RESUMEN
Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMPâ»Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.
Asunto(s)
Señalización del Calcio , Cerebelo/citología , GMP Cíclico/metabolismo , Ácido Glutámico/metabolismo , Neuronas/citología , Óxido Nítrico/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Cerebelo/metabolismo , GMP Cíclico/análogos & derivados , Transferencia Resonante de Energía de Fluorescencia , Guanilato Ciclasa/metabolismo , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Imagen Óptica , Tionucleótidos/metabolismoRESUMEN
Nitric oxide (NO/cyclic guanosine monophosphate (cGMP)-regulated cellular mechanisms are involved in a variety of (patho-) physiological processes. One of the main effector molecules in this system, proteinkinase G (PKG), serves as a molecular switch by phosphorylating different target proteins and thereby turning them on or off. To date, only a few interaction partners of PKG have been described although the identification of protein-protein interactions (PPI) is indispensable for the understanding of cellular processes and diseases. Conventionally used methods to detect PPIs exhibit several disadvantages, e.g., co-immunoprecipitations, which depend on suitable high-affinity antibodies. Therefore, we established a cell-based protein-fragment complementation assay (PCA) for the identification of PKG target proteins. Here, a reporter protein (click beetle luciferase) is split into two fragments and fused to two different possible interaction partners. If interaction occurs, the reporter protein is functionally complemented and the catalyzed reaction can then be quantitatively measured. By using this technique, we confirmed the regulator of G-Protein signaling 2 (RGS2) as an interaction partner of PKGIα (a PKG-isoform) following stimulation with 8-Br-cGMP and 8-pCPT-cGMP. Hence, our results support the conclusion that the established approach could serve as a novel tool for the rapid, easy and cost-efficient detection of novel PKG target proteins.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Luciferasas/metabolismo , Mapeo de Interacción de Proteínas/métodos , Proteínas RGS/metabolismo , Animales , Células COS , Chlorocebus aethiops , GMP Cíclico/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Ratones , Óxido Nítrico/metabolismo , FosforilaciónRESUMEN
Atrial natriuretic peptide (ANP)/cGMPs cause diuresis and natriuresis. Their downstream effectors beyond cGMP remain unclear. To elucidate a probable function of cGMP-dependent protein kinase II (cGKII), we investigated renal parameters in different conditions (basal, salt diets, starving, water load) using a genetically modified mouse model (cGKII-KO), but did not detect any striking differences between WT and cGKII-KO. Thus, cGKII is proposed to play only a marginal role in the adjustment of renal concentration ability to varying salt loads without water restriction or starving conditions. When WT mice were subjected to a volume load (performed by application of a 10-mM glucose solution (3% of BW) via feeding needle), they exhibited a potent diuresis. In contrast, urine volume was decreased significantly in cGKII-KO. We showed that AQP2 plasma membrane (PM) abundance was reduced for about 50% in WT upon volume load, therefore, this might be a main cause for the enhanced diuresis. In contrast, cGKII-KO mice almost completely failed to decrease AQP2-PM distribution. This significant difference between both genotypes is not induced by an altered p-Ser256-AQP2 phosphorylation, as phosphorylation at this site decreases similarly in WT and KO. Furthermore, sodium excretion was lowered in cGKII-KO mice during volume load. In summary, cGKII is only involved to a minor extent in the regulation of basal renal concentration ability. By contrast, cGKII-KO mice are not able to handle an acute volume load. Our results suggest that membrane insertion of AQP2 is inhibited by cGMP/cGKII.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo II/metabolismo , Diuresis , Eliminación Renal , Animales , Acuaporina 2/genética , Acuaporina 2/metabolismo , Membrana Celular/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo II/genética , Glucosa/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Riñón/fisiología , Ratones , Fosforilación , Transporte de Proteínas , Sodio/metabolismoRESUMEN
Cyclic guanosine monophosphate (cGMP) is synthesized by nitric oxide or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions in the kidney. Hence, integration of cGMP signaling by cGMP-dependent protein kinases (cGKs) might play a critical role in renal physiology; however, detailed renal localization of cGKs is still lacking. Here, we performed an immunohistochemical analysis of cGKIα and cGKIß isozymes in the mouse kidney and found both in arterioles, the mesangium, and within the cortical interstitium. In contrast to cGKIα, the ß-isoform was not detected in the juxtaglomerular apparatus or medullary fibroblasts. Since interstitial fibroblasts play a prominent role in interstitial fibrosis, we focused our study on cGKI function in the interstitium, emphasizing a functional differentiation of both isoforms, and determined whether cGKIs influence renal fibrosis induced by unilateral ureter obstruction. Treatment with the guanylyl cyclase activators YC1 or isosorbide dinitrate showed stronger antifibrotic effects in wild-type than in cGKI-knockout or in smooth muscle-cGKIα-rescue mice, which are cGKI deficient in the kidney except in the renal vasculature. Moreover, fibrosis influenced the mRNA and protein expression levels of cGKIα more strongly than cGKIß. Thus, our results indicate that cGMP, acting primarily through cGKIα, is an important suppressor of kidney fibrosis.
