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1.
Physiol Genomics ; 55(4): 179-193, 2023 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-36912534

RESUMO

The endothelium contains morphologically similar cells throughout the vasculature, but individual cells along the length of a single vascular tree or in different regional circulations function dissimilarly. When observations made in large arteries are extrapolated to explain the function of endothelial cells (ECs) in the resistance vasculature, only a fraction of these observations are consistent between artery sizes. To what extent endothelial (EC) and vascular smooth muscle cells (VSMCs) from different arteriolar segments of the same tissue differ phenotypically at the single-cell level remains unknown. Therefore, single-cell RNA-seq (10x Genomics) was performed using a 10X Genomics Chromium system. Cells were enzymatically digested from large (>300 µm) and small (<150 µm) mesenteric arteries from nine adult male Sprague-Dawley rats, pooled to create six samples (3 rats/sample, 3 samples/group). After normalized integration, the dataset was scaled before unsupervised cell clustering and cluster visualization using UMAP plots. Differential gene expression analysis allowed us to infer the biological identity of different clusters. Our analysis revealed 630 and 641 differentially expressed genes (DEGs) between conduit and resistance arteries for ECs and VSMCs, respectively. Gene ontology analysis (GO-Biological Processes, GOBP) of scRNA-seq data discovered 562 and 270 pathways for ECs and VSMCs, respectively, that differed between large and small arteries. We identified eight and seven unique ECs and VSMCs subpopulations, respectively, with DEGs and pathways identified for each cluster. These results and this dataset allow the discovery and support of novel hypotheses needed to identify mechanisms that determine the phenotypic heterogeneity between conduit and resistance arteries.


Assuntos
Células Endoteliais , Transcriptoma , Ratos , Animais , Transcriptoma/genética , Células Endoteliais/metabolismo , Ratos Sprague-Dawley , Endotélio Vascular/metabolismo , Músculo Liso Vascular/metabolismo , Artérias Mesentéricas , Perfilação da Expressão Gênica
2.
Am J Physiol Regul Integr Comp Physiol ; 323(6): R900-R909, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36250874

RESUMO

Hypoxia is the reduction of alveolar partial pressure of oxygen ([Formula: see text]). Military members and people who practice recreational activities from moderate to high altitudes are at risk for hypoxic exposure. Hypoxemia's signs and symptoms vary from asymptomatic to severe responses, such as excessive hypoxic ventilatory responses and residual neurobehavioral impairment. Therefore, it is essential to identify hypoxia-induced biomarkers to indicate people with exposure to hypoxia. Advances have been made in understanding physiological responses to hypoxia, including elevations in circulating levels of endothelin 1 (ET-1) and microRNA 21 (miR-21) and reduction in circulating levels of hydrogen sulfide (H2S). Although the levels of these factors change upon exposure to hypoxia, it is unclear if these changes are sustained on return to normoxia. We hypothesize that hypoxia-induced ET-1 and miR-21 remain elevated, whereas hypoxia-reduction in H2S sustains after returning to normoxic conditions. To test this hypothesis, we exposed male rats to 6 h of 12% O2 and measured circulating levels of ET-1 and miR-21, pre, during, and posthypoxia. We found that ET-1 plasma levels increased in response to hypoxia but returned to normal levels within 30 min after the restoration of normoxia. miR-21 plasma levels and transdermal H2S emissions decreased in response to hypoxia, remaining decreased on return to normoxia, thus following the biomarker criteria. Therefore, this study supports a unique role for plasma miR21 and transdermal H2S as hypoxia biomarkers that could be used to identify individuals after exposure to hypoxia.


Assuntos
Sulfeto de Hidrogênio , MicroRNAs , Masculino , Ratos , Animais , Hipóxia , Oxigênio , Endotelina-1 , Biomarcadores , MicroRNAs/genética
3.
Am J Physiol Heart Circ Physiol ; 320(2): H511-H519, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33275519

