RESUMEN
Aneurismal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by the rupture of the cerebral arteries and the extravasation of blood to the subarachnoid space and is accompanied by severe comorbidities. Secondarily associated vasospasm is one of the main side effects after hydrocephalus and possible rebleeding. Here, we analyze the alterations in function in the arteries of a rat model of SAH. For this, autologous blood was injected into the cisterna magna. We performed electrophysiological, microfluorimetric, and molecular biology experiments at different times after SAH to determine the functional and molecular changes induced by the hemorrhage. Our results confirmed that in SAH animals, arterial myocytes were depolarized on days 5 and 7, had higher [Ca2+]i on baseline, peaks and plateaus, and were more excitable at low levels of depolarization on day 7, than in the control and sham animals. Microarray analysis showed that, on day 7, the sets of genes related to voltage-dependent Ca2+ channels and K+ dynamics in SAH animals decreased, while the voltage-independent Ca2+ dynamics genes were over-represented. In conclusion, after SAH, several mechanisms involved in arterial reactivity were altered in our animal model, suggesting that there is no unique cause of vasospasm and alterations in several signaling pathways are involved in its development.
Asunto(s)
Modelos Animales de Enfermedad , Hemorragia Subaracnoidea , Vasoespasmo Intracraneal , Animales , Hemorragia Subaracnoidea/fisiopatología , Hemorragia Subaracnoidea/metabolismo , Hemorragia Subaracnoidea/patología , Masculino , Vasoespasmo Intracraneal/fisiopatología , Vasoespasmo Intracraneal/metabolismo , Vasoespasmo Intracraneal/etiología , Vasoespasmo Intracraneal/patología , Señalización del Calcio , Factores de Tiempo , Arterias Cerebrales/metabolismo , Arterias Cerebrales/fisiopatología , Arterias Cerebrales/patología , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Músculo Liso Vascular/patología , Ratas Sprague-Dawley , Regulación de la Expresión Génica , Canales de Calcio/metabolismo , Canales de Calcio/genética , RatasRESUMEN
Aneurysmal subarachnoid hemorrhage (aSAH) is a neurovascular disease produced by extravasation of blood to the subarachnoid space after rupture of the cerebral vessels. After bleeding, the immune response is activated. The role of peripheral blood mononuclear cells (PBMCs) in this response is a current subject of research. We have analysed the changes in PBMCs of patients with aSAH and their interaction with the endothelium, focusing on their adhesion and the expression of adhesion molecules. Using an in vitro adhesion assay, we observed that the adhesion of PBMCs of patients with aSAH is increased. Flow cytometry analysis shows that monocytes increased significantly in patients, especially in those who developed vasospasm (VSP). In aSAH patients, the expression of CD162, CD49d, CD62L and CD11a in T lymphocytes and of CD62L in monocytes increased. However, the expression of CD162, CD43, and CD11a decreased in monocytes. Furthermore, monocytes from patients who developed arteriographic VSP had lower expression of CD62L. In conclusion, our results confirm that after aSAH, monocyte count and adhesion of PBMCs increase, especially in patients with VSP, and that the expression of several adhesion molecules is altered. These observations can help predict VSP and to improve the treatment of this pathology.
Asunto(s)
Hemorragia Subaracnoidea , Vasoespasmo Intracraneal , Humanos , Leucocitos Mononucleares , Hemorragia Subaracnoidea/complicaciones , Vasoespasmo Intracraneal/etiología , Monocitos , AngiografíaRESUMEN
Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating disease with high morbidity and mortality rates. Within 24 hours after aSAH, monocytes are recruited and enter the subarachnoid space, where they mature into macrophages, increasing the inflammatory response and contributing, along with other factors, to delayed neurological dysfunction and poor outcomes. High-density lipoproteins (HDL) are lipid-protein complexes that exert anti-inflammatory effects but under pathological conditions undergo structural alterations that have been associated with loss of functionality. Plasma HDL were isolated from patients with aSAH and analyzed for their anti-inflammatory activity and protein composition. HDL isolated from patients lost the ability to prevent VCAM-1 expression in endothelial cells (HUVEC) and subsequent adhesion of THP-1 monocytes to the endothelium. Proteomic analysis showed that HDL particles from patients had an altered composition compared to those of healthy subjects. We confirmed by western blot that low levels of apolipoprotein A4 (APOA4) and high of serum amyloid A1 (SAA1) in HDL were associated with the lack of anti-inflammatory function observed in aSAH. Our results indicate that the study of HDL in the pathophysiology of aSAH is needed, and functional HDL supplementation could be considered a novel therapeutic approach to the treatment of the inflammatory response after aSAH.
