RESUMEN
As the development of combination antiretroviral therapy (cART) against human immunodeficiency virus (HIV) drastically improves the lifespan of individuals with HIV, many are now entering the prime age when Alzheimer's disease (AD)-like symptoms begin to manifest. It has been shown that hyperphosphorylated tau, a known AD pathological characteristic, is prematurely increased in the brains of HIV-infected individuals as early as in their 30s and that its levels increase with age. This suggests that HIV infection might lead to accelerated AD phenotypes. However, whether HIV infection causes AD to develop more quickly in the brain is not yet fully determined. Interestingly, we have previously revealed that the viral glycoproteins HIV gp120 and feline immunodeficiency virus (FIV) gp95 induce neuronal hyperexcitation via cGMP-dependent kinase II (cGKII; also known as PRKG2) activation in cultured hippocampal neurons. Here, we use cultured mouse cortical neurons to demonstrate that the presence of HIV gp120 and FIV gp95 are sufficient to increase cellular tau pathology, including intracellular tau hyperphosphorylation and tau release to the extracellular space. We further reveal that viral glycoprotein-induced cellular tau pathology requires cGKII activation. Taken together, HIV infection likely accelerates AD-related tau pathology via cGKII activation.
Asunto(s)
Enfermedad de Alzheimer , Infecciones por VIH , Virus de la Inmunodeficiencia Felina , Enfermedad de Alzheimer/patología , Animales , Gatos , Glicoproteínas , Virus de la Inmunodeficiencia Felina/fisiología , Ratones , Neuronas/patología , Proteínas tau/genéticaRESUMEN
The L-type Cav1.2 calcium channel is present throughout the animal kingdom and is essential for some aspects of CNS function, cardiac and smooth muscle contractility, neuroendocrine regulation, and multiple other processes. The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel properties have been analyzed in heterologous expression systems including regulation of the L-type CaV1.2 channel by Ca(2+) itself and protein kinases. However, targeted mutations of the calcium channel genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine showed that ß-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of the CaVß2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca(2+)-dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2α1 gene. This article summarizes recent findings on the in vivo relevance of well-established in vitro results.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Calcio/metabolismo , Animales , Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/genética , Humanos , Cinética , Fosforilación/fisiología , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca2+ activity in neurons, stimulating various pathways to damage synaptic functions. We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95's stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Felino/virología , VIH-1/fisiología , Virus de la Inmunodeficiencia Felina/fisiología , Sinapsis/metabolismo , Animales , Calcio/metabolismo , Gatos , Quimiocina CXCL12/farmacología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/metabolismo , Hipocampo/patología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Subunidades de Proteína/metabolismo , Receptores AMPA/metabolismo , Proteínas Virales/metabolismoRESUMEN
Agonist-triggered downregulation of ß-adrenergic receptors (ARs) constitutes vital negative feedback to prevent cellular overexcitation. Here, we report a novel downregulation of ß2AR signaling highly specific for Cav1.2. We find that ß2-AR binding to Cav1.2 residues 1923-1942 is required for ß-adrenergic regulation of Cav1.2. Despite the prominence of PKA-mediated phosphorylation of Cav1.2 S1928 within the newly identified ß2AR binding site, its physiological function has so far escaped identification. We show that phosphorylation of S1928 displaces the ß2AR from Cav1.2 upon ß-adrenergic stimulation rendering Cav1.2 refractory for several minutes from further ß-adrenergic stimulation. This effect is lost in S1928A knock-in mice. Although AMPARs are clustered at postsynaptic sites like Cav1.2, ß2AR association with and regulation of AMPARs do not show such dissociation. Accordingly, displacement of the ß2AR from Cav1.2 is a uniquely specific desensitization mechanism of Cav1.2 regulation by highly localized ß2AR/cAMP/PKA/S1928 signaling. The physiological implications of this mechanism are underscored by our finding that LTP induced by prolonged theta tetanus (PTT-LTP) depends on Cav1.2 and its regulation by channel-associated ß2AR.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Procesamiento Proteico-Postraduccional , Receptores Adrenérgicos beta 2/metabolismo , Animales , Ratones , FosforilaciónRESUMEN
The cyclic guanosine monophosphate (cGMP) signaling system is one of the most prominent regulators of a variety of physiological and pathophysiological processes in many mammalian and non-mammalian tissues. Targeting this pathway by increasing cGMP levels has been a very successful approach in pharmacology as shown for nitrates, phosphodiesterase (PDE) inhibitors and stimulators of nitric oxide-guanylyl cyclase (NO-GC) and particulate GC (pGC). This is an introductory review to the cGMP signaling system intended to introduce those readers to this system, who do not work in this area. This article does not intend an in-depth review of this system. Signal transduction by cGMP is controlled by the generating enzymes GCs, the degrading enzymes PDEs and the cGMP-regulated enzymes cyclic nucleotide-gated ion channels, cGMP-dependent protein kinases and cGMP-regulated PDEs. Part A gives a very concise introduction to the components. Part B gives a very concise introduction to the functions modulated by cGMP. The article cites many recent reviews for those who want a deeper insight.
