Mouse screen reveals multiple new genes underlying mouse and human hearing loss.

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.

Ecto-5'-nucleotidase (CD73) regulates peripheral chemoreceptor activity and cardiorespiratory responses to hypoxia.

KEY POINTS: Carotid body dysfunction is recognized as a cause of hypertension in a number of cardiorespiratory diseases states and has therefore been identified as a potential therapeutic target. Purinergic transmission is an important element of the carotid body chemotransduction pathway. We show that inhibition of ecto-5'-nucleotidase (CD73) in vitro reduces carotid body basal discharge and responses to hypoxia and mitochondrial inhibition. Additionally, inhibition of CD73 in vivo decreased the hypoxic ventilatory response, reduced the hypoxia-induced heart rate elevation and exaggerated the blood pressure decrease in response to hypoxia. Our data show CD73 to be a novel regulator of carotid body sensory function and therefore suggest that this enzyme may offer a new target for reducing carotid body activity in selected cardiovascular diseases. ABSTRACT: Augmented sensory neuronal activity from the carotid body (CB) has emerged as a principal cause of hypertension in a number of cardiovascular related pathologies, including obstructive sleep apnoea, heart failure and diabetes. Development of new targets and pharmacological treatment strategies aiming to reduce CB sensory activity may thus improve outcomes in these key patient cohorts. The present study investigated whether ecto-5'-nucleotidase (CD73), an enzyme that generates adenosine, is functionally important in modifying CB sensory activity and cardiovascular respiratory responses to hypoxia. Inhibition of CD73 by α,ß-methylene ADP (AOPCP) in the whole CB preparation in vitro reduced basal discharge frequency by 76 ± 5% and reduced sensory activity throughout graded hypoxia. AOPCP also significantly attenuated elevations in sensory activity evoked by mitochondrial inhibition. These effects were mimicked by antagonism of adenosine receptors with 8-(p-sulfophenyl) theophylline. Infusion of AOPCP in vivo significantly decreased the hypoxic ventilatory response (Δ V̇E control 74 ± 6%, Δ V̇E AOPCP 64 ± 5%, P < 0.05). AOPCP also modified cardiovascular responses to hypoxia, as indicated by reduced elevations in heart rate and exaggerated changes in femoral vascular conductance and mean arterial blood pressure. Thus we identify CD73 as a novel regulator of CB sensory activity. Future investigations are warranted to clarify whether inhibition of CD73 can effectively reduce CB activity in CB-mediated cardiovascular pathology.

Key roles for AMP-activated protein kinase in the function of the carotid body?

The carotid bodies play a critical role in initiating compensatory ventilatory responses to hypoxia. However, the complete mechanism by which hypoxia excites the oxygen-sensing carotid body type 1 cells has not been fully defined. We have previously proposed that the enzyme adenosine monophosphate-activated protein kinase (AMPK) may couple hypoxic inhibition of mitochondrial oxidative phosphorylation to carotid body type I cell excitation (Evans, Mustard, Wyatt, Peers, Dipp, Kumar, Kinnear and Hardie 2005). Here we discuss evidence that AMPK is a key requirement for hypoxic chemotransduction by the carotid body. In addition, we postulate upon a role for AMPK in the plasticity observed in the carotid body during both chronic and chronic intermittent hypoxia.

Peptidergic activation of locomotor pattern generators in the neonatal spinal cord.

The development of motor networks in the spinal cord is partly activity-dependent. We have observed receptor-mediated excitatory effects of two peptides, arginine vasopressin (AVP) and oxytocin (OXT), on motor network activity in the neonate. With the use of an en bloc in vitro preparation of mouse spinal cord (2-3 d old), which either was isolated completely or had muscles of the hindlimb left intact, we show that the bath application of AVP or OXT can evoke an increase in population bursting of motoneurons recorded from the lumbar ventral roots. By using antagonists for AVP and OXT, we found that these peptides were binding primarily to V1a and OXT receptors, respectively. Western blot analysis revealed a 48 kDa V1a and a 55 kDa OXT receptor immunoreactive band that was expressed in tissue obtained from L1-L6 sections of spinal cord. AVP, but not OXT, could, on occasion, evoke sustained periods of locomotor-like activity. In addition, when we applied AVP or OXT in combination with a 5-HT2 agonist, bouts of locomotor-like activity could be observed in a majority of preparations. Collectively, these data point to a novel role for AVP and OXT in the activation of spinal motor networks.

AMP-activated protein kinase mediates carotid body excitation by hypoxia.

Early detection of an O2 deficit in the bloodstream is essential to initiate corrective changes in the breathing pattern of mammals. Carotid bodies serve an essential role in this respect; their type I cells depolarize when O2 levels fall, causing voltage-gated Ca2+ entry. Subsequent neurosecretion elicits increased afferent chemosensory fiber discharge to induce appropriate changes in respiratory function (1). Although depolarization of type I cells by hypoxia is known to arise from K+ channel inhibition, the identity of the signaling pathway has been contested, and the coupling mechanism is unknown (2). We tested the hypothesis that AMP-activated protein kinase (AMPK) is the effector of hypoxic chemotransduction. AMPK is co-localized at the plasma membrane of type I cells with O2-sensitive K+ channels. In isolated type I cells, activation of AMPK using 5-aminoimidazole-4-carboxamide riboside (AICAR) inhibited O2-sensitive K+ currents (carried by large conductance Ca2+-activated (BKCa) channels and TASK (tandem pore, acid-sensing potassium channel)-like channels, leading to plasma membrane depolarization, Ca2+ influx, and increased chemosensory fiber discharge. Conversely, the AMPK antagonist compound C reversed the effects of hypoxia and AICAR on type I cell and carotid body activation. These results suggest that AMPK activation is both sufficient and necessary for the effects of hypoxia. Furthermore, AMPK activation inhibited currents carried by recombinant BKCa channels, whereas purified AMPK phosphorylated thealpha subunit of the channel in immunoprecipitates, an effect that was stimulated by AMP and inhibited by compound C. Our findings demonstrate a central role for AMPK in stimulus-response coupling by hypoxia and identify for the first time a link between metabolic stress and ion channel regulation in an O2-sensing system.