Asunto(s)
Hemorragia Subaracnoidea , Humanos , Lipoproteínas HDL , Células Endoteliales/patología , Proteómica , Antiinflamatorios , Proteína Amiloide A SéricaRESUMEN
SCN5A gene variants are associated with both Brugada syndrome and conduction disturbances, sometimes expressing an overlapping phenotype. Functional consequences of SCN5A variants assessed by patch-clamp electrophysiology are particularly beneficial for correct pathogenic classification and are related to disease penetrance and severity. Here, we identify a novel SCN5A loss of function variant, p.1449Y>H, which presented with high penetrance and complete left bundle branch block, totally masking the typical findings on the electrocardiogram. We highlight the possibility of this overlap combination that makes impossible an electrocardiographic diagnosis and, through a functional analysis, associate the p.1449Y>H variant to SCN5A pathogenicity.
Asunto(s)
Síndrome de Brugada , Síndrome de Brugada/diagnóstico , Síndrome de Brugada/genética , Bloqueo de Rama/diagnóstico , Bloqueo de Rama/genética , Electrocardiografía , Humanos , Mutación , Canal de Sodio Activado por Voltaje NAV1.5/genéticaRESUMEN
BACKGROUND: Since 1994, over 50 families affected by the episodic ataxia type 1 disease spectrum have been described with mutations in KCNA1, encoding the voltage-gated K+ channel subunit Kv1.1. All of these mutations are either transmitted in an autosomal-dominant mode or found as de novo events. METHODS: A patient presenting with a severe combination of dyskinesia and neonatal epileptic encephalopathy was sequenced by whole-exome sequencing (WES). A candidate variant was tested using cellular assays and patch-clamp recordings. RESULTS: WES revealed a homozygous variant (p.Val368Leu) in KCNA1, involving a conserved residue in the pore domain, close to the selectivity signature sequence for K+ ions (TVGYG). Functional analysis showed that mutant protein alone failed to produce functional channels in homozygous state, while coexpression with wild-type produced no effects on K+ currents, similar to wild-type protein alone. Treatment with oxcarbazepine, a sodium channel blocker, proved effective in controlling seizures. CONCLUSION: This newly identified variant is the first to be reported to act in a recessive mode of inheritance in KCNA1. These findings serve as a cautionary tale for the diagnosis of channelopathies, in which an unreported phenotypic presentation or mode of inheritance for the variant of interest can hinder the identification of causative variants and adequate treatment choice.
Asunto(s)
Ataxia/genética , Discinesias/genética , Epilepsia/genética , Canal de Potasio Kv.1.1/genética , Miocimia/genética , Ataxia/diagnóstico , Ataxia/tratamiento farmacológico , Ataxia/patología , Canalopatías/diagnóstico , Canalopatías/tratamiento farmacológico , Canalopatías/genética , Canalopatías/patología , Niño , Preescolar , Discinesias/diagnóstico , Discinesias/tratamiento farmacológico , Discinesias/patología , Epilepsia/diagnóstico , Epilepsia/tratamiento farmacológico , Epilepsia/patología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Homocigoto , Humanos , Lactante , Recién Nacido , Canal de Potasio Kv.1.1/ultraestructura , Masculino , Mutación/genética , Miocimia/diagnóstico , Miocimia/tratamiento farmacológico , Miocimia/patología , Oxcarbazepina/administración & dosificación , Oxcarbazepina/efectos adversos , Linaje , Secuenciación del ExomaRESUMEN
BACKGROUND/AIMS: Protein kinase C (PKC)- and RhoA/Rho-associated kinase (ROCK) play important roles in arterial sustained contraction. Although depolarization-elicited RhoA/ROCK activation is accepted, the role of PKC in depolarized vascular smooth muscle cells (VSMCs) is a subject of controversy. Our aim was to study the role of PKC in arterial contraction and its interaction with RhoA/ROCK. METHODS: Mass spectrometry was used to identify the PKC isoenzymes. PKCα levels and RhoA activity were analyzed by western blot and G-LISA, respectively, and isometric force was measured in arterial rings. RESULTS: In depolarized VSMCs RhoA and PKCα were translocated to the plasma membrane, where they colocalize and coimmunoprecipitate. Interestingly, depolarization-induced RhoA activation was downregulated by PKCα, effect reverted by PKCα inhibition. Phorbol 12,13-dibutyrate (PDBu) induced the translocation of PKCα to the plasma membrane, increased the level of RhoA in the cytosol and reduced RhoA/ROCK activity. These effects were reverted when PKC was inhibited. Pharmacological or siRNA inhibition of PKCα synergistically potentiated the vasorelaxant effect of RhoA/ROCK inhibition. CONCLUSION: The present study provides the first evidence that RhoA activity is downregulated by PKCα in depolarized and PDBu treated freshly isolated VSMCs and arteries, with an important physiological role on arterial contractility.
