RESUMEN
We previously reported that the sustained component of contraction induced by depolarizing stimulation by high K+ concentration in rat caudal arterial smooth muscle involves a Ca2+-induced Ca2+ sensitization mechanism whereby Ca2+ entry through voltage-gated Ca2+ channels activates proline-rich tyrosine kinase 2 (Pyk2), leading to activation of RhoA/Rho-associated kinase (ROCK). In the present study, we investigated a potential role for Pyk2-mediated RhoA/ROCK activation in contraction mediated by elevation of cytosolic free Ca2+ concentration ([Ca2+]i) induced by a Ca2+ ionophore, ionomycin, rather than by depolarizing stimulation. Ionomycin (60 µM) induced slow and sustained contraction of rat caudal arterial smooth muscle due to influx of Ca2+. Pre-treatment with a myosin light chain kinase (MLCK) inhibitor, ML-9 (30 µM), inhibited both the early phase (4 min) and the sustained phase (30 min) of ionomycin-induced contraction. On the other hand, a ROCK inhibitor, HA-1077 (3 µM), and Pyk2 inhibitors, sodium salicylate (10 mM) and PF-431396 (3 µM), suppressed only the sustained phase of ionomycin-induced contraction. A calmodulin (CaM) inhibitor, W-7 (150 µM), but not W-5 (150 µM), suppressed the early phase of contraction. Early or sustained increase of ionomycin-induced 20 kDa light chain of myosin (LC20) phosphorylation was inhibited by each inhibitor in a manner similar to the attenuation of contraction. These results indicate that the early phase of ionomycin-induced contraction is mediated by MLCK activation by [Ca2+]i elevation, whereas the sustained phase of ionomycin-induced contraction involves RhoA/ROCK activation and inhibition of myosin light chain phosphatase (MLCP) through CaM-independent Pyk2 activation by [Ca2+]i elevation.
Asunto(s)
Calcio , Ionomicina , Contracción Muscular , Quinasas Asociadas a rho , Animales , Ionomicina/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Calcio/metabolismo , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Ratas , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Ionóforos de Calcio/farmacología , Proteína de Unión al GTP rhoA/metabolismo , Ratas Sprague-Dawley , Ratas Wistar , Calmodulina/metabolismoRESUMEN
Since COPD is a heterogeneous disease, a specific anti-inflammatory therapy for this disease has not been established yet. Oxidative stress is recognized as a major predisposing factor to COPD related inflammatory responses, resulting in pathological features of small airway fibrosis and emphysema. However, little is known about effects of oxidative stress on airway smooth muscle. Cigarette smoke increases intracellular Ca2+ concentration and enhances response to muscarinic agonists in human airway smooth muscle. Cigarette smoke also enhances proliferation of these cells with altered mitochondrial protein. Hydrogen peroxide and 8-isoprostans are increased in the exhaled breath condensate in COPD. These endogenous oxidants cause contraction of tracheal smooth muscle with Ca2+ dynamics through Ca2+ channels and with Ca2+ sensitization through Rho-kinase. TNF-α and growth factors potentiate proliferation of these cells by synthesis of ROS. Oxidative stress can alter the function of airway smooth muscle through Ca2+ signaling. These phenotype changes are associated with manifestations (dyspnea, wheezing) and pathophysiology (airflow limitation, airway remodeling, airway hyperresponsiveness). Therefore, airway smooth muscle is a therapeutic target against COPD; oxidative stress should be included in treatable traits for COPD to advance precision medicine. Research into Ca2+ signaling related to ROS may contribute to the development of a novel agent for COPD.
RESUMEN
Vascular smooth muscle contraction is intimately tied to membrane potential and the rise in intracellular Ca2+ enabled by the opening of L-type Ca2+ channels. While voltage is often viewed as the single critical factor gating these channels, research is starting to reveal a more intricate scenario whereby their function is markedly tuned. This emerging concept will be the focus of this three-part review, the first part articulating the mechanistic foundation of contractile development in vascular smooth muscle. Part two will extend this foundational knowledge, introducing readers to functional coupling and how neighboring L-type Ca2+ channels work cooperatively through signaling protein complexes, to facilitate their open probability. The final aspect of this review will discuss the impact of L-type Ca2+ channel trafficking, a process tied to cytoskeleton dynamics. Cumulatively, this brief manuscript provides new insight into how voltage, along with channel cooperativity and number, work in concert to tune Ca2+ responses and smooth muscle contraction.
