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1.
Sci Signal ; 15(727): eabl5405, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35349300

RESUMO

The dense network of capillaries composed of capillary endothelial cells (cECs) and pericytes lies in close proximity to all neurons, ideally positioning it to sense neuron- and glial-derived compounds that enhance regional and global cerebral perfusion. The membrane potential (VM) of vascular cells serves as the physiological bridge that translates brain activity into vascular function. In other beds, the ATP-sensitive K+ (KATP) channel regulates VM in vascular smooth muscle, which is absent in the capillary network. Here, with transgenic mice that expressed a dominant-negative mutant of the pore-forming Kir6.1 subunit specifically in brain cECs or pericytes, we demonstrated that KATP channels were present in both cell types and robustly controlled VM. We further showed that the signaling nucleotide adenosine acted through A2A receptors and the Gαs/cAMP/PKA pathway to activate capillary KATP channels. Moreover, KATP channel stimulation in vivo increased cerebral blood flow (CBF), an effect that was blunted by expression of the dominant-negative Kir6.1 mutant in either capillary cell type. These findings establish an important role for KATP channels in cECs and pericytes in the regulation of CBF.


Assuntos
Células Endoteliais , Pericitos , Adenosina , Trifosfato de Adenosina/metabolismo , Animais , Capilares/metabolismo , Células Endoteliais/metabolismo , Canais KATP/genética , Canais KATP/metabolismo , Camundongos , Pericitos/metabolismo
2.
Function (Oxf) ; 2(3)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34568829

RESUMO

Trauma can lead to widespread vascular dysfunction, but the underlying mechanisms remain largely unknown. Inward-rectifier potassium channels (Kir2.1) play a critical role in the dynamic regulation of regional perfusion and blood flow. Kir2.1 channel activity requires phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane phospholipid that is degraded by phospholipase A2 (PLA2) in conditions of oxidative stress or inflammation. We hypothesized that PLA2-induced depletion of PIP2 after trauma impairs Kir2.1 channel function. A fluid percussion injury model of traumatic brain injury (TBI) in rats was used to study mesenteric resistance arteries 24 hours after injury. The functional responses of intact arteries were assessed using pressure myography. We analyzed circulating PLA2, hydrogen peroxide (H2O2), and metabolites to identify alterations in signaling pathways associated with PIP2 in TBI. Electrophysiology analysis of freshly-isolated endothelial and smooth muscle cells revealed a significant reduction of Ba2+-sensitive Kir2.1 currents after TBI. Additionally, dilations to elevated extracellular potassium and BaCl2- or ML 133-induced constrictions in pressurized arteries were significantly decreased following TBI, consistent with an impairment of Kir2.1 channel function. The addition of a PIP2 analog to the patch pipette successfully rescued endothelial Kir2.1 currents after TBI. Both H2O2 and PLA2 activity were increased after injury. Metabolomics analysis demonstrated altered lipid metabolism signaling pathways, including increased arachidonic acid, and fatty acid mobilization after TBI. Our findings support a model in which increased H2O2-induced PLA2 activity after trauma hydrolyzes endothelial PIP2, resulting in impaired Kir2.1 channel function.


Assuntos
Lesões Encefálicas Traumáticas , Peróxido de Hidrogênio , Ratos , Animais , Hemodinâmica , Transdução de Sinais
3.
Dis Model Mech ; 14(7)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34318329

RESUMO

Benign prostatic hyperplasia/lower urinary tract dysfunction (LUTD) affects nearly all men. Symptoms typically present in the fifth or sixth decade and progressively worsen over the remainder of life. Here, we identify a surprising origin of this disease that traces back to the intrauterine environment of the developing male, challenging paradigms about when this disease process begins. We delivered a single dose of a widespread environmental contaminant present in the serum of most Americans [2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), 1 µg/kg], and representative of a broader class of environmental contaminants, to pregnant mice and observed an increase in the abundance of a neurotrophic factor, artemin, in the developing mouse prostate. Artemin is required for noradrenergic axon recruitment across multiple tissues, and TCDD rapidly increases prostatic noradrenergic axon density in the male fetus. The hyperinnervation persists into adulthood, when it is coupled to autonomic hyperactivity of prostatic smooth muscle and abnormal urinary function, including increased urinary frequency. We offer new evidence that prostate neuroanatomical development is malleable and that intrauterine chemical exposures can permanently reprogram prostate neuromuscular function to cause male LUTD in adulthood.


