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1.
Int J Mol Sci ; 23(19)2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-36232616

RESUMO

This study was designed to connect aortic stiffness to vascular contraction in young male and female Wistar rats. We hypothesized that female animals display reduced intrinsic media-layer stiffness, which associates with improved vascular function. Atomic force microscopy (AFM)-based nanoindentation analysis was used to derive stiffness (Young's modulus) in biaxially (i.e., longitudinal and circumferential) unloaded aortic rings. Reactivity studies compatible with uniaxial loading (i.e., circumferential) were used to assess vascular responses to a selective α1 adrenergic receptor agonist in the presence or absence of extracellular calcium. Elastin and collagen levels were indirectly evaluated with fluorescence microscopy and a picrosirius red staining kit, respectively. We report that male and female Wistar rats display similar AFM-derived aortic media-layer stiffness, even though female animals withstand higher aortic intima-media thickness-to-diameter ratio than males. Female animals also present reduced phenylephrine-induced aortic force development in concentration-response and time-force curves. Specifically, we observed impaired force displacement in both parts of the contraction curve (Aphasic and Atonic) in experiments conducted with and without extracellular calcium. Additionally, collagen levels were lower in female animals without significant elastin content and fragmentation changes. In summary, sex-related functional differences in isolated aortas appear to be related to dissimilarities in the dynamics of vascular reactivity and extracellular matrix composition rather than a direct response to a shift in intrinsic media-layer stiffness.


Assuntos
Elastina , Rigidez Vascular , Agonistas Adrenérgicos , Animais , Cálcio , Espessura Intima-Media Carotídea , Colágeno , Feminino , Masculino , Fenilefrina/farmacologia , Ratos , Ratos Wistar
2.
Biomolecules ; 12(8)2022 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-36009019

RESUMO

Aging impairs the expression of HSP70, an emergent player in vascular biology. However, it is unknown if age-related alterations in HSP70 are linked to a decline in arterial function. In this study, we test the hypothesis that the contributions of HSP70 to vascular contraction are diminished in middle-aged animals. We determined the basal levels of HSP70 in the aorta of young and middle-aged Sprague Dawley male rats using Western blotting. Functional studies were performed in a wire myograph system. Force development in response to phenylephrine was assessed in the presence or absence of extracellular calcium (Ca2+), and in aortic rings treated or non-treated with an HSP70 inhibitor. Fluorescent probes were used to evaluate vascular oxidative stress and nitric oxide levels. We report that middle-aged rats have significantly lower levels of HSP70. Blockade of HSP70 attenuated vascular phasic and tonic contraction in isolated aortas. It appears that a functional HSP70 is required for proper Ca2+ handling as inhibition of this protein led to reduced force-displacement in response to Ca2+ dynamics. Furthermore, middle-aged aortic rings exposed to the HSP70 inhibitor display higher reactive oxygen species levels without changes in nitric oxide. In summary, we show that middle-aged animals have lower levels of HSP70 in aortas, which associates with an age-related decline in vascular responses to α-1 adrenergic stimulation.


Assuntos
Aorta , Óxido Nítrico , Animais , Aorta/metabolismo , Proteínas de Choque Térmico HSP70 , Masculino , Óxido Nítrico/metabolismo , Fenilefrina/farmacologia , Ratos , Ratos Sprague-Dawley
4.
Sci Rep ; 11(1): 1420, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446873