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Enhanced vasoconstriction is increasingly identified as an important contributor to the development of pulmonary hypertension. Chronic hypoxia results in enhanced Rho kinase mediated Ca2+ sensitization contributing to pressure-dependent pulmonary arterial tone as well as augmented vasoconstriction to endothelin-1 and depolarizing stimuli. We sought to investigate the interaction between these vasoconstrictor stimuli in isolated, pressurized, pulmonary arteries. We used the K+ ionophore, valinomycin, to clamp membrane potential (Vm) to investigate the role of membrane depolarization in endothelin-1 and pressure-dependent constriction, and endothelin-1 receptor inhibitors to determine whether membrane depolarization or stretch signal through endothelin-1 receptors. Clamping Vm prevented pressure-dependent tone, but not enhanced vasoconstriction to endothelin-1 following chronic hypoxia. Furthermore, endothelin-1 receptor inhibition had no effect on either pressure-dependent tone or vasoconstriction to KCl. As Src kinases contribute to both pressure-dependent tone and enhanced endothelin-1 vasoconstriction following chronic hypoxia, we further investigated their role in depolarization-induced vasoconstriction. Inhibition of Src kinases attenuated enhanced vasoconstriction to KCl. We conclude that membrane depolarization contributes to pressure-dependent tone but not enhanced vasoconstriction to ET-1, and that Src kinases serve as upstream mediators facilitating enhanced Rho kinase-dependent vasoconstriction following chronic hypoxia.
RESUMEN
Coordinating blood flow to active tissue requires vasomotor responses to conduct among resistance arteries. Vasomotor spread is governed by the electrical and mechanical properties of vessels; the latter being linked to the sigmoid relations between membrane potential (VM), [Ca2+], and smooth muscle contractility. Proteins guiding electrical-to-tone translation are subject to regulation; thus, vasomotor conduction could be viewed as "pliant" to the current regulatory state. Using simple in silico approaches, we explored vasomotor pliancy and how the regulation of contractility impacts conduction along a skeletal muscle artery and a branching cerebrovascular network. Initial simulations revealed how limited electromechanical linearity affects the translation of electrical spread into arterial tone. Subtle changes to the VM-[Ca2+] or [Ca2+]-diameter relationship, akin to regulatory alterations in Ca2+ influx and Ca2+ sensitivity, modified the distance and amplitude of the conducted vasomotor response. Simultaneous changes to both relationships, consistent with agonist stimulation, augmented conduction although the effect varied with stimulus strength and polarity (depolarization vs hyperpolarization). Final simulations using our cerebrovascular network revealed how localized changes to the VM-[Ca2+] or [Ca2+]-diameter relationships could regionally shape conduction without interfering with the electrical spread. We conclude that regulatory changes to key effector proteins (e.g. L-type Ca2+ channels, myosin light chain phosphatase), integral to voltage translation, not only impact conducted vasomotor tone but likely blood flow delivery to active tissues.
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The objective of this project was to find a bronchodilatory compound from herbs and clarify the mechanism. We found that the ethanol extract of Folium Sennae (EEFS) can relax airway smooth muscle (ASM). EEFS inhibited ASM contraction, induced by acetylcholine, in mouse tracheal rings and lung slices. High-performance liquid chromatography assay showed that EEFS contained emodin. Emodin had a similar reversal action. Acetylcholine-evoked contraction was also partially reduced by nifedipine (a selective inhibitor of L-type voltage-dependent Ca2+ channels, LVDCCs), YM-58483 (a selective inhibitor of store-operated Ca2+ entry, SOCE), as well as Y-27632 (an inhibitor of Rho-associated protein kinase). In addition, LVDCC- and SOCE-mediated currents and cytosolic Ca2+ elevations were inhibited by emodin. Emodin reversed acetylcholine-caused increases in phosphorylation of myosin phosphatase target subunit 1. Furthermore, emodin, in vivo, inhibited acetylcholine-induced respiratory system resistance in mice. These results indicate that EEFS-induced relaxation results from emodin inhibiting LVDCC, SOCE, and Ca2+ sensitization. These findings suggest that Folium Sennae and emodin may be new sources of bronchodilators.