Assuntos
Dibenzodioxinas Policloradas , Sistema Urinário , Adulto , Animais , Feminino , Humanos , Masculino , Camundongos , Dibenzodioxinas Policloradas/toxicidade , Gravidez , Próstata , Ratos , Ratos Sprague-Dawley
4.
Am J Physiol Heart Circ Physiol ; 316(6): H1309-H1322, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30848676

RESUMO

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Histonas/toxicidade , Artérias Mesentéricas/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Animais , Pressão Arterial , Morte Celular , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Artérias Mesentéricas/metabolismo , Artérias Mesentéricas/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Receptor 4 Toll-Like/metabolismo , Resistência Vascular
5.
J Physiol ; 597(3): 935-950, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30536555

RESUMO

KEY POINTS: KV 7 channels are a family of voltage-dependent K+ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability. Drugs that target KV 7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how. In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow. Urinary bladder smooth muscle had no measurable KV 7 channel currents. However, the KV 7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that KV 7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness. ABSTRACT: KV 7 channels are voltage-dependent K+ channels that open in response to membrane depolarization to regulate cell excitability. KV 7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of KV 7 channels as regulators of murine urinary bladder function. The KV 7 activator retigabine (10 µM) had no effect on voltage-dependent K+ currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine-sensitive KV 7 channels. The KV 7 inhibitor XE-991 (10 µM) inhibited UBSM K+ currents; the properties of these currents, however, were typical of KV 2 channels and not KV 7 channels. Retigabine inhibited voltage-dependent Ca2+ channel (VDCC) currents and reduced steady-state contractions to 60 mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co-incubation with XE-991, suggesting specific activation of KV 7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non-specific effects on VDCCs and KV 2 channels.


Assuntos
Carbamatos/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Bexiga Urinária/efeitos dos fármacos , Animais , Contração Isométrica/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular/efeitos dos fármacos , Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Neurônios Aferentes/metabolismo , Bexiga Urinária/metabolismo
6.
J Biol Chem ; 293(43): 16791-16802, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30206122

RESUMO

The type I cGMP-dependent protein kinase (PKG I) is an essential regulator of vascular tone. It has been demonstrated that the type Iα isoform can be constitutively activated by oxidizing conditions. However, the amino acid residues implicated in this phenomenon are not fully elucidated. To investigate the molecular basis for this mechanism, we studied the effects of oxidation using recombinant WT, truncated, and mutant constructs of PKG I. Using an in vitro assay, we observed that oxidation with hydrogen peroxide (H2O2) resulted in constitutive, cGMP-independent activation of PKG Iα. PKG Iα C42S and a truncation construct that does not contain Cys-42 (Δ53) were both constitutively activated by H2O2 In contrast, oxidation of PKG Iα C117S maintained its cGMP-dependent activation characteristics, although oxidized PKG Iα C195S did not. To corroborate these results, we also tested the effects of our constructs on the PKG Iα-specific substrate, the large conductance potassium channel (KCa 1.1). Application of WT PKG Iα activated by either cGMP or H2O2 increased the open probabilities of the channel. Neither cGMP nor H2O2 activation of PKG Iα C42S significantly increased channel open probabilities. Moreover, cGMP-stimulated PKG Iα C117S increased KCa 1.1 activity, but this effect was not observed under oxidizing conditions. Finally, we observed that PKG Iα C42S caused channel flickers, indicating dramatically altered KCa 1.1 channel characteristics compared with channels exposed to WT PKG Iα. Cumulatively, these results indicate that constitutive activation of PKG Iα proceeds through oxidation of Cys-117 and further suggest that the formation of a sulfur acid is necessary for this phenotype.


Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Cisteína/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Proteína Quinase Dependente de GMP Cíclico Tipo I/química , Cisteína/química , Modelos Moleculares , Óxido Nítrico/metabolismo , Oxirredução , Fosforilação , Conformação Proteica , Homologia de Sequência
7.
Physiol Rep ; 5(16)2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28830977

RESUMO

The aim of this study was to investigate mechanisms by which adiponectin influences vascular Ca2+ signaling, K+ channel activity and thus contractile tone of small arteries. Vasodilation to adiponectin was studied in mesenteric resistance arteries constricted with intraluminal pressure. Ca2+ signals were characterized using high speed confocal microscopy of intact arteries. Patch clamp investigated the effect of adiponectin on individual VSMC potassium (K+) channel currents. Adiponectin dilated arteries constricted with pressure-induced tone by approximately 5% and the induced vasodilation was only transient. The dilation to adiponectin was reduced by pharmacological interruption of the Ca2+ spark/large conductance activated K+ (BK) channel pathway but from a physiological perspective, interpretation of the data was limited by the small effect. Neither Adiponectin nor the presence of intact perivascular adipose tissue (PVAT) influenced Ca2+ spark or Ca2+ wave frequency or characteristics. Studied using a perforated patch approach, Adiponectin marginally increased current through the VSMC BK channel but this effect was lost using the whole cell technique with dialysis of the cytoplasm. Adiponectin did not change the frequency or amplitude of Ca2+ spark-induced transient outward currents (STOC). Overall, our study shows that Adiponectin induces only a small and transient dilation of pressure constricted mesenteric arteries. This vasodilatory effect is likely to be independent of Ca2+ sparks or direct BK channel activation.


Assuntos
Adiponectina/farmacologia , Artérias/metabolismo , Sinalização do Cálcio , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição , Animais , Artérias/citologia , Artérias/fisiologia , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia
8.
Am J Physiol Renal Physiol ; 312(5): F836-F847, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28148533

RESUMO

Prolonged decreases in urinary bladder blood flow are linked to overactive and underactive bladder pathologies. However, the mechanisms regulating bladder vascular reactivity are largely unknown. To investigate these mechanisms, we examined myogenic and vasoactive properties of mouse bladder feed arterioles (BFAs). Unlike similar-sized arterioles from other vascular beds, BFAs failed to constrict in response to increases in intraluminal pressure (5-80 mmHg). Consistent with this lack of myogenic tone, arteriolar smooth muscle cell membrane potential was hyperpolarized (-72.8 ± 1.4 mV) at 20 mmHg and unaffected by increasing pressure to 80 mmHg (-74.3 ± 2.2 mV). In contrast, BFAs constricted to the thromboxane analog U-46619 (100 nM), the adrenergic agonist phenylephrine (10 µM), and KCl (60 mM). Inhibition of nitric oxide synthase or intermediate- and small-conductance Ca2+-activated K+ channels did not alter arteriolar diameter, indicating that the dilated state of BFAs is not attributable to overactive endothelium-dependent dilatory influences. Myocytes isolated from BFAs exhibited BaCl2 (100 µM)-sensitive K+ currents consistent with strong inward-rectifier K+ (KIR) channels. Notably, block of these KIR channels "restored" pressure-induced constriction and membrane depolarization. This suggests that these channels, in part, account for hyperpolarization and associated absence of tone in BFAs. Furthermore, smooth muscle-specific knockout of KIR2.1 caused significant myogenic tone to develop at physiological pressures. This suggests that 1) the regulation of vascular tone in the bladder is independent of pressure, insofar as pressure-induced depolarizing conductances cannot overcome KIR2.1-mediated hyperpolarization; and 2) maintenance of bladder blood flow during bladder filling is likely controlled by neurohumoral influences.


Assuntos
Arteríolas/efeitos dos fármacos , Pressão Sanguínea , Mecanotransdução Celular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Bexiga Urinária/irrigação sanguínea , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Animais , Arteríolas/metabolismo , Genótipo , Técnicas In Vitro , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/genética , Vasodilatadores/farmacologia
9.
Sci Signal ; 9(449): ra100, 2016 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-27729550