RESUMO

In sleep apnea, airway obstruction causes intermittent hypoxia (IH). In animal studies, IH-dependent hypertension is associated with loss of vasodilator hydrogen sulfide (H2S), and increased H2S activation of sympathetic nervous system (SNS) activity in the carotid body. We previously reported that inhibiting cystathionine γ-lyase (CSE) to prevent H2S synthesis augments vascular resistance in control rats. The goal of this study was to evaluate the contribution of IH-induced changes in CSE signaling to increased blood pressure and vascular resistance. We hypothesized that chronic IH exposure eliminates CSE regulation of blood pressure (BP) and vascular resistance. In rats instrumented with venous catheters, arterial telemeters, and flow probes on the main mesenteric artery, the CSE inhibitor dl-propargylglycine (PAG, 50 mg/kg/day i.v. for 5 days) increased BP in Sham rats but decreased BP in IH rats [in mmHg, Sham (n = 11): 114 ± 4 to 131 ± 6; IH (n = 8): 131 ± 8 to 115 ± 7 mmHg, P < 0.05]. PAG treatment increased mesenteric vascular resistance in Sham rats but decreased it in IH rats (day 5/day 1: Sham: 1.50 ± 0.07; IH: 0.85 ± 0.19, P < 0.05). Administration of the ganglionic blocker hexamethonium (to evaluate SNS activity) decreased mesenteric resistance in PAG-treated Sham rats more than in saline-treated Sham rats or PAG-treated IH rats. CSE immunoreactivity in IH carotid bodies compared with those from Sham rats. However, CSE staining in small mesenteric arteries was less in arteries from IH than in Sham rats but not different in larger arteries (inner diameter > 200 µm). These results suggest endogenous H2S regulates blood pressure and vascular resistance, but this control is lost after IH exposure with decreased CSE expression in resistance size arteries. IH exposure concurrently increases carotid body CSE expression and relative SNS control of blood pressure, suggesting both vascular and carotid body H2S generation contribute to blood pressure regulation.NEW & NOTEWORTHY These results suggest that CSE's protective role in the vasculature is impaired by simulated sleep apnea, which also upregulates CSE in the carotid body. Thus, this enzyme system can exert both pro- and antihypertensive effects and may contribute to elevated SNS outflow in sleep apnea.


Assuntos
Circulação Sanguínea , Pressão Sanguínea , Gasotransmissores/metabolismo , Sulfeto de Hidrogênio/metabolismo , Síndromes da Apneia do Sono/metabolismo , Alcinos/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Corpo Carotídeo/efeitos dos fármacos , Corpo Carotídeo/metabolismo , Corpo Carotídeo/fisiopatologia , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Gasotransmissores/sangue , Glicina/análogos & derivados , Glicina/farmacologia , Hexametônio/farmacologia , Sulfeto de Hidrogênio/sangue , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Ratos , Ratos Sprague-Dawley , Síndromes da Apneia do Sono/fisiopatologia , Resistência Vascular
4.
Am J Physiol Heart Circ Physiol ; 321(1): H77-H111, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33989082

RESUMO

The measurement of vascular function in isolated vessels has revealed important insights into the structural, functional, and biomechanical features of the normal and diseased cardiovascular system and has provided a molecular understanding of the cells that constitutes arteries and veins and their interaction. Further, this approach has allowed the discovery of vital pharmacological treatments for cardiovascular diseases. However, the expansion of the vascular physiology field has also brought new concerns over scientific rigor and reproducibility. Therefore, it is appropriate to set guidelines for the best practices of evaluating vascular function in isolated vessels. These guidelines are a comprehensive document detailing the best practices and pitfalls for the assessment of function in large and small arteries and veins. Herein, we bring together experts in the field of vascular physiology with the purpose of developing guidelines for evaluating ex vivo vascular function. By using this document, vascular physiologists will have consistency among methodological approaches, producing more reliable and reproducible results.


Assuntos
Artérias/fisiologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Veias/fisiologia , Animais , Endotélio Vascular/fisiologia , Microscopia/métodos , Miografia/métodos , Reprodutibilidade dos Testes
5.
Am J Respir Cell Mol Biol ; 62(6): 732-746, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32048876

RESUMO

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.


Assuntos
Hipertensão Pulmonar/fisiopatologia , Hipóxia/fisiopatologia , Mitocôndrias/fisiologia , Proteína Quinase C beta/fisiologia , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Células Cultivadas , Óxidos N-Cíclicos/farmacologia , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/metabolismo , Sequestradores de Radicais Livres/farmacologia , Humanos , Hipertensão Pulmonar/etiologia , Hipóxia/complicações , Hipóxia/enzimologia , Indóis/farmacologia , Masculino , Maleimidas/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/enzimologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Canais de Potássio/metabolismo , Mapeamento de Interação de Proteínas , Artéria Pulmonar/enzimologia , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Síndromes da Apneia do Sono/fisiopatologia , Marcadores de Spin , Acetato de Tetradecanoilforbol/farmacologia
6.
Am J Physiol Lung Cell Mol Physiol ; 319(6): L968-L980, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32997513

RESUMO

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.


