RESUMEN
Calcium (Ca2+) is a secondary messenger that regulates various cellular processes. However, Ca2+ mishandling could lead to pathological conditions. Orai1 is a Ca2+channel contributing to the store-operated calcium entry (SOCE) and plays a critical role in Ca2+ homeostasis in several cell types. Dysregulation of Orai1 contributed to severe combined immune deficiency syndrome, some cancers, pulmonary arterial hypertension (PAH), and other cardiorespiratory diseases. During its activation process, Orai1 is mainly regulated by stromal interacting molecule (STIM) proteins, especially STIM1; however, many other regulatory partners have also been recently described. Increasing knowledge about these regulatory partners provides a better view of the downstream signalling pathways of SOCE and offers an excellent opportunity to decipher Orai1 dysregulation in these diseases. These proteins participate in other cellular functions, making them attractive therapeutic targets. This review mainly focuses on Orai1 regulatory partners in the physiological and pathological conditions of the pulmonary circulation and inflammation.
Asunto(s)
Proteína ORAI1 , Humanos , Proteína ORAI1/metabolismo , Animales , Moléculas de Interacción Estromal/metabolismo , Señalización del Calcio , Calcio/metabolismo , Enfermedades Vasculares/metabolismo , Enfermedades Vasculares/patologíaRESUMEN
BACKGROUND: Stromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated. AIM: To evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats. METHODS: Rat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot. MAIN OUTCOME MEASURES: Functional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES. RESULTS: Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients. CLINICAL TRANSLATION: Targeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED. STRENGTHS AND LIMITATIONS: Improving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability. CONCLUSIONS: STIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients. Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733-1749.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Disfunción Eréctil , Moléculas de Interacción Estromal , Animales , Humanos , Masculino , Ratas , Adrenérgicos/metabolismo , Adrenérgicos/farmacología , Adrenérgicos/uso terapéutico , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Tipo 2/complicaciones , Disfunción Eréctil/tratamiento farmacológico , Disfunción Eréctil/etiología , Erección Peniana , Pene/irrigación sanguínea , Inhibidores de Fosfodiesterasa 5/uso terapéutico , Moléculas de Interacción Estromal/metabolismo , Tromboxanos/metabolismo , Tromboxanos/farmacología , Tromboxanos/uso terapéuticoRESUMEN
Sustained and intermittent hypoxia produce vasoconstriction, arterial remodeling, and hypertension in the lung. Stromal interaction molecule (STIM)-activated transient receptor potential channels (TRPC) and calcium release-activated calcium channel protein (ORAI) channels (STOC) play key roles in the progression of pulmonary hypertension in pre-clinical models of animals subjected to sustained and intermittent hypoxia. The available evidence supports the theory that oxidative stress and hypoxic inducible factors upregulate and activate STIM-activated TRPC-ORAI Ca2+ channels, contributing to the pulmonary remodeling and hypertension induced by sustained hypoxia. However, less is known about the effects of oxidative stress and hypoxic inducible factors on the modulation of STIM-activated TRPC-ORAI channels following chronic intermittent hypoxia. In this review, we examined the emerging evidence supporting the theory that oxidative stress and hypoxic inducible factors induced by intermittent hypoxia upregulate and activate STIM-activated TRPC-ORAI Ca2+ channels. In addition, we used bioinformatics tools to search public databases for the genes involved in the upregulation of STIMactivated TRPC-ORAI Ca2+ channels and compare the differential gene expression and biological processes induced by intermittent and sustained hypoxia in lung cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Hipertensión Pulmonar , Hipertensión , Moléculas de Interacción Estromal , Canales de Potencial de Receptor Transitorio , Animales , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Hipertensión/metabolismo , Hipertensión Pulmonar/etiología , Hipoxia/complicaciones , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Moléculas de Interacción Estromal/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Alzheimer's disease (AD) is a progressive neurological disease that slowly causing memory impairments with no effective treatment. We have recently reported that kisspeptin-13 (KP-13) ameliorates Aß toxicity-induced memory deficit in rats. Here, the possible cellular impact of kisspeptin receptor activation in a rat model of the early stage AD was assessed using whole-cell patch-clamp recording from CA1 pyramidal neurons and molecular approaches. Compared to neurons from the control group, cells from the Aß-treated group displayed spontaneous and evoked hyperexcitability with lower spike frequency adaptation. These cells had also a lower sag ratio in response to hyperpolarizing prepulse current delivered before a depolarizing current injection. Neurons from the Aß-treated group exhibited short spike onset latency, lower rheobase and short utilization time compared with those in the control group. Furthermore, phase plot analysis of action potential showed that Aß treatment affected the action potential features. These electrophysiological changes induced by Aß were associated with increased expression of stromal interaction molecules (STIMs), particularly (STIM2) and decreased pCREB/CREB ratio. Treatment with KP-13 following Aß injection into the entorhinal cortex, however, prevented the excitatory effect of Aß on spontaneous and evoked neuronal activity, increased the latency of onset, enhanced the sag ratio, increased the rheobase and utilization time, and prevented the changes induced Aß on spike parameters. In addition, the KP-13 application after Aß treatment reduced the expression of STIMs and increased the pCREB/CREB ratio compared to those receiving Aß treatment alone. In summary, these results provide evidence that activation of kisspeptin receptor may be effective against pathology of Aß.
Asunto(s)
Enfermedad de Alzheimer , Moléculas de Interacción Estromal , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Hipocampo/metabolismo , Kisspeptinas/efectos adversos , Kisspeptinas/metabolismo , Trastornos de la Memoria/inducido químicamente , Fragmentos de Péptidos/toxicidad , Células Piramidales , Ratas , Ratas Wistar , Moléculas de Interacción Estromal/metabolismoRESUMEN
Cardiac fibrotization is a well-known process characteristic of many cardiac pathological conditions. The key element is excessive activation of cardiac fibroblasts, their transdifferentiation into myofibroblasts, increased production, and accumulation of extracellular matrix proteins, resulting in cardiac stiffness. The exact cellular mechanisms and molecular components involved in the process are not fully elucidated, but the SOCE mechanism could play an important role. Its key molecules are the molecular sensor of calcium in ER/SR - STIM and the highly selective calcium channels Orai located in the plasma membrane. This study aims to evaluate selected SOCE-associated genes in the activation of HCF cell culture by several known substances (phenylephrine, isoprenaline) that represent cardiovascular overload. After cell cultivation, cell medium was collected to measure the soluble collagen content. From the harvested cells, qRT-PCR was performed to determine the mRNA levels of the corresponding genes. The activation of cells was based on changes in the relative expression of collagen genes as well as the collagen content in the medium of the cell culture. We detected an increase in the expression of the Orai2 isoform, a change in the Orai1/Orai3 ratio and also an increase in the expression of the STIM2 isoform. These results suggest an increased activation of the SOCE mechanism under stress conditions of fibroblasts, which supports the hypothesis of fibroblast activation in pathological processes by altering calcium homeostasis through the SOCE mechanism.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Fibroblastos/metabolismo , Miocardio/metabolismo , Moléculas de Interacción Estromal/metabolismo , Células Cultivadas , Humanos , Miocardio/citología , Isoformas de Proteínas/metabolismoRESUMEN
In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.