Asunto(s)
Emodina/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Acetilcolina/efectos adversos , Acetilcolina/farmacología , Animales , Broncodilatadores/metabolismo , Broncodilatadores/farmacología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Contracción Muscular/fisiología , Músculo Liso/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosfatasa de Miosina de Cadena Ligera/fisiología , Extractos Vegetales/farmacología , Senna/metabolismoRESUMEN
The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.
Asunto(s)
Acetaminofén/farmacología , Antipiréticos/farmacología , Asma/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Acetilcolina/química , Acetilcolina/farmacología , Animales , Asma/tratamiento farmacológico , Bronquios/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Nifedipino/farmacología , Potasio/metabolismoRESUMEN
Prostaglandin D2 (PGD2) is one of the key lipid mediators of allergic airway inflammation, including bronchial asthma. However, the role of PGD2 in the pathogenesis of asthma is not fully understood. In the present study, the effect of PGD2 on smooth muscle contractility of the airways was determined to elucidate its role in the development of airway hyperresponsiveness (AHR). In isolated bronchial smooth muscles (BSMs) of naive mice, application of PGD2 (10-9â»10-5 M) had no effect on the baseline tension. However, when the tissues were precontracted partially with 30 mM K⺠(in the presence of 10-6 M atropine), PGD2 markedly augmented the contraction induced by the high K⺠depolarization. The PGD2-induced augmentation of contraction was significantly inhibited both by 10-6 M laropiprant (a selective DP1 antagonist) and 10-7 M Y-27632 (a Rho-kinase inhibitor), indicating that a DP1 receptor-mediated activation of Rho-kinase is involved in the PGD2-induced BSM hyperresponsiveness. Indeed, the GTP-RhoA pull-down assay revealed an increase in active form of RhoA in the PGD2-treated mouse BSMs. On the other hand, in the high Kâº-depolarized cultured human BSM cells, PGD2 caused no further increase in cytosolic Ca2+ concentration. These findings suggest that PGD2 causes RhoA/Rho-kinase-mediated Ca2+ sensitization of BSM contraction to augment its contractility. Increased PGD2 level in the airways might be a cause of the AHR in asthma.
Asunto(s)
Bronquios/metabolismo , Calcio/metabolismo , Citosol/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Prostaglandina D2/farmacología , Animales , Atropina/farmacología , Hiperreactividad Bronquial/metabolismo , Humanos , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Potasio/farmacología , Cultivo Primario de Células , Receptores de Prostaglandina/efectos de los fármacosRESUMEN
KEY POINTS: Activation of focal adhesion kinase (FAK) by integrin signalling facilitates smooth muscle contraction by transmitting the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here we report that electrical field stimulation (EFS) of cholinergic motor neurons activates FAK in gastric fundus smooth muscles, and that FAK activation by EFS is atropine-sensitive but nicardipine-insensitive. PDBu and calyculin A contracted gastric fundus muscles Ca2+ -independently and also activated FAK. Inhibition of FAK activation inhibits the contractile responses evoked by EFS, and inhibits CPI-17 phosphorylation at T38. This study indicates that mechanical force or tension is sufficient to activate FAK, and that FAK appears to be involved in the activation of the protein kinase C-CPI-17 Ca2+ sensitization pathway in gastric fundus smooth muscles. These results reveal a novel role for FAK in gastric fundus smooth muscle contraction by facilitating CPI-17 phosphorylation. ABSTRACT: Smooth muscle contraction involves regulating myosin light chain phosphorylation and dephosphorylation by myosin light chain kinase and myosin light chain phosphatase. C-kinase potentiated protein phosphatase-1 inhibitor of 17 kDa (CPI-17) and myosin phosphatase targeting subunit of myosin light-chain phosphatase (MYPT1) are crucial for regulating gastrointestinal smooth muscle contraction by inhibiting myosin light chain phosphatase. Integrin signalling involves the dynamic recruitment of several proteins, including focal adhesion kinase (FAK), to focal adhesions. FAK tyrosine kinase activation is involved in cell adhesion to the extracellular matrix via integrin signalling. FAK participates in linking the force generated by myofilament activation to the extracellular matrix and throughout the smooth muscle tissue. Here, we show that cholinergic stimulation activates FAK in gastric fundus smooth muscles. Electrical field stimulation