Asunto(s)
GMP Cíclico/metabolismo , Animales , Guanilato Ciclasa/metabolismo , Humanos , Óxido Nítrico/metabolismo , Transducción de SeñalRESUMEN
The underlying cause of cardiac hypertrophy, fibrosis, and heart failure has been investigated in great detail using different mouse models. These studies indicated that cGMP and cGMP-dependent protein kinase type I (cGKI) may ameliorate these negative phenotypes in the adult heart. Recently, evidence has been published that cardiac mitochondrial BKCa channels are a target for cGKI and that activation of mitoBKCa channels may cause some of the positive effects of conditioning in ischemia/reperfusion injury. It will be pointed out that most studies could not present convincing evidence that it is the cGMP level and the activity cGKI in specific cardiac cells that reduces hypertrophy or heart failure. However, anti-fibrotic compounds stimulating nitric oxide-sensitive guanylyl cyclase may be an upcoming therapy for abnormal cardiac remodeling.
Asunto(s)
Cardiomegalia/enzimología , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Insuficiencia Cardíaca/enzimología , Daño por Reperfusión Miocárdica/enzimología , Miocardio/enzimología , Remodelación Ventricular , Animales , Cardiomegalia/tratamiento farmacológico , Cardiomegalia/patología , Cardiomegalia/fisiopatología , Fármacos Cardiovasculares/uso terapéutico , Fibrosis , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Miocardio/patología , Sistemas de Mensajero Secundario , Remodelación Ventricular/efectos de los fármacosRESUMEN
OBJECTIVES: Stimulators of soluble guanylate cyclase (sGC) are currently investigated in clinical trials for the treatment of fibrosis in systemic sclerosis (SSc). In this study, we aim to investigate the role of protein kinases G (PKG) as downstream mediators of sGC-cyclic guanosine monophosphate (cGMP) in SSc. METHODS: Mice with combined knockout of PKG1 and 2 were challenged with bleomycin and treated with the sGC stimulator BAY 41-2272. Fibroblasts were treated with BAY 41-2272 and with the PKG inhibitor KT 5823. RESULTS: PKG1 and 2 are upregulated in SSc in a transforming growth factor-ß1 (TGFß1)-dependent manner, as an attempt to compensate for the decreased signalling through the sGC-cGMP-PKG pathway. Inhibition or knockout of PKG1 and 2 abrogates the inhibitory effects of sGC stimulation on fibroblast activation in a SMAD-independent, but extracellular signal-regulated kinase (ERK)-dependent manner. In vivo, sGC stimulation fails to prevent bleomycin-induced fibrosis in PKG1 and 2 knockout mice. CONCLUSIONS: Our data provide evidence that PKGs are essential mediators of the antifibrotic effects of sGC stimulators through interfering with non-canonical TGFß signalling. TGFß1 promotes its profibrotic effects through inhibition of sGC-cGMP-PKG signalling, sGC stimulation exerts its antifibrotic effects by inhibition of TGFß1-induced ERK phosphorylation.