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1.
Adv Exp Med Biol ; 1131: 183-213, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646511

RESUMO

Ca2+ binding proteins (CBP) are of key importance for calcium to play its role as a pivotal second messenger. CBP bind Ca2+ in specific domains, contributing to the regulation of its concentration at the cytosol and intracellular stores. They also participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e. decoding Ca2+ signals. Since CBP are integral to normal physiological processes, possible roles for them in a variety of diseases has attracted growing interest in recent years. In addition, research on CBP has been reinforced with advances in the structural characterization of new CBP family members. In this chapter we have updated a previous review on CBP, covering in more depth potential participation in physiopathological processes and candidacy for pharmacological targets in many diseases. We review intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins, calcineurin and neuronal Ca2+ sensor proteins (NCS). We also address intracellular CBP lacking the EF-hand domain: annexins, CBP within intracellular Ca2+ stores (paying special attention to calreticulin and calsequestrin), proteins that contain a C2 domain (such as protein kinase C (PKC) or synaptotagmin) and other proteins of interest, such as regucalcin or proprotein convertase subtisilin kexins (PCSK). Finally, we summarise the latest findings on extracellular CBP, classified according to their Ca2+ binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) ɣ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca2+-dependent (C)-type lectin-like domains; (vi) Ca2+-binding pockets of family C G-protein-coupled receptors.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo
2.
Ann Rheum Dis ; 77(3): 431-440, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29259049

RESUMO

OBJECTIVES: Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-ß) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-ß and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. METHODS: PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast-endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. RESULTS: PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-ß in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-ß receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. CONCLUSIONS: Our data reveal a new pathway by which TGF-ß suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

3.
Cell Death Dis ; 8(11): e3163, 2017 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-29095440

RESUMO

The voltage-gated K+ channel has key roles in the vasculature and in atrial excitability and contributes to apoptosis in various tissues. In this study, we have explored its regulation by carbon monoxide (CO), a product of the cytoprotective heme oxygenase enzymes, and a recognized toxin. CO inhibited recombinant Kv1.5 expressed in HEK293 cells in a concentration-dependent manner that involved multiple signalling pathways. CO inhibition was partially reversed by superoxide dismutase mimetics and by suppression of mitochondrial reactive oxygen species. CO also elevated intracellular nitric oxide (NO) levels. Prevention of NO formation also partially reversed CO inhibition of Kv1.5, as did inhibition of soluble guanylyl cyclase. CO also elevated intracellular peroxynitrite levels, and a peroxynitrite scavenger markedly attenuated the ability of CO to inhibit Kv1.5. CO caused nitrosylation of Kv1.5, an effect that was also observed in C331A and C346A mutant forms of the channel, which had previously been suggested as nitrosylation sites within Kv1.5. Augmentation of Kv1.5 via exposure to hydrogen peroxide was fully reversed by CO. Native Kv1.5 recorded in HL-1 murine atrial cells was also inhibited by CO. Action potentials recorded in HL-1 cells were increased in amplitude and duration by CO, an effect mimicked and occluded by pharmacological inhibition of Kv1.5. Our data indicate that Kv1.5 is a target for modulation by CO via multiple mechanisms. This regulation has important implications for diverse cellular functions, including excitability, contractility and apoptosis.


Assuntos
Monóxido de Carbono/farmacologia , Canal de Potássio Kv1.5/metabolismo , Animais , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Peróxido de Hidrogênio/toxicidade , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Metaloporfirinas/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutagênese Sítio-Dirigida , Óxido Nítrico/metabolismo , Ácido Peroxinitroso/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação
4.
J Physiol ; 594(15): 4119-29, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-26804000

RESUMO

Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H2 S is a modulator of low voltage-activated T-type Ca(2+) channels, and discriminates between the different subtypes of T-type Ca(2+) channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2 S augments Cav3.2 currents, an observation which has led to the suggestion that H2 S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2 S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox-sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H2 S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H2 S modulation of T-type Ca(2+) channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T-type Ca(2+) channel modulation by H2 S might be exploited as a novel approach to pain management.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Sulfeto de Hidrogênio/metabolismo , Nociceptividade/fisiologia , Animais , Humanos
5.
Adv Exp Med Biol ; 860: 291-300, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26303493