RESUMO

Heat-shock protein 70 (HSP70) is a ubiquitously expressed molecular chaperone with various biological functions. Recently, we demonstrated that HSP70 is key for adequate vascular reactivity. However, the specific mechanisms targeted by HSP70 to assist in this process remain elusive. Since there is a wealth of evidence connecting HSP70 to calcium ([Formula: see text]), a master regulator of contraction, we designed this study to investigate whether blockade of HSP70 disrupts vascular contraction via impairment of [Formula: see text] handling mechanisms. We performed functional studies in aortas isolated from male Sprague Dawley rats in the presence or absence of exogenous [Formula: see text], and we determined the effects of VER155008, an inhibitor of HSP70, on [Formula: see text] handling as well as key mechanisms that regulate vascular contraction. Changes in the intracellular concentration of [Formula: see text] were measured with a biochemical assay kit. We report that blockade of HSP70 leads to [Formula: see text] mishandling in aorta stimulated with phenylephrine, decreasing both phasic and tonic contractions. Importantly, in [Formula: see text] free Krebs' solution, inhibition of HSP70 only reduced the [Formula: see text] of the phasic contraction if the protein was blocked before IP3r-mediated [Formula: see text] release, suggesting that HSP70 has a positive effect towards this receptor. Corroborating this statement, VER155008 did not potentiate an IP3r inhibitor's outcomes, even with partial blockade. In another set of experiments, the inhibition of HSP70 attenuated the amplitude of the tonic contraction independently of the moment VER155008 was added to the chamber (i.e., whether it was before or after IP3r-mediated phasic contraction). More compelling, following re-addition of [Formula: see text], VER155008 amplified the inhibitory effects of a voltage-dependent [Formula: see text] channel blocker, but not of a voltage-independent [Formula: see text] channel inhibitor, indicating that HSP70 has a positive impact on the latter. Lastly, the mechanism by which HSP70 modulates vascular contraction does not involve the [Formula: see text] sensitizer protein, Rho-kinase, nor the SERCA pump, as blockade of these proteins in the presence of VER155008 almost abolished contraction. In summary, our findings shed light on the processes targeted by HSP70 during vascular contraction and open research avenues for potential new mechanisms in vascular diseases.


Assuntos
Aorta/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Vasoconstrição , Animais , Masculino , Ratos , Ratos Sprague-Dawley
5.
Life Sci ; 191: 219-226, 2017 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-29032115

RESUMO

AIM: Angiotensin II (AngII), a corpus cavernosum (CC) constrictor peptide, modulates Toll like receptor (TLR) expression, a key element of the innate immune system, contributing to impaired vascular function in pathological conditions. However, it is unknown whether TLR4 is involved in AngII-induced erectile dysfunction. In this study, we investigated whether TLR4 plays a role in cavernosal dysfunction caused by AngII upregulation. MATERIAL AND METHODS: Cavernosal smooth muscle cells (CSMC) from C57/BL6 mice were treated with AngII (0.1µM) or bacterial LPS (50ng/ml) for 12-24h and TLR4 expression was assessed. Mice were infused with AngII (90ng/min, 28days) and treated with anti-TLR4 antibody (0.1mg/daily, i.p.) for the last 14days of the treatment. CC tissue was used for functional studies and for Western blotting. Nitric Oxide Synthase (NOS) activity was measured by conversion of [3H]-l-arginine to [3H]-l-citrulline, systemic TNF-α levels by ELISA, and reactive oxygen species (ROS) by immunofluorescence. KEY FINDINGS: We report upregulation of TLR4 in CSMC following AngII or LPS stimulation. In AngII-infused mice, chronic treatment with anti-TLR4 antibody (28±2.1%) attenuates adrenergic CC contraction, which also ameliorates nitrergic (68.90±0.21 vs. 51.07±0.63, 8Hz, AngII-infused mice treated vs. non-treated). Decreased endothelial NOS expression, reduced NOS activity, and augmented levels of TNF-α, and ROS were found following AngII-infusion. These alterations were prevented, or at least decreased by anti-TLR4 antibody treatment. SIGNIFICANCE: Inhibition of TLR4 ameliorates AngII-impaired cavernosal relaxation, decreases TNF-α levels, and restores NO bioavailability, demonstrating that TLR4 partly mediates AngII-induced cavernosal dysfunction.