in the presence of Nω -nitro-l-arginine methyl ester and MRS2500 contracted gastric fundus smooth muscle strips and increased FAK Y397 phosphorylation (pY397). Atropine blocked the contractions and prevented the increase in pY397. The FAK inhibitor PF-431396 inhibited the contractions and the increase in pY397. PF-431396 also inhibited the electrical field stimulation-induced increase in CPI-17 T38 phosphorylation, and reduced MYPT1 T696 and T853, and myosin light chain S19 phosphorylation. Ca2+ influx was unaffected by PF-431396. Nicardipine inhibited the contractions but had no effect on the increase in pY397. Phorbol 12,13-dibutyrate or calyculin A contracted gastric fundus smooth muscle strips Ca2+ independently and increased pY397. Our findings suggest that FAK is activated by mechanical forces during contraction and reveal a novel role of FAK in the regulation of CPI-17 phosphorylation.
Asunto(s)
Quinasa 1 de Adhesión Focal/metabolismo , Quinasa 2 de Adhesión Focal/metabolismo , Fundus Gástrico/fisiología , Contracción Muscular , Proteínas Musculares/metabolismo , Músculo Liso/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Neuronas Colinérgicas/citología , Neuronas Colinérgicas/fisiología , Estimulación Eléctrica , Fundus Gástrico/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Músculo Liso/citología , Fosforilación , Transducción de SeñalRESUMEN
The effects of hypertonic solution on airway smooth muscle (ASM) contraction and the underlying mechanisms are largely unknown. We found that hypertonic saline (HS) inhibited acetylcholine (ACh)-induced contraction of ASM from the mouse trachea and human bronchi. In single mouse ASM cells (ASMCs), ACh induced an increase in intracellular Ca2+ that was further enhanced by 5% NaCl, indicating that the HS-induced inhibition of ASM contraction was not mediated by a decrease in cytosolic Ca2+ . The Rho-associated kinase (ROCK) inhibitor Y-27632 relaxed ACh-induced precontraction of mouse tracheal rings. However, such inhibition was not observed after the relaxation induced by 5% NaCl. Moreover, the incubation of mouse tracheal rings with 5% NaCl decreased ACh-induced phosphorylation of myosin light chain 20 and myosin phosphatase target subunit 1. These data indicate that HS inhibits the contraction of ASM by inhibiting Ca2+ sensitization, not by decreasing intracellular Ca2+ .
Asunto(s)
Calcio/metabolismo , Pulmón/fisiología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Solución Salina Hipertónica/farmacología , Acetilcolina/farmacología , Animales , Asma/metabolismo , Asma/patología , Asma/fisiopatología , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Pulmón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Músculo Liso/citología , Músculo Liso/metabolismoRESUMEN
Prolonged bed rest (PBR) causes orthostatic hypotension (OH). Rapid constriction of splanchnic resistance arteries in response to a sudden increase in sympathetic tone contributes to the recovery of orthostatic arterial pressure upon standing. However, the molecular mechanism of PBR-induced dysfunction in arterial constriction is not fully understood. Previously, we showed that CPI-17, a regulatory protein for myosin phosphatase, mediates α1A-adrenergic receptor-induced rapid contraction of small mesenteric arteries. Here, we tested whether PBR associated with OH affects the α1-adrenergic receptor-induced CPI-17 signaling pathway in mesenteric arteries using rats treated by head-down tail-suspension hindlimb unloading (HDU), an experimental OH model. In normal anesthetized rats, mean arterial pressure (MAP) rapidly reduced upon 90° head-up tilt from supine position and then immediately recovered without change in heart rate, suggesting a rapid arterial constriction. On the other hand, after a 4-week HDU treatment, the fast orthostatic MAP recovery failed for 1 min. Alpha1A subtype-specific antagonist suppressed the orthostatic MAP recovery with a small decrease in basal blood pressure, whereas non-specific α1-antagonist prazosin strongly reduced both basal MAP and orthostatic recovery. The HDU treatment resulted in 68% reduction in contraction in parallel with 83% reduction in CPI-17 phosphorylation in denuded mesenteric arteries 10 s after α1-agonist stimulation. The treatment with either Ca2+-release channel opener or PKC inhibitor mimicked the deficiency in HDU arteries. These results suggest that an impairment of the rapid PKC/CPI-17 signaling pathway downstream of α1A-adrenoceptors in peripheral arterial constriction, as an end organ of orthostatic blood pressure reflex, is associated with OH in prolonged bed rest patients.
