RESUMO
Arteriolar smooth muscle cells (SMCs) and capillary pericytes dynamically regulate blood flow in the central nervous system in the face of fluctuating perfusion pressures. Pressure-induced depolarization and Ca2+ elevation provide a mechanism for regulation of SMC contraction, but whether pericytes participate in pressure-induced changes in blood flow remains unknown. Here, utilizing a pressurized whole-retina preparation, we found that increases in intraluminal pressure in the physiological range induce contraction of both dynamically contractile pericytes in the arteriole-proximate transition zone and distal pericytes of the capillary bed. We found that the contractile response to pressure elevation was slower in distal pericytes than in transition zone pericytes and arteriolar SMCs. Pressure-evoked elevation of cytosolic Ca2+ and contractile responses in SMCs were dependent on voltage-dependent Ca2+ channel (VDCC) activity. In contrast, Ca2+ elevation and contractile responses were partially dependent on VDCC activity in transition zone pericytes and independent of VDCC activity in distal pericytes. In both transition zone and distal pericytes, membrane potential at low inlet pressure (20 mmHg) was approximately -40 mV and was depolarized to approximately -30 mV by an increase in pressure to 80 mmHg. The magnitude of whole-cell VDCC currents in freshly isolated pericytes was approximately half that measured in isolated SMCs. Collectively, these results indicate a loss of VDCC involvement in pressure-induced constriction along the arteriole-capillary continuum. They further suggest that alternative mechanisms and kinetics of Ca2+ elevation, contractility, and blood flow regulation exist in central nervous system capillary networks, distinguishing them from neighboring arterioles.
Assuntos
Cálcio , Pericitos , Pericitos/metabolismo , Cálcio/metabolismo , Canais de Cálcio Tipo L , Arteríolas/fisiologia , Sistema Nervoso Central/metabolismo , Cálcio da DietaRESUMO
Capillaries, composed of electrically coupled endothelial cells and overlying pericytes, constitute the vast majority of blood vessels in the brain. The most arteriole-proximate three to four branches of the capillary bed are covered by α-actin-expressing, contractile pericytes. These mural cells have a distinctive morphology and express different markers compared with their smooth muscle cell (SMC) cousins but share similar excitation-coupling contraction machinery. Despite this similarity, pericytes are considerably more depolarized than SMCs at low intravascular pressures. We have recently shown that pericytes, such as SMCs, possess functional voltage-dependent Ca2+ channels and ATP-sensitive K+ channels. Here, we further investigate the complement of pericyte ion channels, focusing on members of the K+ channel superfamily. Using NG2-DsRed-transgenic mice and diverse configurations of the patch-clamp technique, we demonstrate that pericytes display robust inward-rectifier K+ currents that are primarily mediated by the Kir2 family, based on their unique biophysical characteristics and sensitivity to micromolar concentrations of Ba2+. Moreover, multiple lines of evidence, including characteristic kinetics, sensitivity to specific blockers, biophysical attributes, and distinctive single-channel properties, established the functional expression of two voltage-dependent K+ channels: KV1 and BKCa. Although these three types of channels are also present in SMCs, they exhibit distinctive current density and kinetics profiles in pericytes. Collectively, these findings underscore differences in the operation of shared molecular features between pericytes and SMCs and highlight the potential contribution of these three K+ ion channels in setting pericyte membrane potential, modulating capillary hemodynamics, and regulating cerebral blood flow.