Assuntos
Angiotensina II/imunologia , Disfunção Erétil/imunologia , Óxido Nítrico/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Pressão Sanguínea , Disfunção Erétil/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/patologia , Óxido Nítrico Sintase Tipo III/imunologia , Pênis/imunologia , Pênis/fisiopatologia , Fator de Necrose Tumoral alfa/imunologia
6.
Diabetes ; 65(12): 3754-3764, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27650857

RESUMO

Diabetic bladder dysfunction (DBD) is a common urological complication of diabetes. Innate immune system activation via Toll-like receptor 4 (TLR4) leads to inflammation and oxidative stress and was implicated in diabetes pathophysiology. We hypothesized that bladder hypertrophy and hypercontractility in DBD is mediated by TLR4 activation. Wild-type (WT) and TLR4 knockout (TLR4KO) mice were made diabetic by streptozotocin (STZ) treatment, and bladder contractile function and TLR4 pathway expression were evaluated. Immunohistochemistry confirmed the expression of TLR4 in human and mouse bladder. Recombinant high-mobility group box protein 1 (HMGB1) increased bladder TLR4 and MyD88 expression and enhanced contractile response to electrical field stimulation. Bladder expression of TLR4 and MyD88 and serum expression of HMGB1 were increased in STZ compared with control mice. Carbachol (CCh)-mediated contraction was increased in bladders from STZ mice, and TLR4 inhibitor CLI-095 attenuated this increase. Induction of diabetes by STZ in WT mice increased bladder weight and contractile responses to CCh and to electrical field stimulation. TLR4KO mice were not protected from STZ-induced diabetes; however, despite levels of hyperglycemia similar to those of WT STZ mice, TLR4KO STZ mice were protected from diabetes-induced bladder hypertrophy and hypercontractility. These data suggest that TLR4 activation during diabetes mediates DBD-associated bladder hypertrophy and hypercontractility.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Nefropatias Diabéticas/metabolismo , Receptor 4 Toll-Like/metabolismo , Bexiga Urinária/metabolismo , Animais , Carbacol/farmacologia , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animais de Doenças , Proteína HMGB1/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/genética , Bexiga Urinária/efeitos dos fármacos
7.
PLoS One ; 8(8): e72277, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23977269

RESUMO

BACKGROUND: Elevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice. METHODS AND RESULTS: Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser(1177) (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks. CONCLUSIONS: Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.


Assuntos
Aorta/enzimologia , Arginase/metabolismo , Diabetes Mellitus Tipo 1/enzimologia , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/enzimologia , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Arginase/antagonistas & inibidores , Arginase/genética , Arginina/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Ácidos Borônicos/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Humanos , Masculino , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Pênis/irrigação sanguínea , Pênis/efeitos dos fármacos , Pênis/fisiopatologia
8.
Front Immunol ; 4: 219, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23908657

RESUMO

Enhanced arginase (ARG) activity has been identified as a factor that reduces nitric oxide production and impairs endothelial function in vascular pathologies. Using a gene deletion model, we investigated involvement of arginase isoforms arginase 1 and 2 (ARG1 and ARG2) in hypertension and endothelial dysfunction in a mineralocorticoid-salt mouse model. Hypertension was induced in wild type (WT), partial ARG1(+/-) knockout (KO), and complete ARG2(-/-) KO mice by uninephrectomy and deoxycorticosterone acetate (DOCA)-salt treatment for 6-weeks. (Control uninephrectomized mice drank tap water.) After 2 weeks of DOCA-salt treatment, systolic blood pressure (SBP) was increased by ∼15 mmHg in all mouse genotypes. SBP continued to rise in DOCA-salt WT and ARG2(-/-) mice to ∼130 mmHg at 5-6 weeks, whereas in ARG1(+/-) mice SBP waned toward control levels by 6 weeks (109 ± 4 vs. 101 ± 3 mmHg, respectively). DOCA-salt treatment in WT mice increased vascular ARG activity (aorta by 1.5-fold; mesenteric artery (MA) by 2.6-fold and protein levels of ARG1 (aorta: 1.49-fold and MA: 1.73-fold) vs. WT Sham tissues. ARG2 protein increased in WT-DOCA MA (by 2.15-fold) but not in aorta compared to those of WT Sham tissues. Maximum endothelium-dependent vasorelaxation to acetylcholine was significantly reduced in DOCA-salt WT mice and largely or partially maintained in DOCA ARG1(+/-) and ARG2(-/-) mice vs. their Sham controls. DOCA-salt augmented contractile responses to phenylephrine in aorta of all mouse genotypes. Additionally, treatment of aorta or MA from WT-DOCA mice with arginase inhibitor (100 µM) improved endothelium-mediated vasorelaxation. DOCA-salt-induced coronary perivascular fibrosis (increased by 2.1-fold) in WT was prevented in ARG1(+/-) and reduced in ARG2(-/-) mice. In summary, ARG is involved in murine DOCA-salt-induced impairment of vascular function and hypertension and may represent a novel target for antihypertensive therapy.