Assuntos
Colágeno Tipo V/metabolismo , Hipertensão Pulmonar/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Animais , Núcleo Celular/metabolismo , Imunidade Celular/fisiologia , Transplante de Pulmão/métodos
7.
Am J Physiol Renal Physiol ; 316(5): F1041-F1052, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30810064

RESUMO

Obstructive sleep apnea is characterized by recurrent episodes of pharyngeal collapse during sleep, resulting in intermittent hypoxia (IH), and is associated with a high incidence of hypertension and accelerated renal failure. In rodents, endothelin (ET)-1 contributes to IH-induced hypertension, and ET-1 levels inversely correlate with glomerular filtration rate in patients with end-stage chronic kidney disease (CKD). Therefore, we hypothesized that a dual ET receptor antagonist, macitentan (Actelion Pharmaceuticals), will attenuate and reverse hypertension and renal dysfunction in a rat model of combined IH and CKD. Male Sprague-Dawley rats received one of three diets (control, 0.2% adenine, and 0.2% adenine + 30 mg·kg-1·day-1 macitentan) for 2 wk followed by 2 wk of recovery diet. Rats were then exposed for 4 wk to air or IH (20 short exposures/h to 5% O2-5% CO2 7 h/day during sleep). Macitentan prevented the increases in mean arterial blood pressure caused by CKD, IH, and the combination of CKD + IH. However, macitentan did not improve kidney function, fibrosis, and inflammation. After CKD was established, rats were exposed to air or IH for 2 wk, and macitentan feeding continued for 2 more wk. Macitentan reversed the hypertension in IH, CKD, and CKD + IH groups without improving renal function. Our data suggest that macitentan could be an effective antihypertensive in patients with CKD and irreversible kidney damage as a way to protect the heart, brain, and eyes from elevated arterial pressure, but it does not reverse toxin-induced tubule atrophy.


Assuntos
Anti-Hipertensivos/farmacologia , Pressão Arterial/efeitos dos fármacos , Antagonistas do Receptor de Endotelina A/farmacologia , Antagonistas do Receptor de Endotelina B/farmacologia , Hipertensão/prevenção & controle , Rim/efeitos dos fármacos , Pirimidinas/farmacologia , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/tratamento farmacológico , Síndromes da Apneia do Sono/complicações , Síndromes da Apneia do Sono/tratamento farmacológico , Sulfonamidas/farmacologia , Animais , Modelos Animais de Doenças , Endotelina-1/genética , Endotelina-1/metabolismo , Taxa de Filtração Glomerular/efeitos dos fármacos , Hipertensão/etiologia , Hipertensão/fisiopatologia , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Masculino , Ratos Sprague-Dawley , Insuficiência Renal Crônica/patologia , Insuficiência Renal Crônica/fisiopatologia , Síndromes da Apneia do Sono/fisiopatologia
8.
Am J Physiol Heart Circ Physiol ; 317(5): H1157-H1165, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31625777

