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OBJECTIVE: This study tested the hypothesis that administration of the KCa channel activator SKA-31 restores endothelium-dependent vasodilation in vivo in Type 2 Diabetic (T2D) rats. BACKGROUND: Acute treatment of isolated resistance arteries from T2D rats and humans with SKA-31 significantly improved endothelium-dependent vasodilation. However, it is unknown whether these in situ actions translate to intact vascular beds in vivo. METHODS: Male Sprague Dawley (SD) and T2D Goto-Kakizaki (GK) rats (26-32â¯weeks of age) were injected intraperitoneally with either drug vehicle or 10â¯mg/kg SKA-31. Doppler ultrasound imaging was used to record reactive hyperemia/flow-mediated dilation (FMD) in the femoral artery following release of an occlusion cuff on the distal hind limb, along with diameter changes in the left main coronary artery in response to inhaled isoflurane (2â¯%â¯ââ¯5â¯%). RESULTS: Vehicle treated SD rats exhibited a robust and reversible FMD response, the magnitude and time course of which did not differ in SD rats treated with SKA-31. In contrast, only a weak FMD response was observed in vehicle-treated T2D GK rats, whereas prior SKA-31 administration restored FMD to the level observed in control SD rats. Exposure of SD rats to 5â¯% isoflurane caused robust coronary artery dilation, which was not altered by prior treatment with SKA-31. In T2D GK rats, 5â¯% isoflurane inhalation alone did not increase coronary artery diameter, however, a strong vasodilatory response was observed following SKA-31 treatment. SKA-31 administration did not modify intrinsic heart rate responses in either protocol. CONCLUSIONS: Enhancement of KCa channel activity in vivo restores endothelium-dependent vasodilation in T2D rats that exhibit peripheral endothelial dysfunction.
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Elevated levels of cholesterol in the blood can induce endothelial dysfunction, a condition characterized by impaired nitric oxide production and decreased vasodilatory capacity. Endothelial dysfunction can promote vascular disease, such as atherosclerosis, where macrophages accumulate in the vascular intima and fatty plaques form that impair normal blood flow in conduit arteries. Current pharmacological strategies to treat atherosclerosis mostly focus on lipid lowering to prevent high levels of plasma cholesterol that induce endothelial dysfunction and atherosclerosis. While this approach is effective for most patients with atherosclerosis, for some, lipid lowering is not enough to reduce their cardiovascular risk factors associated with atherosclerosis (e.g., hypertension, cardiac dysfunction, stroke, etc.). For such patients, additional strategies targeted at reducing endothelial dysfunction may be beneficial. One novel strategy to restore endothelial function and mitigate atherosclerosis risk is to enhance the activity of Ca2+-activated K+ (KCa) channels in the endothelium with positive gating modulator drugs. Here, we review the mechanism of action of these small molecules and discuss their ability to improve endothelial function. We then explore how this strategy could mitigate endothelial dysfunction in the context of atherosclerosis by examining how KCa modulators can improve cardiovascular function in other settings, such as aging and type 2 diabetes. Finally, we consider questions that will need to be addressed to determine whether KCa channel activation could be used as a long-term add-on to lipid lowering to augment atherosclerosis treatment, particularly in patients where lipid-lowering is not adequate to improve their cardiovascular health.