9.
J Sex Med ; 10(6): 1502-15, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23566117

RESUMO

INTRODUCTION: Activated RhoA/Rho kinase (ROCK) has been implicated in diabetes-induced erectile dysfunction. Earlier studies have demonstrated involvement of ROCK pathway in the activation of arginase in endothelial cells. However, signaling pathways activated by ROCK in the penis remain unclear. AIM: We tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes. METHODS: Eight weeks after streptozotocin-induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho-p38 MAPK, p38 MAPK, phospho-MYPT-1(Thr850), MYPT-1 and arginase levels were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT + D), partial ROCK 2(+/-) knockout (KO), and ROCK 2(+/-) KO + D mice. MAIN OUTCOME MEASURES: The expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT-1(Thr850) and p38 MAPK, arginase activity/expression, endothelial- and nitrergic-dependent relaxation of CC was assayed. RESULTS: Diabetes significantly reduced maximum relaxation (Emax ) to both endothelium-dependent acetylcholine (WT + D: Emax; 61 ± 4% vs. WT: Emax; 75 ± 2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho-MYPT-1(Thr850), phospho-p38 MAPK, arginase II, and activity of corporal arginase (1.6-fold) in WT diabetic CC. However, this impairment in CC of WT + D mice was absent in heterozygous ROCK 2(+/-) KO + D mice for acetylcholine (Emax : 80 ± 5%) and attenuated for nitrergic nerve-induced relaxation. CC of ROCK 2(+/-) KO + D mice showed much less ROCK activity, did not exhibit p38 MAPK activation, and had reduced arginase activity and arginase II expression. These findings indicate that ROCK 2 mediates diabetes-induced elevation of arginase activity. Additionally, pretreatment of WT diabetic CC with inhibitors of arginase (ABH) or p38 MAPK (SB203580) partially prevented impairment of ACh- and nitrergic nerve-induced relaxation and elevation of arginase activity. CONCLUSION: ROCK 2, p38 MAPK and arginase play key roles in diabetes-induced impairment of CC relaxation.


Assuntos
Arginase/metabolismo , Diabetes Mellitus Tipo 1/complicações , Angiopatias Diabéticas/etiologia , Pênis/fisiopatologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Arginase/antagonistas & inibidores , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/fisiopatologia , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/fisiopatologia , Angiopatias Diabéticas/enzimologia , Angiopatias Diabéticas/fisiopatologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Ativação Enzimática , Haploinsuficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinase de Cadeia Leve de Miosina/metabolismo , Fosfatase de Miosina-de-Cadeia-Leve , Ereção Peniana , Pênis/efeitos dos fármacos , Pênis/inervação , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/deficiência , Quinases Associadas a rho/genética , Proteína rhoA de Ligação ao GTP
10.
Toxicon ; 69: 152-9, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23583324