RESUMO

Hydrogen sulfide (H2S) dilates isolated arteries, and knockout of the H2S-synthesizing enzyme cystathionine γ-lyase (CSE) increases blood pressure. However, the contributions of endogenously produced H2S to blood flow regulation in specific vascular beds are unknown. Published studies in isolated arteries show that CSE production of H2S influences vascular tone more in small mesenteric arteries than in renal arteries or the aorta. Therefore, the goal of this study was to evaluate H2S regulation of blood pressure, vascular resistance, and regional blood flows using chronically instrumented rats. We hypothesized that during whole animal CSE inhibition, vascular resistance would increase more in the mesenteric than the renal circulation. Under anesthesia, CSE inhibition [ß-cyanoalanine (BCA), 30 mg/kg bolus + 5 mg·kg-1·min-1 for 20 min iv) rapidly increased mean arterial pressure (MAP) more than saline administration (%Δ: saline -1.4 ± 0.75 vs. BCA 7.1 ± 1.69, P < 0.05) but did not change resistance (MAP/flow) in either the mesenteric or renal circulation. In conscious rats, BCA infusion similarly increased MAP (%Δ: saline -0.8 ± 1.18 vs. BCA 8.2 ± 2.6, P < 0.05, n = 7) and significantly increased mesenteric resistance (saline 0.9 ± 3.1 vs. BCA 15.6 ± 6.5, P < 0.05, n = 12). The H2S donor Na2S (50 mg/kg) decreased blood pressure and mesenteric resistance ,but the fall in resistance was not significant. Inhibiting CSE for multiple days with dl-proparglycine (PAG, 50 mg·kg-1·min-1 iv bolus for 5 days) significantly increased vascular resistance in both mesenteric (ratio of day 1: saline 0.86 ± 0.033 vs. PAG 1.79 ± 0.38) and renal circulations (ratio of day 1: saline 1.26 ± 0.22 vs. 1.98 ± 0.14 PAG). These results support our hypothesis that CSE-derived H2S is an important regulator of blood pressure and vascular resistance in both mesenteric and renal circulations. Furthermore, inhalation anesthesia diminishes the effect of CSE inhibition on vascular tone.NEW & NOTEWORTHY These results suggest that CSE-derived H2S has a prominent role in regulating blood pressure and blood flow under physiological conditions, which may have been underestimated in prior studies in anesthetized subjects. Therefore, enhancing substrate availability or enzyme activity or dosing with H2S donors could be a novel therapeutic approach to treat cardiovascular diseases.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Artérias Mesentéricas/metabolismo , Artéria Renal/metabolismo , Circulação Renal , Circulação Esplâncnica , Alanina/análogos & derivados , Alanina/farmacologia , Animais , Pressão Arterial , Velocidade do Fluxo Sanguíneo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Ratos Sprague-Dawley , Artéria Renal/efeitos dos fármacos , Circulação Renal/efeitos dos fármacos , Circulação Esplâncnica/efeitos dos fármacos , Sulfetos/farmacologia , Resistência Vascular
9.
Am J Physiol Renal Physiol ; 315(4): F927-F941, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29897288

RESUMO

Kidney injury and sleep apnea (SA) are independent risk factors for hypertension. Exposing rats to intermittent hypoxia (IH) to simulate SA increases blood pressure whereas adenine feeding causes persistent kidney damage to model chronic kidney disease (CKD). We hypothesized that exposing CKD rats to IH would exacerbate the development of hypertension and renal failure. Male Sprague-Dawley rats were fed a 0.2% adenine diet or control diet (Control) until blood urea nitrogen was >120 mg/dl in adenine-fed rats (14 ± 4 days, mean ± SE). After 2 wk of recovery on normal chow, rats were exposed to IH (20 exposures/h of 5% O2-5% CO2 7 h/day) or control conditions (Air) for 6 wk. Mean arterial pressure (MAP) was monitored with telemeters, and plasma and urine samples were collected weekly to calculate creatinine clearance as an index of glomerular filtration rate (GFR). Prior to IH, adenine-fed rats had higher blood pressure than rats on control diet. IH treatment increased MAP in both groups, and after 6 wk, MAP levels in the CKD/IH rats were greater than those in the CKD/Air and Control/IH rats. MAP levels in the Control/Air rats were lower than those in the other three groups. Kidney histology revealed crystalline deposits, tubule dilation, and interstitial fibrosis in both CKD groups. IH caused no additional kidney damage. Plasma creatinine was similarly increased in both CKD groups throughout whereas IH alone increased plasma creatinine. IH increases blood pressure further in CKD rats without augmenting declines in GFR but appears to impair GFR in healthy rats. We speculate that treating SA might decrease hypertension development in CKD patients and protect renal function in SA patients.


Assuntos
Pressão Sanguínea/fisiologia , Hipertensão/fisiopatologia , Hipóxia/fisiopatologia , Insuficiência Renal Crônica/fisiopatologia , Animais , Pressão Arterial/fisiologia , Doenças Cardiovasculares/fisiopatologia , Rim/fisiopatologia , Testes de Função Renal , Masculino , Ratos Sprague-Dawley , Insuficiência Renal Crônica/complicações
10.
Am J Physiol Cell Physiol ; 312(5): C537-C549, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148499