RESUMO

Activation of Ca2+-sensitive, large-conductance potassium (BK) channels in vascular smooth muscle cells (VSMCs) by local, ryanodine receptor-mediated Ca2+ signals (Ca2+ sparks) acts as a brake on pressure-induced (myogenic) vasoconstriction-a fundamental mechanism that regulates blood flow in small resistance arteries. We report that physiological intraluminal pressure within resistance arteries activated cGMP-dependent protein kinase (PKG) in VSMCs through oxidant-induced formation of an intermolecular disulfide bond between cysteine residues. Oxidant-activated PKG was required to trigger Ca2+ sparks, BK channel activity, and vasodilation in response to pressure. VSMCs from arteries from mice expressing a form of PKG that could not be activated by oxidants showed reduced Ca2+ spark frequency, and arterial preparations from these mice had decreased pressure-induced activation of BK channels. Thus, the absence of oxidative activation of PKG disabled the BK channel-mediated negative feedback regulation of vasoconstriction. Our results support the concept of a negative feedback control mechanism that regulates arterial diameter through mechanosensitive production of oxidants to activate PKG and enhance Ca2+ sparks.


Assuntos
Pressão Sanguínea/fisiologia , Sinalização do Cálcio/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Mecanotransdução Celular/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição/fisiologia , Animais , Proteínas Quinases Dependentes de GMP Cíclico/genética , Canais de Potássio Ativados por Cálcio de Condutância Alta/genética , Camundongos , Camundongos Mutantes
10.
J Cereb Blood Flow Metab ; 36(7): 1195-201, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27052838

RESUMO

Transient global cerebral ischemia is often followed by delayed disturbances of cerebral blood flow, contributing to neuronal injury. The pathophysiological processes underlying such disturbances are incompletely understood. Here, using an established model of transient global cerebral ischemia, we identify dramatically impaired neurovascular coupling following ischemia. This impairment results from the loss of functional inward rectifier potassium (KIR) channels in the smooth muscle of parenchymal arterioles. Therapeutic strategies aimed at protecting or restoring cerebrovascular KIR channel function may therefore improve outcomes following ischemia.


Assuntos
Arteríolas/metabolismo , Ataque Isquêmico Transitório/fisiopatologia , Músculo Liso Vascular/metabolismo , Acoplamento Neurovascular/fisiologia , Tecido Parenquimatoso/irrigação sanguínea , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Arteríolas/fisiopatologia , Circulação Cerebrovascular/fisiologia , Endotélio Vascular , Ataque Isquêmico Transitório/metabolismo , Masculino , Músculo Liso Vascular/fisiopatologia , Ratos Sprague-Dawley
11.
J Physiol ; 594(12): 3271-85, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-26840527

RESUMO

KEY POINTS: Increase in endothelial cell (EC) calcium activates calcium-sensitive intermediate and small conductance potassium (IK and SK) channels, thereby causing hyperpolarization and endothelium-dependent vasodilatation. Endothelial cells express inward rectifier potassium (Kir) channels, but their role in endothelium-dependent vasodilatation is not clear. In the mesenteric arteries, only ECs, but not smooth muscle cells, displayed Kir currents that were predominantly mediated by the Kir2.1 isoform. Endothelium-dependent vasodilatations in response to muscarinic receptor, TRPV4 (transient receptor potential vanilloid 4) channel and IK/SK channel agonists were highly attenuated by Kir channel inhibitors and by Kir2.1 channel knockdown. These results point to EC Kir channels as amplifiers of vasodilatation in response to increases in EC calcium and IK/SK channel activation and suggest that EC Kir channels could be targeted to treat endothelial dysfunction, which is a hallmark of vascular disorders. ABSTRACT: Endothelium-dependent vasodilators, such as acetylcholine, increase intracellular Ca(2+) through activation of transient receptor potential vanilloid 4 (TRPV4) channels in the plasma membrane and inositol trisphosphate receptors in the endoplasmic reticulum, leading to stimulation of Ca(2+) -sensitive intermediate and small conductance K(+) (IK and SK, respectively) channels. Although strong inward rectifier K(+) (Kir) channels have been reported in the native endothelial cells (ECs) their role in EC-dependent vasodilatation is not clear. Here, we test the idea that Kir channels boost the EC-dependent vasodilatation of resistance-sized arteries. We show that ECs, but not smooth muscle cells, of small mesenteric arteries have Kir currents, which are substantially reduced in EC-specific Kir2.1 knockdown (EC-Kir2.1(-/-) ) mice. Elevation of extracellular K(+) to 14 mm caused vasodilatation of pressurized arteries, which was prevented by endothelial denudation and Kir channel inhibitors (Ba(2+) , ML-133) or in the arteries from EC-Kir2.1(-/-) mice. Potassium-induced dilatations were unaffected by inhibitors of TRPV4, IK and SK channels. The Kir channel blocker, Ba(2+) , did not affect currents through TRPV4, IK or SK channels. Endothelial cell-dependent vasodilatations in response to activation of muscarinic receptors, TRPV4 channels or IK/SK channels were reduced, but not eliminated, by Kir channel inhibitors or EC-Kir2.1(-/-) . In angiotensin II-induced hypertension, the Kir channel function was not altered, although the endothelium-dependent vasodilatation was severely impaired. Our results support the concept that EC Kir2 channels boost vasodilatory signals that are generated by Ca(2+) -dependent activation of IK and SK channels.


