglandin F2alpha receptor mediating contraction in human umbilical vein.

Our purposes were to perform the pharmacological characterization of PGF(2alpha) receptor (prostanoid FP-receptor) involved in human umbilical vein contraction and confirm its expression in this tissue. Umbilical cords from healthy patients after full-term deliveries were employed. The vein was dissected out of cords and used for either isolated organ bath or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assays. The natural prostanoid FP-receptor agonist, PGF(2alpha), and its selective analogues, latanoprost and bimatoprost free acids are full agonists (produce more than 80% of the maximal contractile response to 5-HT) in human umbilical vein. The agonist potency (pEC(50)) order was PGF(2alpha) (6.01+/-0.05)>latanoprost free acid (5.65+/-0.07)=bimatoprost free acid (5.59+/-0.08). The contractile effects of PGF(2alpha) and latanoprost free acid were blocked competitively by the prostanoid FP-receptor antagonist, AL-8810. The antagonist potencies (pK(B)) of AL-8810 vs. PGF(2alpha) (5.93+/-0.05) and vs. latanoprost free acid (6.40+/-0.08) in human umbilical vein are in good agreement with its ability to antagonize prostanoid FP receptors of rat, mouse and human cells. In all samples, clear signal was detected for cDNA amplification of prostanoid FP receptor and the specific prostanoid FP-receptor antibody recognized a protein of approximately 64 kDa. In conclusion, taking into account the obtained functional and biochemical data, we propose for the first time that human umbilical vein express prostanoid FP-receptors and these receptors could be involved in the vasoconstriction action of PGF(2alpha) in this tissue.

Genetic architecture of mouse skin inflammation and tumour susceptibility.

Germline polymorphisms in model organisms and humans influence susceptibility to complex trait diseases such as inflammation and cancer. Mice of the Mus spretus species are resistant to tumour development, and crosses between M. spretus and susceptible Mus musculus strains have been used to map locations of genetic variants that contribute to skin cancer susceptibility. We have integrated germline polymorphisms with gene expression in normal skin from a M. musculus x M. spretus backcross to generate a network view of the gene expression architecture of mouse skin. Here we demonstrate how this approach identifies expression motifs that contribute to tissue organization and biological functions related to inflammation, haematopoiesis, cell cycle control and tumour susceptibility. Motifs associated with inflammation, epidermal barrier function and proliferation are differentially regulated in backcross mice susceptible or resistant to tumour development. The intestinal stem cell marker Lgr5 is identified as a candidate master regulator of the hair follicle, and the vitamin D receptor (Vdr) is linked to coordinated control of epidermal barrier function, inflammation and tumour susceptibility.

The endocannabinoid anandamide inhibits kinin B1 receptor sensitization through cannabinoid CB1 receptor stimulation in human umbilical vein.

The possible inhibition of kinin B(1) receptor up-regulation by arachidonoylethanolamide (anandamide) was evaluated in isolated human umbilical vein. Anandamide and its metabolically stable analogue, R-N-(2-Hydroxy-1-methylethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (R-(+)-methanandamide), produced a selective and dose-dependent inhibition of kinin B(1) receptor-sensitized contractile responses. The inhibitory effect of anandamide on B(1) receptor-sensitized responses failed to be modified either by 5-biphenyl-4-ylmethyl-tetrazole-1-carboxylic acid dimethylamide (LY2183240), a selective anandamide uptake inhibitor, or 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-y l](4-methoxyphenyl) methanone (AM630), selective cannabinoid CB(2) receptor antagonist. However, the cannabinoid CB(1) receptor antagonist, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophen yl)-4-methyl-1H-pyrazole-3-carboxamide (AM251), abolished anandamide effects on kinin B(1) receptor sensitization. The present results provide strong pharmacological evidence indicating that endocannabinoid anandamide inhibits kinin B(1) receptor up-regulation through cannabinoid CB(1) receptor stimulation in human umbilical vein.

Potentiation of adrenaline vasoconstrictor response by sub-threshold concentrations of U-46619 in human umbilical vein: involvement of smooth muscle prostanoid TP(alpha) receptor isoform.