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Fibroblastos/metabolismo , Esclerodermia Sistémica/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Adulto , Anciano , Animales , Bleomicina/farmacología , Western Blotting , Carbazoles/farmacología , Técnicas de Cultivo de Célula , Femenino , Fibroblastos/efectos de los fármacos , Fibrosis/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Pirazoles/farmacología , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Esclerodermia Sistémica/patología , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Signaling through cGMP has therapeutic potential in the colon, where it has been implicated in the suppression of colitis and colon cancer. In this study, we tested the ability of cGMP and type 2 cGMP-dependent protein kinase (PKG2) to activate forkhead box O (FoxO) in colon cancer cells and in the colon epithelium of mice. We show that activation of PKG2 in colon cancer cells inhibited cell proliferation, inhibited AKT, and activated FoxO. Treatment of colon explants with 8Br-cGMP also activated FoxO target gene expression at both RNA and protein levels, and reduced epithelial reduction-oxidation (redox) stress. FoxO3a was the most prominent isoform in the distal colon epithelium, with prominent luminal staining. FoxO3a levels were reduced in Prkg2-/- animals, and FoxO target genes were unaffected by 8Br-cGMP challenge in vitro. Treatment of mice with the phosphodiesterase-5 inhibitor vardenafil (Levitra) mobilized FoxO3a to the nucleus of luminal epithelial cells, which corresponded to increased FoxO target gene expression, reduced redox stress, and increased epithelial barrier integrity. Treatment of human colonic biopsy specimens with 8Br-cGMP also activated catalase and manganese superoxide dismutase expression, indicating that this pathway is conserved in humans. Taken together, these results identify a novel signaling pathway in the colon epithelium, where FoxO tumor suppressors could provide protection from redox stress. Moreover, this pathway is regulated by endogenous cGMP/PKG2 signaling, and can be targeted using phosphodiesterase-5 inhibitors.
Asunto(s)
Antioxidantes/metabolismo , Neoplasias del Colon/metabolismo , Proteína Forkhead Box O3/metabolismo , Mucosa Intestinal/metabolismo , Transducción de Señal/fisiología , Animales , Western Blotting , Línea Celular Tumoral , GMP Cíclico/metabolismo , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TranscriptomaRESUMEN
Gene knockout (KO) does not always result in phenotypic changes, possibly due to mechanisms of functional compensation. We have studied mice lacking cGMP-dependent kinase II (cGKII), which phosphorylates GluA1, a subunit of AMPA receptors (AMPARs), and promotes hippocampal long-term potentiation (LTP) through AMPAR trafficking. Acute cGKII inhibition significantly reduces LTP, whereas cGKII KO mice show no LTP impairment. Significantly, the closely related kinase, cGKI, does not compensate for cGKII KO. Here, we describe a previously unidentified pathway in the KO hippocampus that provides functional compensation for the LTP impairment observed when cGKII is acutely inhibited. We found that in cultured cGKII KO hippocampal neurons, cGKII-dependent phosphorylation of inositol 1,4,5-trisphosphate receptors was decreased, reducing cytoplasmic Ca(2+) signals. This led to a reduction of calcineurin activity, thereby stabilizing GluA1 phosphorylation and promoting synaptic expression of Ca(2+)-permeable AMPARs, which in turn induced a previously unidentified form of LTP as a compensatory response in the KO hippocampus. Calcineurin-dependent Ca(2+)-permeable AMPAR expression observed here is also used during activity-dependent homeostatic synaptic plasticity. Thus, a homeostatic mechanism used during activity reduction provides functional compensation for gene KO in the cGKII KO hippocampus.