Assuntos
Células Endoteliais/fisiologia , Artérias Mesentéricas/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/fisiologia , Acetilcolina/farmacologia , Animais , Cálcio/fisiologia , Células Endoteliais/efeitos dos fármacos , Fatores Relaxantes Dependentes do Endotélio/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Vasodilatação
12.
Am J Physiol Heart Circ Physiol ; 309(4): H592-604, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26092991

RESUMO

Diabetes in rat pregnancy is associated with impaired vasodilation of the maternal uteroplacental vasculature. In the present study, we explored the role of endothelial cell (EC) Ca(2+)-activated K(+) channels of small conductance (SKCa channels) and intermediate conductance (IKCa channels) in diabetes-induced uterine vascular dysfunction. Diabetes was induced by injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with citrate buffer. Changes in smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and vasodilation induced by SKCa/IKCa channel activators were studied in uteroplacental arteries of control and diabetic rats. The impact of diabetes on SKCa- and IKCa-mediated currents was explored in freshly dissociated ECs. NS309 evoked a potent vasodilation that was effectively inhibited by TRAM-34 but not by apamin. NS309-induced smooth muscle cell intracellular Ca(2+) concentration, membrane potential, and dilator responses were significantly diminished by diabetes; N-cyclohexyl-N-2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA)-evoked responses were not affected. Ca(2+)-activated ion currents in ECs were insensitive to paxilline, markedly inhibited by charybdotoxin (ChTX), and diminished by apamin. NS309-induced EC currents were generated mostly due to activation of ChTX-sensitive channels. Maternal diabetes resulted in a significant reduction in ChTX-sensitive currents with no effect on apamin-sensitive or CyPPA-induced currents. We concluded that IKCa channels play a prevalent role over SKCa channels in the generation of endothelial K(+) currents and vasodilation of uteroplacental arteries. Impaired function of IKCa channels importantly contributes to diabetes-induced uterine endothelial dysfunction. Therapeutic restoration of IKCa channel function may be a novel strategy for improvement of maternal uteroplacental blood flow in pregnancies complicated by diabetes.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Circulação Placentária , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Potenciais de Ação , Animais , Cálcio/farmacologia , Células Cultivadas , Diabetes Mellitus Experimental/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/fisiopatologia , Feminino , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Gravidez , Ratos , Ratos Sprague-Dawley , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Vasodilatação
13.
Proc Natl Acad Sci U S A ; 112(7): E796-805, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25646445

