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1.
Am J Emerg Med ; 41: 104-109, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33421674

RESUMO

INTRODUCTION: Early recognition and management of hemorrhage, damage control resuscitation, and blood product administration have optimized management of severe trauma. Recent data suggest hypocalcemia exacerbates the ensuing effects of coagulopathy in trauma. OBJECTIVE: This narrative review of available literature describes the physiology and role of calcium in trauma resuscitation. Authors did not perform a systematic review or meta-analysis. DISCUSSION: Calcium is a divalent cation found in various physiologic forms, specifically the bound, inactive state and the unbound, physiologically active state. While calcium plays several important physiologic roles in multiple organ systems, the negative hemodynamic effects of hypocalcemia are crucial to address in trauma patients. The negative ramifications of hypocalcemia are intrinsically linked to components of the lethal triad of acidosis, coagulopathy, and hypothermia. Hypocalcemia has direct and indirect effects on each portion of the lethal triad, supporting calcium's potential position as a fourth component in this proposed lethal diamond. Trauma patients often present hypocalcemic in the setting of severe hemorrhage secondary to trauma, which can be worsened by necessary transfusion and resuscitation. The critical consequences of hypocalcemia in the trauma patient have been repeatedly demonstrated with the associated morbidity and mortality. It remains poorly defined when to administer calcium, though current data suggest that earlier administration may be advantageous. CONCLUSIONS: Calcium is a key component of trauma resuscitation and the coagulation cascade. Recent data portray the intricate physiologic reverberations of hypocalcemia in the traumatically injured patient; however, future research is needed to further guide the management of these patients.


Assuntos
Hipocalcemia/etiologia , Ressuscitação , Ferimentos e Lesões/complicações , Ferimentos e Lesões/terapia , Cálcio/fisiologia , Humanos , Ferimentos e Lesões/mortalidade
2.
Environ Health Prev Med ; 25(1): 70, 2020 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-33160329

RESUMO

BACKGROUND: Resveratrol has been shown to inhibit platelet aggregation. However, the mechanism for this action of resveratrol remains to be clarified. The purpose of this study was to elucidate the Ca2+-related mechanism for the inhibitory action of resveratrol on platelet aggregation. METHODS: Ca2+ entry and subsequent aggregation of human platelets induced by different stimulants including thrombin, thapsigargin, and 1-oleoyl-2-acetylglycerol (OAG) were measured by the fluorescence method and light transmittance method, respectively. Each stimulant was added to a nominally Ca2+-free medium containing platelets, and then CaCl2 was added to the medium to induce Ca2+ influx into platelets. RESULTS: Thapsigargin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 6.25 µM or higher concentrations, while OAG-induced Ca2+ entry and subsequent platelet aggregation were not affected by resveratrol at concentrations up to 50 µM. In the nominally Ca2+-free medium, thrombin induced a small transient increase in intracellular Ca2+ concentrations, which was attenuated in the presence of resveratrol at 12.5 µM or higher concentrations. Thrombin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 12.5 µM or higher concentrations. CONCLUSIONS: The results suggest that resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca2+ release from its stores and inhibiting store-operated Ca2+ influx into platelets.


Assuntos
Cálcio/fisiologia , Inibidores da Agregação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Resveratrol/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antioxidantes/administração & dosagem , Humanos , Inibidores da Agregação de Plaquetas/administração & dosagem , Resveratrol/administração & dosagem
3.
Folia Biol (Praha) ; 66(3): 91-103, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33069188

RESUMO

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.


Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/fisiologia , Genes Fúngicos , Acetiltransferases/deficiência , Acetiltransferases/genética , Acetiltransferases/fisiologia , Antifúngicos/farmacologia , Sistemas CRISPR-Cas , Cálcio/fisiologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/patogenicidade , Cátions/farmacologia , Adesão Celular , Ciclo Celular , Parede Celular/efeitos dos fármacos , Quitinases/farmacologia , Dano ao DNA , Proteínas Fúngicas/genética , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/farmacologia , Hifas/crescimento & desenvolvimento , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/fisiologia , Fases de Leitura Aberta , Reprodução Assexuada , Fatores Associados à Proteína de Ligação a TATA/deficiência , Fatores Associados à Proteína de Ligação a TATA/genética , Fatores Associados à Proteína de Ligação a TATA/fisiologia , Virulência/genética
4.
Proc Natl Acad Sci U S A ; 117(35): 21731-21739, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32801213