Considering the potential physiological, pharmacological and therapeutic relevance of synergistic interaction of thromboxane A(2) with adrenaline at postjunctional receptor sites, we examined whether sub-threshold concentrations of thromboxane A(2) mimetic U-46619 (9,11-dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha)) could amplify adrenaline-induced contraction in human umbilical vein. The receptor involved in U-46619-induced potentiation of adrenaline contractility was also investigated. Umbilical cords (n=125) from healthy patients after full-term vaginal or caesarean deliveries were employed. The vein was dissected out of cords and rings used for isolated organ bath experiments or reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Presence of endothelium did not modify U-46619-induced contraction in human umbilical vein. Prostanoid TP-selective receptor antagonist, SQ-29548 (7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-[1S(1alpha,2alpha(Z),3alpha,4alpha)]-5-Heptenoic acid), inhibited U-46619-induced contraction (pA(2)=8.22+/-0.11). U-46619 sub-threshold concentrations (0.1-0.3 nM) potentiated adrenaline-vasoconstriction response in a concentration-dependent manner. SQ-29548 (0.1 microM) abolished this potentiation. Using RT-PCR, we found that human umbilical vein rings with or without endothelium express the prostanoid TP(alpha), but not the prostanoid TP(beta) receptor isoform. Western blot allowed the identification of proteins with an electrophoretic mobility (47- and 55-kDa) indistinguishable from human platelet prostanoid TP receptor, a rich source of prostanoid TP(alpha) receptor isoform. Collectively, present results demonstrate that prostanoid TP(alpha) is the major receptor isoform localized on smooth muscle cells which participate in both direct vasoconstriction and potentiating effects of U-46619 on adrenaline contractions in human umbilical vein. These results suggest that thromboxane A(2) may interact synergistically with adrenaline in pathophysiological situations that lead to an increase of its umbilical venous levels (e.g. preeclampsia associated with fetal distress) raising the possibility of vasoconstriction affecting fetal blood flow.

Functional evidence of des-Arg10-kallidin enzymatic inactivating pathway in isolated human umbilical vein.

It has been known for many years that plasma and tissues contain a variety of enzymes capable of metabolizing kinins. The aim of the present study was to evaluate, by means of functional studies in a capacitance vessel such as the human umbilical vein (HUV), the possible role played by the metallopeptidases angiotensin-converting enzyme (ACE), neutral endopeptidase (NEP), and aminopeptidase M (APM) as an inactivating pathway of the B(1) receptor endogenous agonist des-Arg(10)-kallidin (DAKD). In HUV rings with and without endothelium, concentration-response curves (CRCs) to DAKD were determined after a 300-min incubation period, and enzymatic inhibitors were added to the organ baths 30 min before construction of the CRC. Presence of endothelial layer was confirmed by histological studies. There was a significant leftward shift observed in control HUV rings devoid of endothelium compared with intact tissues. Exposure to 1 microM captopril (ACE inhibitor) potentiated DAKD-elicited vasoconstrictor responses in HUV rings with endothelium while no such effect was observed in tissues devoid of endothelium. Application of 10 microM amastatin (APM inhibitor) induced a leftward shift of DAKD-elicited contractile responses in HUV with and without endothelium. On the other hand, 10 microM phosphoramidon (NEP inhibitor) showed no potentiating effect in HUV rings either with or without endothelium. However, under concurrent inhibition of ACE, NEP and APM, there was a higher potentiation of DAKD-elicited contractile responses compared with the effect observed with combined inhibition of ACE and APM. Moreover, when we evaluated contractile responses induced by Sar(0)-D-Phe(8)-des-Arg(9)-BK (a metabolically protected B(1) receptor agonist), no potentiating effect was observed under triple enzymatic inhibition. In conclusion, in the present study for the first time, we demonstrated in a capacitance vessel, HUV, that metallopeptidases ACE, NEP and APM represent a relevant functional inactivation pathway of DAKD.

Neutral endopeptidase up-regulation in isolated human umbilical artery: involvement in desensitization of bradykinin-induced vasoconstrictor effects.

Previous reports show that bradykinin B(2) receptors mediate contractile responses induced by bradykinin (BK) in human umbilical artery (HUA). However, although it has been reported that BK-induced responses can desensitize in several inflammatory models, the effects of prolonged in vitro incubation on BK-induced vasoconstriction in HUA have not been studied. In isolated HUA rings, BK-induced responses after a 5-h in vitro incubation showed a marked desensitization compared with responses at 2 h. Inhibition of either angiotensin-converting enzyme (ACE) or neutral endopeptidase (NEP), both BK-inactivating enzymes, failed to modify responses to BK at 2 h. After 5 h, ACE inhibition produced only a slight potentiation of BK-induced responses. In contrast, BK-induced vasoconstriction at 5 h was markedly potentiated by NEP inhibition. Moreover, NEP activity, measured by hydrolysis of its synthetic substrate (Z-Ala-Ala-Leu-p-nitroanilide), showed a 2.4-fold increase in 5-h incubated versus 2-h incubated tissues, which was completely reversed by cycloheximide (CHX) treatment. Furthermore, CHX significantly potentiated BK-induced responses, suggesting that NEP-mediated kininase activity increase at 5 h depends on de novo protein synthesis. In addition, under NEP inhibition, CHX treatment failed to produce an additional potentiation of BK-induced vasoconstriction. Still, NEP up-regulation was confirmed by Western blot, showing a 2.1-fold increase in immunoreactive NEP in 5-h incubated versus 2-h incubated HUA. In summary, the present study provides strong pharmacological evidence that NEP is up-regulated and plays a key role in desensitization of BK-induced vasoconstriction after prolonged in vitro incubation in HUA. Our results provide new insights into the possible mechanisms involved in BK-induced response desensitization during sustained inflammatory conditions.