RESUMO

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), caused by dominant mutations in the NOTCH3 receptor in vascular smooth muscle, is a genetic paradigm of small vessel disease (SVD) of the brain. Recent studies using transgenic (Tg)Notch3(R169C) mice, a genetic model of CADASIL, revealed functional defects in cerebral (pial) arteries on the surface of the brain at an early stage of disease progression. Here, using parenchymal arterioles (PAs) from within the brain, we determined the molecular mechanism underlying the early functional deficits associated with this Notch3 mutation. At physiological pressure (40 mmHg), smooth muscle membrane potential depolarization and constriction to pressure (myogenic tone) were blunted in PAs from TgNotch3(R169C) mice. This effect was associated with an ∼ 60% increase in the number of voltage-gated potassium (KV) channels, which oppose pressure-induced depolarization. Inhibition of KV1 channels with 4-aminopyridine (4-AP) or treatment with the epidermal growth factor receptor agonist heparin-binding EGF (HB-EGF), which promotes KV1 channel endocytosis, reduced KV current density and restored myogenic responses in PAs from TgNotch3(R169C) mice, whereas pharmacological inhibition of other major vasodilatory influences had no effect. KV1 currents and myogenic responses were similarly altered in pial arteries from TgNotch3(R169C) mice, but not in mesenteric arteries. Interestingly, HB-EGF had no effect on mesenteric arteries, suggesting a possible mechanistic basis for the exclusive cerebrovascular manifestation of CADASIL. Collectively, our results indicate that increasing the number of KV1 channels in cerebral smooth muscle produces a mutant vascular phenotype akin to a channelopathy in a genetic model of SVD.


Assuntos
Encéfalo/fisiopatologia , Transtornos Cerebrovasculares/genética , Canais de Potássio/genética , 4-Aminopiridina/farmacologia , Animais , Encéfalo/irrigação sanguínea , Transtornos Cerebrovasculares/fisiopatologia , Modelos Animais de Doenças , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/fisiologia , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Receptor Notch3 , Receptores Notch/genética , Receptores Notch/fisiologia
14.
Sci Signal ; 7(333): ra66, 2014 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-25005230

RESUMO

Endothelial cell dysfunction, characterized by a diminished response to endothelial cell-dependent vasodilators, is a hallmark of hypertension. TRPV4 channels play a major role in endothelial-dependent vasodilation, a function mediated by local Ca(2+) influx through clusters of functionally coupled TRPV4 channels rather than by a global increase in endothelial cell Ca(2+). We showed that stimulation of muscarinic acetylcholine receptors on endothelial cells of mouse arteries exclusively activated TRPV4 channels that were localized at myoendothelial projections (MEPs), specialized regions of endothelial cells that contact smooth muscle cells. Muscarinic receptor-mediated activation of TRPV4 depended on protein kinase C (PKC) and the PKC-anchoring protein AKAP150, which was concentrated at MEPs. Cooperative opening of clustered TRPV4 channels specifically amplified Ca(2+) influx at MEPs. Cooperativity of TRPV4 channels at non-MEP sites was much lower, and cooperativity at MEPs was greatly reduced by chelation of intracellular Ca(2+) or AKAP150 knockout, suggesting that Ca(2+) entering through adjacent channels underlies the AKAP150-dependent potentiation of TRPV4 activity. In a mouse model of angiotensin II-induced hypertension, MEP localization of AKAP150 was disrupted, muscarinic receptor stimulation did not activate TRPV4 channels, cooperativity among TRPV4 channels at MEPs was weaker, and vasodilation in response to muscarinic receptor stimulation was reduced. Thus, endothelial-dependent dilation of resistance arteries is enabled by MEP-localized AKAP150, which ensures the proximity of PKC to TRPV4 channels and the coupled channel gating necessary for efficient communication from endothelial to smooth muscle cells in arteries. Disruption of this molecular assembly may contribute to altered blood flow in hypertension.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Endotélio Vascular/metabolismo , Hipertensão/metabolismo , Ativação do Canal Iônico , Canais de Cátion TRPV/metabolismo , Vasodilatação , Proteínas de Ancoragem à Quinase A/genética , Angiotensina II/efeitos adversos , Angiotensina II/farmacologia , Animais , Sinalização do Cálcio , Modelos Animais de Doenças , Endotélio Vascular/patologia , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/patologia , Camundongos , Camundongos Knockout , Canais de Cátion TRPV/genética , Vasoconstritores/efeitos adversos , Vasoconstritores/farmacologia
15.
J Pharmacol Exp Ther ; 350(3): 520-30, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24951278

RESUMO

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.


