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1.
Sci Adv ; 10(12): eadl1126, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38507485

RESUMO

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.


Assuntos
Cálcio , Canal de Liberação de Cálcio do Receptor de Rianodina , Canal de Liberação de Cálcio do Receptor de Rianodina/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Cálcio/metabolismo , Músculo Esquelético/metabolismo , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/metabolismo , Retículo Sarcoplasmático/metabolismo , Contração Muscular/fisiologia
2.
Biochem Biophys Res Commun ; 529(3): 720-725, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32736698

RESUMO

TMEM16E deficiency has been shown to be responsible for human limb-girdle muscular dystrophy LGMD2L. We found that endogenous TMEM16E co-localized with caveolin-3 at cytoplasmic vesicular compartments in a myotube from C2C12 cells (C2C12 myotube) without forming a molecular complex. In contrast, a myotube from murine myoblastic dysferlin-deficient GREG cells (GREG myotube) showed not only co-localization but also constitutive association of caveolin-3 and TMEM16E. GREG myotubes also displayed constitutive association of TMEM16E with DHPRα, which reside in different membrane compartments, indicating increased contact of the different vesicular membrane compartments. Τhese results suggest that a dynamic tethering of different membrane compartments might represent a distorted membrane damage repairing process in the absence of dysferlin.


Assuntos
Anoctaminas/metabolismo , Canais de Cálcio Tipo L/metabolismo , Disferlina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Anoctaminas/análise , Canais de Cálcio Tipo L/análise , Caveolina 3/análise , Caveolina 3/metabolismo , Disferlina/análise , Disferlina/genética , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/citologia
3.
Sci Rep ; 9(1): 7867, 2019 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133706

RESUMO

Pathological cardiac hypertrophy is a debilitating condition characterized by deleterious thickening of the myocardium, dysregulated Ca2+ signaling within cardiomyocytes, and contractile dysfunction. Importantly, the nanoscale organization, localization, and patterns of expression of critical Ca2+ handling regulators including dihydropyridine receptor (DHPR), ryanodine receptor 2 (RyR2), phospholamban (PLN), and sarco/endoplasmic reticulum Ca2+-ATPase 2A (SERCA2A) remain poorly understood, especially during pathological hypertrophy disease progression. In the current study, we induced cardiac pathological hypertrophy via transverse aortic constriction (TAC) on 8-week-old CD1 mice, followed by isolation of cardiac ventricular myocytes. dSTORM super-resolution imaging was then used to visualize proteins at nanoscale resolution at two time points and we quantified changes in protein cluster properties using Voronoi tessellation and 2D Fast Fourier Transform analyses. We showed a decrease in the density of DHPR and RyR2 clusters with pressure-overload cardiac hypertrophy and an increase in the density of SERCA2A protein clusters. PLN protein clusters decreased in density in 2-week TAC but returned to sham levels by 4-week TAC. Furthermore, 2D-FFT analysis revealed changes in molecular organization during pathological hypertrophy, with DHPR and RyR2 becoming dispersed while both SERCA2A and PLN sequestered into dense clusters. Our work reveals molecular adaptations that occur in critical SR proteins at a single molecule during pressure overload-induced cardiomyopathy. Nanoscale alterations in protein localization and patterns of expression of crucial SR proteins within the cardiomyocyte provided insights into the pathogenesis of cardiac hypertrophy, and specific evidence that cardiomyocytes undergo significant structural remodeling during the progression of pathological hypertrophy.


Assuntos
Cardiomegalia/patologia , Miócitos Cardíacos/patologia , Retículo Sarcoplasmático/patologia , Animais , Canais de Cálcio Tipo L/análise , Proteínas de Ligação ao Cálcio/análise , Cardiomegalia/diagnóstico por imagem , Células Cultivadas , Análise de Fourier , Camundongos , Microscopia , Imagem Óptica , Pressão , Canal de Liberação de Cálcio do Receptor de Rianodina/análise , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/análise
4.
Neurochem Int ; 114: 108-119, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29425964