Asunto(s)
Reposo en Cama/efectos adversos , Hipotensión Ortostática/metabolismo , Arterias Mesentéricas/metabolismo , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Animales , Presión Arterial/fisiología , Femenino , Inclinación de Cabeza/efectos adversos , Inclinación de Cabeza/fisiología , Frecuencia Cardíaca/fisiología , Hipotensión Ortostática/etiología , Masculino , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Airway hyperresponsiveness (AHR) and inflammation are key pathophysiological features of asthma. Enhanced contraction of bronchial smooth muscle (BSM) is one of the causes of the AHR. It is thus important for development of asthma therapy to understand the change in the contractile signaling of airway smooth muscle cells associated with the AHR. In addition to the Ca2+-mediated phosphorylation of myosin light chain (MLC), contractile agonists also enhance MLC phosphorylation level, Ca2+-independently, by inactivating MLC phosphatase (MLCP), called Ca2+ sensitization of contraction, in smooth muscle cells including airways. To date, involvements of RhoA/ROCKs and PKC/Ppp1r14a (also called as CPI-17) pathways in the Ca2+ sensitization have been identified. Our previous studies revealed that the agonist-induced Ca2+ sensitization of contraction is markedly augmented in BSMs of animal models of allergen-induced AHR. In BSMs of these animal models, the expression of RhoA and CPI-17 proteins were significantly increased, indicating that both the Ca2+ sensitizing pathways are augmented. Interestingly, incubation of BSM cells with asthma-associated cytokines, such as interleukin-13 (IL-13), IL-17, and tumor necrosis factor-α (TNF-α), caused up-regulations of RhoA and CPI-17 in BSM cells of naive animals and cultured human BSM cells. In addition to the transcription factors such as STAT6 and NF-κB activated by these inflammatory cytokines, an involvement of down-regulation of miR-133a, a microRNA that negatively regulates RhoA translation, has also been suggested in the IL-13- and IL-17-induced up-regulation of RhoA. Thus, the Ca2+ sensitizing pathways and the cytokine-mediated signaling including microRNAs in BSMs might be potential targets for treatment of allergic asthma, especially the AHR.
Asunto(s)
Asma/etiología , Asma/fisiopatología , Bronquios/fisiopatología , Contracción Muscular , Músculo Liso/fisiopatología , Hipersensibilidad Respiratoria/etiología , Animales , Asma/terapia , Calcio/metabolismo , Células Cultivadas , Citocinas/fisiología , Humanos , Mediadores de Inflamación/fisiología , Péptidos y Proteínas de Señalización Intracelular , MicroARNs , Terapia Molecular Dirigida , Proteínas Musculares , Cadenas Ligeras de Miosina/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Hipersensibilidad Respiratoria/terapia , Transducción de Señal/fisiología , Quinasas Asociadas a rho/metabolismo , Quinasas Asociadas a rho/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain phosphatase by phosphorylating its targeting subunit myosin phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8-10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18-20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.