Assuntos
Agonistas dos Canais de Cálcio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Animais , Agonistas dos Canais de Cálcio/química , Feminino , Cobaias , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Masculino , Contração Muscular/fisiologia , Relaxamento Muscular/fisiologia , Técnicas de Cultura de Órgãos , Bexiga Urinária/fisiologia
16.
Am J Physiol Heart Circ Physiol ; 304(7): H935-45, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23376827

RESUMO

Diabetes mellitus in pregnancy is associated with impaired endothelium-mediated dilatation of maternal arteries, although the underlying cellular mechanisms remain unknown. In this study, we hypothesized that diabetes during rat gestation attenuates agonist-induced uterine vasodilation through reduced endothelial cell (EC) Ca(2+) elevations and impaired smooth muscle cell (SMC) hyperpolarization and SMC intracellular Ca(2+) concentration ([Ca(2+)]i) responses. Diabetes was induced by an injection of streptozotocin to second-day pregnant rats and confirmed by the development of maternal hyperglycemia. Control rats were injected with a citrate buffer. Fura-2-based measurements of SMC [Ca(2+)]i or microelectrode recordings of SMC membrane potential were performed concurrently with dilator responses to ACh in uteroplacental arteries from control and diabetic pregnant rats. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations in pressurized vessels and small EC sheets were studied as well. Diabetes reduced ACh-induced vasodilation due to a markedly impaired EDHF-mediated response. Diminished vasodilation to ACh was associated with attenuated SMC hyperpolarization and [Ca(2+)]i responses. Basal levels of EC [Ca(2+)]i and ACh-induced EC [Ca(2+)]i elevations were significantly reduced by diabetes. In conclusion, these data demonstrate that reduced endothelium-mediated hyperpolarization contributes to attenuated uteroplacental vasodilation and SMC [Ca(2+)]i responses to ACh in diabetic pregnancy. Impaired endothelial Ca(2+) signaling is in part responsible for endothelial dysfunction in the uterine resistance vasculature of diabetic rats. Pharmacological improvement of EC Ca(2+) handling may provide an important strategy for the restoration of endothelial function and enhancement of maternal blood flow in human pregnancies complicated by diabetes.


Assuntos
Sinalização do Cálcio , Diabetes Gestacional/metabolismo , Diabetes Gestacional/fisiopatologia , Células Endoteliais/metabolismo , Artéria Uterina/fisiopatologia , Vasodilatação , Acetilcolina/farmacologia , Animais , Fatores Biológicos/farmacologia , Cálcio/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiopatologia , Feminino , Hiperglicemia/induzido quimicamente , Hiperglicemia/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Placenta/irrigação sanguínea , Circulação Placentária , Gravidez , Ratos , Ratos Sprague-Dawley , Artéria Uterina/metabolismo , Útero/irrigação sanguínea
17.
Acta Neurochir Suppl ; 115: 167-71, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-22890664

RESUMO

The matching of blood flow to regional brain function, called functional hyperemia or neurovascular coupling, involves the coordinated activity of neurons, astrocytes, and parenchymal arterioles. Under physiological conditions, localized neuronal activation leads to elevated astrocyte endfoot Ca(2+) and vasodilation, resulting in an increase in cerebral blood flow. In this study, we examined the impact of subarachnoid hemorrhage (SAH) on neurovascular coupling. SAH model rats received two injections of autologous blood into the cisterna magna 24 h apart. Cortical brain slices from SAH model animals were prepared 4 days after the initial blood injection. Arteriolar diameter and astrocyte endfoot Ca(2+) were simultaneously measured using two-photon microscopy. As expected, neuronal activity evoked by electrical field stimulation (EFS) caused an elevation in endfoot Ca(2+) and vasodilation in brain slices from control animals. However, in brain slices from SAH animals, EFS induced a similar increase in astrocyte endfoot Ca(2+) that caused arteriolar constriction rather than vasodilation. Vasoconstriction was observed in approximately 90% of brain slices from SAH animals in response to EFS, with 40% exhibiting a sustained vasoconstriction, 30% exhibiting a transient vasoconstriction -(diameter restored within 1 min after EFS), and 20% responded with a biphasic response (brief vasodilation followed by -vasoconstriction). This inversion of neurovascular coupling may play a role in the development of neurological deficits following SAH.