Asunto(s)
Membrana Celular/enzimología , Músculo Liso Vascular/enzimología , Miocitos del Músculo Liso/enzimología , Proteína Quinasa C-alfa/metabolismo , Vasodilatación , Proteínas de Unión al GTP rho/metabolismo , Animales , Masculino , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Forbol 12,13-Dibutirato/farmacología , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Quinasas Asociadas a rho/metabolismoRESUMEN
Aims: Urocortin-2 (Ucn-2) is a potent cardioprotector against Ischemia and Reperfusion (I/R) injuries. However, little is known about its role in the regulation of intracellular Ca2+ concentration ([Ca2+]i) under I/R. Here, we examined whether the addition of Ucn-2 in reperfusion promotes cardioprotection focusing on ([Ca2+]i handling. Methods and Results: Cardiac Wistar rat model of I/R was induced by transient ligation of the left coronary artery and experiments were conducted 1 week after surgery in tissue and adult cardiomyocytes isolated from risk and remote zones. We observed that I/R promoted significant alteration in cardiac contractility as well as an increase in hypertrophy and fibrosis in both zones. The study of confocal [Ca2+]i imaging in adult cardiomyocytes revealed that I/R decreased the amplitude of [Ca2+]i transient and cardiomyocytes contraction in risk and remote zones. Interestingly, intravenous infusion of Ucn-2 before heart's reperfusion recovered significantly cardiac contractility and prevented fibrosis, but it didn't affect cardiac hypertrophy. Moreover, Ucn-2 recovered the amplitude of [Ca2+]i transient and modulated the expression of several proteins related to [Ca2+]i homeostasis, such as TRPC5 and Orai1 channels. Using Neonatal Rat Ventricular Myocytes (NRVM) we demonstrated that Ucn-2 blunted I/R-induced Store Operated Ca2+ Entry (SOCE), decreased the expression of TRPC5 and Orai1 as well as their interaction in reperfusion. Conclusion: Our study provides the first evidences demonstrating that Ucn-2 addition at the onset of reperfusion attenuates I/R-induced adverse cardiac remodeling, involving the [Ca2+]i handling and inhibiting the expression and interaction between TRPC5 and Orai1.
RESUMEN
Evidence has shown that vascular smooth muscle cells (VSMCs) of spontaneously hypertensive rats (SHRs) are depolarized and that the expression of L-type Ca2+ channels (LTCCs) and the sarcoplasmic reticulum (SR) Ca2+ buffering system are upregulated. Arterial rings exposed to high K+ solutions develop a contraction with two components, namely, an initial or phasic component and a sustained or tonic component. Because LTCCs and SR have different functions in the phasic and tonic components of depolarization-induced contraction, this study investigated the role of LTCC-SR coupling in depolarized arterial rings of SHRs. In the absence of extracellular Ca2+, high external K+ or LTCC agonists elicited a transitory contraction, which was sensitive to nifedipine and was potentiated in SHRs. In the presence of extracellular Ca2+, cyclopiazonic acid (CPA), an SR Ca2+-ATPase (SERCA) inhibitor, evoked a transient contraction that was significantly increased in SHRs. Although the phasic and tonic components were markedly increased in depolarized arterial rings of SHRs, they showed different voltage-dependence and sensitivity to SERCA inhibition. The tonic component was more sensitive to moderate depolarizations, and CPA selectively reduced the tonic component to the level observed in WKY rats. These results suggested that LTCC-SR coupling is potentiated in the sustained contraction of hypertensive VSMCs.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Hipertensión/fisiopatología , Retículo Sarcoplasmático/fisiología , Vasoconstricción/fisiología , Animales , Calcio/metabolismo , Masculino , Cloruro de Potasio/farmacología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKYRESUMEN
BACKGROUND AND PURPOSE: Rho-kinase, an effector of RhoA, is associated with various cardiovascular diseases in circulating blood cells. However, the role of RhoA/Rho-kinase in peripheral blood mononuclear cells from patients with spontaneous aneurysmal subarachnoid hemorrhage (aSAH) has not yet been studied in relation to the severity of this disease. Therefore, we analyzed the expression and activity of RhoA as a possible biomarker in aSAH. METHODS: Twenty-four patients with aSAH and 15 healthy subjects were examined. Peripheral blood mononuclear cells were collected, and RhoA activity and expression were determined by RhoA activation assay kit (G-LISA) and enzyme-linked immunosorbent assay tests, respectively. The severity of aSAH was determined from the World Federation of Neurological Surgeon scale, and vasospasm was evaluated using clinical symptoms, arteriography, and sonography. RESULTS: RhoA expression was significantly increased in peripheral blood mononuclear cells from patients on days 0, 2, and 4 after aSAH versus healthy subjects (P=0.036, 0.010, and 0.018, respectively, by U Mann-Whitney analysis). There was a significant correlation between RhoA expression and injury severity on days 2 and 4 (Spearman test, day 2: r=0.682, n=14, P=0.007; day 4: r=0.721, n=14, P=0.004). No significant correlation was observed on day 0 (day 0: r=0.131, n=6, P=0.805). Active RhoA was not significantly different in patients and healthy subjects on days 0, 2, and 4 (P=0.243, 0.222, and 0.600, respectively) nor did it increase significantly on days 0 and 2 in patients with vasospasm versus patients without vasospasm (P=0.064 and 0.519, respectively). In contrast, active RhoA was significantly higher on day 4 in patients who developed vasospasm versus patients without vasospasm (P=0.028). CONCLUSIONS: Our preliminary results indicate that RhoA expression and activity in peripheral blood mononuclear cells might be related with aSAH severity and cerebral vasospasm. RhoA is a potential biomarker of the risks associated with aSAH.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Hemorragia Subaracnoidea/metabolismo , Vasoespasmo Intracraneal/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Biomarcadores/sangre , Angiografía Cerebral/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Hemorragia Subaracnoidea/complicaciones , Vasoespasmo Intracraneal/diagnósticoRESUMEN
The role of L-type Ca2+ channels (LTCCs) and RhoA/Rho kinase (ROCK) on depolarization-induced sustained arterial contraction lasting several minutes is already known. However, in vivo, vascular smooth muscle cells can be depolarized for longer periods, inducing substantial inactivation of LTCCs and markedly reducing Ca2+ influx into the myocytes. We have examined, in femoral arterial rings, the role of LTCCs and RhoA/ROCK during long-lasting depolarization. Our results reveal a new vasoreactive response after 20-30min of depolarization in 2.5mM external Ca2+ that has not been identified previously with shorter stimuli. Prolonged depolarization-induced arterial contraction was permanently abolished when arterial rings were treated with 100nM external Ca2+ or 20nM nifedipine. However, when Ca2+ influx was restricted, applying ~7µM external Ca2+ solution or 3nM nifedipine, vasorelaxation was transient, and isometric force slowly increased after 30min and maintained its level until the end of the stimulus. Under these conditions, arterial contraction showed the same temporal course of RhoA activity and was sensitive to fasudil, nifedipine and cyclopiazonic acid. Ca2+-response curve in ß-escin permeabilized arteries was also sensitive to ROCK inhibitors. Thus, although long-lasting depolarization inactivates LTCCs, the reduced Ca2+ entry can induce a detectable arterial contraction via RhoA/ROCK activation.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Señalización del Calcio , Calcio/metabolismo , Músculo Liso Vascular/enzimología , Vasoconstricción , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/efectos de los fármacos , Señalización del Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Arteria Femoral/enzimología , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Músculo Liso Vascular/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Ratas Wistar , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasodilatadores/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Voltage-dependent CaV1.2 L-type Ca2+ channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2+ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2+ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2+ concentration ([Ca2+]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2+ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.