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Actinas/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Fosfatasa de Miosina de Cadena Ligera/antagonistas & inhibidores , Vasoconstricción , Animales , Calcio/metabolismo , Arterias Cerebrales/fisiopatología , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , Polimerizacion , Ratas , Ratas Endogámicas , Quinasas Asociadas a rhoRESUMEN
Long-acting muscarinic antagonists (LAMAs) and short-acting ß2-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca2+ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 µM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca2+-activated K⺠(KCa) channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca2+ sensitization mediated by PKC and Ca2+ dynamics mediated by KCa channels. PKC and KCa channels may be molecular targets for cross talk between ß2-adrenoceptors and muscarinic receptors.
RESUMEN
Formaldehyde, a common indoor air pollutant, exacerbates asthma and synergizes with allergen to induce airway hyperresponsiveness (AHR) in animal models. The mechanisms mediating formaldehyde-induced AHR remain poorly understood. We posit that formaldehyde modulates agonist-induced contractile response of human airway smooth muscle (HASM) cells to elicit AHR. HASM cells were exposed to formaldehyde or vehicle and agonist-induced intracellular Ca2+ ([Ca2+]i) and myosin light-chain phosphatase (MYPT1) phosphorylation were determined. Air-liquid interface-differentiated human bronchial epithelial (HBE) cells were exposed to formaldehyde or vehicle and cocultured with HASM cells. Agonist-induced [Ca2+]i and MYPT1 phosphorylation were determined in the cocultured HASM cells. Precision-cut human lung slices were exposed to PBS or varying concentrations of formaldehyde, and then carbachol-induced airway narrowing was determined 24 hours after exposure. HASM cells were transfected with nontargeting or nuclear factor erythroid-derived 2, like 2 (Nrf-2)-targeting small interfering RNA and exposed to formaldehyde or vehicle, followed by determination of antioxidant response (quinone oxido-reductase 1 and thioredoxin 1) and basal and agonist-induced MYPT1 phosphorylation. Formaldehyde enhanced the basal Rho-kinase activity and MYPT1 phosphorylation with little effect on agonist-induced [Ca2+]i in HASM cells. Formaldehyde induced Nrf-2-dependent antioxidant response in HASM cells, although the MYPT1 phosphorylation was independent of Nrf-2 induction. Although HBE cells exposed to formaldehyde had little effect on agonist-induced [Ca2+]i or MYPT1 phosphorylation in cocultured HASM cells, formaldehyde enhanced carbachol-induced airway responsiveness in precision-cut human lung slices. In conclusion, formaldehyde induces phosphorylation of the regulatory subunit of MYPT1, independent of formaldehyde-induced Nrf-2 activation in HASM cells. The findings suggest that the Rho kinase-dependent Ca2+ sensitization pathway plays a role in formaldehyde-induced AHR.
RESUMEN
This study is the first pharmacological characterization of the novel chemical entity, ORM-3819 (L-6-{4-[N'-(4-Hydroxi-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one), focusing primarily on its cardiotonic effects. ORM-3819 binding to cardiac troponin C (cTnC) was confirmed by nuclear magnetic resonance spectroscopy, and a selective inhibition of the phosphodiesterase III (PDE III) isozyme (IC50=3.88±0.3 nM) was revealed during in vitro enzyme assays. The Ca(2+)-sensitizing effect of ORM-3819 was demonstrated in vitro in permeabilized myocyte-sized preparations from left ventricles (LV) of guinea pig hearts (ΔpCa50=0.12±0.01; EC50=2.88±0.14 µM). ORM-3819 increased the maximal rate of LV pressure development (+dP/dtmax) (EC50=8.9±1.7 nM) and LV systolic pressure (EC50=7.63±1.74 nM) in Langendorff-perfused guinea pig hearts. Intravenous administration of ORM-3819 increased LV+dP/dtmax (EC50=0.13±0.05 µM/kg) and improved the rate of LV pressure decrease (-dP/dtmax); (EC50=0.03±0.02 µM/kg) in healthy guinea pigs. In an in vivo dog model of myocardial stunning, ORM-3819 restored the depressed LV+dP/dtmax and improved % segmental shortening (%SS) in the ischemic area (to 18.8±3), which was reduced after the ischaemia-reperfusion insult (from 24.1±2.1 to 11.0±2.4). Our data demonstrate ORM-3819 as a potent positive inotropic agent exerting its cardiotonic effect by a cTnC-dependent Ca(2+)-sensitizing mechanism in combination with the selective inhibition of the PDE III isozyme. This dual mechanism of action results in the concentration-dependent augmentation of the contractile performance under control conditions and in the postischemic failing myocardium.