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Fibronectin (FN) enhances K+ channel activity by integrin-mediated mechanisms. As vascular smooth muscle (VSM) K+ channels mediate vasodilation, we hypothesized that modification of fibronectin, via advanced non-enzymatic glycation, would alter signaling of this extracellular matrix protein through these channels. Bovine FN (1 mg/ml) was glycated (gFN) for 5 days using methylglyoxal (50 mM), and albumin was similarly glycated as a non-matrix protein control. VSM cells were isolated from rat cerebral arteries for measurement of macroscopic K+ channel activity using whole cell patch clamp methodology. Pharmacological inhibitors, iberiotoxin (0.1 µM) and 4-aminopyridine (0.1 mM), were used to identify contributions of large-conductance, Ca2+-activated, K+ channels and voltage-gated K+ channels, respectively. Compared with baseline, native FN enhanced whole cell K+ current in a concentration-dependent manner, whereas gFN inhibited basal current. Furthermore, native albumin did not enhance basal K+ current, but the glycated form (gAlb) caused inhibition. gFN was shown to impair both the Kv and BKCa components of total macroscopic K+ current. Anti-integrin α5 and ß1 antibodies attenuated the effects of both FN and gFN on macroscopic K+ current at +70 mV. Consistent with an action on BKCa activity, FN increased, whereas gFN decreased the frequency of spontaneous transient outward current (STOCs). In contrast, gAlb inhibited whole cell K+ current predominantly through Kv, showing little effect on STOCs. A function-blocking, anti-RAGE antibody partially reversed the inhibitory effects of gFN, suggesting involvement of this receptor. Further, gFN caused production of reactive oxygen species (ROS) by isolated VSMCs as revealed by the fluorescent indicator, DHE. Evoked ROS production was attenuated by the RAGE blocking antibody. Collectively, these studies identify ion channel-related mechanisms (integrin and ROS-mediated) by which protein glycation may modify VSMC function.
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Reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide, are reported to contribute to the dynamic regulation of contractility in various arterial preparations, however, the situation in pressurized, myogenically active resistance arteries is much less clear. In the present study, we have utilized established pharmacological inhibitors of NADPH oxidase activity to examine the potential contribution of ROS to intrinsic myogenic contractility in adult Sprague-Dawley rat resistance arteries and responses to vasoactive agents acting via the endothelium (i.e., acetylcholine, SKA-31) or smooth muscle (i.e., sodium nitroprusside, phenylephrine). In cannulated and pressurized cremaster skeletal muscle and middle cerebral arteries, the NOX inhibitors 2-acetylphenothiazine (2-APT) and VAS2870, selective for NOX1 and NOX2, respectively, evoked concentration-dependent inhibition of basal myogenic tone in a reversible and irreversible manner, respectively, whereas the non-selective inhibitor apocynin augmented myogenic contractility. The vasodilatory actions of 2-APT and VAS2870 occurred primarily via the vascular endothelium and smooth muscle, respectively. Functional responses to established endothelium-dependent and -independent vasoactive agents were largely unaltered in the presence of either 2-APT or apocynin. In cremaster arteries from Type 2 Diabetic (T2D) Goto-Kakizaki rats with endothelial dysfunction, treatment with either 2-APT or apocynin did not modify stimulus-evoked vasoactive responses, but did affect basal myogenic tone. These same NOX inhibitors produced robust inhibition of total NADPH oxidase activity in aortic tissue homogenates from control and T2D rats, and NOX isozymes 1, 2 and 4, along with superoxide dismutase 1, were detected by qPCR in cremaster arteries and aorta from both species. Based on the diverse effects that we observed for established, chemically distinct NOX inhibitors, the functional contribution of vascular NADPH oxidase activity to stimulus-evoked vasoactive signaling in myogenically active, small resistance arteries remains unclear.