RESUMO

Ca2+ uptake by mitochondria regulates bioenergetics, apoptosis, and Ca2+ signaling. The primary pathway for mitochondrial Ca2+ uptake is the mitochondrial calcium uniporter (MCU), a Ca2+-selective ion channel in the inner mitochondrial membrane. MCU-mediated Ca2+ uptake is driven by the sizable inner-membrane potential generated by the electron-transport chain. Despite the large thermodynamic driving force, mitochondrial Ca2+ uptake is tightly regulated to maintain low matrix [Ca2+] and prevent opening of the permeability transition pore and cell death, while meeting dynamic cellular energy demands. How this is accomplished is controversial. Here we define a regulatory mechanism of MCU-channel activity in which cytoplasmic Ca2+ regulation of intermembrane space-localized MICU1/2 is controlled by Ca2+-regulatory mechanisms localized across the membrane in the mitochondrial matrix. Ca2+ that permeates through the channel pore regulates Ca2+ affinities of coupled inhibitory and activating sensors in the matrix. Ca2+ binding to the inhibitory sensor within the MCU amino terminus closes the channel despite Ca2+ binding to MICU1/2. Conversely, disruption of the interaction of MICU1/2 with the MCU complex disables matrix Ca2+ regulation of channel activity. Our results demonstrate how Ca2+ influx into mitochondria is tuned by coupled Ca2+-regulatory mechanisms on both sides of the inner mitochondrial membrane.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Apoptose , Transporte Biológico , Cálcio/fisiologia , Canais de Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/fisiologia , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Citoplasma/metabolismo , Citosol/metabolismo , Células HEK293 , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/fisiologia , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/fisiologia , Oxirredução , Multimerização Proteica , Transdução de Sinais
5.
Proc Natl Acad Sci U S A ; 117(35): 21701-21710, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817426

RESUMO

Rod photoreceptors are composed of a soma and an inner segment (IS) connected to an outer segment (OS) by a thin cilium. OSs are composed of a stack of ∼800 lipid discs surrounded by the plasma membrane where phototransduction takes place. Intracellular calcium plays a major role in phototransduction and is more concentrated in the discs, where it can be incorporated and released. To study calcium dynamics in rods, we used the fluorescent calcium dye CaSiR-1 AM working in the near-infrared (NIR) (excitation at 650 and emission at 664 nm), an advantage over previously used dyes. In this way, we investigated calcium dynamics with an unprecedented accuracy and most importantly in semidark-adapted conditions. We observed light-induced drops in [Ca2+]i with kinetics similar to that of photoresponses recorded electrophysiologically. We show three properties of the rods. First, intracellular calcium and key proteins have concentrations that vary from the OS base to tip. At the OS base, [Ca2+]i is ∼80 nM and increases up to ∼200 nM at the OS tip. Second, there are spontaneous calcium flares in healthy and functional rod OSs; these flares are highly localized and are more pronounced at the OS tip. Third, a bright flash of light at 488 nm induces a drop in [Ca2+]i at the OS base but often a flare at the OS tip. Therefore, rod OSs are not homogenous structures but have a structural and functional gradient, which is a fundamental aspect of transduction in vertebrate photoreceptors.


Assuntos
Cálcio/metabolismo , Transdução de Sinal Luminoso/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Cálcio/fisiologia , Membrana Celular/metabolismo , Citoplasma/metabolismo , Feminino , Cinética , Masculino , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Segmento Externo da Célula Bastonete/fisiologia , Xenopus laevis
6.
Proc Natl Acad Sci U S A ; 117(35): 21711-21722, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817431

RESUMO

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+ alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+ activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.


Assuntos
Cálcio/metabolismo , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/ultraestrutura , Regulação Alostérica/fisiologia , Sítios de Ligação/genética , Cálcio/fisiologia , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Ligantes , Conformação Molecular , Receptores de Detecção de Cálcio/fisiologia , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais
7.
J Vis Exp ; (160)2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32628156

RESUMO

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.