Asunto(s)
Calcio/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Hipocampo/enzimología , Receptores AMPA/metabolismo , Animales , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Homeostasis/efectos de los fármacos , Potenciación a Largo Plazo , Ratones , Ratones Noqueados , Fosforilación , Sinapsis/enzimología , Sinapsis/metabolismo , Tetrodotoxina/farmacologíaRESUMEN
Beta-adrenergic stimulation of the heart increases ICa. PKA dependent phosphorylation of several amino acids (among them Ser 1700 and Thr 1704 in the carboxy-terminus of the Cav1.2 α1c subunit) has been implicated as decisive for the ß-adrenergic up-regulation of cardiac ICa. Mutation of Ser 1700 and Thr 1704 to alanine results in the Cav1.2PKA_P2-/- mice. Cav1.2PKA_P2-/- mice display reduced cardiac L-type current. Fractional shortening and ejection fraction in the intact animal and ICa in isolated cardiomyocytes (CM) are stimulated by isoproterenol. Cardiac specific expression of the mutated Cav1.2PKA_P2-/- gene reduces Cav1.2 α1c protein concentration, ICa, and the ß-adrenergic stimulation of L-type ICa in CMs. Single channels were not detected on the CM surface of the cCav1.2PKA_P2-/- hearts. This outcome supports the notion that S1700/1704 is essential for expression of the Cav1.2 channel and that isoproterenol stimulates ICa in Cav1.2PKA_P2-/- CMs.
Asunto(s)
Canales de Calcio Tipo L/genética , Mutación/genética , Miocardio/metabolismo , Receptores Adrenérgicos beta/metabolismo , Animales , Activación del Canal Iónico/efectos de los fármacos , Isoproterenol/farmacología , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Fenotipo , Tamoxifeno/farmacologíaRESUMEN
Conflicting results have been reported for the roles of cGMP and cGMP-dependent protein kinase I (cGKI) in various pathological conditions leading to cardiac hypertrophy and fibrosis. A cardioprotective effect of cGMP/cGKI has been reported in whole animals and isolated cardiomyocytes, but recent evidence from a mouse model expressing cGKIß only in smooth muscle (ßRM) but not in cardiomyocytes, endothelial cells, or fibroblasts has forced a reevaluation of the requirement for cGKI activity in the cardiomyocyte antihypertrophic effects of cGMP. In particular, ßRM mice developed the same hypertrophy as WT controls when subjected to thoracic aortic constriction or isoproterenol infusion. Here, we challenged ßRM and WT (Ctr) littermate control mice with angiotensin II (AII) infusion (7 d; 2 mg â kg(-1) â d(-1)) to induce hypertrophy. Both genotypes developed cardiac hypertrophy, which was more pronounced in Ctr animals. Cardiomyocyte size and interstitial fibrosis were increased equally in both genotypes. Addition of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, in the drinking water had a small effect in reducing myocyte hypertrophy in WT mice and no effect in ßRM mice. However, sildenafil substantially blocked the increase in collagen I, fibronectin 1, TGFß, and CTGF mRNA in Ctr but not in ßRM hearts. These data indicate that, for the initial phase of AII-induced cardiac hypertrophy, lack of cardiomyocyte cGKI activity does not worsen hypertrophic growth. However, expression of cGKI in one or more cell types other than smooth muscle is necessary to allow the antifibrotic effect of sildenafil.
Asunto(s)
Angiotensina II/farmacología , Cardiomegalia/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 5/metabolismo , Hipertensión/metabolismo , Animales , Cardiomegalia/inducido químicamente , GMP Cíclico/metabolismo , Fibrosis/inducido químicamente , Fibrosis/metabolismo , Marcadores Genéticos , Hipertensión/inducido químicamente , Ratones , Músculo Liso/metabolismo , Contracción Miocárdica/efectos de los fármacos , Contracción Miocárdica/fisiología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Inhibidores de Fosfodiesterasa 5/farmacología , Piperazinas/farmacología , Purinas/farmacología , Citrato de Sildenafil , Sulfonas/farmacología , Vasoconstrictores/farmacologíaRESUMEN
The nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (cGK) signaling pathway regulates the clustering and the recruitment of proteins and vesicles to the synapse, thereby adjusting the exoendocytic cycle to the intensity of activity. Accordingly, this pathway can accelerate endocytosis following large-scale exocytosis, and pre-synaptic cGK type II (cGKII) plays a major role in this process, controlling the homeostatic balance of vesicle exocytosis and endocytosis. We have studied synaptic vesicle recycling in cerebellar granule cells from mice lacking cGKII under strong and sustained stimulation, combining imaging techniques and ultrastructural analyses. The ultrastructure of synapses in the adult mouse cerebellar cortex was also examined in these animals. The lack of cGKII provokes structural changes to synapses in cultured cells and in the cerebellar cortex. Moreover, endocytosis is slowed down in a subset of boutons in these cells when they are stimulated strongly. In addition, from the results obtained with the selective inhibitor of cGKs, KT5823, it can be concluded that cGKI also regulates some aspects of vesicle cycling. Overall, these results confirm the importance of the cGMP pathway in the regulation of vesicle cycling following strong stimulation of cerebellar granule cells.