Assuntos
Encéfalo , Capilares , Pericitos , Pericitos/metabolismo , Pericitos/citologia , Animais , Capilares/metabolismo , Capilares/citologia , Camundongos , Encéfalo/irrigação sanguínea , Encéfalo/citologia , Encéfalo/metabolismo , Canais de Potássio/metabolismo , Camundongos Transgênicos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Capillaries are equipped to sense neurovascular coupling agents released onto the outer wall of a capillary, translating these external signals into electrical/Ca2+ changes that play a crucial role in blood flow regulation and ensuring that neuronal demands are met. However, control mechanisms attributable to forces imposed onto the lumen are less clear. Here, we show that Piezo1 channels act as mechanosensors in central nervous system capillaries. Electrophysiological analyses confirmed expression and function of Piezo1 channels in brain cortical and retinal capillaries. Activation of Piezo1 channels evoked currents that were sensitive to endothelial cell-specific Piezo1 deletion. Using genetically encoded Ca2+ indicator mice and an ex vivo pressurized retina preparation, we found that activation of Piezo1 channels by mechanical forces triggered Ca2+ signals in capillary endothelial cells. Collectively, these findings indicate that Piezo1 channels are capillary mechanosensors that initiate crucial Ca2+ signals and could, therefore, have a profound impact on central nervous system blood flow control.
Assuntos
Capilares , Canais Iônicos , Acoplamento Neurovascular , Animais , Sistema Nervoso Central/irrigação sanguínea , Células Endoteliais/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , CamundongosRESUMO
The accepted role of the protein Kv2.1 in arterial smooth muscle cells is to form K+ channels in the sarcolemma. Opening of Kv2.1 channels causes membrane hyperpolarization, which decreases the activity of L-type CaV1.2 channels, lowering intracellular Ca2+ ([Ca2+]i) and causing smooth muscle relaxation. A limitation of this model is that it is based exclusively on data from male arterial myocytes. Here, we used a combination of electrophysiology as well as imaging approaches to investigate the role of Kv2.1 channels in male and female arterial myocytes. We confirmed that Kv2.1 plays a canonical conductive role but found it also has a structural role in arterial myocytes to enhance clustering of CaV1.2 channels. Less than 1% of Kv2.1 channels are conductive and induce membrane hyperpolarization. Paradoxically, by enhancing the structural clustering and probability of CaV1.2-CaV1.2 interactions within these clusters, Kv2.1 increases Ca2+ influx. These functional impacts of Kv2.1 depend on its level of expression, which varies with sex. In female myocytes, where expression of Kv2.1 protein is higher than in male myocytes, Kv2.1 has conductive and structural roles. Female myocytes have larger CaV1.2 clusters, larger [Ca2+]i, and larger myogenic tone than male myocytes. In contrast, in male myocytes, Kv2.1 channels regulate membrane potential but not CaV1.2 channel clustering. We propose a model in which Kv2.1 function varies with sex: in males, Kv2.1 channels control membrane potential but, in female myocytes, Kv2.1 plays dual electrical and CaV1.2 clustering roles. This contributes to sex-specific regulation of excitability, [Ca2+]i, and myogenic tone in arterial myocytes.
Assuntos
Artérias/metabolismo , Canais de Cálcio Tipo L/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio Shab/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Células Cultivadas , Feminino , Masculino , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Potássio Shab/genéticaRESUMO
The essential function of the circulatory system is to continuously and efficiently supply the O2 and nutrients necessary to meet the metabolic demands of every cell in the body, a function in which vast capillary networks play a key role. Capillary networks serve an additional important function in the central nervous system: acting as a sensory network, they detect neuronal activity in the form of elevated extracellular K+ and initiate a retrograde, propagating, hyperpolarizing signal that dilates upstream arterioles to rapidly increase local blood flow. Yet, little is known about how blood entering this network is distributed on a branch-to-branch basis to reach specific neurons in need. Here, we demonstrate that capillary-enwrapping projections of junctional, contractile pericytes within a postarteriole transitional region differentially constrict to structurally and dynamically determine the morphology of capillary junctions and thereby regulate branch-specific blood flow. We further found that these contractile pericytes are capable of receiving propagating K+-induced hyperpolarizing signals propagating through the capillary network and dynamically channeling red blood cells toward the initiating signal. By controlling blood flow at junctions, contractile pericytes within a functionally distinct postarteriole transitional region maintain the efficiency and effectiveness of the capillary network, enabling optimal perfusion of the brain.