RESUMO

Voltage-dependent calcium channels (VDCC) have been shown to regulate neuronal excitability and their antagonists have been used clinically for the control of seizures. While functional studies of VDCC in epileptogenesis in the CA1 area of hippocampus or the dentate gyrus have been done, few studies were carried out in the CA3 area. Given the bursting characteristics of the CA3 neurons, we speculated that VDCC in the CA3 area might play an important role in the epileptogenesis. In the present study in the mouse pilocarpine model of temporal lobe epilepsy, we investigated the alterations of alpha 1 subunits of L-type VDCC in the CA3 area of the hippocampus at different stages of epileptogenesis, i.e., acute stage from 10 min to 1 day during and after pilocapine-induced status epilepticus (SE), latent period at 1 week, and chronic stage with spontaneous recurrent seizures at 2 months after SE. We found that an immediate redistribution of alpha 1 subunits in the CA3 area occurred during SE which might be involved in the seizure occurrence indicated by the Racine score record. Alterations of alpha 1 subunits were also demonstrated in the latent period and chronic stage, which might be related to the epileptogenesis and occurrence of epilepsy. Cav1.3, but not Cav1.2, was expressed in reactive astrocytes of the CA3 area, indicating the involvement of Cav1.3 in the modulation of astrocytic Ca2+ homeostasis during epileptogenesis.


Assuntos
Região CA3 Hipocampal/metabolismo , Canais de Cálcio Tipo L/biossíntese , Epilepsia/induzido quimicamente , Epilepsia/metabolismo , Pilocarpina/toxicidade , Animais , Região CA3 Hipocampal/química , Região CA3 Hipocampal/efeitos dos fármacos , Canais de Cálcio Tipo L/análise , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Subunidades Proteicas/análise , Subunidades Proteicas/biossíntese
5.
Int J Mol Med ; 41(5): 2909-2916, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29436604

RESUMO

Ghrelin influences pancreatic endocrine and exocrine functions, regulates intracellular calcium [Ca2+]i levels, and has an anti-inflammatory role in acute pancreatitis. This study investigated the role of endogenous ghrelin in the expression of Cav 1.2 (L-type of Ca2+ channel) and Cav 2.2 (N-type of Ca2+ channel) in acute pancreatitis. For this purpose, acute edematous pancreatitis (AEP) and acute necrotizing pancreatitis (ANP) rat models were established. Cav 1.2 and Cav 2.2 expression was assessed by immunohistochemistry in the pancreatic tissues of rats; ghrelin, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) serum levels were detected using ELISA. Next, in AR42J cells with either knock-out or overexpression of ghrelin, Cav 1.2 and Cav 2.2 expression was examined using western blot analysis, and intracellular calcium [Ca2+]i was detected with confocal microscopy. In this study, the ghrelin serum level was highest in the ANP group and was higher in the AEP group than the normal group. Expression of Cav 1.2 and Cav 2.2 in the ANP and AEP groups was higher than in the respective control groups. The serum IL-1ß and TNF-α levels were significantly higher in the ANP group compared to the other groups. Cav 1.2 and Cav 2.2 expression and [Ca2+]i decreased in ghrelin knockdown AR42J cells but increased in ghrelin overexpressing cells. In conclusion, Cav 1.2 and Cav 2.2 expression increased in ANP. The [Ca2+]i level, which is mediated by Cav 1.2 and Cav 2.2 expression, is directly regulated by ghrelin in pancreatic acinar cells, and serum ghrelin levels may be involved in the severity of acute pancreatitis.


Assuntos
Células Acinares/patologia , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo N/análise , Grelina/metabolismo , Pâncreas/patologia , Pancreatite Necrosante Aguda/patologia , Células Acinares/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo N/genética , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica , Grelina/sangue , Grelina/genética , Masculino , Pâncreas/metabolismo , Pancreatite Necrosante Aguda/sangue , Pancreatite Necrosante Aguda/genética , Ratos , Ratos Sprague-Dawley , Transfecção
6.
Sci Rep ; 8(1): 3244, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29459735