Asunto(s)
Cardiotónicos/farmacología , Hidrazonas/farmacología , Contracción Miocárdica/efectos de los fármacos , Inhibidores de Fosfodiesterasa 3/farmacología , Piridazinas/farmacología , Animales , Calcio/fisiología , Perros , Femenino , Cobayas , Ventrículos Cardíacos/citología , Técnicas In Vitro , Masculino , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Troponina C/metabolismo , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
Defective protein kinase C (PKC) signaling has been suggested to contribute to abnormal vascular contraction in disease conditions including hypertension and diabetes. Our previous work on agonist and pressure-induced cerebral vasoconstriction implicated PKC as a major contributor to force production in a myosin light chain (LC20) phosphorylation-independent manner. Here, we used phorbol dibutyrate to selectively induce a PKC-dependent constriction in rat middle cerebral arteries and delineate the relative contribution of different contractile mechanisms involved. Specifically, we employed an ultra-sensitive 3-step western blotting approach to detect changes in the content of phosphoproteins that regulate myosin light chain phosphatase (MLCP) activity, thin filament activation, and actin cytoskeleton reorganization. Data indicate that PKC activation evoked a greater constriction at a similar level of LC20 phosphorylation achieved by 5-HT. PDBu-evoked constriction persisted in the presence of Gö6976, a selective inhibitor of Ca(2+)-dependent PKC, and in the absence of extracellular Ca(2+). Biochemical evidence indicates that either + or - extracellular Ca(2+), PDBu (i) inhibits MLCP activity via the phosphorylation of myosin targeting subunit of myosin phosphatase (MYPT1) and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17), (ii) increases the phosphorylation of paxillin and heat shock protein 27 (HSP27), and reduces G-actin content, and (iii) does not change the phospho-content of the thin filament proteins, calponin and caldesmon. PDBu-induced constriction was more sensitive to disruption of actin cytoskeleton compared to inhibition of cross-bridge cycling. In conclusion, this study provided evidence for the pivotal contribution of cytoskeletal actin polymerization in force generation following PKC activation in cerebral resistance arteries.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Encéfalo/irrigación sanguínea , Cadenas Ligeras de Miosina/metabolismo , Proteína Quinasa C/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Activación Enzimática , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Masculino , Arteria Cerebral Media/efectos de los fármacos , Arteria Cerebral Media/fisiología , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Polimerizacion , Proteína Quinasa C/antagonistas & inhibidores , Ratas Sprague-Dawley , Serotonina/farmacología , Tiazolidinas/farmacología , Resistencia Vascular , Vasoconstricción/efectos de los fármacosRESUMEN
Retinal relaxing factor (RRF) has recently been identified as a novel paracrine regulator of retinal circulation acting differently from well known mediators of the endothelium and the retina. Herein, we aimed to characterize the relaxing mechanism of the retina, i.e. RRF, by evaluating the role of Ca(+2)-dependent and -independent signaling mechanisms as well as inward rectifier K(+) (Kir) channels. Retinal relaxation was determined by placing a piece of retinal tissue just on top of the precontracted bovine retinal arteries mounted in a wire myograph. The retina produced a complete relaxation response, which display a biphasic character, in depolarized arteries contracted by L-type Ca(2+) channel agonist, Bay k 8644. Blockade of L-type Ca(2+) channel by nifedipine, inhibition of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid or removal of extracellular Ca(2+) did not influence the prominent relaxation to the retina. Originally, retinal relaxation was found to be unaffected from the inhibition of myosin light chain kinase by ML7, whereas, completely abolished in the presence of myosin light chain phosphatase (MLCP) inhibitor, Calyculin A. Moreover, the inhibition of Rho kinase by its putative inhibitor, Y-27632 displayed comparable relaxant effects to RRF in retinal arteries precontracted either by prostaglandin F2α or K(+), and augmented the moderate response to the retina in K(+) precontracted arteries. In addition, retinal relaxation was significantly inhibited and lost its biphasic character in the presence of Kir channel blocker, Ba(2+). Our results suggested that inhibition of Ca(2+) sensitization through the activation of MLCP, possibly via interfering with Rho kinase, and the opening of Kir channels are likely to be involved in the inhibitory influence of RRF on the retinal arteries.