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BACKGROUND: Endothelial dysfunction is an early pathogenic event in the progression of cardiovascular disease in patients with Type 2 Diabetes (T2D). Endothelial KCa2.3 and KCa3.1 K+ channels are important regulators of arterial diameter, and we thus hypothesized that SKA-31, a small molecule activator of KCa2.3 and KCa3.1, would positively influence agonist-evoked dilation in myogenically active resistance arteries in T2D. METHODOLOGY: Arterial pressure myography was utilized to investigate endothelium-dependent vasodilation in isolated cremaster skeletal muscle resistance arteries from 22 to 24â¯week old T2D Goto-Kakizaki rats, age-matched Wistar controls, and small human intra-thoracic resistance arteries from T2D subjects. Agonist stimulated changes in cytosolic free Ca2+ in acutely isolated, single endothelial cells from Wistar and T2D Goto-Kakizaki cremaster and cerebral arteries were examined using Fura-2 fluorescence imaging. MAIN FINDINGS: Endothelium-dependent vasodilation in response to acetylcholine (ACh) or bradykinin (BK) was significantly impaired in isolated cremaster arteries from T2D Goto-Kakizaki rats compared with Wistar controls, and similar results were observed in human intra-thoracic arteries. In contrast, inhibition of myogenic tone by sodium nitroprusside, a direct smooth muscle relaxant, was unaltered in both rat and human T2D arteries. Treatment with a threshold concentration of SKA-31 (0.3⯵M) significantly enhanced vasodilatory responses to ACh and BK in arteries from T2D Goto-Kakizaki rats and human subjects, whereas only modest effects were observed in non-diabetic arteries of both species. Mechanistically, SKA-31 enhancement of evoked dilation was independent of vascular NO synthase and COX activities. Remarkably, SKA-31 treatment improved agonist-stimulated Ca2+ elevation in acutely isolated endothelial cells from T2D Goto-Kakizaki cremaster and cerebral arteries, but not from Wistar control vessels. In contrast, SKA-31 treatment did not affect intracellular Ca2+ release by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor cyclopiazonic acid. CONCLUSIONS: Collectively, our data demonstrate that KCa channel modulation can acutely restore endothelium-dependent vasodilatory responses in T2D resistance arteries from rats and humans, which appears to involve improved endothelial Ca2+ mobilization.
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Artérias/metabolismo , Cálcio/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Acetilcolina/farmacologia , Animais , Artérias/efeitos dos fármacos , Bradicinina/farmacologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Músculo Esquelético/irrigação sanguínea , Ratos , Ratos Wistar , Vasodilatação/efeitos dos fármacosRESUMO
Aging represents an independent risk factor for the development of cardiovascular disease, and is associated with complex structural and functional alterations in the vasculature, such as endothelial dysfunction. Small- and intermediate-conductance, Ca2+-activated K+ channels (KCa2.3 and KCa3.1, respectively) are prominently expressed in the vascular endothelium, and pharmacological activators of these channels induce robust vasodilation upon acute exposure in isolated arteries and intact animals. However, the effects of prolonged in vivo administration of such compounds are unknown. In our study, we hypothesized that such treatment would ameliorate aging-related cardiovascular deficits. Aged (â¼18 months) male Sprague Dawley rats were treated daily with either vehicle or the KCa channel activator SKA-31 (10â¯mg/kg, intraperitoneal injection; nâ¯=â¯6/group) for 8 weeks, followed by echocardiography, arterial pressure myography, immune cell and plasma cytokine characterization, and tissue histology. Our results show that SKA-31 administration improved endothelium-dependent vasodilation, reduced agonist-induced vascular contractility, and prevented the aging-associated declines in cardiac ejection fraction, stroke volume and fractional shortening, and further improved the expression of endothelial KCa channels and associated cell signalling components to levels similar to those observed in young male rats (â¼5 months at end of study). SKA-31 administration did not promote pro-inflammatory changes in either T cell populations or plasma cytokines/chemokines, and we observed no overt tissue histopathology in heart, kidney, aorta, brain, liver and spleen. SKA-31 treatment in young rats had little to no effect on vascular reactivity, select protein expression, tissue histology, plasma cytokines/chemokines or immune cell properties. Collectively, these data demonstrate that administration of the KCa channel activator SKA-31 improved aging-related cardiovascular function, without adversely affecting the immune system or promoting tissue toxicity.