Asunto(s)
Cerebelo/citología , Cerebelo/metabolismo , GMP Cíclico/metabolismo , Neuronas/metabolismo , Proteínas Quinasas/metabolismo , Membranas Sinápticas/metabolismo , Animales , Endocitosis , Exocitosis , Técnica del Anticuerpo Fluorescente , Potenciales de la Membrana , Ratones , Ratones Noqueados , Imagen Molecular , Neuronas/ultraestructura , Proteínas Quinasas/genética , Vesículas Secretoras/metabolismo , Membranas Sinápticas/ultraestructuraRESUMEN
Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO hippocampus is increased as a functional compensation for gene deletion, while such compensation is absent in the prefrontal cortex. Thus, there are brain region-specific effects of cGKII KO on AMPAR trafficking, which could affect animal behavior. Here, we show that GluA1 phosphorylation levels differ in various brain regions, and specific behaviors are altered according to region-specific changes in GluA1 phosphorylation. Moreover, we identified distinct regulations of phosphatases in different brain regions, leading to regional heterogeneity of GluA1 phosphorylation in the KO brain. Our work demonstrates region-specific changes in GluA1 phosphorylation in cGKII KO mice and corresponding effects on cognitive performance. We also reveal distinct regulation of phosphatases in different brain region in which region-specific effects of kinase gene KO arise and can selectively alter animal behavior.
Asunto(s)
Conducta Animal/fisiología , Encéfalo/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Transporte de Proteínas , Receptores AMPA/metabolismo , Amígdala del Cerebelo/metabolismo , Animales , Condicionamiento Clásico , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Depresión/fisiopatología , Miedo/fisiología , Hipocampo/metabolismo , Masculino , Ratones Endogámicos C57BL , Bulbo Olfatorio/metabolismo , Fosforilación , Corteza Prefrontal/metabolismo , Olfato/fisiologíaRESUMEN
Cav1.2 and Cav1.3 are the major L-type voltage-gated Ca(2+) channels in the CNS. Yet, their individual in vivo functions are largely unknown. Both channel subunits are expressed in the auditory brainstem, where Cav1.3 is essential for proper maturation. Here, we investigated the role of Cav1.2 by targeted deletion in the mouse embryonic auditory brainstem. Similar to Cav1.3, loss of Cav1.2 resulted in a significant decrease in the volume and cell number of auditory nuclei. Contrary to the deletion of Cav1.3, the action potentials of lateral superior olive (LSO) neurons were narrower compared with controls, whereas the firing behavior and neurotransmission appeared unchanged. Furthermore, auditory brainstem responses were nearly normal in mice lacking Cav1.2. Perineuronal nets were also unaffected. The medial nucleus of the trapezoid body underwent a rapid cell loss between postnatal days P0 and P4, shortly after circuit formation. Phosphorylated cAMP response element-binding protein (CREB), nuclear NFATc4, and the expression levels of p75NTR, Fas, and FasL did not correlate with cell death. These data demonstrate for the first time that both Cav1.2 and Cav1.3 are necessary for neuronal survival but are differentially required for the biophysical properties of neurons. Thus, they perform common as well as distinct functions in the same tissue.