Asunto(s)
Señalización del Calcio/fisiología , Canales de Potasio/fisiología , Retina/fisiología , Arteria Retiniana/fisiología , Análisis de Varianza , Animales , Bloqueadores de los Canales de Calcio/farmacología , Bovinos , Modelos Animales , Bloqueadores de los Canales de Potasio/farmacología , Retina/efectos de los fármacos , Arteria Retiniana/efectos de los fármacos , Transducción de Señal/fisiología , Vasodilatación/fisiología , Quinasas Asociadas a rho/antagonistas & inhibidores , Quinasas Asociadas a rho/fisiologíaRESUMEN
The structural and functional integrity of the brain, and therefore, cognition, are critically dependent on the appropriate control of blood flow within the cerebral circulation. Inadequate flow leads to ischemia, whereas excessive flow causes small vessel rupture and (or) blood-brain-barrier disruption. Cerebral blood flow is controlled through the interplay of several physiological mechanisms that regulate the contractile state of vascular smooth muscle cells (VSMCs) within the walls of cerebral resistance arteries and arterioles. The myogenic response of cerebral VSMCs is a key mechanism that is responsible for maintaining constant blood flow during variations in systemic pressure, i.e., flow autoregulation. Inappropriate myogenic control of cerebral blood flow is associated with, and prognostic of, neurological deterioration and poor outcome in patients with several conditions, including type 2 diabetes. Here, we review recent advances in our understanding of the role of inappropriate Rho-associated kinase activity as a cause of impaired myogenic regulation of cerebral arterial diameter in type 2 diabetes.
Asunto(s)
Arterias Cerebrales/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Miogenina/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Circulación Cerebrovascular , HumanosRESUMEN
Aortocaval fistula (ACF)-induced volume overload (VO) heart failure (HF) results in progressive left ventricular (LV) dysfunction. Hemodynamic load reversal during pre-HF (4 wk post-ACF; REV) results in rapid structural but delayed functional recovery. This study investigated myocyte and myofilament function in ACF and REV and tested the hypothesis that a myofilament Ca(2+) sensitizer would improve VO-induced myofilament dysfunction in ACF and REV. Following the initial sham or ACF surgery in male Sprague-Dawley rats (200-240 g) at week 0, REV surgery and experiments were performed at weeks 4 and 8, respectively. In ACF, decreased LV function is accompanied by impaired sarcomeric shortening and force generation and decreased Ca(2+) sensitivity, whereas, in REV, impaired LV function is accompanied by decreased Ca(2+) sensitivity. Intravenous levosimendan (Levo) elicited the best inotropic and lusitropic responses and was selected for chronic oral studies. Subsets of ACF and REV rats were given vehicle (water) or Levo (1 mg/kg) in drinking water from weeks 4-8. Levo improved systolic (% fractional shortening, end-systolic elastance, and preload-recruitable stroke work) and diastolic (τ, dP/dtmin) function in ACF and REV. Levo improved Ca(2+) sensitivity without altering the amplitude and kinetics of the intracellular Ca(2+) transient. In ACF-Levo, increased cMyBP-C Ser-273 and Ser-302 and cardiac troponin I Ser-23/24 phosphorylation correlated with improved diastolic relaxation, whereas, in REV-Levo, increased cMyBP-C Ser-273 phosphorylation and increased α-to-ß-myosin heavy chain correlated with improved diastolic relaxation. We concluded that Levo improves LV function, and myofilament composition and regulatory protein phosphorylation likely play a key role in improving function.