Assuntos
Capilares/fisiologia , Circulação Cerebrovascular , Microcirculação , Pericitos/fisiologia , Animais , Arteríolas/fisiologia , Canais de Cálcio/metabolismo , Veias Cerebrais/fisiologia , CamundongosRESUMO
Hyperglycemia exacerbates edema formation and worsens neurological outcome in ischemic stroke. Edema formation in the early hours of stroke involves transport of ions and water across an intact blood-brain barrier (BBB), and swelling of astrocytes. We showed previously that high glucose (HG) exposures of 24 hours to 7 days increase abundance and activity of BBB Na+-K+-2Cl- cotransport (NKCC) and Na+/H+ exchange 1 (NHE1). Further, bumetanide and HOE-642 inhibition of these transporters significantly reduces edema and infarct following middle cerebral artery occlusion in hyperglycemic rats, suggesting that NKCC and NHE1 are effective therapeutic targets for reducing edema in hyperglycemic stroke. The mechanisms underlying hyperglycemia effects on BBB NKCC and NHE1 are not known. In the present study we investigated whether serum-glucocorticoid regulated kinase 1 (SGK1) and protein kinase C beta II (PKCßII) are involved in HG effects on BBB NKCC and NHE1. We found transient increases in phosphorylated SGK1 and PKCßII within the first hour of HG exposure, after 5-60 min for SGK1 and 5 min for PKCßII. However, no changes were observed in cerebral microvascular endothelial cell SGK1 or PKCßII abundance or phosphorylation (activity) after 24 or 48 h HG exposures. Further, we found that HG-induced increases in NKCC and NHE1 abundance were abolished by inhibition of SGK1 but not PKCßII, whereas the increases in NKCC and NHE activity were abolished by inhibition of either kinase. Finally, we found evidence that STE20/SPS1-related proline/alanine-rich kinase and oxidative stress-responsive kinase-1 (SPAK/OSR1) participate in the HG-induced effects on BBB NKCC.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glucose/toxicidade , Proteínas Imediatamente Precoces/metabolismo , Proteína Quinase C beta/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Animais , Barreira Hematoencefálica/enzimologia , Barreira Hematoencefálica/patologia , Bovinos , Células Cultivadas , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Ativação Enzimática , Humanos , Fosforilação , Transdução de Sinais , Fatores de TempoRESUMO
BACKGROUND: Spontaneous deep intracerebral hemorrhage (ICH) is a devastating subtype of stroke without specific treatments. It has been thought that smooth muscle cell (SMC) degeneration at the site of arteriolar wall rupture may be sufficient to cause hemorrhage. However, deep ICHs are rare in some aggressive small vessel diseases that are characterized by significant arteriolar SMC degeneration. Here we hypothesized that a second cellular defect may be required for the occurrence of ICH. METHODS: We studied a genetic model of spontaneous deep ICH using Col4a1+/G498V and Col4a1+/G1064D mouse lines that are mutated for the α1 chain of collagen type IV. We analyzed cerebroretinal microvessels, performed genetic rescue experiments, vascular reactivity analysis, and computational modeling. We examined postmortem brain tissues from patients with sporadic deep ICH. RESULTS: We identified in the normal cerebroretinal vasculature a novel segment between arterioles and capillaries, herein called the transitional segment (TS), which is covered by mural cells distinct from SMCs and pericytes. In Col4a1 mutant mice, this TS was hypermuscularized, with a hyperplasia of mural cells expressing more contractile proteins, whereas the upstream arteriole exhibited a loss of SMCs. TSs mechanistically showed a transient increase in proliferation of mural cells during postnatal maturation. Mutant brain microvessels, unlike mutant arteries, displayed a significant upregulation of SM genes and Notch3 target genes, and genetic reduction of Notch3 in Col4a1+/G498V mice protected against ICH. Retina analysis showed that hypermuscularization of the TS was attenuated, but arteriolar SMC loss was unchanged in Col4a1+/G498V, Notch3+/- mice. Moreover, hypermuscularization of the retinal TS increased its contractility and tone and raised the intravascular pressure in the upstream feeding arteriole. We similarly found hypermuscularization of the TS and focal arteriolar SMC loss in brain tissues from patients with sporadic deep ICH. CONCLUSIONS: Our results suggest that hypermuscularization of the TS, through increased Notch3 activity, is involved in the occurrence of ICH in Col4a1 mutant mice, by raising the intravascular pressure in the upstream feeding arteriole and promoting its rupture at the site of SMC loss. Our human data indicate that these 2 mutually reinforcing vascular defects may represent a general mechanism of deep ICH.