Asunto(s)
Señalización del Calcio/fisiología , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Retículo Endoplásmico/metabolismo , Animales , Sitios de Unión , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Humanos , Microdominios de Membrana/metabolismo , Modelos Biológicos , Sistemas de Mensajero Secundario/fisiología , Análisis Espacio-Temporal , Moléculas de Interacción Estromal/metabolismo , Canales Catiónicos TRPC/metabolismoRESUMEN
The amyloid-beta (Aß) fibrillation process seems to execute a principal role in the neuropathology of Alzheimer's disease (AD). Accordingly, novel therapeutic plans have concentrated on the inhibition or degradation of Aß oligomers and fibrils. Biocompatible nanoparticles (NPs), e.g., gold and iron oxide NPs, take a unique capacity in redirecting Aß fibrillation kinetics; nevertheless, their impacts on AD-related memory impairment have not been adequately evaluated in vivo. Here, we examined the effect of commercial PEGylated superparamagnetic iron oxide nanoparticles (SPIONs) on the learning and memory of an AD-animal model. The outcomes demonstrated the dose-dependent effect of SPIONs on Aß fibrillation and learning and memory processes. In vitro and in vivo findings revealed that Low doses of SPIONs inhibited Aß aggregation and ameliorated learning and memory deficit in the AD model, respectively. Enhanced level of hippocampal proteins, including brain-derived neurotrophic factor, BDNF, phosphorylated-cAMP response element-binding protein, p-CREB, and stromal interaction molecules, e.g., STIM1 and STIM2, were also observed. However, at high doses, SPIONs did not improve the detrimental impacts of Aß fibrillation on spatial memory and hippocampal proteins expression. Overall, we revealed the potential capacity of SPIONs on retrieval of behavioral and molecular manifestations of AD in vivo, which needs further investigations to determine the mechanistic effect of SPIONs in the AD conundrum.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Aprendizaje/efectos de los fármacos , Nanopartículas Magnéticas de Óxido de Hierro/administración & dosificación , Trastornos de la Memoria/tratamiento farmacológico , Polietilenglicoles/administración & dosificación , Moléculas de Interacción Estromal , Enfermedad de Alzheimer/inducido químicamente , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Reacción de Fuga/efectos de los fármacos , Reacción de Fuga/fisiología , Aprendizaje/fisiología , Masculino , Trastornos de la Memoria/metabolismo , Fragmentos de Péptidos/toxicidad , Ratas , Ratas Wistar , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Moléculas de Interacción Estromal/metabolismoRESUMEN
Voltage-gated L-type Ca2+ channel (Cav1.2) blockers (LCCBs) are major drugs for treating hypertension, the preeminent risk factor for heart failure. Vascular smooth muscle cell (VSMC) remodeling is a pathological hallmark of chronic hypertension. VSMC remodeling is characterized by molecular rewiring of the cellular Ca2+ signaling machinery, including down-regulation of Cav1.2 channels and up-regulation of the endoplasmic reticulum (ER) stromal-interacting molecule (STIM) Ca2+ sensor proteins and the plasma membrane ORAI Ca2+ channels. STIM/ORAI proteins mediate store-operated Ca2+ entry (SOCE) and drive fibro-proliferative gene programs during cardiovascular remodeling. SOCE is activated by agonists that induce depletion of ER Ca2+, causing STIM to activate ORAI. Here, we show that the three major classes of LCCBs activate STIM/ORAI-mediated Ca2+ entry in VSMCs. LCCBs act on the STIM N terminus to cause STIM relocalization to junctions and subsequent ORAI activation in a Cav1.2-independent and store depletion-independent manner. LCCB-induced promotion of VSMC remodeling requires STIM1, which is up-regulated in VSMCs from hypertensive rats. Epidemiology showed that LCCBs are more associated with heart failure than other antihypertensive drugs in patients. Our findings unravel a mechanism of LCCBs action on Ca2+ signaling and demonstrate that LCCBs promote vascular remodeling through STIM-mediated activation of ORAI. Our data indicate caution against the use of LCCBs in elderly patients or patients with advanced hypertension and/or onset of cardiovascular remodeling, where levels of STIM and ORAI are elevated.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Hipertensión/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Moléculas de Interacción Estromal/metabolismo , Remodelación Vascular/fisiología , Animales , Antihipertensivos/farmacología , Calcio/metabolismo , Canales de Calcio Tipo L/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular , Proliferación Celular , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Técnicas de Inactivación de Genes , Células HEK293 , Insuficiencia Cardíaca , Humanos , Proteínas de la Membrana/genética , Miocitos del Músculo Liso , Proteínas de Neoplasias , Proteína ORAI1/genética , Ratas , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 2/genéticaRESUMEN
Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Moléculas de Interacción Estromal/metabolismo , Animales , HumanosRESUMEN
