RESUMEN
The pharmacological targeting of the α7 nicotinic acetylcholine receptor (α7) is a promising strategy in the development of new drugs for neurologic diseases. Because α7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α7 receptor. Both influx of calcium through the α7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α7D44A or α7345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α7 receptor allosteric modulation on both local and global calcium dynamics.
Asunto(s)
Calcio/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Señalización del Calcio/efectos de los fármacos , Señalización del Calcio/fisiología , Línea Celular Tumoral , Citoplasma/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Isoxazoles/farmacología , Células PC12 , Compuestos de Fenilurea/farmacología , RatasRESUMEN
α7 nicotinic acetylcholine receptors (nAChRs) play an important role in synaptic transmission and inflammation. In response to ligands, this receptor channel opens to conduct cations into the cell but desensitizes rapidly. In recent studies we show that α7 nAChRs bind signaling proteins such as heterotrimeric GTP-binding proteins (G proteins). Here, we demonstrate that direct coupling of α7 nAChRs to G proteins enables a downstream calcium signaling response that can persist beyond the expected time course of channel activation. This process depends on a G protein-binding cluster (GPBC) in the M3-M4 loop of the receptor. A mutation of the GPBC in the α7 nAChR (α7345-348A) abolishes interaction with Gαq as well as Gßγ while having no effect on receptor synthesis, cell-surface trafficking, or α-bungarotoxin binding. Expression of α7345-348A, however, did significantly attenuate the α7 nAChR-induced Gαq calcium signaling response as evidenced by a decrease in PLC-ß activation and IP3R-mediated calcium store release in the presence of the α7 selective agonist choline. Taken together, the data provides new evidence for the existence of a GPBC in nAChRs serving to promote intracellular signaling.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/metabolismo , Ratones , Datos de Secuencia Molecular , Células PC12 , Ratas , Homología de Secuencia de Aminoácido , Receptor Nicotínico de Acetilcolina alfa 7/químicaRESUMEN
Nicotinic acetylcholine receptors (nAChRs) modulate the growth and structure of neurons throughout the nervous system. Ligand stimulation of the α7 nAChR has been shown to regulate the large heterotrimeric GTP-binding protein (G protein) signaling in various types of cells. Here, we demonstrate a role for α7 nAChR/G protein interaction in the activation of the small (monomeric) RhoA GTPase leading to cytoskeletal changes during neurite growth. Treatment of PC12 cells with the α7 nAChR agonist choline or PNU-282987 was associated with an increase in RhoA activity and an inhibition in neurite growth. Specifically, choline treatment was found to attenuate the velocity of microtubule growth at the growth cone and decrease the rate of actin polymerization throughout the cell. The effects of α7 nAChR activation were abolished by expression of a dominant negative α7 nAChR (α7345-348A ) deficient in G protein coupling. Proteomic analysis of immunoprecipitated α7 nAChR complexes from differentiating PC12 cells and synaptic fractions of the developing mouse hippocampus revealed the existence of Rho GTPase-regulating guanine nucleotide exchange factors within α7 nAChR interactomes. These findings underscore the role of α7 nAChR/G protein in cytoskeletal regulation during neurite growth. This image depicts the hypothesized interaction of the traditionally ionotropic α7 nicotinic acetylcholine receptor (α7 nAChR) and its ability to interact and signal through both large and small G proteins, leading to the regulation of cytoskeletal growth. Using differentiated PC12 cells, and the specific agonist choline, it was shown that α7 nAChR/G protein interactions mediate both short- and long-term neurite growth dynamics through increased RhoA activation. Activation of RhoA was shown to decrease actin polymerization, and lead to an overall decrease in neurite growth via regulation of the microtubule network. Cover Image for this issue: doi: 10.1111/jnc.13330.
Asunto(s)
Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Benzamidas/farmacología , Compuestos Bicíclicos con Puentes/farmacología , Señalización del Calcio/fisiología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Activación Enzimática , Femenino , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Masculino , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Agonistas Nicotínicos/farmacología , Células PC12 , Ratas , Receptor Nicotínico de Acetilcolina alfa 7/efectos de los fármacosRESUMEN
Chronic smoking is a primary risk factor for breast cancer due to the presence of various toxins and carcinogens within tobacco products. Nicotine is the primary addictive component of tobacco products and has been shown to promote breast cancer cell proliferation and metastases. Nicotine activates nicotinic acetylcholine receptors (nAChRs) that are expressed in cancer cell lines. Here, we examine the role of the α7 nAChR in coupling to heterotrimeric G proteins within breast cancer MCF-7 cells. Pharmacological activation of the α7 nAChR using choline or nicotine was found to increase proliferation, motility, and calcium signaling in MCF-7 cells. This effect of α7 nAChR on cell proliferation was abolished by application of Gαi/o and Gαq protein blockers. Specifically, application of the Gαi/o inhibitor pertussis toxin was found to abolish choline-mediated cell proliferation and intracellular calcium transient response. These findings were corroborated by expression of a G protein binding dominant negative nAChR subunit (α7345-348A), which resulted in significantly attenuating calcium signaling and cellular proliferation in response to choline. Our study shows a new role for G protein signaling in the mechanism of α7 nAChR-associated breast cancer growth.