RESUMO

Mechanisms underlying ectopic activity in the pulmonary vein (PV) which triggers paroxysmal atrial fibrillation are unknown. Although several studies have suggested that calcium signalling might be involved in these arrhythmias, little is known about calcium cycling in PV cardiomyocytes (CM). We found that individual PV CM showed a wide range of transverse tubular incidence and organization, going from their virtual absence, as described in atrial CM, to well transversally organised tubular systems, like in ventricular CM. These different types of CM were found in groups scattered throughout the tissue. The variability of the tubular system was associated with cell to cell heterogeneity of calcium channel (Cav1.2) localisation and, thereby, of Cav1.2-Ryanodine receptor coupling. This was responsible for multiple forms of PV CM calcium transient. Spontaneous calcium sparks and waves were not only more abundant in PV CM than in LA CM but also associated with a higher depolarising current. In conclusion, compared with either the atrium or the ventricle, PV myocardium presents marked structural and functional heterogeneity.


Assuntos
Sinalização do Cálcio , Átrios do Coração/citologia , Miócitos Cardíacos/fisiologia , Veias Pulmonares/citologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/análise , Miócitos Cardíacos/enzimologia , Veias Pulmonares/fisiologia , Ratos
7.
Life Sci ; 194: 139-149, 2018 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-29288765

RESUMO

Voltage-gated calcium channels (VGCCs) are the predominant source of calcium influx in the heart leading to calcium-induced calcium release and ultimately excitation-contraction coupling. In the heart, VGCCs are modulated by the ß-adrenergic signaling. Signaling through ß-adrenergic receptors (ßARs) and modulation of VGCCs by ß-adrenergic signaling in the heart are critical signaling and changes to these have been significantly implicated in heart failure. However, data related to calcium channel dysfunction in heart failure is divergent and contradictory ranging from reduced function to no change in the calcium current. Many recent studies have highlighted the importance of functional and spatial microdomains in the heart and that may be the key to answer several puzzling questions. In this review, we have briefly discussed the types of VGCCs found in heart tissues, their structure, and significance in the normal and pathological condition of the heart. More importantly, we have reviewed the modulation of VGCCs by ßARs in normal and pathological conditions incorporating functional and structural aspects. There are different types of ßARs, each having their own significance in the functioning of the heart. Finally, we emphasize the importance of location of proteins as it relates to their function and modulation by co-signaling molecules. Its implication on the studies of heart failure is speculated.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Insuficiência Cardíaca/patologia , Miocárdio/patologia , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/análise , Insuficiência Cardíaca/metabolismo , Humanos , Miocárdio/metabolismo , Receptores Adrenérgicos beta/análise
8.
Biochemistry ; 56(28): 3669-3681, 2017 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-28613835

RESUMO

The voltage-gated L-type Ca2+ channel CaV1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of CaV1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of CaV1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal CaV1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous CaV1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to CaV1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α11.2 subunit of CaV1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant CaV1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α11.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity.


Assuntos
Actinina/metabolismo , Canais de Cálcio Tipo L/metabolismo , Animais , Sítios de Ligação , Canais de Cálcio Tipo L/análise , Células HEK293 , Humanos , Ligação Proteica , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismo , Transporte Proteico , Coelhos
9.
Ann Thorac Surg ; 101(5): 1789-95, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26707005