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Envelhecimento , Pressão Arterial/efeitos dos fármacos , Benzotiazóis/farmacologia , Coração/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/agonistas , Envelhecimento/efeitos dos fármacos , Animais , Células Cultivadas , Coração/fisiologia , Masculino , Canais de Potássio Cálcio-Ativados/metabolismo , Ratos Sprague-Dawley , Volume Sistólico/efeitos dos fármacos , Vasodilatação/efeitos dos fármacosRESUMO
Pancreatic islets adapt to the increase in insulin demand during pregnancy by upregulating ß-cell number, insulin synthesis, and secretion. These changes require prolactin receptor (PrlR) signaling, as mice with PrlR deletion are glucose intolerant with a lower ß-cell mass. Prolactin also prevents ß-cell apoptosis. Many genes participate in these adaptive changes in the islet, and Lrrc55 is one of the most upregulated genes with unknown function in islets. Because Lrrc55 expression increases in parallel to the increase in ß-cell number and insulin production during pregnancy, we hypothesize that Lrrc55 might regulate ß-cell proliferation/apoptosis (thus ß-cell number) and insulin synthesis. Here, we found that Lrrc55 expression was upregulated by >60-fold during pregnancy in a PrlR-dependent manner, and this increase was restricted only to the islets. Overexpression of Lrrc55 in ß-cells had minimal effect on ß-cell proliferation and glucose-stimulated insulin secretion but protected ß-cells from glucolipotoxicity-induced reduction in insulin gene expression. Moreover, Lrrc55 protects ß-cells from glucolipotoxicity-induced apoptosis, with upregulation of prosurvival signals and downregulation of proapoptotic signals of the endoplasmic reticulum (ER) stress pathway. Furthermore, Lrrc55 attenuated calcium depletion induced by glucolipotoxicity, which may contribute to its antiapoptotic effect. Hence our findings suggest that Lrrc55 is a novel prosurvival factor that is upregulated specifically in islets during pregnancy, and it prevents conversion of adaptive unfolded protein response to unresolved ER stress and apoptosis in ß-cells. Lrrc55 could be a potential therapeutic target in diabetes by reducing ER stress and promoting ß-cell survival.
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Células Secretoras de Insulina/fisiologia , Ilhotas Pancreáticas/fisiologia , Proteínas de Membrana/fisiologia , Animais , Apoptose/genética , Apoptose/fisiologia , Cálcio/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Diabetes Mellitus Experimental/genética , Feminino , Insulina/biossíntese , Insulina/genética , Secreção de Insulina/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Transdução de Sinais/genética , Resposta a Proteínas não Dobradas/genética , Regulação para CimaRESUMO
Systemic hypertension is a major risk factor for the development of cardiovascular disease and is often associated with endothelial dysfunction. KCa2.3 and KCa3.1 channels are expressed in the vascular endothelium and contribute to stimulus-evoked vasodilation. We hypothesized that acute treatment with SKA-31, a selective activator of KCa2.x and KCa3.1 channels, would improve endothelium-dependent vasodilation and transiently lower mean arterial pressure (MAP) in male, spontaneously hypertensive rats (SHRs). Isolated vascular preparations exhibited impaired vasodilation in response to bradykinin (i.e., endothelial dysfunction) compared with Wistar controls, which was associated with decreased bradykinin receptor expression in mesenteric arteries. In contrast, similar levels of endothelial KCa channel expression were observed, and SKA-31 evoked vasodilation was comparable in vascular preparations from both strains. Addition of a low concentration of SKA-31 (i.e., 0.2-0.3 µM) failed to augment bradykinin-induced vasodilation in arteries from SHRs. However, responses to acetylcholine were enhanced. Surprisingly, acute bolus administration of SKA-31 in vivo (30 mg/kg, i.p. injection) modestly elevated MAP compared with vehicle injection. In summary, pharmacological targeting of endothelial KCa channels in SHRs did not readily reverse endothelial dysfunction in situ, or lower MAP in vivo. SHRs thus appear to be less responsive to endothelial KCa channel activators, which may be related to their vascular pathology.