RESUMO

The use of natural substances for the treatment of diseases or injuries is an ancient practice of many cultures. According to folklore, natural aphrodisiacs may help to raise libido and increase desire. The supposed aphrodisiacs mainly include a plethora of preparations of plants, among other substances. However, the real boundary between myth and reality has not been established yet in most cases and such boundaries must be drawn by scientific methods. A growing interest of the scientific community has been focused on animal venoms, especially those from arthropods, i.e. spiders and scorpions, which cause priapism, a prolonged and painful erection. This review highlights the studies that have been performed with venoms and toxins from arthropods known to cause priapism, among other toxic symptoms, pointing out some pharmacological approaches for better understanding this effect. To date, the venom of some spiders, mainly Phoneutria nigriventer, and scorpions, such as the yellow South American scorpion Tityus serrulatus, among others, have been known to cause priapism. Since erectile dysfunction (ED) is a growing health problem in the world, more common in patients with vascular diseases as diabetes and hypertension, the use of animal venoms and toxins as pharmacological tools could not only shed light to the mechanisms involved in erectile function, but also represent a possible model for new drugs to treat ED. Unfortunately, attempts to correlate the structure of those priapism-related toxins were unfruitful. Such difficulties lie firstly on the poor data concerning purified priapism-related toxins, instead of whole venoms and/or semi-purified fractions, and secondly, on the scarce available primary sequences and structural data, mainly from spider toxins. It has been shown that all these toxins modify the sodium (Na(+)) channel activity, mostly slowing down its inactivation current. Improving the knowledge on the tertiary structure of these toxins could provide a key in the search of a new drug for ED treatment.


Assuntos
Ereção Peniana/efeitos dos fármacos , Peptídeos/farmacologia , Venenos de Escorpião/farmacologia , Venenos de Aranha/farmacologia , Sequência de Aminoácidos , Animais , Disfunção Erétil/fisiopatologia , Disfunção Erétil/terapia , Humanos , Masculino , Dados de Sequência Molecular , Peptídeos/química , Priapismo/induzido quimicamente , Priapismo/patologia , Venenos de Escorpião/química , Escorpiões , Venenos de Aranha/química , Aranhas
11.
J Sex Med ; 9(10): 2574-81, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22925420

RESUMO

INTRODUCTION: Age-associated erectile dysfunction (ED) involves a decrease in nitric oxide (NO) availability and impaired relaxation. PnTx2-6, a toxin from the Phoneutria nigriventer spider, has been demonstrated to improve erectile function via NO/cyclic guanosine monophosphate (cGMP) pathway. This spider's venom is characterized by several symptoms, including erection. PnTx2-6 has been implicated in this phenomenon. Animal venoms have been postulated as potential drugs to treat ED. AIM: PnTx2-6 toxin improves erectile function in aged rats via NO/cGMP. We investigated the effect of PnTx2-6 in the erectile function of aged rats. MAIN OUTCOME MEASURES: ED was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio during electrical field stimulation (EFS) of the pelvic ganglion of aged and adult rats (70 vs. 14 weeks). In functional studies, EFS-induced relaxation of corpus cavernosum (CC) strips were performed with or without PnTx2-6 (10-8M). RESULTS: The decrease in erectile function associated with age was partially restored 15-20 minutes after injection of PnTx2-6 and further improved by sildenafil. PnTx2-6 enhanced EFS-induced relaxation, as well as cGMP levels in CC, from young and aged rats. Relaxation due to PnTx2-6 was further increased after 30 minutes incubation with Y-27632, a Rho-kinase inhibitor (10-6 M), in aging CC. Nitric oxide synthase (NOS) activity in aged and young cavernosal tissue was increased by incubation with PnTx2-6 (10 minutes). However, this toxin did not modify NOS expression. CONCLUSION: PnTx2-6 improves penile relaxation in aged rats, via increased NOS activity and NO release, resulting in enhanced cGMP levels.