RESUMO

BACKGROUND: Graft spasm remains challenging in coronary artery bypass grafting (CABG). Calcium antagonists are commonly used in patients with coronary artery disease. This study investigated the inhibitory effect of third-generation dihydropyridine calcium channel antagonist benidipine on the vasoconstriction induced by various vasoconstrictors in the human internal mammary artery (IMA). METHODS: Isolated human IMA rings (N = 65, taken from 37 patients undergoing CABG) were studied in a myograph in 2 ways: the relaxing effect of benidipine on vasoconstrictor-induced precontraction by KCl and U46619 and the depressing effect of benidipine at plasma concentrations on the contraction. Enzyme-linked immunosorbent assay (ELISA) was used to measure the change of the protein related to the L-type calcium channel. RESULTS: Benidipine caused more relaxation in KCl-contracted (86.7% ± 3.3%; n = 12) than in U46619-contracted (63.8% ± 5.3%; n = 8; p < 0.001) IMA rings. Pretreatment of IMA with plasma concentrations of benidipine (-6.92 log M) significantly depressed subsequent contraction by KCl (from 17.3 ± 2.7 mN to 7.4 ± 1.2 mN; n = 6; p < 0.05) but did not significantly affect the contraction caused by U46619. Benidipine also caused a decrease of caveolin (CaV)1.2 protein content (0.55 ± 0.02 versus 0.63 ± 0.02 mg/mL; p < 0.05). CONCLUSIONS: We conclude that in human IMA, the third-generation dihydropyridine calcium channel antagonist benidipine has a potent inhibitory effect on the vasoconstriction mediated by a variety of vasoconstrictors. Use of benidipine in patients undergoing CABG may provide vasorelaxant or antispastic effects in the grafts.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Artéria Torácica Interna/efeitos dos fármacos , Vasodilatadores/farmacologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/antagonistas & inibidores , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacologia , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Técnicas In Vitro , Miografia , Cloreto de Potássio/antagonistas & inibidores , Cloreto de Potássio/farmacologia , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia
10.
Circulation ; 132(25): 2372-84, 2015 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-26450916

RESUMO

BACKGROUND: Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs). METHODS AND RESULTS: Isolated rat and human AMs were characterized by scanning ion conductance, confocal, and electron microscopy. Half of AMs possessed T-tubules and structured topography, proportional to cell width. A bigger proportion of myocytes in the left atrium had organized T-tubules and topography than in the right atrium. Super-resolution scanning patch clamp showed that LTCCs distribute equally in T-tubules and crest areas of the sarcolemma, whereas, in ventricular myocytes, LTCCs primarily cluster in T-tubules. Rat, but not human, T-tubule LTCCs had open probability similar to crest LTCCs, but exhibited ≈ 40% greater current. Optical mapping of Ca(2+) transients revealed that rat AMs presented ≈ 3-fold as many spontaneous Ca(2+) release events as ventricular myocytes. Occurrence of crest LTCCs and spontaneous Ca(2+) transients were eliminated by either a caveolae-targeted LTCC antagonist or disrupting caveolae with methyl-ß-cyclodextrin, with an associated ≈ 30% whole-cell ICa,L reduction. Heart failure (16 weeks post-myocardial infarction) in rats resulted in a T-tubule degradation (by ≈ 40%) and significant elevation of spontaneous Ca(2+) release events. Although heart failure did not affect LTCC occurrence, it led to ≈ 25% decrease in T-tubule LTCC amplitude. CONCLUSIONS: We provide the first direct evidence for the existence of 2 distinct subpopulations of functional LTCCs in rat and human AMs, with their biophysical properties modulated in heart failure in a microdomain-specific manner.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Átrios do Coração , Microdomínios da Membrana/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Canais de Cálcio Tipo L/análise , Sinalização do Cálcio/fisiologia , Átrios do Coração/química , Humanos , Microdomínios da Membrana/química , Miócitos Cardíacos/química , Ratos , Especificidade da Espécie
11.
Med Oncol ; 32(9): 228, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26255063

RESUMO

Androgens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100 nM of 5α-dihydrotestosterone (DHT) for 24-72 h. The obtained results showed that 1 nM DHT up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Di-Hidrotestosterona/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/genética , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Di-Hidrotestosterona/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/análise , Peptídeos e Proteínas de Sinalização Intracelular/genética
12.
Methods Enzymol ; 556: 455-74, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25857795

RESUMO

Skeletal muscle excitation-contraction coupling is triggered by the concerted action of two enormous Ca(2+) channel complexes, the dihydropyridine receptor and the type 1 ryanodine receptor. Recent advances in our understanding of the structure of these large Ca(2+) channels have been driven by fluorescence resonance energy transfer (FRET)-based analysis. A methodological challenge in conducting these FRET measurements is the ability to site-specifically label these huge ion channels with donor and acceptor fluorophores capable of undergoing energy transfer. In this chapter, we detail specific protocols for tagging large membrane proteins with these fluorescent probes using three orthogonal labeling methods: fluorescent protein fusions, biarsenical reagents directed to engineered tetracysteine tags, and Cy3/5 nitrilotriacetic acid conjugates that bind to poly-histidine tags.