Assuntos
Cálcio/fisiologia , Técnicas Eletrofisiológicas Cardíacas , Função Ventricular , Ventrículos do Coração , Humanos , Técnicas In Vitro , Potenciais da Membrana , Microtomia
8.
J Neurosci ; 40(27): 5247-5263, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32503886

RESUMO

The accessory olfactory bulb (AOB), the first neural circuit in the mouse accessory olfactory system, is critical for interpreting social chemosignals. Despite its importance, AOB information processing is poorly understood compared with the main olfactory bulb (MOB). Here, we sought to fill gaps in the understanding of AOB interneuron function. We used 2-photon GCaMP6f Ca2+ imaging in an ex vivo preparation to study chemosensory tuning in AOB external granule cells (EGCs), interneurons hypothesized to broadly inhibit activity in excitatory mitral cells (MCs). In ex vivo preparations from mice of both sexes, we measured MC and EGC tuning to natural chemosignal blends and monomolecular ligands, finding that EGC tuning was sparser, not broader, than upstream MCs. Simultaneous electrophysiological recording and Ca2+ imaging showed no differences in GCaMP6f-to-spiking relationships in these cell types during simulated sensory stimulation, suggesting that measured EGC sparseness was not due to cell type-dependent variability in GCaMP6f performance. Ex vivo patch-clamp recordings revealed that EGC subthreshold responsivity was far broader than indicated by GCaMP6f Ca2+ imaging, and that monomolecular ligands rarely elicited EGC spiking. These results indicate that EGCs are selectively engaged by chemosensory blends, suggesting different roles for EGCs than analogous interneurons in the MOB.SIGNIFICANCE STATEMENT The mouse accessory olfactory system (AOS) interprets social chemosignals, but we poorly understand AOS information processing. Here, we investigate the functional properties of external granule cells (EGCs), a major class of interneurons in the accessory olfactory bulb (AOB). We hypothesized that EGCs, which are densely innervated by excitatory mitral cells (MCs), would show broad chemosensory tuning, suggesting a role in divisive normalization. Using ex vivo GCaMP6f imaging, we found that EGCs were instead more sparsely tuned than MCs. This was not due to weaker GCaMP6f signaling in EGCs than in MCs. Instead, we found that many MC-activating chemosignals caused only subthreshold EGC responses. This indicates a different role for AOB EGCs compared with analogous cells in the main olfactory bulb.


Assuntos
Bulbo Olfatório/citologia , Bulbo Olfatório/fisiologia , Olfato/fisiologia , Animais , Cálcio/fisiologia , Grânulos Citoplasmáticos , Fenômenos Eletrofisiológicos/fisiologia , Feminino , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroimagem , Odorantes , Técnicas de Patch-Clamp , Órgão Vomeronasal/citologia , Órgão Vomeronasal/fisiologia
9.
PLoS Pathog ; 16(5): e1008214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379809

RESUMO

Calcium signaling plays a key role in many essential processes in almost all eukaryotic systems. It is believed that it may also be an important signaling system of the protist parasite Entamoeba histolytica. Motility, adhesion, cytolysis, and phagocytosis/trogocytosis are important steps in invasion and pathogenesis of E. histolytica, and Ca2+ signaling is thought to be associated with these processes leading to tissue invasion. There are a large number of Ca2+-binding proteins (CaBPs) in E. histolytica, and a number of these proteins appear to be associated with different steps in pathogenesis. The genome encodes 27 EF-hand-containing CaBPs in addition to a number of other Ca2+-binding domain/motif-containing proteins, which suggest intricate calcium signaling network in this parasite. Unlike other eukaryotes, a typical calmodulin-like protein has not been seen in E. histolytica. Though none of the CaBPs display sequence similarity with a typical calmodulin, extensive structural similarity has been seen in spite of lack of significant functional overlap with that of typical calmodulins. One of the unique features observed in E. histolytica is the identification of CaBPs (EhCaBP1, EhCaBP3) that have the ability to directly bind actin and modulate actin dynamics. Direct interaction of CaBPs with actin has not been seen in any other system. Pseudopod formation and phagocytosis are some of the processes that require actin dynamics, and some of the amoebic CaBPs (EhC2Pk, EhCaBP1, EhCaBP3, EhCaBP5) participate in this process. None of these E. histolytica CaBPs have any homolog in organisms other than different species of Entamoeba, suggesting a novel Ca2+ signaling pathway that has evolved in this genus.