Assuntos
Córtex Cerebral/irrigação sanguínea , Hemorragia Subaracnóidea/patologia , Hemorragia Subaracnóidea/fisiopatologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologia , Animais , Cálcio/metabolismo , Córtex Cerebral/fisiopatologia , Circulação Cerebrovascular/fisiologia , Modelos Animais de Doenças , Estimulação Elétrica , Potenciais Evocados , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley
18.
Proc Natl Acad Sci U S A ; 109(21): E1387-95, 2012 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-22547803

RESUMO

The cellular events that cause ischemic neurological damage following aneurysmal subarachnoid hemorrhage (SAH) have remained elusive. We report that subarachnoid blood profoundly impacts communication within the neurovascular unit-neurons, astrocytes, and arterioles-causing inversion of neurovascular coupling. Elevation of astrocytic endfoot Ca(2+) to ∼400 nM by neuronal stimulation or to ∼300 nM by Ca(2+) uncaging dilated parenchymal arterioles in control brain slices but caused vasoconstriction in post-SAH brain slices. Inhibition of K(+) efflux via astrocytic endfoot large-conductance Ca(2+)-activated K(+) (BK) channels prevented both neurally evoked vasodilation (control) and vasoconstriction (SAH). Consistent with the dual vasodilator/vasoconstrictor action of extracellular K(+) ([K(+)](o)), [K(+)](o) <10 mM dilated and [K(+)](o) >20 mM constricted isolated brain cortex parenchymal arterioles with or without SAH. Notably, elevation of external K(+) to 10 mM caused vasodilation in brain slices from control animals but caused a modest constriction in brain slices from SAH model rats; this latter effect was reversed by BK channel inhibition, which restored K(+)-induced dilations. Importantly, the amplitude of spontaneous astrocytic Ca(2+) oscillations was increased after SAH, with peak Ca(2+) reaching ∼490 nM. Our data support a model in which SAH increases the amplitude of spontaneous astrocytic Ca(2+) oscillations sufficiently to activate endfoot BK channels and elevate [K(+)](o) in the restricted perivascular space. Abnormally elevated basal [K(+)](o) combined with further K(+) efflux stimulated by neuronal activity elevates [K(+)](o) above the dilation/constriction threshold, switching the polarity of arteriolar responses to vasoconstriction. Inversion of neurovascular coupling may contribute to the decreased cerebral blood flow and development of neurological deficits that commonly follow SAH.


Assuntos
Sangue/metabolismo , Encéfalo/irrigação sanguínea , Encéfalo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Alta/fisiologia , Hemorragia Subaracnóidea/fisiopatologia , Animais , Arteríolas/citologia , Arteríolas/fisiologia , Astrócitos/citologia , Astrócitos/fisiologia , Sinalização do Cálcio/fisiologia , Comunicação Celular/fisiologia , Modelos Animais de Doenças , Gliose/fisiopatologia , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/fisiologia , Vasoespasmo Intracraniano/fisiopatologia
19.
Science ; 336(6081): 597-601, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22556255

RESUMO

Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca(2+)) signals ("sparklets") in the vascular endothelium of resistance arteries that represent Ca(2+) influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca(2+)-sensitive potassium (K(+)) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca(2+) influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca(2+) sensitivity of IK and SK channels to cause vasodilation.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Células Endoteliais/metabolismo , Artérias Mesentéricas/metabolismo , Canais de Cátion TRPV/metabolismo , Vasodilatação , Animais , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ativação do Canal Iônico , Leucina/análogos & derivados , Leucina/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-Clamp , Receptores Muscarínicos/metabolismo , Transdução de Sinais , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo , Sulfonamidas/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/antagonistas & inibidores
20.
Am J Physiol Heart Circ Physiol ; 302(3): H594-602, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22140050

RESUMO

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.


Assuntos
Sinalização do Cálcio/fisiologia , Artérias Mesentéricas/inervação , Artérias Mesentéricas/fisiologia , Sistema Nervoso Simpático/fisiologia , Vasoconstrição/fisiologia , Animais , Cálcio/metabolismo , Conexinas/genética , Endotélio Vascular/metabolismo , Retroalimentação Fisiológica/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Músculo Liso Vascular/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Proteína alfa-5 de Junções Comunicantes
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