Assuntos
GMP Cíclico/metabolismo , Disfunção Erétil/tratamento farmacológico , Óxido Nítrico/metabolismo , Ereção Peniana/efeitos dos fármacos , Peptídeos/farmacologia , Venenos de Aranha/farmacologia , Animais , Pressão Sanguínea , Disfunção Erétil/fisiopatologia , Masculino , Ereção Peniana/fisiologia , Ratos , Ratos Wistar
12.
J Sex Med ; 8(12): 3335-44, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21995824

RESUMO

INTRODUCTION: Increased arginase activity (AA) has been implicated in hypertension and diabetes-induced endothelial dysfunction by reducing L-arginine availability and nitric oxide production. Higher levels of active extracellular signal-regulated kinase (ERK) have been found in patients with erectile dysfunction (ED) compared to patients without it. Both ERK and arginase have been reported to affect the expression and activity of nitric oxide synthase (NOS) and consequently penile erection. Nevertheless, signaling pathways activated by ERK in the penis are not well known. AIM: We hypothesized that inhibition of ERK by ERK inhibitor PD98059 decreases AA and thus improves cavernosal relaxation in streptozotocin (STZ)-diabetic mice. METHODS: The AA, ERK, eNOS, and arginase I and II expressions were examined through Western blot, and functional response of cavernosal tissue were determined. Control and diabetic cavernosal tissues were pretreated with PD98059 (10(-5) M) and arginase inhibitor ((S)-(2-boronoethyl)-L-cysteine hydrochloride, [BEC]10(-4) M]). MAIN OUTCOME MEASURES: Diabetes increased AA significantly (twofold) over control mice and this effect was blocked by acute treatment with PD98059. Cavernosal strips from diabetic mice exhibited decreased relaxation (STZ-diabetic vs. control, respectively) to both the endothelium-dependent agonist acetylcholine (38.0 ± 5% vs. 82.5 ± 7%) and nitrergic stimulation (27 ± 2% vs. 76 ± 6%) by electrical field stimulation (EFS, 1-32 Hz). However, this impairment in cavernosal relaxation from diabetic mice was attenuated by treatment with PD98059 in nitrergic (27 ± 2% vs. 60 ± 4%) and endothelium-dependent relaxation responses (38.0 ± 5% vs. 67.5 ± 6%). Acute treatment with the arginase inhibitor BEC (10(-4) M) also improves EFS-induced relaxation in diabetic mice (31 ± 3% vs. 49 ± 2%). Moreover, vascular expression of activated ERK was increased in diabetic over control mice. CONCLUSION: These data suggest that ERK inhibition prevents elevation of penile AA and protects against ED caused by diabetes.


Assuntos
Arginase/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Flavonoides/uso terapêutico , Impotência Vasculogênica/tratamento farmacológico , Ereção Peniana/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Análise de Variância , Animais , Arginase/biossíntese , Arginase/metabolismo , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Endotélio Vascular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Masculino , Camundongos , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/irrigação sanguínea , Pênis/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
13.
J Sex Med ; 7(12): 3879-88, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20722794

RESUMO

INTRODUCTION: Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na(+) ) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. AIM: We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. MAIN OUTCOME MEASURES: PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. METHODS: Rat cavernosal strips were incubated with bretylium (3 × 10(-5) M) and contracted with phenylephrine (PE; 10(-5) M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10(-8) M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10(-6) M), a muscarinic receptor antagonist, N-type Ca(2+) channel blockers (ω-conotoxin GVIA, 10(-6) M) and sildenafil (3 × 10(-8) M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. RESULTS: Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca(2+) channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. CONCLUSION: We show that PnTx2-6 slows Na(+) channels inactivation in nitrergic neurons, allowing Ca(2+) influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction.