Assuntos
Cálcio/metabolismo , Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Actinas/metabolismo , Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Fagocitose , Proteínas de Protozoários/metabolismo
10.
J Smooth Muscle Res ; 56(0): 19-28, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32350168

RESUMO

Blebbistatin, a potent inhibitor of myosin II, is known to suppress smooth muscle contraction without affecting myosin light chain phosphorylation level. In order to clarify the regulatory mechanisms of blebbistatin on phasic and tonic smooth muscles in detail, we examined the effects of blebbistatin on relaxation process by Ca2+ removal after Ca2+-induced contraction of ß-escin skinned (cell membrane permeabilized) trachea and taenia cecum preparations from guinea pigs. Blebbistatin significantly suppressed the force during relaxation both in skinned trachea and taenia cecum. The data fitting analysis of the relaxation processes indicates that blebbistatin accelerates slow (latch-like) bridge dissociation.


Assuntos
Ceco/citologia , Ceco/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Traqueia/citologia , Traqueia/efeitos dos fármacos , Animais , Cálcio/fisiologia , Membrana Celular , Células Cultivadas , Escina , Cobaias , Masculino
11.
Adv Immunol ; 145: 187-241, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32081198

RESUMO

Metals are essential components in all forms of life required for the function of nearly half of all enzymes and are critically involved in virtually all fundamental biological processes. Especially, the transition metals iron (Fe), zinc (Zn), manganese (Mn), nickel (Ni), copper (Cu) and cobalt (Co) are crucial micronutrients known to play vital roles in metabolism as well due to their unique redox properties. Metals carry out three major functions within metalloproteins: to provide structural support, to serve as enzymatic cofactors, and to mediate electron transportation. Metal ions are also involved in the immune system from metal allergies to nutritional immunity. Within the past decade, much attention has been drawn to the roles of metal ions in the immune system, since increasing evidence has mounted to suggest that metals are critically implicated in regulating both the innate immune sensing of and the host defense against invading pathogens. The importance of ions in immunity is also evidenced by the identification of various immunodeficiencies in patients with mutations in ion channels and transporters. In addition, cancer immunotherapy has recently been conclusively demonstrated to be effective and important for future tumor treatment, although only a small percentage of cancer patients respond to immunotherapy because of inadequate immune activation. Importantly, metal ion-activated immunotherapy is becoming an effective and potential way in tumor therapy for better clinical application. Nevertheless, we are still in a primary stage of discovering the diverse immunological functions of ions and mechanistically understanding the roles of these ions in immune regulation. This review summarizes recent advances in the understanding of metal-controlled immunity. Particular emphasis is put on the mechanisms of innate immune stimulation and T cell activation by the essential metal ions like calcium (Ca2+), zinc (Zn2+), manganese (Mn2+), iron (Fe2+/Fe3+), and potassium (K+), followed by a few unessential metals, in order to draw a general diagram of metalloimmunology.


Assuntos
Imunidade Inata , Metais/metabolismo , Transdução de Sinais/imunologia , Animais , Cálcio/química , Cálcio/metabolismo , Cálcio/fisiologia , Enzimas , Humanos , Imunoterapia , Íons/química , Íons/metabolismo , Ferro/metabolismo , Ferro/fisiologia , Manganês/metabolismo , Manganês/fisiologia , Metais/química , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/imunologia , Potássio/química , Potássio/metabolismo , Potássio/fisiologia , Zinco/química , Zinco/deficiência , Zinco/fisiologia
12.
Circ Res ; 126(7): 889-906, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32070187

RESUMO

RATIONALE: Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes. OBJECTIVE: To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes. METHODS AND RESULTS: Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K+] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca2+ overload and increased generation of both spontaneous Ca2+ waves and delayed afterdepolarizations. However, similar Ca2+-dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na+-K+ ATPase and Na+-Ca2+ exchanger proteins within these structures, as reduction in Na+ pump activity locally inhibited Ca2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na+ current, as they were rapidly blocked by tetrodotoxin. Na+ current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K+ current (IKur); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes. CONCLUSIONS: Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.