Asunto(s)
Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Enfermedades Renales/prevención & control , Riñón/enzimología , Animales , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/deficiencia , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Modelos Animales de Enfermedad , Activación Enzimática , Activadores de Enzimas/farmacología , Fibroblastos/enzimología , Fibroblastos/patología , Fibronectinas/genética , Fibronectinas/metabolismo , Fibrosis , Regulación Enzimológica de la Expresión Génica , Guanilato Ciclasa/metabolismo , Isoenzimas , Riñón/efectos de los fármacos , Riñón/patología , Enfermedades Renales/enzimología , Enfermedades Renales/etiología , Enfermedades Renales/patología , Ratones , Ratones de la Cepa 129 , ARN Mensajero/metabolismo , Proteína de Unión al Calcio S100A4 , Proteínas S100/genética , Proteínas S100/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Obstrucción Ureteral/complicaciones , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Adiponectin is an adipocyte-derived protein with atheroprotective and immunoregulatory function. Adiponectin and activin A reduce foam cell formation and adiponectin activates the p38 MAPK pathway that is well described to induce activin A. Therefore, it was analyzed whether adiponectin alters activin A in primary human monocytes. Adiponectin dose- and time-dependently induced activin A in the supernatant, and the maximal amount was observed after 12h of incubation. Adiponectin-stimulated release of activin A was blocked by a p38 MAPK inhibitor. Metformin and pioglitazone are drugs frequently used to treat diabetic patients and metformin slightly reduced monocytic activin A release whereas pioglitazone had no effect. Type 2 diabetes is associated with elevated inflammatory systemic cytokines but activin A serum levels were similar in slim probands, overweight controls and type 2 diabetic patients. Furthermore, activin A did not correlate to systemic adiponectin, body mass index, waist to hip ratio or C-reactive protein. These findings indicate that adiponectin upregulates monocytic activin A release via the p38 MAPK pathway, and this may in part explain the immunoregulatory and antiatherosclerotic effects of this adipokine.
Asunto(s)
Activinas/metabolismo , Adiponectina/farmacología , Diabetes Mellitus Tipo 2/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Obesidad/metabolismo , Anciano , Células Cultivadas , Humanos , Hipoglucemiantes/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Metformina/farmacología , Persona de Mediana Edad , Monocitos/citología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Pharmacological inhibition of the renin-angiotensin-aldosterone system (RAAS) is, in combination with diuretics, the first-choice treatment for hypertension, although 10-20% of patients do not respond adequately. Next to the RAAS, the nitric oxide/cGMP/protein kinase G (PKG) system is the second fundamental blood pressure regulator. Whether both systems influence each other is not well-studied. It has been shown that nitric oxide (NO) supports renin recruitment via activation of soluble guanylate cyclase (sGC) and subsequent generation of cGMP. Whether this leads to an ensuing activation of PKGs in this context is not known. PKGIα, as well as PKGII, is expressed in renin-producing cells. Hence, we analyzed whether these enzymes play a role regarding renin synthesis, secretion, or recruitment. We generated renin-cell-specific PKGI-knockout mice and either stimulated or inhibited the renin system in these mice by salt diets. To exclude the possibility that one kinase isoform can compensate the lack of the other, we also studied double-knockout animals with a conditional knockout of PKGI in juxtaglomerular cells (JG cells) and a ubiquitous knockout of PKGII. We analyzed blood pressure, renin mRNA and renal renin protein content as well as plasma renin concentration. Furthermore, we stimulated the cGMP system in these mice using BAY 41-8543, an sGC stimulator, and examined renin regulation either after acute administration or after 7 days (application once daily). We did not reveal any striking differences regarding long-term renin regulation in the studied mouse models. Yet, when we studied the acute effect of BAY 41-8543 on renin secretion in isolated perfused kidneys as well as in living animals, we found that the administration of the substance led to a significant increase in plasma renin concentration in control animals. This effect was completely abolished in double-knockout animals. However, after 7 days of once daily application, we did not detect a persistent increase in renin mRNA or protein in any studied genotype. Therefore, we conclude that in mice, cGMP and PKG are involved in the acute regulation of renin release but have no influence on long-term renin adjustment.
RESUMEN
Agents that enhance production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) ameliorate the progression of renal fibrosis. However, the molecular mechanism of this process is not fully understood. We hypothesize that the antifibrotic effects of cGMP and cGMP-dependent kinase I (cGKI) are mediated via regulation of the TGFß signalling pathway, both via ERK and the Smad-dependent route. Kidney fibrosis was induced by unilateral ureter obstruction (UUO) in wild-type and cGKI-deficient (cGKI-KO) mice. The cGMP/cGKI signalling pathway was activated by application of the soluble guanylate cyclase (sGC) stimulator BAY 41-8543 (BAY), beginning 1 day after UUO. After 7 days, the antifibrotic effects of BAY were analysed by measuring mRNA and protein expression of characteristic fibrotic biomarkers. The effects of cGMP/TGFß on cultured fibroblasts were also analysed in vitro. BAY application influenced the activity of the extracellular matrix (ECM)-degrading matrix metalloproteases (MMP2 and MMP9) and their inhibitor tissue inhibitors of metalloproteinase-1, the secretion of cytokines (e.g. IL-6) and the expression pattern of ECM proteins (e.g. collagen, fibronectin) and profibrotic mediators (e.g. connective tissue growth factors and plasminogen-activator inhibitor-1). Activation of the cGMP/cGKI signalling pathway showed protective effects against fibrosis which were mediated by inhibition of P-Erk1/2 and translocation of P-smad3. The elucidation of these signalling mechanisms might support the development of new therapeutic options regarding cGMP/cGKI-mediated antifibrotic actions.