RESUMO

T-type Ca(2+) channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe(2+) and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca(2+) channels.Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca(2+) channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC(50) values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca(2+) ](i) in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015).Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca(2+) channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca(2+) channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca(2+) channels.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Monóxido de Carbono/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo T/análise , Proliferação de Células , Células HEK293 , Heme Oxigenase-1/fisiologia , Humanos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia
6.
Adv Exp Med Biol ; 860: 353-60, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26303500

RESUMO

T-type Ca(2+) channels are a distinct family of low voltage-activated Ca(2+) channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H(2)S) was more recently discovered as an important signalling molecule involved in many functions, including O(2) sensing. Since ion channels are emerging as an important family of target proteins for modulation by H(2)S, and both T-type Ca(2+) channels and H(2)S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca(2+) channels are a target for modulation by H(2)S. Using patch-clamp electrophysiology, we demonstrate that the H(2)S donor, NaHS, selectively inhibits Cav3.2 T-type Ca(2+) channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. Sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) using TPEN prevented channel inhibition by H(2)S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H(2)S regulation, the T-type Ca(2+) channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Etilenodiaminas/farmacologia , Células HEK293 , Humanos
7.
Biochem Biophys Res Commun ; 461(4): 659-64, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-25918023

RESUMO

T-type Ca(2+) channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca(2+) channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca(2+) channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca(2+) channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca(2+) channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca(2+) channel-mediated proliferation by H2S is independent of the channels' sensitivity to H2S.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Cálcio/metabolismo , Proliferação de Células/fisiologia , Regulação da Expressão Gênica/fisiologia , Sulfeto de Hidrogênio/administração & dosagem , Ativação do Canal Iônico/fisiologia , Miócitos de Músculo Liso/fisiologia , Animais , Canais de Cálcio Tipo T/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos
9.
Pflugers Arch ; 467(2): 415-27, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24744106

RESUMO

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca(2+) channels in HEK293 cells raised basal [Ca(2+)]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca(2+)]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca(2+) currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca(2+) channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Monóxido de Carbono/farmacologia , Proliferação de Células , Heme Oxigenase-1/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células HEK293 , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/fisiologia , Ratos
10.
FASEB J ; 28(12): 5376-87, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25183670

RESUMO

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T-type Ca(2+) channels. Using patch-clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 µM-1 mM) selectively inhibits Cav3.2 T-type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox-sensitive extracellular residue H191, which is also required for tonic binding of Zn(2+) to this channel. Chelation of Zn(2+) with N,N,N',N'-tetra-2-picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn(2+) binding. Inhibition of native T-type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T-type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T-type Ca(2+) channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Animais , Western Blotting , Linhagem Celular , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
J Biol Chem ; 289(23): 16421-9, 2014 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-24719320

RESUMO

Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na(+) channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na(+) current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor N(ω)-nitro-L-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na(+) current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.


Assuntos
Monóxido de Carbono/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Células HEK293 , Humanos , Oxirredução
12.
Vascul Pharmacol ; 60(3): 95-101, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24469067

RESUMO

Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Vasodilatação/fisiologia , Animais , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos WKY , Vasodilatação/efeitos dos fármacos
13.
FASEB J ; 27(8): 3395-407, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23671274

RESUMO

T-type Ca(2+) channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1) is often found. Here, we report regulation of T-type Ca(2+) channels by carbon monoxide (CO) a HO-1 by-product. CO (applied as CORM-2) caused a concentration-dependent, poorly reversible inhibition of all T-type channel isoforms (Cav3.1-3.3, IC50 ∼3 µM) expressed in HEK293 cells, and native T-type channels in NG108-15 cells and primary rat sensory neurons. No recognized CO-sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox-sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2-5 µM) reduced Cav3.2 currents and their CO sensitivity by >50% but increased sensitivity to dithiothreitol ∼3-fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO-1-derived CO disrupts this regulation, causing channel inhibition. CO modulation of T-type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation.