Assuntos
Canais de Cálcio Tipo L/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Músculo Esquelético/química , Canal de Liberação de Cálcio do Receptor de Rianodina/análise , Animais , Canais de Cálcio Tipo L/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Histidina/metabolismo , Humanos , Modelos Moleculares , Músculo Esquelético/metabolismo , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo
13.
Theriogenology ; 83(3): 421-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25459425

RESUMO

It is well known that insemination of cryopreserved semen always results in lower fertility when compared with fresh semen, but there is an increased interest and demand for frozen equine semen by the major breeder associations because of the utility arising from semen already "on hand" at breeding time. In this article, we report that equine sperm cells express L-type voltage-gated calcium channels; their localization is restricted to sperm neck and to the principal piece of the tail in both fresh and frozen-thawed spermatozoa. We also studied the causes of cryoinjury at the membrane level focusing on the function of L-type calcium channels. We report that in cryopreserved spermatozoa the mean basal value of [Ca(2+)]i is higher than that of spermatozoa from fresh semen (447.130 vs. 288.3 nM; P < 0.001) and L-type channels function differently in response to their agonist and antagonist in relation to semen condition (fresh or frozen-thawed). We found that on addition of agonist to the culture medium, the increase in intracellular calcium concentrations ([Ca(2+)]i) was greater in frozen semen than in fresh semen (Δ[Ca(2+)]i = 124.59 vs. 16.04 nM; P < 0.001), whereas after the addition of antagonist the decrease in [Ca(2+)]i was lower in frozen semen than in fresh semen (Δ[Ca(2+)]i = 32.5 vs. 82.5 nM; P < 0.001). In this article, we also discuss the impact of cryopreservation on sperm physiology.


Assuntos
Canais de Cálcio Tipo L/análise , Cálcio/metabolismo , Criopreservação/veterinária , Cavalos/metabolismo , Preservação do Sêmen/veterinária , Espermatozoides/metabolismo , Animais , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/metabolismo , Masculino , Preservação do Sêmen/efeitos adversos , Espermatozoides/fisiologia
14.
Cell Tissue Res ; 357(2): 463-76, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24996399

RESUMO

The use of specific activators and inhibitors that penetrate the central nervous system has suggested an essential functional role of L-type calcium channels (LTCC) in several important physiological processes of the brain, including the modulation of the mesoaccumbal dopamine signalling pathway, synaptic transmission of auditory stimuli and synaptic plasticity of neutral and aversive learning and memory processes. However, the lack of selectivity of available pharmacological agents towards the most prominent LTCC isoforms in the brain, namely Cav1.2 and Cav1.3, has hampered the elucidation of the precise contribution made by each specific channel isoform within these specific physiological processes. Modern genetic approaches, both in rodents and in human, have recently enhanced our understanding of the selective functional roles of Cav1.2 and Cav1.3 channels. In rodents, the characterisation of global and conditional isoform-specific knockouts suggests a contribution of Cav1.2 channels in spatial memory formation, whereas Cav1.3 channels seem to be involved in the consolidation of fear memories and in neurodegenerative mechanisms associated with the development of Parkinson's disease. With regard to the molecular mechanisms underlying drug addiction, Cav1.3 channels are necessary for the development and Cav1.2 channels for the expression of cocaine and amphetamine behavioural sensitisation. In humans, both the identification of naturally occurring LTCC variants ("channelopathies") and unbiased genome-wide association studies have linked LTCCs to working memory performance in healthy individuals and schizophrenic patients. Individually, CACNA1C polymorphisms and CACNA1D variants have been linked to a variety of psychiatric diseases and to congenital deafness, respectively. However, the contribution of individual LTCCs and their polymorphisms to human brain function and diseases remains unclear, necessitating the use of isoform-specific pharmacological agents.