Assuntos
Arritmias Cardíacas/metabolismo , Fibrilação Atrial/metabolismo , Cálcio/metabolismo , Hipopotassemia/metabolismo , Miócitos Cardíacos/metabolismo , Potenciais de Ação , Animais , Arritmias Cardíacas/fisiopatologia , Fibrilação Atrial/fisiopatologia , Cálcio/fisiologia , Células Cultivadas , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Humanos , Potássio/metabolismo , Ratos , Sódio/metabolismo , Trocador de Sódio e Cálcio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo
13.
Proc Natl Acad Sci U S A ; 117(8): 4281-4291, 2020 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-32047033

RESUMO

VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of our findings in PD, we identify a missense mutation of Parkin discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.


Assuntos
Apoptose , Mitofagia , Ubiquitina-Proteína Ligases/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismo , Animais , Cálcio/fisiologia , Drosophila/genética , Drosophila/metabolismo , Feminino , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Canal de Ânion 1 Dependente de Voltagem/genética
14.
Dtsch Med Wochenschr ; 145(3): 171-174, 2020 02.
Artigo em Alemão | MEDLINE | ID: mdl-32018291

RESUMO

Calcium is pivotal for neuromuscular function, coagulation, and signal transduction. An imbalance of enteral calcium uptake, deposition in and resorption from bones, and renal calcium elimination causes hypercalcemia. The dissociation between total serum calcium and ionized calcium has important implications in diagnosing hypercalcemia. The calcium sensing receptor (CaSR) regulates parathyroid hormone release and renal calcium reabsorption. Knowing the actions of the CaSR is important for diagnosing and treating patients with hyperparathyroidism. Diuretics can cause hypercalcemia, but also provide a clinical tool to lower serum calcium.


Assuntos
Hipercalcemia , Cálcio/metabolismo , Cálcio/fisiologia , Humanos , Hormônio Paratireóideo/metabolismo , Hormônio Paratireóideo/fisiologia , Receptores de Detecção de Cálcio/metabolismo , Receptores de Detecção de Cálcio/fisiologia
15.
J Ethnopharmacol ; 252: 112559, 2020 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31935497

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Crataegus leaves, flowers and fruits have been traditionally used to improve blood circulation, numerous preclinical and clinical studies supporting the cardiovascular benefits of Crataegus preparations. In this respect, there is very limited data on Crataegus pentagyna; in addition, the chemical profile of this species is still incompletely elucidated. AIM OF THE STUDY: The objective of this study was to examine the cardiovascular benefits of Crataegus pentagyna Waldst. et Kit. ex Willd. (small-flowered black hawthorn, Rosaceae) extracts (leaf, flower and fruit ethyl acetate extracts) and the underlying mechanisms. We hypothesized that C. pentagyna extracts might exert vasodilatory effects and inhibit arginase activity due, in large part, to their polyphenolic constituents. MATERIALS AND METHODS: C. pentagyna extracts induced-relaxation and the mechanisms involved were studied ex vivo in isolated aortic rings from Sprague-Dawley rats. The inhibitory effects on bovine liver arginase I were assessed by an in vitro assay. Metabolite profiling of C. pentagyna extracts was performed and the most endothelium- and nitric oxide synthase-dependent; flower extract additionally reduced Ca2+ entry and, to a lesser extent, Ca2+ release from the sarcoplasmic reticulum. C. pentagyna proved to be an important source of arginase inhibitors with potential benefits in endothelial dysfunction that remains to be explored.


