RESUMEN
Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst-evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC ß1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer-based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.
Asunto(s)
Guanilato Ciclasa/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patología , Óxido Nítrico/metabolismo , Ruido/efectos adversos , Receptores de Superficie Celular/metabolismo , Animales , Femenino , Células Ciliadas Auditivas Internas/efectos de los fármacos , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Morfolinas/farmacología , Pirimidinas/farmacología , Ratas , Ratas Wistar , Receptores de Superficie Celular/agonistas , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/patologíaRESUMEN
In the inner ear, cyclic guanosine monophosphate (cGMP) signaling has been described as facilitating otoprotection, which was previously observed through elevated cGMP levels achieved by phosphodiesterase 5 inhibition. However, to date, the upstream guanylyl cyclase (GC) subtype eliciting cGMP production is unknown. Here, we show that mice with a genetic disruption of the gene encoding the cGMP generator GC-A, the receptor for atrial and B-type natriuretic peptides, display a greater vulnerability of hair cells to hidden hearing loss and noise- and age-dependent hearing loss. This vulnerability was associated with GC-A expression in spiral ganglia and outer hair cells (OHCs) but not in inner hair cells (IHCs). GC-A knockout mice exhibited elevated hearing thresholds, most pronounced for the detection of high-frequency tones. Deficits in OHC input-output functions in high-frequency regions were already present in young GC-A-deficient mice, with no signs of an accelerated progression of age-related hearing loss or higher vulnerability to acoustic trauma. OHCs in these frequency regions in young GC-A knockout mice exhibited diminished levels of KCNQ4 expression, which is the dominant K+ channel in OHCs, and decreased activation of poly (ADP-ribose) polymerase-1, an enzyme involved in DNA repair. Further, GC-A knockout mice had IHC synapse impairments and reduced amplitudes of auditory brainstem responses that progressed with age and with acoustic trauma, in contrast to OHCs, when compared to GC-A wild-type littermates. We conclude that GC-A/cGMP-dependent signaling pathways have otoprotective functions and GC-A gene disruption differentially contributes to hair-cell damage in a healthy, aged, or injured system. Thus, augmentation of natriuretic peptide GC-A signaling likely has potential to overcome hidden and noise-induced hearing loss, as well as presbycusis.
RESUMEN
Mechanisms that limit thrombosis are poorly defined. One of the few known endogenous platelet inhibitors is nitric oxide (NO). NO activates NO sensitive guanylyl cyclase (NO-GC) in platelets, resulting in an increase of cyclic guanosine monophosphate (cGMP). Here we show, using cGMP sensor mice to study spatiotemporal dynamics of platelet cGMP, that NO-induced cGMP production in pre-activated platelets is strongly shear-dependent. We delineate a new mode of platelet-inhibitory mechanotransduction via shear-activated NO-GC followed by cGMP synthesis, activation of cGMP-dependent protein kinase I (cGKI), and suppression of Ca2+ signaling. Correlative profiling of cGMP dynamics and thrombus formation in vivo indicates that high cGMP concentrations in shear-exposed platelets at the thrombus periphery limit thrombosis, primarily through facilitation of thrombus dissolution. We propose that an increase in shear stress during thrombus growth activates the NO-cGMP-cGKI pathway, which acts as an auto-regulatory brake to prevent vessel occlusion, while preserving wound closure under low shear.
Asunto(s)
Plaquetas/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , GMP Cíclico/metabolismo , Óxido Nítrico/metabolismo , Trombosis/metabolismo , Animales , Calcio/metabolismo , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos , Ratones Transgénicos , Activación Plaquetaria , Transducción de Señal , Estrés Mecánico , Trombosis/fisiopatologíaRESUMEN
The original version of this Article contained an error in the description of Supplementary Movie 4, in which the final sentence was inadvertently truncated. The HTML has been updated to include a corrected version of the 'Description of Additional Supplementary Files' file.
RESUMEN
Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.