Asunto(s)
Neoplasias de la Mama , Proteínas de Unión al GTP Heterotriméricas , Receptores Nicotínicos , Humanos , Femenino , Nicotina/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Señalización del Calcio , Receptores Nicotínicos/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Proliferación Celular , Colina/farmacología , Calcio/metabolismoRESUMEN
True aneurysms of the anterior tibial artery are rare with less than 20 published reports in the literature. We report an urgent endovascular repair of a true anterior tibial artery aneurysm in a patient with Ehlers-Danlos type IV, vascular type. This approach resulted in an uneventful recovery without the elevated risks associated with open vascular repair in the setting of connective tissue disorder. Continuous follow-up in the subsequent 4 years has demonstrated durability and efficacy of the original intervention.
Asunto(s)
Aneurisma/terapia , Síndrome de Ehlers-Danlos/complicaciones , Embolización Terapéutica/instrumentación , Arterias Tibiales , Adulto , Aneurisma/diagnóstico por imagen , Aneurisma/etiología , Síndrome de Ehlers-Danlos/diagnóstico , Humanos , Masculino , Arterias Tibiales/diagnóstico por imagen , Resultado del TratamientoRESUMEN
The α7 nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel that plays an important role in cellular calcium signaling contributing to synaptic development and plasticity, and is a key drug target for the treatment of neurodegenerative conditions such as Alzheimer's disease. Here we show that α7 nAChR mediated calcium signals in differentiating PC12 cells activate the proteolytic enzyme calpain leading to spectrin breakdown, microtubule retraction, and attenuation in neurite growth. Imaging in growth cones confirms that α7 activation decreases EB3 comet motility in a calcium dependent manner as demonstrated by the ability of α7 nAChR, ryanodine, or IP3 receptor antagonists to block the effect of α7 nAChR on growth. α7 nAChR mediated EB3 comet motility, spectrin breakdown, and neurite growth was also inhibited by the addition of the selective calpain blocker calpeptin and attenuated by the expression of an α7 subunit unable to bind Gαq and activate calcium store release. The findings indicate that α7 nAChRs regulate cytoskeletal dynamics through local calcium signals for calpain protease activity.
Asunto(s)
Calcio/metabolismo , Calpaína/metabolismo , Conos de Crecimiento/metabolismo , Neuritas/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Calpaína/antagonistas & inhibidores , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Conos de Crecimiento/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neuritas/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Neurogénesis/fisiología , Neurotransmisores/farmacología , Células PC12 , Ratas , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Espectrina/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidoresRESUMEN
Acetylcholine activation of α7 nicotinic acetylcholine receptors (α7 nAChRs) in microglia attenuates neuroinflammation and regulates TNF-α release. We used lipopolysaccharide to model inflammation in the microglial cell line EOC20 and examined signaling by the α7 nAChR. Co-immunoprecipitation experiments confirm that α7 nAChRs bind heterotrimeric G proteins in EOC20 cells. Interaction with Gαi mediates α7 nAChR signaling via enhanced intracellular calcium release and a decrease in cAMP, p38 phosphorylation, and TNF-α release. These α7 nAChR effects were blocked by the inhibition of Gαi signaling via pertussis toxin, PLC activity with U73122, and α7 nAChR channel activity with the selective antagonist α-bungarotoxin. Moreover, α7 nAChR signaling in EOC20 cells was significantly diminished by the expression of a dominant-negative α7 nAChR, α7345-8A, shown to be impaired in G protein binding. These findings indicate an essential role for G protein coupling in α7 nAChR function in microglia leading to the regulation of inflammation in the nervous system.
RESUMEN
Although menthol, a common flavoring additive to cigarettes, has been found to impact the addictive properties of nicotine cigarettes in smokers little is known about its pharmacological and molecular actions in the brain. Studies were undertaken to examine whether the systemic administration of menthol would modulate nicotine pharmacokinetics, acute pharmacological effects (antinociception and hypothermia) and withdrawal in male ICR mice. In addition, we examined changes in the brain levels of nicotinic receptors of rodents exposed to nicotine and menthol. Administration of i.p. menthol significantly decreased nicotine's clearance (2-fold decrease) and increased its AUC compared to i.p. vehicle treatment. In addition, menthol pretreatment prolonged the duration of nicotine-induced antinociception and hypothermia (2.5 mg/kg, s.c.) for periods up to 180 min post-nicotine administration. Repeated administration of menthol with nicotine increased the intensity of mecamylamine-precipitated withdrawal signs in mice exposed chronically to nicotine. The potentiation of withdrawal intensity by menthol was accompanied by a significant increase in nicotine plasma levels in these mice. Western blot analyses of α4 and ß2 nAChR subunit expression suggests that chronic menthol impacts the levels and distribution of these nicotinic subunits in various brain regions. In particular, co-administration of menthol and nicotine appears to promote significant increase in ß2 and α4 nAChR subunit expression in the hippocampus, prefrontal cortex and striatum of mice. Surprisingly, chronic injections of menthol alone to mice caused an upregulation of ß2 and α4 nAChR subunit levels in these brain regions. Because the addition of menthol to tobacco products has been suggested to augment their addictive potential, the current findings reveal several new pharmacological molecular adaptations that may contribute to its unique addictive profile.