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Endotélio Vascular/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/agonistas , Acetilcolina/farmacologia , Animais , Benzotiazóis/farmacologia , Benzotiazóis/uso terapêutico , Pressão Sanguínea , Bradicinina/farmacologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Hipertensão/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Masculino , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Receptores da Bradicinina/genética , Receptores da Bradicinina/metabolismo , VasodilataçãoRESUMO
Rhythmical contractility of blood vessels was first observed in bat wing veins by Jones (Philos Trans R Soc Lond 1852:142, 131-136), and subsequently described in arteries and arterioles of multiple vascular beds in several species. Despite an abundance of descriptive literature regarding the presence of vasomotion, to date we do not have an accurate picture of the cellular and ionic basis of these oscillations in tone, or the physiological relevance of the changes in pulsatile blood flow arising from vasomotion. This chapter reviews our current understanding of the cellular and ionic mechanisms underlying vasomotion in resistance arteries and arterioles. Focus is directed to the ion channels, changes in cytosolic Ca2+ concentration, and involvement of intercellular gap junctions in the development and synchronization of rhythmic changes in membrane potential and cytosolic Ca2+ concentration within the vessel wall that contribute to vasomotion. The physiological consequences of vasomotion are discussed with a focus on the cerebral vasculature, as recent advances show that rhythmic oscillations in cerebral arteriolar diameter appear to be entrained by cortical neural activity to increase the local supply of blood flow to active regions of the brain.
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Artérias/fisiologia , Sinalização do Cálcio , Canais Iônicos/fisiologia , Músculo Liso Vascular/fisiologia , Animais , Arteríolas , Junções Comunicantes , Potenciais da Membrana , Fluxo PulsátilRESUMO
We have systematically investigated how vascular smooth muscle α1 -adrenoceptor activation impacts endothelium-mediated vasodilation in isolated, myogenically active, rat cremaster muscle 1A arteries. Cannulated cremaster arteries were pressurized intraluminally to 70 mmHg to induce myogenic tone, and exposed to vasoactive agents via bath superfusion at 34°C. Smooth muscle membrane potential was measured via sharp microelectrode recordings in pressurized, myogenic arteries. The α1 -adrenergic agonist phenylephrine (25-100 nmol/L) produced further constriction of myogenic arteries, but did not alter the vasorelaxant responses to acetylcholine (0.3 µmol/L), SKA-31 (an activator of endothelial Ca2+ -dependent K+ channels) (3 µmol/L) or sodium nitroprusside (10 µmol/L). Exposure to 0.25-1 µmol/L phenylephrine or 1 µmol/L norepinephrine generated more robust constrictions, and also enhanced the vasodilations evoked by acetylcholine and SKA-31, but not by sodium nitroprusside. In contrast, the thromboxane receptor agonist U46619 (250 nmol/L) dampened responses to all three vasodilators. Phenylephrine exposure depolarized myogenic arteries, and mimicking this effect with 4-aminopyridine (1 mmol/L) was sufficient to augment the SKA-31-evoked vasodilation. Inhibition of L-type Ca2+ channels by 1 µmol/L nifedipine decreased myogenic tone, phenylephrine-induced constriction and prevented α1 -adrenergic enhancement of endothelium-evoked vasodilation; these latter deficits were overcome by exposure to 3 and 10 µmol/L phenylephrine. Mechanistically, augmentation of ACh-evoked dilation by phenylephrine was dampened by eNOS inhibition and abolished by blockade of endothelial KCa channels. Collectively, these data suggest that increasing α1 -adrenoceptor activation beyond a threshold level augments endothelium-evoked vasodilation, likely by triggering transcellular signaling between smooth muscle and the endothelium. Physiologically, this negative feedback process may serve as a "brake" to limit the extent of vasoconstriction in the skeletal microcirculation evoked by the elevated sympathetic tone.