Assuntos
Encéfalo/fisiologia , Encéfalo/fisiopatologia , Canais de Cálcio Tipo L/metabolismo , Animais , Encéfalo/metabolismo , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/genética , Cognição , Humanos , Memória , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Plasticidade Neuronal , Polimorfismo Genético , Esquizofrenia/genética , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia
15.
J Cell Biol ; 205(6): 829-46, 2014 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-24934157

RESUMO

Voltage-gated calcium channel (Cav) ß subunits are auxiliary subunits to Cavs. Recent reports show Cavß subunits may enter the nucleus and suggest a role in transcriptional regulation, but the physiological relevance of this localization remains unclear. We sought to define the nuclear function of Cavß in muscle progenitor cells (MPCs). We found that Cavß1a is expressed in proliferating MPCs, before expression of the calcium conducting subunit Cav1.1, and enters the nucleus. Loss of Cavß1a expression impaired MPC expansion in vitro and in vivo and caused widespread changes in global gene expression, including up-regulation of myogenin. Additionally, we found that Cavß1a localizes to the promoter region of a number of genes, preferentially at noncanonical (NC) E-box sites. Cavß1a binds to a region of the Myog promoter containing an NC E-box, suggesting a mechanism for inhibition of myogenin gene expression. This work indicates that Cavß1a acts as a Cav-independent regulator of gene expression in MPCs, and is required for their normal expansion during myogenic development.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mioblastos/metabolismo , Miogenina/metabolismo , Animais , Canais de Cálcio Tipo L/análise , Canais de Cálcio Tipo L/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Camundongos , Miogenina/genética
16.
Curr Protoc Neurosci ; Chapter 2: Unit 2.18, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23315944

RESUMO

At synapses in the central nervous system, precisely localized assemblies of presynaptic proteins, neurotransmitter-filled vesicles, and postsynaptic receptors are required to communicate messages between neurons. Our understanding of synaptic function has been significantly advanced using electrophysiological methods, but the dynamic spatial behavior and real-time organization of synapses remains poorly understood. In this unit, we describe a method for labeling individual presynaptic calcium channels with photostable quantum dots for single-particle tracking analysis. We have used this technique to examine the mobility of L-type calcium channels in the presynaptic membrane of rod and cone photoreceptors in the retina. These channels control release of glutamate-filled synaptic vesicles at the ribbon synapses in photoreceptor terminals. This technique offers the advantage of providing a real-time biophysical readout of ion channel mobility and can be manipulated by pharmacological or electrophysiological methods. For example, the combination of electrophysiological and single-particle tracking experiments has revealed that fusion of nearby vesicles influences calcium channel mobility and changes in channel mobility can influence release. These approaches can also be readily adapted to examine membrane proteins in other systems.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Rastreamento de Células/métodos , Proteínas do Olho/fisiologia , Microscopia de Fluorescência/métodos , Células Fotorreceptoras de Vertebrados/fisiologia , Pontos Quânticos , Transmissão Sináptica , Ambystoma , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Biotinilação , Canais de Cálcio/imunologia , Canais de Cálcio Tipo L/análise , Epitopos/imunologia , Proteínas do Olho/análise , Proteínas do Olho/imunologia , Ácido Glutâmico , Imuno-Histoquímica , Dados de Sequência Molecular , Células Fotorreceptoras de Vertebrados/química , Células Fotorreceptoras de Vertebrados/ultraestrutura , Terminações Pré-Sinápticas/fisiologia , Terminações Pré-Sinápticas/ultraestrutura , Estrutura Terciária de Proteína , Estreptavidina
17.
J Pharmacol Sci ; 115(3): 364-73, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21350314