Assuntos
Canais de Cálcio Tipo T/fisiologia , Dióxido de Carbono/metabolismo , Ativação do Canal Iônico/fisiologia , Tiorredoxinas/metabolismo , Animais , Auranofina/farmacologia , Western Blotting , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Ditiotreitol/farmacologia , Células HEK293 , Heme Oxigenase-1/metabolismo , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/genética , Potenciais da Membrana/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Neurônios/fisiologia , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacologia , Oxirredução/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Tiorredoxina Dissulfeto Redutase/antagonistas & inibidores , Tiorredoxina Dissulfeto Redutase/metabolismo
14.
Vascul Pharmacol ; 58(1-2): 98-104, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22960580

RESUMO

Despite a large number of studies, the mechanism by which 3',5'-cyclic monophosphate (cAMP) induces vasorelaxation is not fully understood. The comparison between results obtained in different vessels or species has often been the source of conflicting reports. In order to shed more light onto this mechanism, we studied the effects of forskolin in phenylephrine-pre-contracted endothelium-denuded rat aorta and measured cAMP levels in rat aortic myocytes by enzyme-immunoassay. Nanomolar forskolin relaxed phenylephrine-induced contractions. This effect was mimicked by dibutyryl-cAMP and was potentiated by rolipram or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB-203580). Nifedipine and verapamil partially relaxed phenylephrine-induced contractions, while further application of cAMP-elevating agents fully relaxed these contractions. In Ca(2+)-free extracellular solution, forskolin reduced phenylephrine-induced transient contractions and reduced the Ca(2+)-induced contraction after depletion of intracellular stores. Nanomolar concentrations of forskolin increased basal cAMP levels only in the presence of rolipram or phenylephrine, which did not modify intracellular levels of cAMP by themselves. In conclusion, relaxation by cAMP is mediated in part by decrease of depletion of intracellular Ca(2+) stores and inhibition of capacitative calcium entry. This study provides the first evidence that inhibition of PDE4 or p38-MAPK potentiates the vasodilator effect of cAMP-elevating agents in rat aortic myocytes.


Assuntos
Aorta Torácica/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Colforsina/farmacologia , Imidazóis/farmacologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nifedipino/farmacologia , Fenilefrina/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Piridinas/farmacologia , Ratos , Ratos Endogâmicos WKY , Rolipram/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Verapamil/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
15.
Am J Respir Crit Care Med ; 186(7): 648-56, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22822026

RESUMO

RATIONALE: Clinical reports describe life-threatening cardiac arrhythmias after environmental exposure to carbon monoxide (CO) or accidental CO poisoning. Numerous case studies describe disruption of repolarization and prolongation of the QT interval, yet the mechanisms underlying CO-induced arrhythmias are unknown. OBJECTIVES: To understand the cellular basis of CO-induced arrhythmias and to identify an effective therapeutic approach. METHODS: Patch-clamp electrophysiology and confocal Ca(2+) and nitric oxide (NO) imaging in isolated ventricular myocytes was performed together with protein S-nitrosylation to investigate the effects of CO at the cellular and molecular levels, whereas telemetry was used to investigate effects of CO on electrocardiogram recordings in vivo. MEASUREMENTS AND MAIN RESULTS: CO increased the sustained (late) component of the inward Na(+) current, resulting in prolongation of the action potential and the associated intracellular Ca(2+) transient. In more than 50% of myocytes these changes progressed to early after-depolarization-like arrhythmias. CO elevated NO levels in myocytes and caused S-nitrosylation of the Na(+) channel, Na(v)1.5. All proarrhythmic effects of CO were abolished by the NO synthase inhibitor l-NAME, and reversed by ranolazine, an inhibitor of the late Na(+) current. Ranolazine also corrected QT variability and arrhythmias induced by CO in vivo, as monitored by telemetry. CONCLUSIONS: Our data indicate that the proarrhythmic effects of CO arise from activation of NO synthase, leading to NO-mediated nitrosylation of Na(V)1.5 and to induction of the late Na(+) current. We also show that the antianginal drug ranolazine can abolish CO-induced early after-depolarizations, highlighting a novel approach to the treatment of CO-induced arrhythmias.