RESUMO

Hydrogen sulfide (H2S) is a ubiquitous signaling molecule with important functions in many mammalian organs and systems. Observations in the 1990s ascribed physiological actions to H2S in the nervous system, proposing that this gasotransmitter acts as a neuromodulator. Soon after that, the vasodilating properties of H2S were demonstrated. In the past decade, H2S was shown to exert a multitude of physiological effects in the vessel wall. H2S is produced by vascular cells and exhibits antioxidant, antiapoptotic, anti-inflammatory, and vasoactive properties. In this concise review, we have focused on the impact of H2S on vascular structure and function with an emphasis on angiogenesis, vascular tone, vascular permeability and atherosclerosis. H2S reduces arterial blood pressure, limits atheromatous plaque formation, and promotes vascularization of ischemic tissues. Although the beneficial properties of H2S are well established, mechanistic insights into the molecular pathways implicated in disease prevention and treatment remain largely unexplored. Unraveling the targets and downstream effectors of H2S in the vessel wall in the context of disease will aid in translation of preclinical observations. In addition, acute regulation of H2S production is still poorly understood and additional work delineating the pathways regulating the enzymes that produce H2S will allow pharmacological manipulation of this pathway. As the field continues to grow, we expect that H2S-related compounds will find their way into clinical trials for diseases affecting the blood vessels.


Assuntos
Artérias/fisiopatologia , Aterosclerose/fisiopatologia , Sulfeto de Hidrogênio/metabolismo , Neovascularização Patológica/fisiopatologia , Acoplamento Neurovascular , Sistema Vasomotor/fisiopatologia , Animais , Humanos , Modelos Cardiovasculares , Transdução de Sinais , Vasoconstrição
11.
Am J Physiol Heart Circ Physiol ; 312(4): H791-H799, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28130342

RESUMO

Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O2-5% CO2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H2S)-induced vasodilation in rats. The enzyme that produces H2S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca2+ concentration ([Ca2+]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca2+] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P < 0.05), and fewer EC were NFATc3 nuclear positive in IH rat arteries than in sham rat arteries (IH = 13 ± 3, sham = 59 ± 11%, P < 0.05). H2S production was also lower in mesenteric tissue from IH rats vs. sham rats. Endothelium-dependent vasodilation to acetylcholine (ACh) was lower in mesenteric arteries from IH rats than in arteries from sham rats, and inhibiting CSE with ß-cyanoalanine diminished ACh-induced vasodilation in arteries from sham but not IH rats but did not affect dilation to the H2S donor NaHS. Thus, IH lowers EC [Ca2+], NFAT activity, CSE expression and activity, and H2S production while inhibiting NFAT activation lowers CSE expression. The observations that IH exposure decreases NFATc3 activation and CSE-dependent vasodilation support a role for NFAT in regulating endothelial H2S production.NEW & NOTEWORTHY This study identifies the calcium-regulated transcription factor nuclear factor of activated T cells as a novel regulator of cystathionine γ-lyase (CSE). This pathway is basally active in mesenteric artery endothelial cells, but, after exposure to intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation.


Assuntos
Cistationina gama-Liase/biossíntese , Células Endoteliais/metabolismo , Hipóxia/metabolismo , Fatores de Transcrição NFATC/metabolismo , Acetilcolina/farmacologia , Animais , Sequência de Bases , Calcineurina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Cistationina gama-Liase/genética , Sulfeto de Hidrogênio/metabolismo , Hipóxia/enzimologia , Masculino , Artérias Mesentéricas/citologia , Artérias Mesentéricas/metabolismo , Ratos , Ratos Sprague-Dawley , Síndromes da Apneia do Sono/genética , Síndromes da Apneia do Sono/fisiopatologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
14.
Am J Physiol Heart Circ Physiol ; 311(6): H1437-H1444, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27765747