Asunto(s)
Aorta/metabolismo , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/fisiología , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Proteína ORAI1/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Aorta/citología , Calcio/metabolismo , Canales de Calcio Tipo L/genética , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Proteína ORAI1/genética , Serotonina/metabolismo , Canales Catiónicos TRPC/genética , Vasoconstricción/fisiologíaRESUMEN
BACKGROUND: Episodic ataxia type 1 is a rare autosomal dominant neurological disorder caused by mutations in the KCNA1 gene that encodes the α subunit of voltage-gated potassium channel Kv1.1. The functional consequences of identified mutations on channel function do not fully correlate with the clinical phenotype of patients. METHODS: A clinical and genetic study was performed in a family with 5 patients with episodic ataxia type 1, with concurrent epilepsy in 1 of them. Protein expression, modeling, and electrophysiological analyses were performed to study Kv1.1 function. RESULTS: Whole-genome linkage and candidate gene analyses revealed the novel heterozygous mutation p.Arg324Thr in the KCNA1 gene. The encoded mutant Kv1.1 channel displays reduced currents and altered activation and inactivation. CONCLUSIONS: Taken together, we provide genetic and functional evidence that mutation p.Arg324Thr in the KCNA1 gene is pathogenic and results in episodic ataxia type 1 through a dominant-negative effect. © 2016 International Parkinson and Movement Disorder Society.
Asunto(s)
Ataxia/genética , Ataxia/fisiopatología , Canal de Potasio Kv.1.1/genética , Miocimia/genética , Miocimia/fisiopatología , Epilepsia/genética , Epilepsia/fisiopatología , Femenino , Humanos , Masculino , LinajeRESUMEN
We have previously described that L-type Ca(2+) channels' (LTCCs) activation and metabotropic Ca(2+) release from the sarcoplasmic reticulum (SR) regulate RhoA/Rho kinase (ROCK) activity and sustained arterial contraction. We have investigated whether this signaling pathway can be altered in a new experimental model of subarachnoid hemorrhage (SAH). For this purpose, arterial reactivity was evaluated on days 1 to 5 after surgery. A significant increase of basal tone, measured 4 and 60min after normalization, was observed on day 5 after SAH and at 60min on days 2 and 3 after SAH. This phenomenon was suppressed with LTCCs and ROCK inhibitors. We have also studied arterial rings vasoreactivity in response to high K(+) solutions. Interestingly, there were no significant differences in the phasic component of the high K(+)-induced contraction between sham and SAH groups, whereas a significant increase in the sustained contraction was observed on day 5 after SAH. This latter component was sensitive to fasudil, and selectively reduced by low nifedipine concentration, and phospholipase C and SR-ATPase inhibitors. Therefore, our data suggest that the metabotropic function of LTCCs is potentiated in SAH. Our results could provide a new strategy to optimize the pharmacological treatment of this pathological process.
Asunto(s)
Arteria Basilar/metabolismo , Canales de Calcio Tipo L/metabolismo , Contracción Muscular/fisiología , Retículo Sarcoplasmático/metabolismo , Hemorragia Subaracnoidea/metabolismo , Vasoconstricción/fisiología , Quinasas Asociadas a rho/metabolismo , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Arteria Basilar/efectos de los fármacos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Masculino , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Nifedipino/farmacología , Ratas , Ratas Wistar , Retículo Sarcoplasmático/efectos de los fármacos , Hemorragia Subaracnoidea/tratamiento farmacológico , Vasoconstricción/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