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Endotélio Vascular/fisiologia , Músculo Liso Vascular/fisiologia , Receptores Adrenérgicos alfa 1/fisiologia , Vasodilatação , Acetilcolina/fisiologia , Agonistas de Receptores Adrenérgicos alfa 1/administração & dosagem , Animais , Canais de Cálcio Tipo L/fisiologia , Endotélio Vascular/efeitos dos fármacos , Masculino , Potenciais da Membrana , Músculo Liso Vascular/irrigação sanguínea , Músculo Liso Vascular/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/fisiologia , Norepinefrina/administração & dosagem , Fenilefrina/administração & dosagem , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Ratos Sprague-Dawley , Vasodilatação/efeitos dos fármacosRESUMO
It is now well recognized that endothelial KCa2.3 and KCa3.1 channel activities contribute to dilation of resistance arteries via endothelium-mediated hyperpolarization and vascular smooth muscle relaxation. In this study, we have investigated the functional effect of the KCa channel activator SKA-31 in third order rat mesenteric arteries using arterial pressure myography. Isolated arteries were cannulated, pressurized intraluminally to 70â¯mmHg at 36⯰C and then constricted with 1⯵M phenylephrine. Acute bath exposure to SKA-31 evoked a robust and reversible inhibition of developed tone (IC50 = 0.22⯵M). The vasodilatory effects of SKA-31 and acetylcholine were blunted in the presence of KCa2.3 and KCa3.1 channel antagonists, and were largely prevented following endothelial denudation. Western blot and q-PCR analyses of isolated mesenteric arteries revealed KCa2.3 and KCa3.1 channel expression at the protein and mRNA levels, respectively. Penitrem-A, an inhibitor of KCa1.1 channels, decreased vasodilatory responses to acetylcholine, sodium nitroprusside and NS-1619, but had little effect on SKA-31. Similarly, bath exposure to the eNOS inhibitor L-NAME did not alter SKA-31 and acetylcholine-mediated vasodilation. Collectively, these data highlight the major cellular mechanisms by which the endothelial KCa channel activator SKA-31 inhibits agonist-evoked vasoconstriction in rat small mesenteric arteries.
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Benzotiazóis/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/agonistas , Artérias Mesentéricas/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/agonistas , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Relação Dose-Resposta a Droga , Técnicas In Vitro , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Masculino , Artérias Mesentéricas/metabolismo , Miografia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
Endothelial small and intermediate-conductance, Ca2+-activated K+ channels (KCa2.3 and KCa3.1, respectively) play an important role in the regulation of vascular function and systemic blood pressure. Growing evidence indicates that they are intimately involved in agonist-evoked vasodilation of small resistance arteries throughout the circulation. Small molecule activators of KCa2.x and 3.1 channels, such as SKA-31, can acutely inhibit myogenic tone in isolated resistance arteries, induce effective vasodilation in intact vascular beds, such as the coronary circulation, and acutely decrease systemic blood pressure in vivo. The blood pressure-lowering effect of SKA-31, and early indications of improvement in endothelial dysfunction suggest that endothelial KCa channel activators could eventually be developed into a new class of endothelial targeted agents to combat hypertension or atherosclerosis. This review summarises recent insights into the activation of endothelial Ca2+ activated K+ channels in various vascular beds, and how tools, such as SKA-31, may be beneficial in disease-related conditions.
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Benzotiazóis/farmacologia , Sistema Cardiovascular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Animais , Sistema Cardiovascular/metabolismo , Humanos , Canais de Potássio Cálcio-Ativados/metabolismoRESUMO
As the primary interface between the blood and various tissues of the body, the vascular endothelium exhibits a diverse range of roles and activities, all of which contribute to the overall health and function of the cardiovascular system. In this focused review, we discuss several key aspects of endothelial function, how this may be compromised and subsequent consequences. Specifically, we examine the dynamic regulation of arterial contractility and distribution of blood flow through the generation of chemical and electrical signaling events that impinge upon vascular smooth muscle. The endothelium can generate a diverse range of vasoactive compounds and signals, most of which act locally to adjust blood flow in a dynamic fashion to match tissue metabolism. Disruption of these vascular signaling processes (e.g. reduced nitric oxide bioavailability) is typically referred to as endothelial dysfunction, which is a recognized risk factor for cardiovascular disease in patients and occurs early in the development and progression of hypertension, atherosclerosis and tissue ischemia. Endothelial dysfunction is also associated with type-2 Diabetes and aging and increased mechanistic knowledge of the cellular changes contributing to these effects may provide important clues for interventional strategies. The endothelium also serves as the initial site of interaction for immune cells entering tissues in response to damage and acts to facilitate the actions of both the innate and acquired immune systems to interact with the vascular wall. In addition to representing the main cell type responsible for the formation of new blood vessels (i.e. angiogenesis) within the vasculature, the endothelium is also emerging as a source of extracellular vesicle or microparticles for the transport of signaling molecules and other cellular materials to nearby, or remote, sites in the body. The characteristics of released microparticles appear to change with the functional status of the endothelium; thus, these microparticles may represent novel biomarkers of endothelial health and more serious cardiovascular disease.