Assuntos
Neuropeptídeos/farmacologia , Neurotoxinas/farmacologia , Óxido Nítrico/metabolismo , Pênis/efeitos dos fármacos , Venenos de Aranha/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Atropina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/efeitos dos fármacos , Estimulação Elétrica , Masculino , Antagonistas Muscarínicos/farmacologia , Doadores de Óxido Nítrico/farmacologia , Nitroprussiato/farmacologia , Pênis/irrigação sanguínea , Pênis/inervação , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Purinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Citrato de Sildenafila , Sulfonas/farmacologia , Tecnécio , ômega-Conotoxina GVIA/farmacologia
14.
J Sex Med ; 5(12): 2793-807, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18823320

RESUMO

INTRODUCTION: The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells not only respond to but also synthesize ET-1. AIM: Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, we hypothesized that activation of the ET-1/ET(A) receptor pathway contributes to erectile dysfunction (ED) associated with mineralocorticoid hypertension. METHODS: Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treatment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water. Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ET(A) receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ET(B) receptor agonist (IRL-1620) and electric field stimulation (EFS) were evaluated in vitro. Expression of ROCKalpha, ROCKbeta, myosin phosphatase target subunit 1 (MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1 and ET(A) receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction. Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluate erectile function in vivo. MAIN OUTCOME MEASURE: ET(A) receptor blockade prevents DOCA-salt-associated ED. RESULTS: Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractile responses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE were enhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated with increased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completely prevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completely preventing ED in DOCA-salt rats. CONCLUSION: Activation of the ET-1/ET(A) pathway contributes to mineralocorticoid hypertension-associated ED. ET(A) receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitive hypertension and in pathological conditions where increased levels of ET-1 are present.


Assuntos
Endotelina-1/genética , Disfunção Erétil/genética , Hipertensão/genética , Receptor de Endotelina A/genética , Animais , Atrasentana , Pressão Sanguínea/efeitos dos fármacos , Desoxicorticosterona , Relação Dose-Resposta a Droga , Antagonistas do Receptor de Endotelina B , Endotelina-1/antagonistas & inibidores , Endotelina-1/fisiologia , Disfunção Erétil/induzido quimicamente , Disfunção Erétil/fisiopatologia , Expressão Gênica/efeitos dos fármacos , Hipertensão/induzido quimicamente , Hipertensão/fisiopatologia , Técnicas In Vitro , Masculino , Proteína Quinase 3 Ativada por Mitógeno/genética , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Pênis/irrigação sanguínea , Pirrolidinas/farmacologia , RNA Mensageiro/genética , Ratos , Receptor de Endotelina B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quinases Associadas a rho/genética
15.
Can J Physiol Pharmacol ; 86(6): 320-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18516094

RESUMO

The penis is kept in the flaccid state mainly via a tonic activity of norepinephrine and endothelins (ETs). ET-1 is important in salt-sensitive forms of hypertension. We hypothesized that cavernosal responses to ET-1 are enhanced in deoxycorticosterone acetate (DOCA)-salt mice and that blockade of ETA receptors prevents abnormal responses of the corpus cavernosum in DOCA-salt hypertension. Male C57BL/6 mice were unilaterally nephrectomized and treated for 5 weeks with both DOCA and water containing 1% NaCl and 0.2% KCl. Control mice were uninephrectomized and received tap water with no added salt. Animals received either the ETA antagonist atrasentan (5 mg x day(-1) x kg(-1) body weight) or vehicle. DOCA-salt mice displayed increased systolic blood pressure (SBP), and treatment with atrasentan decreased SBP in DOCA-salt mice. Contractile responses in cavernosal strips from DOCA-salt mice were enhanced by ET-1, phenylephrine, and electrical field stimulation (EFS) of adrenergic nerves, whereas relaxations were not altered by IRL-1620 (an ETB agonist), acetylcholine, sodium nitroprusside, and EFS of nonadrenergic noncholinergic nerves. PD59089 (an ERK1/2 inhibitor), but not Y-27632 (a Rho-kinase inhibitor), abolished enhanced contractions to ET-1 in cavernosum from DOCA-salt mice. Treatment of DOCA-salt mice with atrasentan did not normalize cavernosal responses. In summary, DOCA-salt treatment in mice enhances cavernosal reactivity to contractile, but not to relaxant, stimuli, via ET-1/ETA receptor-independent mechanisms.