VGF gene encodes for a neuropeptide precursor of 68 kDa composed by 615 (human) and 617 (rat, mice) residues, expressed prevalently in the central nervous system (CNS), but also in the peripheral nervous system (PNS) and in various endocrine cells. This precursor undergoes proteolytic cleavage, generating a family of peptides different in length and biological activity. Among them, TLQP-21, a peptide of 21 amino acids, has been widely investigated for its relevant endocrine and extraendocrine activities. The complement complement C3a receptor-1 (C3aR1) has been suggested as the TLQP-21 receptor and, in different cell lines, its activation by TLQP-21 induces an increase of intracellular Ca2+. This effect relies both on Ca2+ release from the endoplasmic reticulum (ER) and extracellular Ca2+ entry. The latter depends on stromal interaction molecules (STIM)-Orai1 interaction or transient receptor potential channel (TRPC) involvement. After Ca2+ entry, the activation of outward K+-Ca2+-dependent currents, mainly the KCa3.1 currents, provides a membrane polarizing influence which offset the depolarizing action of Ca2+ elevation and indirectly maintains the driving force for optimal Ca2+ increase in the cytosol. In this review, we address the main endocrine and extraendocrine actions displayed by TLQP-21, highlighting recent findings on its mechanism of action and its potential in different pathological conditions.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Neuropéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Animales , Citosol/efectos de los fármacos , Citosol/metabolismo , Humanos , Moléculas de Interacción Estromal/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Store-operated calcium entry (SOCE) is one of regulatory mechanisms which regulates Ca2+ cycling in the heart. SOCE alterations in pathological conditions contribute to progression of heart failure and cardiac hypertrophy by multiple signaling pathways such as Cn/NFAT and CaMKII/MEF2. Several components mediating SOCE have been identified, such as STIM and Orai. Different isoforms of both Orai and STIM have been detected in animal studies, exhibiting distinct functional properties. This study is focused on the analysis of STIM and Orai isoforms expression in the end-stage human failing myocardium. Left ventricle samples isolated from 43 explanted hearts from patients undergoing heart transplant and from 5 healthy donor hearts were used to determine the mRNA levels of Orai1, Orai2 and Orai3, STIM1, STIM2 and STIM2.1 by qRT-PCR. The expression was further analyzed for connection with gender, related co-morbidities, pathoetiology, clinical data and biochemical parameters. We show that Orai1 expression is decreased by 30 % in failing myocardium, even though we detected no significant changes in expression of Orai2 or Orai3. Interestingly, this decrease in Orai1 was gender-specific and was present only in men, with no change in women. The ratio Orai1/Orai3 was significantly lower in males as well. The novel STIM2.1 isoform was detected both in healthy and failing human myocardium. In the end-stage heart failure, the expression of STIM2.1 was significantly decreased. The lower ratio of STIM2.1/STIM2 in failing hearts indicates a switch from SOCE-inhibiting STIM2.1 isoform to stimulatory STIM2.2. STIM1 mRNA levels were not significantly changed. These observed alterations in Orai and STIM expression were independent of functional heart parameters, clinical or biochemical patient characteristics. These results provide detailed insight into the alterations of SOCE regulation in human failing myocardium. Gender-specific change in Orai1 expression might represent a possible mechanism of cardioprotective effects of estrogens. The switch from STIM2.1 to STIM2.2 indicates an amplification of SOCE and could contribute to the hypertrophy development in the filing heart.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Miocardio/metabolismo , Moléculas de Interacción Estromal/metabolismo , Adulto , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Isoformas de Proteínas/metabolismo , Caracteres Sexuales , Remodelación VentricularRESUMEN
Effective therapy of hypertension represents a key strategy for reducing the burden of cardiovascular disease and its associated mortality. The significance of voltage dependent L-type Ca²âº channels to Ca²âº influx, and of their regulatory mechanisms in the development of heart disease, is well established. A wide variety of L-type Ca²âº channel inhibitors and Ca²âº antagonists have been found to be beneficial not only in the treatment of hypertension, but also in myocardial infarction and heart failure. Over the past two decades, another class of Ca²âº channel - the voltage independent store-operated Ca²âº channel - has been implicated in the regulation and fine tuning of Ca²âº entry in both cardiac and smooth muscle cells. Store-operated Ca²âº channels are activated by the depletion of Ca²âº stores within the endoplasmic/sarcoplasmic reticulum, or by low levels of cytosolic Ca²âº, thereby facilitating agonist-induced Ca²âº influx. Store-operated Ca²âº entry through this pivotal pathway involves both stromal interaction molecule (STIM) and Orai channels. Different degrees of changes in these proteins are considered to promote Ca²âº entry and hence contribute to the pathogenesis of cardiovascular dysfunction. Several blockers of store-operated Ca²âº channels acting at the level of both STIM and Orai channels have been shown to depress Ca²âº influx and lower blood pressure. However, their specificity, safety, and clinical significance remain to be established. Thus, there is an ongoing challenge in the development of selective inhibitors of store-operated Ca²âº channels that act in vascular smooth muscles for the improved treatment of hypertension.