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Endotélio Vascular , Animais , Fármacos Cardiovasculares , Endotélio Vascular/imunologia , Endotélio Vascular/fisiologia , Endotélio Vascular/fisiopatologia , Humanos , Camundongos , Óxido Nítrico/metabolismo , Ratos , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
Large conductance, Ca2+-activated K+ (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 µM) and sensitive to the BK channel inhibitor, penitrem-A (100 nM). Fostriecin (30 nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30 nM), a phosphodiesterase 5 (PDE5) inhibitor. Using proximity ligation assay (PLA)-based co-immunostaining, BK channels were observed to co-localize with PP2A, PDE5, and cGMP-dependent protein kinase (cGKI) (spatial restriction < 40 nm); cGKI co-localization increased following SNP exposure. SNP (10 µM) reversibly inhibited myogenic tone in cannulated cerebral arteries, which was augmented by either fostriecin or sildenafil and inhibited by penitrem-A. Collectively, these data suggest that (1) cGKI, PDE5, and PP2A are compartmentalized with cerebrovascular BK channels and determine the extent of BK current augmentation by NO/cGMP signaling, and (2) the dynamic regulation of BK activity by co-localized signaling enzymes modulates NO-evoked dilation of cerebral resistance arteries.
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Artérias Cerebrais/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/análise , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/análise , Masculino , Proteína Fosfatase 2/análise , Proteína Fosfatase 2/metabolismo , Ratos , Ratos Sprague-Dawley , VasodilataçãoRESUMO
Local blood flow autoregulation in response to intraluminal pressure requires small artery myogenic vasoconstriction, the extent of which is thought to be governed by a feedback process that depends on Ca2+ signaling. In this issue of Science Signaling, Khavandi et al suggest a role for cyclic guanosine monophosphate (cGMP)-dependent protein kinase G Iα (PKGIα) activated by oxidants in a cGMP-independent manner.
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Arteríolas/metabolismo , Sinalização do Cálcio/fisiologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Animais , Velocidade do Fluxo Sanguíneo/fisiologia , Proteína Quinase Dependente de GMP Cíclico Tipo I/genética , Humanos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genéticaRESUMO
SKA-31, an activator of endothelial KCa2.3 and KCa3.1 channels, reduces systemic blood pressure in mice and dogs, however, its effects in larger mammals are not well known. We therefore examined the hemodynamic effects of SKA-31, along with sodium nitroprusside (SNP), in anesthetized, juvenile male domestic pigs. Experimentally, continuous measurements of left ventricular (LV), aortic and inferior vena cava (IVC) pressures, along with flows in the ascending aorta, carotid artery, left anterior descending coronary artery and renal artery, were performed during acute administration of SKA-31 (0.1, 0.3, 1.0, 3.0 and 5.0mg/ml/kg) and a single dose of SNP (5.0 µg/ml/kg). SKA-31 dose-dependently reduced mean aortic pressure (mPAO), with the highest dose decreasing mPAO to a similar extent as SNP (-23 ± 3 and -28 ± 4 mmHg, respectively). IVC pressure did not change. Systemic conductance and conductance in coronary and carotid arteries increased in response to SKA-31 and SNP, but renal artery conductance was unaffected. There was no change in either LV stroke volume (SV) or heart rate (versus the preceding control) for any infusion. With no change in SV, drug-evoked decreases in LV stroke work (SW) were attributed to reductions in mPAO (SW vs. mPAO, r(2)=0.82, P<0.001). In summary, SKA-31 dose-dependently reduced mPAO by increasing systemic and arterial conductances. Primary reductions in mPAO by SKA-31 largely account for associated decreases in SW, implying that SKA-31 does not directly impair cardiac contractility.