RESUMO

Hydrogen sulfide (H2S) is a recently described gaseous vasodilator produced within the vasculature by the enzymes cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase. Previous data demonstrate that endothelial cells (EC) are the source of endogenous H2S production and are required for H2S-induced dilation. However, the signal transduction pathway activated by H2S within EC has not been elucidated. TRPV4 and large-conductance Ca2+-activated K channels (BK channels) are expressed in EC. H2S-induced dilation is inhibited by luminal administration of iberiotoxin and disruption of the endothelium. Calcium influx through TRPV4 may activate these endothelial BK channels (eBK). We hypothesized that H2S-mediated vasodilation involves activation of TRPV4 within the endothelium. In pressurized, phenylephrine-constricted mesenteric arteries, H2S elicited a dose-dependent vasodilation blocked by inhibition of TRPV4 channels (GSK2193874A, 300 nM). H2S (1 µM) increased TRPV4-dependent (1.8-fold) localized calcium events in EC of pressurized arteries loaded with fluo-4 and Oregon Green. In pressurized EC tubes, H2S (1 µM) and the TRPV4 activator, GSK101679A (30 nM), increased calcium events 1.8- and 1.5-fold, respectively. H2S-induced an iberiotoxin-sensitive outward current measured using whole cell patch-clamp techniques in freshly dispersed EC. H2S increased K+ currents from 10 to 30 pA/pF at +150 mV. Treatment with Na2S increased the level of sulfhydration of TRPV4 channels in aortic ECs. These results demonstrate that H2S-mediated vasodilation involves activation of TRPV4-dependent Ca2+ influx and BK channel activation within EC. Activation of TRPV4 channels appears to cause calcium events that result in the opening of eBK channels, endothelial hyperpolarization, and subsequent vasodilation.


Assuntos
Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Gasotransmissores/farmacologia , Sulfeto de Hidrogênio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Leucina/análogos & derivados , Leucina/farmacologia , Masculino , Artérias Mesentéricas/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores
15.
Am J Physiol Heart Circ Physiol ; 309(11): H1915-22, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26408536

RESUMO

Ca(+) sparks are vascular smooth muscle cell (VSMC) Ca(2+)-release events that are mediated by ryanodine receptors (RyR) and promote vasodilation by activating large-conductance Ca(2+)-activated potassium channels and inhibiting myogenic tone. We have previously reported that exposing rats to intermittent hypoxia (IH) to simulate sleep apnea augments myogenic tone in mesenteric arteries through loss of hydrogen sulfide (H2S)-induced dilation. Because we also observed that H2S can increase Ca(2+) spark activity, we hypothesized that loss of H2S after IH exposure reduces Ca(2+) spark activity and that blocking Ca(2+) spark generation reduces H2S-induced dilation. Ca(2+) spark activity was lower in VSMC of arteries from IH compared with sham-exposed rats. Furthermore, depolarizing VSMC by increasing luminal pressure (from 20 to 100 mmHg) or by elevating extracellular [K(+)] increased spark activity in VSMC of arteries from sham rats but had no effect in arteries from IH rats. Inhibiting endogenous H2S production in sham arteries prevented these increases. NaHS or phosphodiesterase inhibition increased spark activity to the same extent in sham and IH arteries. Depolarization-induced increases in Ca(2+) spark activity were due to increased sparks per site, whereas H2S increases in spark activity were due to increased spark sites per cell. Finally, inhibiting Ca(2+) spark activity with ryanodine (10 µM) enhanced myogenic tone in arteries from sham but not IH rats and blocked dilation to exogenous H2S in arteries from both sham and IH rats. Our results suggest that H2S regulates RyR activation and that H2S-induced dilation requires Ca(2+) spark activation. IH exposure decreases endogenous H2S-dependent Ca(2+) spark activation to cause membrane depolarization and enhance myogenic tone in mesenteric arteries.


Assuntos
Sinalização do Cálcio , Hipóxia/metabolismo , Músculo Liso Vascular/metabolismo , Vasodilatação , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Modelos Animais de Doenças , Sulfeto de Hidrogênio/metabolismo , Hipóxia/fisiopatologia , Técnicas In Vitro , Masculino , Potenciais da Membrana , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Inibidores de Fosfodiesterase/farmacologia , Ratos Sprague-Dawley , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacos , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sulfetos/metabolismo , Sulfetos/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia
16.
Am J Physiol Heart Circ Physiol ; 306(5): H667-73, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24414066