Assuntos
Desoxicorticosterona/administração & dosagem , Endotelina-1/administração & dosagem , Pênis/irrigação sanguínea , Pênis/efeitos dos fármacos , Cloreto de Sódio/administração & dosagem , Animais , Atrasentana , Antagonistas do Receptor de Endotelina A , Endotelina-1/antagonistas & inibidores , Endotelina-1/genética , Hipertensão/etiologia , Hipertensão/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Ereção Peniana/efeitos dos fármacos , Ereção Peniana/fisiologia , Pênis/fisiologia , Pirrolidinas/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia
16.
Recent Pat Cardiovasc Drug Discov ; 2(2): 119-32, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18221110

RESUMO

In recent years male sexual research has increasingly centered on molecular mechanisms operating from the central nervous system to peripheral end-organ levels involved in the penile erectile response. Major progress has been made in the field, and currently a whole host of neurotransmitters, chemical effectors, growth factors, second-messenger molecules, ions, intercellular proteins, and hormones have been characterized as components of the complex physiology of erectile function. Foremost among these mediators is nitric oxide (NO), which was initially characterized as a locally released physiologic mediator of the erectile response. Impaired formation and action of NO is closely associated with erectile dysfunction (ED), which may be caused by a variety of pathogenic factors. The impact of this knowledge has been substantial, leading to the development of several NO-based medical approaches for the treatment of ED. This review will focus on recent patents and current clinical trials involving innovative pharmacological and gene therapies in the field of male ED, particularly targeting the NO/intracellular cyclic GMP pathway, which still represents the most promising therapeutic approach to treat patients with ED.


Assuntos
GMP Cíclico/fisiologia , Disfunção Erétil , Óxido Nítrico/fisiologia , Disfunção Erétil/tratamento farmacológico , Disfunção Erétil/metabolismo , Disfunção Erétil/fisiopatologia , Humanos , Masculino , Inibidores da Fosfodiesterase 5
17.
Infect Immun ; 70(11): 6263-72, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12379705

RESUMO

Infection by nematode parasites with a pulmonary migration in their life cycle and allergic asthma are two highly prevalent diseases in humans; therefore, one may expect both may occur concomitantly. There is a predominant and essential role of Th2 lymphocytes in the mechanisms underlying the control of parasite elimination as well as in the pathology observed in the asthmatic lung. The consequences of such situations have been explored, with controversial results, justifying the development of experimental models in which the relationship between allergic airway inflammation and helminth infection might be evaluated. The present work describes the inflammatory, humoral, and functional changes that occur in the lung of rats after single (subcutaneous inoculation of 1,500 L3 larvae) or multiple (five weekly subcutaneous inoculations of 1,500 L3 larvae) Strongyloides venezuelensis infections. The results show that the migration of S. venezuelensis larvae through the lungs of infected rats induces a local eosinophilic inflammation process which is mostly focal and parenchymal for rats infected a single time and which is peribronchial after multiple infections. The inflammatory process is accompanied by mucus hypersecretion, thickening of bronchial epithelial and muscle layers, and local increase in immunoglobulin E concentrations that peak after 5 to 7 days and are resolved after 12 days of single or multiple infections. The peak of lung immunopathologic changes observed in infected rats coincides with lung airway hyperresponsiveness (AHR), a key functional alteration in asthma. We propose that this experimental model is ideal to carry out further studies on immunoprotection against nematode infection versus immunopathology of allergic airway inflammation.


Assuntos
Hiper-Reatividade Brônquica/etiologia , Eosinofilia/etiologia , Imunoglobulina E/biossíntese , Inflamação/etiologia , Pulmão/patologia , Estrongiloidíase/imunologia , Animais , Asma/etiologia , Citocinas/biossíntese , Ratos , Ratos Wistar , Estrongiloidíase/complicações , Estrongiloidíase/patologia
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