RESUMO

In the disease states of urinary frequency and urgency, atropine-resistant contractions are known to be involved, in addition to contractions mediated by cholinergic nerves. This study was undertaken to investigate the mechanism underlying the development of atropine-resistant contractions using the representative antimuscarinic drugs solifenacin and tolterodine and also propiverine that has Ca(2+) channel-antagonizing activity in addition to antimuscarinic activity. Rat models of urinary frequency were established by intravesical infusion of acetylcholine (ACh) (cholinergic nerve-mediated urinary frequency model), acetic acid (AcOH) [non-adrenergic non-cholinergic nerve (NANC)-mediated urinary frequency model], or CaCl(2) (atropine-resistant contractions-mediated urinary frequency model). Cystometrograms were obtained to measure the micturition parameters following oral administration of the aforementioned drugs. Propiverine increased the micturition weight in all the urinary frequency models. Solifenacin and tolterodine increased the micturition weight in the ACh-induced urinary frequency model but neither had any effect in the AcOH- or CaCl(2)-induced urinary frequency models. While antimuscarinic drugs are, in general, effective for the control of urinary frequency and incontinence, use of drugs possessing inhibitory effects on contractions mediated by cholinergic as well as NANC nerve transmission or Ca(2+) influx into smooth muscles is recommended for management of the symptoms in disease states in which atropine-resistant contractions, such as Ca(2+)- and capsaicin-sensitive sensory nerves, are involved.


Assuntos
Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Bexiga Urinária Hiperativa/tratamento farmacológico , Bexiga Urinária/efeitos dos fármacos , Micção/efeitos dos fármacos , Animais , Atropina/metabolismo , Atropina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/análise , Modelos Animais de Doenças , Feminino , Músculo Liso/fisiologia , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/fisiologia , Bexiga Urinária/fisiologia , Incontinência Urinária/tratamento farmacológico
18.
J Matern Fetal Neonatal Med ; 24(3): 437-43, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20873977

RESUMO

OBJECTIVE: To elucidate the underlying mechanisms responsible for ß-cell mass and function in response to intrauterine growth restriction (IUGR). METHODS: Offspring from undernourished mother rats with birth weight < -2 SD (IUGR group) and from standard-nourished rats with birth weight between mean +/- 1SD normal birth weight (NBW group) were examined. Levels of fasting glucose, serum insulin, and insulin-like growth factor-1 (IGF-1) were analyzed. Entire pancreas or islet-like cell clusters (ICCs) were collected to evaluate relative ß-cell mass and determine the genetic and protein profiles of pancreatic and duodenal homeobox 1 (Pdx1), Cacna1c and Cacna1d using real-time PCR, immunohistochemical staining, and western blotting at day (d) of birth and at d21 of age. RESULTS: Fasting serum insulin and IGF-1 concentrations were significantly lower in the IUGR group than in the NBW group at d0 and d21. The levels of Pdx1 and insulin mRNAs in IUGR pancreas were also decreased. At birth, the ratios of ß-cell mass to body weight were not significantly different between the two groups. However, by d21 the relative ß-cell mass in IUGR had not grown to compensate for the increase in body weight, as compared to the NBW group (p < 0.05). The Cacna1c and Cacna1d proteins were significantly higher in the NBW group than that in the IUGR group at birth, but there were no statistical differences at d21. CONCLUSION: Decreased Pdx1 levels and IGF-1 concentration restrain ß-cell mass and insulin expression in rat offspring from undernourished mothers. However, Cacna1c and Cacna1d expression were able to reach normal levels as the IUGR rats were aged, indicating that these factors were not responsible for the IUGR rat phenotype of insulin resistance and ß-cell dysfunction in later life.


Assuntos
Canais de Cálcio Tipo L/sangue , Canais de Cálcio/sangue , Retardo do Crescimento Fetal/patologia , Proteínas de Homeodomínio/sangue , Células Secretoras de Insulina/patologia , Insulina/sangue , Desnutrição/patologia , Transativadores/sangue , Animais , Animais Recém-Nascidos , Peso ao Nascer/fisiologia , Canais de Cálcio/análise , Canais de Cálcio Tipo L/análise , Contagem de Células , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/etiologia , Proteínas de Homeodomínio/análise , Insulina/análise , Células Secretoras de Insulina/metabolismo , Desnutrição/sangue , Desnutrição/complicações , Fenômenos Fisiológicos da Nutrição Materna , Tamanho do Órgão , Gravidez , Ratos , Ratos Sprague-Dawley , Transativadores/análise
19.
Pancreas ; 39(6): 847-55, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20697208