Asunto(s)
Vías Auditivas/citología , Tronco Encefálico/citología , Canales de Calcio Tipo L/fisiología , Potenciales de Acción/fisiología , Animales , Vías Auditivas/metabolismo , Tronco Encefálico/metabolismo , Muerte Celular , Matriz Extracelular/metabolismo , RatonesRESUMEN
The voltage-gated Ca(2+) (CaV) channel acts as a key player in ß cell physiology and pathophysiology. ß cell CaV channels undergo hyperactivation subsequent to exposure to type 1 diabetic (T1D) serum resulting in increased cytosolic free Ca(2+) concentration and thereby Ca(2+)-triggered ß cell apoptosis. The present study was aimed at revealing the subtypes of CaV1 channels hyperactivated by T1D serum as well as the biophysical mechanisms responsible for T1D serum-induced hyperactivation of ß cell CaV1 channels. Patch-clamp recordings and single-cell RT-PCR analysis were performed in pancreatic ß cells from CaV1 channel knockout and corresponding control mice. We now show that functional CaV1.3 channels are expressed in a subgroup of islet ß cells from CaV1.2 knockout mice (CaV1.2(-/-)). T1D serum enhanced whole-cell CaV currents in islet ß cells from CaV1.3 knockout mice (CaV1.3(-/-)). T1D serum increased the open probability and number of functional unitary CaV1 channels in CaV1.2(-/-) and CaV1.3(-/-) ß cells. These data demonstrate that T1D serum hyperactivates both CaV1.2 and CaV1.3 channels by increasing their conductivity and number. These findings suggest CaV1.2 and CaV1.3 channels as potential targets for anti-diabetes therapy.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Diabetes Mellitus Tipo 1/sangre , Células Secretoras de Insulina/metabolismo , Animales , Canales de Calcio Tipo L/genética , Células Cultivadas , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Masculino , Ratones , Ratones NoqueadosRESUMEN
Signaling via cGMP-dependent protein kinase I (cGKI) and canonical transient receptor potential (TRPC) channels appears to be involved in the regulation of cardiac hypertrophy. Recent evidence suggests that TRPC channels are targets for cGKI, and phosphorylation of these channels may mediate the antihypertrophic effects of cGMP signaling. We tested this concept by investigating the role of cGMP/cGKI signaling on angiotensin II (A II)-induced cardiac hypertrophy using a control group (Ctr), trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM mice, and trpc3(-/-)/6(-/-) × ßRM mice. ßRM mice express cGKIß only in the smooth muscle on a cGKI(-/-) background. The control group was composed of littermate mice that contained at least one wild type gene of the respective genotype. A II was infused by minipumps (7 days; 2 mg/kg/day) in Ctr, trpc6(-/-), trpc3(-/-), trpc3(-/-)/6(-/-), ßRM, and trpc3(-/-)/6(-/-) × ßRM mice. Hypertrophy was assessed by measuring heart weight per tibia length (HW/TL) and fibrosis by staining of heart slices. A II-induced increase in HW/TL and fibrosis was absent in trpc3 (-/-) mice, whereas an increase in HW/TL and fibrosis was evident in Ctr and trpc6(-/-), minimal or absent in trpc3(-/-), moderate in ßRM, and dramatic in trpc3(-/-)/6(-/-) ßRM mice. These results suggest that TRPC3 may be necessary for A II-induced cardiac hypertrophy. On the other hand, hypertrophy and fibrosis were massively increased in ßRM mice on a TRPC3/6 × cGKI(-/-)KO background, indicating an "additive" coupling between both signaling pathways.