STIM1 is a ubiquitous Ca2+ sensor of the intracellular, agonist-sensitive, Ca2+ stores that communicates the filling state of the Ca2+ compartments to plasma membrane store-operated Ca2+ (SOC) channels. STIM1 has been presented as a point of convergence between store-operated and voltage-operated Ca2+ influx, both inducing activation of SOC channels while suppressing Cav1.2 channels. Here we report that Homer proteins play a relevant role in the communication between STIM1 and Cav1.2 channels. HEK-293 cells transiently expressing Cav1.2 channel subunits α1, ß2 and α2δ-1 exhibited a significant Ca2+ entry upon treatment with a high concentration of KCl. In Cav1.2-expressing cells, treatment with thapsigargin (TG), to induce passive discharge of the intracellular Ca2+ stores, resulted in Ca2+ influx that was significantly greater than in cells not expressing Cav1.2 channels, a difference that was abolished by nifedipine and diltiazem. Treatment with TG induces co-immunoprecipitation of Homer1 with STIM1 and the Cav1.2 α1 subunit. Impairment of Homer function by introduction of the synthetic PPKKFR peptide into cells, which emulates the proline-rich sequences of the PPXXF motif, or using siRNA Homer1, reduced the association of STIM1 and the Cav1.2 α1 subunit. These findings indicate that Homer is important for the association between both proteins. Finally, treatment with siRNA Homer1 or the PPKKFR peptide enhanced the nifedipine-sensitive component of TG response in Cav1.2-expressing cells. Altogether, these findings provide evidence for a new role of Homer1 supporting the regulation of Cav1.2 channels by STIM1.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Diltiazem/farmacología , Células HEK293 , Proteínas de Andamiaje Homer , Humanos , Inmunoprecipitación , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Datos de Secuencia Molecular , Nifedipino/farmacología , Péptidos/química , Péptidos/farmacología , Unión Proteica/efectos de los fármacos , Subunidades de Proteína/metabolismo , Conejos , Ratas , Molécula de Interacción Estromal 1 , Tapsigargina/farmacologíaRESUMEN
The aim of this study was to investigate the levels of persistent chlorinated pollutants (POPs) in wild birds. The concentrations of multiple POPs, including polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were determined in plasma of White stork nestlings. Blood samples were collected from three breeding colonies located in the West of Spain that were exposed to different environmental conditions and human activities. The concentrations of PCBs were below the limit of detection in all samples. The OCPs Heptaclor, 4,4'-DDE, endosulfan and aldrin were detected most frequently in plasma from White stork nestlings. From these four compounds, 4,4'-DDE and Heptaclor were found in high abundance. OCPs could not be detected in the colony breading nearby a landfill and an intensive agricultural area, indicating that these banned compounds are not taken up by crops growing in this area. However, birds from the colony breading next to a landfill exhibited the highest OCPs levels. These high levels might be due to exposure of the mothers to OCPs during their migration to Africa and subsequent transfer to their eggs.
Asunto(s)
Aves/sangre , Monitoreo del Ambiente/métodos , Contaminantes Ambientales/sangre , Hidrocarburos Clorados/sangre , Plaguicidas/sangre , Adulto , Animales , Femenino , Humanos , EspañaRESUMEN
Cadmium, lead, mercury, selenium, iron, zinc and arsenic levels were measured in blood samples from 59 free-ranging white stork nestlings from colonies located in three different environmental conditions in Western Spain. The reference colony was situated in "Llanos de Cáceres y Sierra de Fuentes", an Area of Special Interest for Bird Protection. A second colony was located close to (4.9 km) an urban landfill and a third one was close to both an intensive agricultural area and an urban landfill (1.5 km). Blood samples were diluted and elemental analysis was performed using inductively coupled plasma mass spectrometer. In all cases, the essential metals zinc and iron were found at the highest mean concentrations followed by lead > selenium > mercury > arsenic > cadmium. Regarding toxic metals, the highest concentrations were found for lead (ranging from 23.27 to 146.4 µg/L) although in all cases the concentrations were lower than those considered to cause subclinical effects. The metals levels detected in the chick's blood were not related to the previously reported levels in the soil next to the colonies, which may indicate that landfills are the main source of metals in white stork nestlings. The present data showed that metal levels in white stork chicks may be influenced by the use of landfills as feeding areas by the parents. However, more studies on the metal content in the feed of white stork and the influence of the distance to the landfill are necessary to establish the causality of these findings.