RESUMO

OBJECTIVES: We investigated the influence of leucine supplementation on insulin secretion and on some proteins related to insulin secretion in malnourished mice. METHODS: Swiss mice (aged 21 days) received isocaloric normo-17% (NP) or 6% low-protein (LP) diet for 120 days. Half of the NP and LP mice received 1.5% leucine in the drinking water during the last 30 days (NPL and LPL, respectively). RESULTS: The LP mice were hypoinsulinemic compared with the NP group, whereas LPL mice exhibited increased insulinemia in the fed state versus LP mice. The LP mouse islets were less responsive to 22.2 mM glucose, 100 microM carbachol (Cch), and 10 mM leucine than the NP group. However, LPL islets were more responsive to all these conditions compared with the LP group. The muscarinic type 3 receptor, (M3R) Cabeta2, and PKC-alpha protein contents were reduced in LP compared with NP islets but significantly higher in LPL than LP islets. The p-AKT/AKT ratio was higher in LPL compared with LP islets. CONCLUSIONS: Leucine supplementation increases insulin secretion in response to glucose and leucine and to agents that potentiate secretion, such as Cch, in malnourished mice. The enhanced levels of M3R, Cabeta2, and PKC-alpha proteins, as well as of the p-AKT/AKT ratio, may play a role in this process.


Assuntos
Suplementos Nutricionais , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Leucina/administração & dosagem , Desnutrição/tratamento farmacológico , Albuminas/análise , Animais , Glicemia/efeitos dos fármacos , Peso Corporal , Canais de Cálcio Tipo L/análise , Carbacol/farmacologia , Colesterol/sangue , Dieta com Restrição de Proteínas , Ácidos Graxos não Esterificados/sangue , Glucose/metabolismo , Glucose/fisiologia , Insulina/sangue , Secreção de Insulina , Desnutrição/metabolismo , Camundongos , Proteína Quinase C/análise , Proteínas Serina-Treonina Quinases/análise , Receptor Muscarínico M3/análise , Triglicerídeos/sangue
20.
PLoS One ; 5(4): e10140, 2010 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-20405051

RESUMO

Clinical trials and epidemiological studies have suggested that dietary fish oil (FO) supplementation can provide an anti-arrhythmic benefit in some patient populations. The underlying mechanisms are not entirely clear. We wanted to understand how FO supplementation (for 4 weeks) affected the action potential configuration/duration of ventricular myocytes, and the ionic mechanism(s)/molecular basis for these effects. The experiments were conducted on adult rabbits, a widely used animal model for cardiac electrophysiology and pathophysiology. We used gas chromatography-mass spectroscopy to confirm that FO feeding produced a marked increase in the content of n-3 polyunsaturated fatty acids in the phospholipids of rabbit hearts. Left ventricular myocytes were used in current and voltage clamp experiments to monitor action potentials and ionic currents, respectively. Action potentials of myocytes from FO-fed rabbits exhibited much more positive plateau voltages and prolonged durations. These changes could be explained by an increase in the L-type Ca current (I(CaL)) and a decrease in the transient outward current (I(to)) in these myocytes. FO feeding did not change the delayed rectifier or inward rectifier current. Immunoblot experiments showed that the FO-feeding induced changes in I(CaL) and I(to) were associated with corresponding changes in the protein levels of major pore-forming subunits of these channels: increase in Cav1.2 and decrease in Kv4.2 and Kv1.4. There was no change in other channel subunits (Cav1.1, Kv4.3, KChIP2, and ERG1). We conclude that long-term fish oil supplementation can impact on cardiac electrical activity at least partially by changing channel subunit expression in cardiac myocytes.


Assuntos
Canais de Cálcio Tipo L/efeitos dos fármacos , Óleos de Peixe/farmacologia , Canais de Potássio/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio Tipo L/análise , Eletrofisiologia Cardíaca , Suplementos Nutricionais , Óleos de Peixe/administração & dosagem , Ventrículos do Coração , Modelos Animais , Células Musculares/química , Células Musculares/fisiologia , Fosfolipídeos/análise , Canais de Potássio/análise , Coelhos , Fatores de Tempo
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