Asunto(s)
Cardiomegalia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Cardiomegalia/patología , Fibrosis , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Canales Catiónicos TRPC/genéticaRESUMEN
α1-Adrenergic stimulation increases blood vessel tone in mammals. This process involves a number of intracellular signaling pathways that include signaling via phospholipase C, diacylglycerol (DAG), and protein kinase C. So far, it is not certain whether signaling via phospholipase D (PLD) and PLD-derived DAG is involved in this process. We asked whether PLD participates in the α1-adrenergic-mediated signaling in vascular smooth muscle. α1-Adrenergic-induced contraction was assessed by myography of isolated aortic rings and by pressure recordings using the hindlimb perfusion model in mice. The effects of the PLD inhibitor 1-butanol (IC50 0.15 vol%) and the inactive congener 2-butanol were comparatively studied. Inhibition of PLD by 1-butanol reduced specifically the α1-adrenergic-induced contraction and the α1-adrenergic-induced pressure increase by 10 and 40% of the maximum, respectively. 1-Butanol did not influence the aortic contractions induced by high extracellular potassium, by the thromboxane analog U46619, or by a phorbol ester. The effects of 1-butanol were absent in mice that lack PLD1 (Pld1(-/-) mice) or that selectively lack the CaV1.2 channel in smooth muscle (sm-CaV1.2(-/-) mice) but still present in the heterozygous control mice. α1-Adrenergic contraction of vascular smooth muscle involves activation of PLD1, which controls a portion of the α1-adrenergic-induced CaV1.2 channel activity.
Asunto(s)
Músculo Liso Vascular/fisiología , Fosfolipasa D/fisiología , Receptores Adrenérgicos alfa 1/fisiología , Animales , Calcio/fisiología , Ratones , Ratones Noqueados , Contracción Muscular , Fosfolipasa D/genéticaRESUMEN
Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I(-/-) mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I(-/-) mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I(-/-) mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Potenciación a Largo Plazo , Nociceptores/metabolismo , Dolor/patología , Aminoquinolinas/farmacología , Animales , Conducta Animal , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I , Proteínas Quinasas Dependientes de GMP Cíclico/genética , Activación Enzimática , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Eliminación de Gen , Guanilato Ciclasa/antagonistas & inhibidores , Guanilato Ciclasa/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Fibras Nerviosas/metabolismo , Fibras Nerviosas/patología , Nociceptores/efectos de los fármacos , Nociceptores/patología , Dolor/metabolismo , Técnicas de Placa-Clamp , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Transducción de Señal , Especificidad por Sustrato , Transmisión SinápticaRESUMEN
Stimulation of renal proximal tubule (PT) transport by angiotensin II (Ang II) is critical for regulation of BP. Notably, in rats, mice, and rabbits, the regulation of PT sodium transport by Ang II is biphasic: transport is stimulated by picomolar to nanomolar concentrations of Ang II but inhibited by nanomolar to micromolar concentrations of Ang II. However, little is known about the effects of Ang II on human PT transport. By functional analysis with isolated PTs obtained from nephrectomy surgery, we found that Ang II induces a dose-dependent profound stimulation of human PT transport by type 1 Ang II receptor (AT1)-dependent phosphorylation of extracellular signal-regulated kinase (ERK). In PTs of wild-type mice, the nitric oxide (NO) /cGMP/cGMP-dependent kinase II (cGKII) pathway mediated the inhibitory effect of Ang II. In PTs of cGKII-deficient mice, the inhibitory effect of Ang II was lost, but activation of the NO/cGMP pathway failed to phosphorylate ERK. Conversely, in human PTs, the NO/cGMP pathway mediated the stimulatory effect of Ang II by phosphorylating ERK independently of cGKII. These contrasting responses to the NO/cGMP pathway may largely explain the different modes of PT transport regulation by Ang II, and the unopposed marked stimulation of PT transport by high intrarenal concentrations of Ang II may be an important factor in the pathogenesis of human hypertension. Additionally, the previously unrecognized stimulatory effect of the NO/cGMP pathway on PT transport may represent a human-specific therapeutic target in hypertension.
Asunto(s)
Angiotensina II/administración & dosificación , Angiotensina II/fisiología , GMP Cíclico/fisiología , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/metabolismo , Óxido Nítrico/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In VitroRESUMEN
Mouse pancreatic ß- and α-cells are equipped with voltage-gated Na(+) currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at -100 mV and -50 mV in ß- and α-cells, respectively). Single-cell PCR analyses show that both α- and ß-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: ß-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na(+) current by 80%. In ß-cells, knockout of Scn9a lowers the Na(+) current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a(-/-) islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na(+) channel α subunit in both α- and ß-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in ß-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.