Asunto(s)
Antihipertensivos/uso terapéutico , Presión Sanguínea/efectos de los fármacos , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Activados por la Liberación de Calcio/antagonistas & inhibidores , Hipertensión/tratamiento farmacológico , Músculo Liso Vascular/efectos de los fármacos , Moléculas de Interacción Estromal/antagonistas & inhibidores , Vasodilatadores/uso terapéutico , Animales , Antihipertensivos/efectos adversos , Bloqueadores de los Canales de Calcio/efectos adversos , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Moléculas de Interacción Estromal/metabolismo , Resultado del Tratamiento , Vasodilatadores/efectos adversosRESUMEN
Calcium (Ca2+) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1⯵M medroxyprogesterone acetate) and cyclic AMP (0.5â¯mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release and IP3R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca2+ release. Finally, intracellular Ca2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs.
Asunto(s)
Calcio/metabolismo , Decidua/citología , Decidua/metabolismo , Endometrio/citología , Endometrio/metabolismo , Células del Estroma/metabolismo , Adulto , Antígenos CD/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Retículo Endoplásmico/metabolismo , Femenino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Moléculas de Interacción Estromal/metabolismoRESUMEN
Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Moléculas de Interacción Estromal/metabolismo , Animales , Canales de Calcio Activados por la Liberación de Calcio/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Moléculas de Interacción Estromal/genéticaRESUMEN
Calcium influx through store-operated Ca2+ entry (SOCE), mediated by STIM-operated Orai channels, is crucial for many cellular functions. To dissect the molecular mechanisms underlying the process of STIM-Orai activation and identify regulators that modify this process, ratiometric imaging of SOCE responses in HEK cells overexpressing STIM and Orai is a routinely used method. Here we describe one commonly used procedure of monitoring SOCE activity with a ratiometric membrane-permeable dye fura-2-AM. Other ratiometric indicators suitable for SOCE measurements are also discussed.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/fisiología , Expresión Génica , Imagen Molecular , Moléculas de Interacción Estromal/genética , Calcio/metabolismo , Señalización del Calcio , Análisis de Datos , Colorantes Fluorescentes , Células HEK293 , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Imagen Molecular/instrumentación , Imagen Molecular/métodos , Programas Informáticos , Moléculas de Interacción Estromal/metabolismoRESUMEN
The characterization of protein-protein interactions through methods such as co-immunoprecipitation, followed by Western blot analysis, is a crucial step in the understanding of protein functions and the biology of the cell. Since the discovery of ORAI and STIM proteins as component of store-operated channel (SOC), overexpressing systems have been used to demonstrate how ORAI and STIM can associate with physiological and pathological conditions. Here we describe a protocol allowing endogenous studies.