Assuntos
Aorta Torácica/efeitos dos fármacos , Arginase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Vasodilatadores/farmacologia , Animais , Aorta Torácica/fisiologia , Cálcio/fisiologia , Crataegus , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Flores , Frutas , Masculino , Folhas de Planta , Canais de Potássio/fisiologia , Ratos Sprague-Dawley
16.
Toxicol Lett ; 323: 1-9, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31982503

RESUMO

Zearalenone (ZEA) is a prevalent non-steroidal estrogenic mycotoxin produced mainly by Fusarium contamination. Our previous study showed that ZEA induces the autophagy of Sertoli cells (SCs). However, the underlying mechanisms are still unknown. Several studies have indicated that the increasing level of cytoplasmic Ca2+ could induce autophagy through CaMKKß and AMPK pathways. Thus in order to investigate the potential mechanism underlying ZEA-induced autophagy, the activity of calmodulin-dependent kinase kinase ß(CaMKKß)and AMP-activated protein kinase (AMPK) signaling pathway in ZEA-infected TM4 cells was studied. In the present study, ZEA activated the CaMKKß and AMPK signaling pathways. The AMPK inhibitor and activator significantly inhibited and stimulated the effect of ZEA on AMPK, the transformation from LC3I to LC3II, and the distribution of LC3 dots. In addition, cytosolic calcium (Ca2+) was increased gradually with the concentration of ZEA. After treatment of ZEA-infected cells with 1, 2-bis (2-aminophenoxy) ethane-N, N, N', N'- tetraacetic acid- tetraac etoxymethyl ester (BAPTA-AM) and 2-aminoethyl diphenylborinate (2-APB), the intracellular concentration of Ca2+ reduced significantly. Also, the activities of CaMKKß and AMPK and subsequent autophagy decreased. Moreover, the antioxidant NAC significantly decreased activities of AMPK and autophagy -related protein. Therefore, it can be speculated that ROS- mediated ER-stress induced by ZEA activates AMPK via Ca2+-CaMKKß leading to autophagy in TM4 cells.


Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Autofagia/efeitos dos fármacos , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/fisiologia , Cálcio/fisiologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Zearalenona/toxicidade , Animais , Células Cultivadas , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologia , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/fisiologia
17.
Am J Respir Cell Mol Biol ; 62(6): 709-718, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31945301

RESUMO

Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. Nicotinamide adenine dinucleotide phosphate oxidase and EGFR (epidermal growth factor receptor) signaling contributes to this response. Caveolin-1 regulates the activity of a variety of proteins, including EGFR and nicotinamide adenine dinucleotide phosphate oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction after CH. Although exposure of rats to CH (4 wk, ∼380 mm Hg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content as assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted pulmonary arteries (∼150 µm inner diameter) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH rats than in those obtained from control rats, and increased superoxide production as assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide antennapedia-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction after CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting that cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction after CH.


Assuntos
Cálcio/fisiologia , Caveolina 1/biossíntese , Colesterol/fisiologia , Hipóxia/fisiopatologia , Lipídeos de Membrana/fisiologia , Músculo Liso Vascular/metabolismo , Artéria Pulmonar/fisiopatologia , Vasoconstrição/fisiologia , Animais , Caveolina 1/genética , Doença Crônica , Receptores ErbB/fisiologia , Hipóxia/metabolismo , Masculino , Potenciais da Membrana , Artéria Pulmonar/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Superóxidos/metabolismo
18.
Science ; 367(6473): 83-87, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31896716

RESUMO

The active electrical properties of dendrites shape neuronal input and output and are fundamental to brain function. However, our knowledge of active dendrites has been almost entirely acquired from studies of rodents. In this work, we investigated the dendrites of layer 2 and 3 (L2/3) pyramidal neurons of the human cerebral cortex ex vivo. In these neurons, we discovered a class of calcium-mediated dendritic action potentials (dCaAPs) whose waveform and effects on neuronal output have not been previously described. In contrast to typical all-or-none action potentials, dCaAPs were graded; their amplitudes were maximal for threshold-level stimuli but dampened for stronger stimuli. These dCaAPs enabled the dendrites of individual human neocortical pyramidal neurons to classify linearly nonseparable inputs-a computation conventionally thought to require multilayered networks.