Asunto(s)
Aves/sangre , Contaminantes Ambientales/sangre , Metaloides/sangre , Metales Pesados/sangre , Instalaciones de Eliminación de Residuos , Animales , Arsénico , Cruzamiento , Cadmio , Monitoreo del Ambiente , Hierro , Plomo , Mercurio , Selenio , España , ZincRESUMEN
Vascular smooth muscle cells (VSMCs) contraction can be evoked by the rise of cytosolic [Ca(2+)] owing to transmembrane Ca(2+) influx or sarcoplasmic reticulum (SR) Ca(2+) release. Although the classical ionotropic role of voltagedependent (L-type) Ca(2+) channels (VGCCs) is known, we review here data suggesting a new metabotropic function of VGCCs in vascular smooth muscle cells. VGCCs can trigger Ca(2+) release from the SR in the absence of extracellular Ca2+. During depolarization, VGCCs can activate G proteins and phospholipase C (PLC)/inositol 1,4,5-trisphosphate (InsP3) pathway leading to Ca2+ release and arterial contraction. This new metabotropic role of VGCCs, referred as calcium channel-induced Ca(2+) release (CCICR), has a major role in tonic VSM contractility, as it links sustained membrane depolarization and Ca(2+) channel activation with metabotropic Ca(2+) release from the sarcoplasmic reticulum (SR) and tonic smooth muscle contraction. This new role of VGCCs could have a wide functional relevance for the pathogenesis of vasospasms mediated by membrane depolarization and vasoactive agents that can activate VGCCs. Precise understanding of CCICR could help to optimize pharmacological treatments for clinical conditions where Ca(2+) channels antagonists are recommended.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Contracción Muscular/fisiología , Músculo Liso Vascular/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Proteínas de Unión al GTP/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/citología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Retículo Sarcoplasmático/metabolismo , Fosfolipasas de Tipo C/metabolismoRESUMEN
KCl-evoked sustained contraction requires L-type Ca(2+) channel activation, metabotropic Ca(2+) release from the sarcoplasmic reticulum (mechanism denoted calcium channel-induced Ca(2+) release) and RhoA/Rho associated kinase activation. Although high K(+) solutions are used to depolarize myocytes, these solutions can stimulate other signaling pathways such as those triggered by the activation of muscarinic and purinergic receptors. The present study examines the functional role of calcium channel-induced Ca(2+) release under pharmacological activation of L-type Ca(2+) channel without significant membrane depolarization. It also analyzes the role of the "steady-state" Ca(2+) influx through L-type Ca(2+) channels on myocyte sustained contraction. Measurement of contractility in arterial rings was done on a vessel myograph. Membrane potential was measured by fluorescence techniques loading intact myocytes with a membrane potential sensitive dye, and a reversible permeabilization method was used to load myocytes in intact arteries with GDPßS and Ca(v)1.2 siRNA. Application of an L-type Ca(2+) channel agonist, without effect on membrane potential, evoked sustained contraction via G-protein induced Ca(2+) release from the sarcoplasmic reticulum and RhoA/Rho associated kinase activation. Tonic myocyte contractions mediated by L-type Ca(2+) channel activation required sustained Ca(2+) influx through the channels and Ca(2+) uptake by the sarcoplasmic reticulum. Because L-type Ca(2+) channels participate in numerous pathophysiological processes mediated by maintained arterial contraction, our data could help to optimize therapeutic treatment of arterial vasospasm.
Asunto(s)
Arteria Basilar/enzimología , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Receptores Acoplados a Proteínas G/metabolismo , Vasoconstricción , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Arteria Basilar/efectos de los fármacos , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/genética , Señalización del Calcio/efectos de los fármacos , Activación Enzimática , Colorantes Fluorescentes , Potenciales de la Membrana , Microscopía Fluorescente , Miografía , Interferencia de ARN , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/efectos de los fármacos , Retículo Sarcoplasmático/enzimología , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/farmacologíaRESUMEN
AIMS: Tungstate reduces blood pressure in experimental animal models of both hypertension and metabolic syndrome, although the underlying mechanisms are not fully understood. Given that the large-conductance voltage- and Ca(2+)-dependent K(+) (BK) channel is a key element in the control of arterial tone, our aim was to evaluate whether BK channel modulation by tungstate can contribute to its antihypertensive effect. METHODS AND RESULTS: Patch-clamp studies of heterologously expressed human BK channels (α + ß(1-4) subunits) revealed that cytosolic tungstate (1 mM) induced a significant left shift (â¼20 mV) in the voltage-dependent activation curve only in BK channels containing αß(1) or αß(4) subunits, but reduced the amplitude of K(+) currents through all BK channels tested. The ß(1)-dependent activation of BK channels by tungstate was enhanced at cytosolic Ca(2+) levels reached during myocyte contraction, and prevented either by removal of cytosolic Mg(2+) or by mutations rendering the channel insensitive to Mg(2+). A lower concentration of tungstate (0.1 mM) induced voltage-dependent activation of the vascular BKαß(1) channel without reducing current amplitude, and consistently exerted a vasodilatory action on wild-type but not on ß(1)-knockout mouse arteries pre-contracted with endothelin-1. CONCLUSION: Tungstate activates BK channels in a ß subunit- and Mg(2+)-dependent manner and induces vasodilatation only in mouse arteries that express the BK ß(1) subunit.