Asunto(s)
Western Blotting , Proteínas Portadoras/metabolismo , Inmunoprecipitación , Mapeo de Interacción de Proteínas , Moléculas de Interacción Estromal/metabolismo , Western Blotting/métodos , Calcio/metabolismo , Canales de Calcio/metabolismo , Línea Celular Tumoral , Humanos , Inmunoprecipitación/métodos , Unión Proteica , Mapeo de Interacción de Proteínas/métodosRESUMEN
The expression of chimeras that consist of a fluorescent protein (FP) conjugated with a protein of interest provides the ability to visualize, track, and quantify the subcellular localization and dynamics of specific proteins in biological samples. Array confocal laser scanning microscopy is an eminently suitable technique for live-cell imaging of FP-tagged fusion proteins. Here, we describe real-time monitoring of the subcellular dynamics of the stromal-interacting molecule 1 (STIM1) and Orai1, the key protagonists of store-operated Ca2+ entry (SOCE) under resting conditions, and upon Ca2+ mobilization from the endoplasmic reticulum (ER).
Asunto(s)
Microscopía Confocal , Imagen Molecular , Moléculas de Interacción Estromal/metabolismo , Calcio/metabolismo , Expresión Génica , Orden Génico , Genes Reporteros , Vectores Genéticos/genética , Procesamiento de Imagen Asistido por Computador , Espacio Intracelular/metabolismo , Imagen Molecular/métodos , Transporte de Proteínas , Programas Informáticos , Moléculas de Interacción Estromal/genética , Imagen de Lapso de Tiempo , TransfecciónRESUMEN
Skeletal muscle contracts or relaxes to maintain the body position and locomotion. For the contraction and relaxation of skeletal muscle, Ca2ï¼ in the cytosol of skeletal muscle fibers acts as a switch to turn on and off a series of contractile proteins. The cytosolic Ca2ï¼ level in skeletal muscle fibers is governed mainly by movements of Ca2ï¼ between the cytosol and the sarcoplasmic reticulum (SR). Store-operated Ca2ï¼ entry (SOCE), a Ca2ï¼ entryway from the extracellular space to the cytosol, has gained a significant amount of attention from muscle physiologists. Orai1 and stromal interaction molecule 1 (STIM1) are the main protein identities of SOCE. This mini-review focuses on the roles of STIM proteins and SOCE in the physiological and pathophysiological functions of skeletal muscle and in their correlations with recently identified proteins, as well as historical proteins that are known to mediate skeletal muscle function. [BMB Reports 2018; 51(8): 378-387].
Asunto(s)
Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Moléculas de Interacción Estromal/metabolismo , Moléculas de Interacción Estromal/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Espacio Extracelular/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/fisiología , Retículo Sarcoplasmático/metabolismoRESUMEN
Electrical pacemaker activity generates phasic contractions and motility patterns such as segmentation and peristalsis in the gastrointestinal tract. Pacemaker currents are generated in interstitial cells of Cajal (ICC), which release Ca2+ from intracellular stores that stimulates Ca2+-activated Cl- channels (CaCCs) in the plasma membrane. Thus, Ca2+ stores must be maintained to sustain pacemaker activity. Store-operated Ca2+ entry (SOCE) facilitates the refilling of Ca2+ stores by a mechanism dependent upon interactions between STIM and Orai proteins. We investigated the role of SOCE in ICC pacemaker activity. Reintroduction of extracellular Ca2+ in store-depleted ICC resulted in CaCC activation. Blocking CaCCs revealed an inwardly rectifying current with properties of a Ca2+ release-activated current (ICRAC). An inhibitory peptide that interfered with the STIM-Orai interaction blocked ICRAC in HEK 293 cells expressing STIM1 and Orai1 and blocked spontaneous transient inward currents (STICs) and slow wave currents in ICC. STICs, which are fundamental pacemaker events in ICC, were blocked by an Orai antagonist. Imaging of Ca2+ transients linked to pacemaker activity in ICC in intact muscles showed that the Orai antagonist blocked Ca2+ transients in ICC. These data suggest that Ca2+ recovery through STIM-Orai interactions is necessary to maintain ICC pacemaker activity.