Assuntos
Arritmias Cardíacas/etiologia , Intoxicação por Monóxido de Carbono/complicações , Monóxido de Carbono/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Canais de Sódio Disparados por Voltagem/efeitos dos fármacos , Acetanilidas/uso terapêutico , Potenciais de Ação/efeitos dos fármacos , Animais , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/fisiopatologia , Sinalização do Cálcio/efeitos dos fármacos , Monóxido de Carbono/efeitos adversos , Intoxicação por Monóxido de Carbono/fisiopatologia , Técnicas de Cultura de Células , Modelos Animais de Doenças , Exposição Ambiental/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Masculino , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Piperazinas/uso terapêutico , Ranolazina , Ratos , Ratos Wistar , Canais de Sódio Disparados por Voltagem/fisiologia
16.
Mol Nutr Food Res ; 55(8): 1237-48, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21710562

RESUMO

SCOPE: The aim of this study was to investigate whether the dietary polyphenol trans-resveratrol (t-Resv) increases [Ca(2+)](c) in endothelial cells, leading to a simultaneous augmentation of nitric oxide (NO) biosynthesis. METHODS AND RESULTS: We have separately and simultaneously measured [Ca(2+)](c) and NO in human endothelial cells using the Ca(2+) indicator fura-2 and the NO-sensitive fluorescent probe 4,5-diaminofluorescein. In ∼30% of cells, t-Resv (30 µM) induced an increase in [Ca(2+)](c) with a transient as well as sustained component and a simultaneous increase in NO biosynthesis. This effect was reduced by non-selective Ca(2+) channel blockers, inhibition of intracellular Ca(2+) release, inhibition of endothelial nitric oxide synthase (eNOS) and, to a lesser extent, inhibition of extracellular signal-regulated kinase 1/2 (ERK 1/2) or 5' adenosine monophosphate-activated protein kinase (AMPK). t-Resv did not modify in vitro eNOS activity, suggesting that the observed stimulation of NO generation proceeds via mobilisation of Ca(2+) and not through direct effects on eNOS. CONCLUSION: We therefore show, for the first time, that t-Resv induces a concentration-dependent, simultaneous increase in [Ca(2+)](c) and NO biosynthesis that could be linked to its endothelium-dependent vasorelaxant effect. Under the assumption that t-Resv exhibits similar behaviour in human blood vessels in vivo, the pharmacological properties described here may contribute to the beneficial cardiovascular effects of this polyphenol by improving endothelial function.


Assuntos
Cálcio/metabolismo , Citoplasma/metabolismo , Células Endoteliais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estilbenos/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Resveratrol
17.
Eur J Pharmacol ; 577(1-3): 91-9, 2007 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-17822692

RESUMO

Although the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cálcio/metabolismo , Miócitos de Músculo Liso/metabolismo , Estilbenos/farmacologia , Angiotensina II/farmacologia , Animais , Arginina Vasopressina/farmacologia , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Corantes Fluorescentes , Fura-2 , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Resveratrol , Rianodina/farmacologia , Estereoisomerismo
18.
Mol Nutr Food Res ; 49(5): 396-404, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15830338

RESUMO

The effects of trans- and cis-resveratrol on cytosolic Ca2+ concentration ([Ca2+]i) were studied using fura-2 in vascular smooth muscle cells (A7r5). Both isomers of resveratrol caused a sustained elevation in [Ca2+]i, cis-resveratrol being significantly more effective than the trans-isomer. The resveratrol-induced increase in [Ca2+]i was significantly potentiated by the previous application of low concentrations of thapsigargin, partially inhibited by nifedipine or Ni2+, and not affected by SKF 96365. In the absence of extracellular Ca2+, both isomers of resveratrol induced a transient, slow increase in [Ca2+]i, which was inhibited by the previous depletion of intracellular stores with thapsigargin and completely blocked by preincubation with TMB-8, an inhibitor of intracellular calcium release. Reintroduction of Ca2+ in the external solution after the resveratrol-induced release of Ca2+ activated the Ca2+ influx through store-operated calcium channels. The resveratrol-induced increase in [Ca2+]i in the absence of extracelullar Ca2+ partially reduced the increase in [Ca2+]i evoked by the subsequent application of thapsigargin. Our results suggest that trans- and cis-resveratrol induce a depletion of Ca2+ from the same intracellular stores released by thapsigargin and subsequent capacitative influx of Ca2+. Additionally, a direct activation of transmembrane Ca2+ influx through another type of channel may be also implicated.


Assuntos
Cálcio/análise , Citoplasma/química , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/ultraestrutura , Estilbenos/farmacologia , Animais , Aorta , Linhagem Celular , Sinergismo Farmacológico , Embrião de Mamíferos , Imidazóis/farmacologia , Níquel/farmacologia , Nifedipino/farmacologia , Ratos , Resveratrol , Estereoisomerismo , Tapsigargina/farmacologia
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