RESUMO

Sleep apnea is associated with cardiovascular disease, and patients with sleep apnea have elevated plasma endothelin (ET)-1 concentrations. Rats exposed to intermittent hypoxia (IH), a model of sleep apnea, also have increased plasma ET-1 concentrations and heightened constriction to ET-1 in mesenteric arteries without an increase in global vascular smooth muscle cell Ca(2+) concentration ([Ca(2+)]). Because ET-1 has been shown to increase the occurrence of propagating Ca(2+) waves, we hypothesized that ET-1 increases Ca(2+) wave activity in mesenteric arteries, rather than global [Ca(2+)], to mediate enhanced vasoconstriction after IH exposure. Male Sprague-Dawley rats were exposed to sham or IH conditions for 7 h/day for 2 wk. Mesenteric arteries from sham- and IH-exposed rats were isolated, cannulated, and pressurized to 75 mmHg to measure ET-1-induced constriction as well as changes in global [Ca(2+)] and Ca(2+) wave activity. A low concentration of ET-1 (1 nM) elicited similar vasoconstriction and global Ca(2+) responses in the two groups. Conversely, ET-1 had no effect on Ca(2+) wave activity in arteries from sham rats but significantly increased wave frequency in arteries from IH-exposed rats. The ET-1-induced increase in Ca(2+) wave frequency in arteries from IH rats was dependent on phospholipase C and inositol 1,4,5-trisphosphate receptor activation, yet inhibition of phospholipase C and the inositol 1,4,5-trisphosphate receptor did not prevent ET-1-mediated vasoconstriction. These results suggest that although ET-1 elevates Ca(2+) wave activity after IH exposure, increases in wave activity are not associated with increased vasoconstriction.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Endotelina-1/farmacologia , Hipóxia/metabolismo , Artérias Mesentéricas/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Pressão Arterial/efeitos dos fármacos , Modelos Animais de Doenças , Hipóxia/fisiopatologia , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/fisiopatologia , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Fatores de Tempo , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo
18.
Am J Physiol Heart Circ Physiol ; 304(11): H1446-54, 2013 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-23525712

RESUMO

We have previously shown that hydrogen sulfide (H2S) reduces myogenic tone and causes relaxation of phenylephrine (PE)-constricted mesenteric arteries. This effect of H2S to cause vasodilation and vascular smooth muscle cell (VSMC) hyperpolarization was mediated by large-conductance Ca(2+)-activated potassium channels (BKCa). Ca(2+) sparks are ryanodine receptor (RyR)-mediated Ca(2+)-release events that activate BKCa channels in VSMCs to cause membrane hyperpolarization and vasodilation. We hypothesized that H2S activates Ca(2+) sparks in small mesenteric arteries. Ca(2+) sparks were measured using confocal microscopy in rat mesenteric arteries loaded with the Ca(2+) indicator fluo-4. VSMC membrane potential (Em) was measured in isolated arteries using sharp microelectrodes. In PE-constricted arteries, the H2S donor NaHS caused vasodilation that was inhibited by ryanodine (RyR blocker), abluminal or luminal iberiotoxin (IbTx, BKCa blocker), endothelial cell (EC) disruption, and sulfaphenazole [cytochrome P-450 2C (Cyp2C) inhibitor]. The H2S donor NaHS (10 µmol/l) increased Ca(2+) sparks but only in the presence of intact EC and this was blocked by sulfaphenazole or luminal IbTx. Inhibiting cystathionine γ-lyase (CSE)-derived H2S with ß-cyano-l-alanine (BCA) also reduced VSMC Ca(2+) spark frequency in mesenteric arteries, as did EC disruption. However, excess CSE substrate homocysteine did not affect spark activity. NaHS hyperpolarized VSMC Em in PE-depolarized mesenteric arteries with intact EC and also hyperpolarized EC Em in arteries cut open to expose the lumen. This hyperpolarization was prevented by ryanodine, sulfaphenazole, and abluminal or luminal IbTx. BCA reduced IbTx-sensitive K(+) currents in freshly dispersed mesenteric ECs. These results suggest that H2S increases Ca(2+) spark activity in mesenteric artery VSMC through activation of endothelial BKCa channels and Cyp2C, a novel vasodilatory pathway for this emerging signaling molecule.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Endotélio Vascular/metabolismo , Sulfeto de Hidrogênio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/efeitos dos fármacos , Artérias Mesentéricas/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Vasodilatadores , Análise de Variância , Compostos de Anilina , Animais , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/metabolismo , Fenômenos Eletrofisiológicos , Endotélio Vascular/efeitos dos fármacos , Corantes Fluorescentes , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Microeletrodos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Xantenos
19.
J Pharmacol Exp Ther ; 344(1): 68-76, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23093023