Assuntos
Pressão Arterial/efeitos dos fármacos , Benzotiazóis/farmacologia , Endotélio Vascular/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/agonistas , Canais de Potássio Ativados por Cálcio de Condutância Baixa/agonistas , Animais , Pressão Arterial/fisiologia , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Masculino , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Sus scrofa , SuínosRESUMO
Cysteine string protein (CSPα) is a presynaptic J protein co-chaperone that opposes neurodegeneration. Mutations in CSPα (i.e., Leu115 to Arg substitution or deletion (Δ) of Leu116) cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly inherited neurodegenerative disease. We have previously demonstrated that CSPα limits the expression of large conductance, calcium-activated K+ (BK) channels in neurons, which may impact synaptic excitability and neurotransmission. Here we show by western blot analysis that expression of the pore-forming BKα subunit is elevated ~2.5 fold in the post-mortem cortex of a 36-year-old patient with the Leu116∆ CSPα mutation. Moreover, we find that the increase in BKα subunit level is selective for ANCL and not a general feature of neurodegenerative conditions. While reduced levels of CSPα are found in some postmortem cortex specimens from Alzheimer's disease patients, we find no concomitant increase in BKα subunit expression in Alzheimer's specimens. Both CSPα monomer and oligomer expression are reduced in synaptosomes prepared from ANCL cortex compared with control. In a cultured neuronal cell model, CSPα oligomers are short lived. The results of this study indicate that the Leu116∆ mutation leads to elevated BKα subunit levels in human cortex and extend our initial work in rodent models demonstrating the modulation of BKα subunit levels by the same CSPα mutation. While the precise sequence of pathogenic events still remains to be elucidated, our findings suggest that dysregulation of BK channels may contribute to neurodegeneration in ANCL.
Assuntos
Doença de Alzheimer/metabolismo , Córtex Cerebral/metabolismo , Proteínas de Choque Térmico HSP40/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Proteínas de Membrana/genética , Lipofuscinoses Ceroides Neuronais/genética , Lipofuscinoses Ceroides Neuronais/metabolismo , Adulto , Idoso , Doença de Alzheimer/genética , Animais , Autopsia , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Neurônios/metabolismo , Sinaptossomos/metabolismoRESUMO
Endothelial KCa2.3 and KCa3.1 channels contribute to the regulation of myogenic tone in resistance arteries by Ca(2+)-mobilizing vasodilatory hormones. To define further the functional role of these channels in distinct vascular beds, we have examined the vasodilatory actions of the KCa channel activator SKA-31 in myogenically active rat cremaster and middle cerebral arteries. Vessels pressurized to 70 mm Hg constricted by 80-100 µm (ie, 25%-45% of maximal diameter). SKA-31 (10 µM) inhibited myogenic tone by 80% in cremaster and â¼65% in middle cerebral arteries, with IC50 values of â¼2 µM in both vessels. These vasodilatory effects were largely prevented by the KCa2.3 blocker UCL1684 and the KCa3.1 blocker TRAM-34 and abolished by endothelial denudation. Preincubation with N(G) nitro L-arginine methyl ester (L-NAME, 0.1 mM) did not affect the inhibitory response to SKA-31, but attenuated the ACh-evoked dilation by â¼45%. Penitrem-A, a blocker of BK(Ca) channels, did not alter SKA-31 evoked vasodilation but did reduce the inhibition of myogenic tone by ACh, the BKCa channel activator NS1619, and sodium nitroprusside. Collectively, these data demonstrate that SKA-31 produces robust inhibition of myogenic tone in resistance arteries isolated from distinct vascular beds in an endothelium-dependent manner.