Assuntos
Potenciais de Ação , Dendritos/fisiologia , Neocórtex/fisiologia , Células Piramidais/fisiologia , Adolescente , Adulto , Idoso , Cálcio/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neocórtex/citologia , Adulto Jovem
19.
Am J Physiol Heart Circ Physiol ; 318(2): H354-H365, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31886723

RESUMO

Rodent models are frequently employed in cardiovascular research, yet our understanding of pediatric cardiac physiology has largely been deduced from more simplified two-dimensional cell studies. Previous studies have shown that postnatal development includes an alteration in the expression of genes and proteins involved in cell coupling, ion channels, and intracellular calcium handling. Accordingly, we hypothesized that postnatal cell maturation is likely to lead to dynamic alterations in whole heart electrophysiology and calcium handling. To test this hypothesis, we employed multiparametric imaging and electrophysiological techniques to quantify developmental changes from neonate to adult. In vivo electrocardiograms were collected to assess changes in heart rate, variability, and atrioventricular conduction (Sprague-Dawley rats). Intact, whole hearts were transferred to a Langendorff-perfusion system for multiparametric imaging (voltage, calcium). Optical mapping was performed in conjunction with an electrophysiology study to assess cardiac dynamics throughout development. Postnatal age was associated with an increase in the heart rate (181 ± 34 vs. 429 ± 13 beats/min), faster atrioventricular conduction (94 ± 13 vs. 46 ± 3 ms), shortened action potentials (APD80: 113 ± 18 vs. 60 ± 17 ms), and decreased ventricular refractoriness (VERP: 157 ± 45 vs. 57 ± 14 ms; neonatal vs. adults, means ± SD, P < 0.05). Calcium handling matured with development, resulting in shortened calcium transient durations (168 ± 18 vs. 117 ± 14 ms) and decreased propensity for calcium transient alternans (160 ± 18- vs. 99 ± 11-ms cycle length threshold; neonatal vs. adults, mean ± SD, P < 0.05). Results of this study can serve as a comprehensive baseline for future studies focused on pediatric disease modeling and/or preclinical testing.NEW & NOTEWORTHY This is the first study to assess cardiac electrophysiology and calcium handling throughout postnatal development, using both in vivo and whole heart models.


Assuntos
Envelhecimento/fisiologia , Cálcio/metabolismo , Cálcio/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Coração/crescimento & desenvolvimento , Coração/fisiologia , Potenciais de Ação/fisiologia , Agonistas Adrenérgicos beta/farmacologia , Animais , Animais Recém-Nascidos , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Circulação Coronária/fisiologia , Eletrocardiografia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Coração/efeitos dos fármacos , Sistema de Condução Cardíaco/crescimento & desenvolvimento , Sistema de Condução Cardíaco/fisiologia , Frequência Cardíaca/fisiologia , Técnicas In Vitro , Isoproterenol/farmacologia , Perfusão , Ratos , Ratos Sprague-Dawley
20.
Neuron ; 105(4): 630-644.e9, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-31859031

RESUMO

Sirtuin 1 (Sirt1) is a NAD+-dependent deacetylase capable of countering age-related neurodegeneration, but the basis of Sirt1 neuroprotection remains elusive. Spinocerebellar ataxia type 7 (SCA7) is an inherited CAG-polyglutamine repeat disorder. Transcriptome analysis of SCA7 mice revealed downregulation of calcium flux genes accompanied by abnormal calcium-dependent cerebellar membrane excitability. Transcription-factor binding-site analysis of downregulated genes yielded Sirt1 target sites, and we observed reduced Sirt1 activity in the SCA7 mouse cerebellum with NAD+ depletion. SCA7 patients displayed increased poly(ADP-ribose) in cerebellar neurons, supporting poly(ADP-ribose) polymerase-1 upregulation. We crossed Sirt1-overexpressing mice with SCA7 mice and noted rescue of neurodegeneration and calcium flux defects. NAD+ repletion via nicotinamide riboside ameliorated disease phenotypes in SCA7 mice and patient stem cell-derived neurons. Sirt1 thus achieves neuroprotection by promoting calcium regulation, and NAD+ dysregulation underlies Sirt1 dysfunction in SCA7, indicating that cerebellar ataxias exhibit altered calcium homeostasis because of metabolic dysregulation, suggesting shared therapy targets.


Assuntos
Cálcio/fisiologia , Homeostase/fisiologia , Neuroproteção/fisiologia , Niacinamida/metabolismo , Sirtuína 1/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Linhagem Celular , Cerebelo/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Transdução de Sinais/fisiologia , Sirtuína 1/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/prevenção & controle
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