RESUMO

Obstructive sleep apnea (OSA) is associated with cardiovascular complications including hypertension. Previous findings from our laboratory indicate that exposure to intermittent hypoxia (IH), to mimic sleep apnea, increases blood pressure in rats. IH also increases endothelin-1 (ET-1) constrictor sensitivity in a protein kinase C (PKC) δ-dependent manner in mesenteric arteries. Because phosphoinositide-dependent kinase-1 (PDK-1) regulates PKCδ activity, we hypothesized that PDK-1 contributes to the augmented ET-1 constrictor sensitivity and elevated blood pressure following IH. Male Sprague-Dawley rats were exposed to either sham or IH (cycles between 21% O(2)/0% CO(2) and 5% O(2)/5% CO(2)) conditions for 7 h/day for 14 or 21 days. The contribution of PKCδ and PDK-1 to ET-1-mediated vasoconstriction was assessed in mesenteric arteries using pharmacological inhibitors. Constrictor sensitivity to ET-1 was enhanced in arteries from IH-exposed rats. Inhibition of PKCδ or PDK-1 blunted ET-1 constriction in arteries from IH but not sham group rats. Western analysis revealed similar levels of total and phosphorylated PDK-1 in arteries from sham and IH group rats but decreased protein-protein interaction between PKCδ and PDK-1 in arteries from IH- compared with sham-exposed rats. Blood pressure was increased in rats exposed to IH, and treatment with the PDK-1 inhibitor OSU-03012 [2-amino-N-{4-[5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]-phenyl}-acetamide] (33 mg/day) lowered blood pressure in IH but not sham group rats. Our results suggest that exposure to IH unmasks a role for PDK-1 in regulating ET-1 constrictor sensitivity and blood pressure that is not present under normal conditions. These novel findings suggest that PDK-1 may be a uniquely effective antihypertensive therapy for OSA patients.


Assuntos
Pressão Sanguínea/efeitos dos fármacos , Endotelina-1/farmacologia , Hipóxia/fisiopatologia , Proteína Quinase C-delta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Vasoconstrição/efeitos dos fármacos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Inibidores Enzimáticos/farmacologia , Imunoprecipitação , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Ratos , Ratos Sprague-Dawley , Síndromes da Apneia do Sono/complicações , Síndromes da Apneia do Sono/fisiopatologia , Sulfonamidas/farmacologia
20.
Circ Res ; 108(12): 1439-47, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21512160

RESUMO

RATIONALE: Myogenic tone, an important regulator of vascular resistance, is dependent on vascular smooth muscle (VSM) depolarization, can be modulated by endothelial factors, and is increased in several models of hypertension. Intermittent hypoxia (IH) elevates blood pressure and causes endothelial dysfunction. Hydrogen sulfide (H(2)S), a recently described endothelium-derived vasodilator, is produced by the enzyme cystathionine γ-lyase (CSE) and acts by hyperpolarizing VSM. OBJECTIVE: Determine whether IH decreases endothelial H(2)S production to increase myogenic tone in small mesenteric arteries. METHODS AND RESULTS: Myogenic tone was greater in mesenteric arteries from IH than sham control rat arteries, and VSM membrane potential was depolarized in IH in comparison with sham arteries. Endothelium inactivation or scavenging of H(2)S enhanced myogenic tone in sham arteries to the level of IH. Inhibiting CSE also enhanced myogenic tone and depolarized VSM in sham but not IH arteries. Similar results were seen in cerebral arteries. Exogenous H(2)S dilated and hyperpolarized sham and IH arteries, and this dilation was blocked by iberiotoxin, paxilline, and KCl preconstriction but not glibenclamide or 3-isobutyl-1-methylxanthine. Iberiotoxin enhanced myogenic tone in both groups but more in sham than IH. CSE immunofluorescence was less in the endothelium of IH than in sham mesenteric arteries. Endogenouse H(2)S dilation was reduced in IH arteries. CONCLUSIONS: IH appears to decrease endothelial CSE expression to reduce H(2)S production, depolarize VSM, and enhance myogenic tone. H(2)S dilatation and hyperpolarization of VSM in small mesenteric arteries requires BK(Ca) channels.


Assuntos
Poluentes Atmosféricos/farmacologia , Endotélio Vascular/metabolismo , Sulfeto de Hidrogênio/farmacologia , Hipóxia/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Artérias Mesentéricas/metabolismo , Vasodilatação/efeitos dos fármacos , 1-Metil-3-Isobutilxantina , Poluentes Atmosféricos/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Endotélio Vascular/patologia , Glibureto/farmacologia , Sulfeto de Hidrogênio/metabolismo , Hipoglicemiantes/farmacologia , Hipóxia/fisiopatologia , Masculino , Artérias Mesentéricas/patologia , Paxilina/farmacologia , Peptídeos/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley
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