Assuntos
Hemorragia Cerebral/etiologia , Hemorragia Cerebral/prevenção & controle , Microvasos/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Biomarcadores , Hemorragia Cerebral/diagnóstico , Hemorragia Cerebral/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Expressão Gênica , Genótipo , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Microvasos/fisiopatologia , Imagem Molecular , Mutação , Miócitos de Músculo Liso/metabolismo , Receptor Notch3/metabolismo , Retina/metabolismo , Retina/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologiaRESUMO
Brain edema forms rapidly in the early hours of ischemic stroke by increased secretion of Na, Cl, and water into the brain across an intact blood-brain barrier (BBB), together with swelling of astrocytes as they take up the ions and water crossing the BBB. Our previous studies provide evidence that luminal BBB Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) participate in ischemia-induced edema formation. NKCC1 and two NHE isoforms, NHE1 and NHE2, reside predominantly at the luminal BBB membrane. NKCC and NHE activities of cerebral microvascular endothelial cells (CMEC) are rapidly stimulated by the ischemic factors hypoxia, aglycemia, and AVP, and inhibition of NKCC and NHE activities by bumetanide and HOE642, respectively, reduces brain Na uptake and edema in the rat middle cerebral artery occlusion model of stroke. The present study was conducted to further explore BBB NHE responses to ischemia. We examined whether ischemic factor-stimulated NHE activity is sustained over several hours, when the majority of edema forms during stroke. We also examined whether ischemic factors alter NHE1 and/or NHE2 protein abundance. Finally, we conducted initial studies of ERK1/2 MAP kinase involvement in BBB NHE and NKCC responses to ischemic factors. We found that hypoxia, aglycemia, and AVP increase CMEC NHE activity through 5 h and that NHE1, but not NHE2, abundance is increased by 1- to 5-h exposures to these factors. Furthermore, we found that these factors rapidly increase BBB ERK1/2 activity and that ERK1/2 inhibition reduces or abolishes ischemic factor stimulation of NKCC and NHE activities.
Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Arginina Vasopressina/metabolismo , Arginina Vasopressina/farmacologia , Bovinos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica , Glucose/deficiência , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Oxigênio/metabolismo , Oxigênio/farmacologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/genéticaRESUMO
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/metabolismoRESUMO
Exergames have been suggested as a possible alternative to traditional exercise in the general population. The purpose of this study was to examine the heart rate (HR) and energy expenditure (EE) of young adults playing several different exergames, while self-selecting the component of the game to play and the intensity. A total of 117 participants, 18-35 years of age, were evaluated on one of four active video games. Participants were free to choose any component of the given game to play and they played at a self-selected intensity. The average HR and EE during the individual games were compared to resting conditions and to the American College of Sports Medicine (ACSM) guidelines. The HR and EE increased above resting conditions during each game (p<0.05). When the results of all games were combined, the HR was 125.4 ± 20.0 bpm and the average EE was 6.7 ± 2.1 kcal/min. This HR represents an average percent of heart rate reserve of 44.6 ± 14.1, high enough to be considered moderate intensity exercise. If performed for 30 minutes a day, five days per week, the average EE would be 1,005 kcals, enough to meet the ACSM recommendations for weekly EE. Therefore, at least some exergames could be a component of an exercise program.