Narong Simakajornboon

Brainstem activation of platelet-derived growth factor-β receptor modulates the late phase of the hypoxic ventilatory response

Abstract: The early phase of the biphasic ventilatory response to hypoxia in mammals is critically dependent on NMDA glutamate receptor activation within the nucleus of the solitary tract. However, the mechanisms underlying the subsequent development of the typical ventilatory roll‐off are unclear and could underlie important roles in the functional and molecular adaptation to oxygen deprivation. Because the growth factor platelet‐derived growth factor (PDGF)‐BB can modulate the open channel probability of NMDA receptors by activating PDGF‐β receptors, its contribution to hypoxic ventilatory roll‐off was examined. Administration of PDGF‐BB, but not PDGF‐AA, in the nucleus of the solitary tract was associated with significant attenuations of the early hypoxic ventilatory response in conscious rats. Furthermore, marked reductions in the magnitude of hypoxic ventilatory roll‐off occurred in mice heterozygous for a mutation in the PDGF‐β receptor. Administration of a PDGF‐β receptor antagonist to wild‐type littermates elicited similar declines in hypoxic ventilatory roll‐off. The relative abundance of PDGF‐β receptors was confirmed in the nucleus of the solitary tract and other nuclei implicated in the hypoxic ventilatory response. In nucleus of the solitary tract lysates, PDGF‐β receptor tyrosine phosphorylation was temporally correlated with hypoxic ventilatory roll‐off formation. Increased PDGF‐B chain mRNA expression was induced by hypoxia in the nucleus of the solitary tract, and PDGF‐B chain immunoreactivity colocalized with ≅40% of nucleus of the solitary tract neurons, demonstrating hypoxia‐induced c‐Fos enhancements. Thus, PDGF‐BB release and PDGF‐β receptor activation in the nucleus of the solitary tract are critical components of hypoxic ventilatory roll‐off and may have important functional implications in processes underlying survival and acclimatization to hypoxic environments.

Signaling pathways of the acute hypoxic ventilatory response in the nucleus tractus solitarius.

The nucleus tractus solitarii (nTS) provides the initial central synaptic relay to peripheral chemoreceptor afferent inputs elicited by changes in oxygen tension. Insofar, the overall cumulative evidence pointing towards the N-methyl-D-aspartate (NMDA) glutamate receptor as the critical receptor underlying the early component of the hypoxic ventilatory response (HVR) is reviewed in detail. In addition, we will present recent findings supporting a role for platelet-derived growth factor (PDGF) beta receptor activation in modulation of the late phase of HVR. This evidence underscores the proposal of a working model for intracellular signaling pathways, downstream to the NMDA glutamate and PDGF-beta receptors in nTS neurons, which may contribute to both the ventilatory characteristics of the acute hypoxic response and to subsequently occurring functional adaptations and synaptic plasticity phenomena.

Hypoxia induces c-Fos protein expression in NMDA but not AMPA glutamate receptor labeled neurons within the nucleus tractus solitarii of the conscious rat.

Glutamatergic transmission within the nucleus tractus solitarii (nTS) is critical to full expression of hypoxia-induced cardiorespiratory responses in the rat. To further examine the role of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) glutamate receptors in these responses, double-labeling studies of immunoreactivity for c-Fos protein and either NMDA (NR1) or AMPA (mGluR2/3) receptor expression were conducted in normoxic rats and in hypoxic rats receiving vehicle, MK801, or NBQX. Hypoxia markedly increased c-Fos immunoreactivity within nTS neurons which in the vast majority co-labeled for NMDA. Furthermore, MK801 markedly attenuated such responses. In contrast, co-localization of AMPA and c-Fos occurred in only a small proportion of neurons, and NBQX failed to modify hypoxia-induced c-Fos enhancements. These data suggest a predominant role for NMDA but not for AMPA glutamate receptors in nTS mediated components of the hypoxic response.

Maturational changes in neuromodulation of central pathways underlying hypoxic ventilatory response

The neuromodulator systems mediating the central component of the hypoxic ventilatory response (HVR) during development are complex and diverse. The early component of the HVR is mediated through N-methyl-D-aspartate (NMDA) glutamate receptors in the caudal brainstem. The intracellular downstream signal transductions of the NMDA receptors involve protein kinase C (PKC), neuronal nitric oxide synthase (nNOS) and tyrosine kinase (TK). Activation of NMDA receptors will also lead to activation of the early gene transcription factors including AP-1 (c-fos, c-jun) and NF-kappaB which may play a role in modulation of the subsequent response to hypoxia. NMDA receptors in the caudal brainstem play a critical role in the development of the HVR and increasing dependency on NMDA receptors emerges over time. Similarly, hypoxia-induced PKC, NOS and c-Fos activation in the caudal brainstem is relatively weak in the immature animals, but this activation increases with age and the strength of the response appears to increase concomitantly with the appearance of NMDA expression. Several neurotransmitters including adenosine, gamma-aminobutyric acid (GABA), serotonin and opioids are involved in the late component of the HVR. In addition, the late phase of the HVR is mediated in part through platelet-derived growth factor (PDGF)-beta receptors. PDGF-beta receptor activation is an important contributor of the hypoxic ventilatory depression at all postnatal ages, but its role is more critical in the developing animals. Maturation of these neuromodulators, especially the NMDA and PDGF-beta receptors-mediated pathways, occurs primarily during the early postnatal period. Perturbation of these developmental processes may result in short-term or sustained alterations to the HVR and may also affect neuronal survival during hypoxia.

Prenatal cigarette smoke exposure selectively alters protein kinase C and nitric oxide synthase expression within the neonatal rat brainstem.

Maternal smoking is a major risk factor for sudden infant death syndrome. Protein kinase C (PKC) and neuronal nitric oxide synthase (NOS) activities within the dorsocaudal brainstem (DB) mediate critical components of respiratory drive and could be implicated in SIDS. Thus, exposure to smoking during fetal life could modify the expression of these kinases in the DB. Rats were exposed to cigarette smoke or room air (Sham) from day 2 to 22 of pregnancy. Immunoblots of DB lysates at 2 days postnatally revealed no differences in PKC-alpha, PKC-beta, and endothelial NOS expression. However, PKC-gamma, PKC-delta, and neuronal NOS immunoreactivities were reduced in the cigarette smoke group. We conclude that gestational smoking is associated with selective reductions in PKC and NOS isoforms within the DB, which could decrease respiratory drive and lead to enhanced hypoxic vulnerability in infants of smoking mothers.

Hypoxia induces selective SAPK/JNK-2-AP-1 pathway activation in the nucleus tractus solitarii of the conscious rat.

Abstract : In the nucleus tractus solitarii, NMDA glutamate receptors are critical to the hypoxic ventilatory response. However, the signal transduction pathways underlying the hypoxic ventilatory response remain undefined. To assess the effect of a moderate hypoxic stimulus (10% O2) on tyrosine phosphorylation of proteins in the nucleus tractus solitarii, tissue lysates were harvested by repeated punch sampling at 0, 1, 10, and 60 min of hypoxia and examined for the presence of phosphorylated tyrosine residues by immunoblotting. Time‐dependent phosphotyrosine increases occurred in proteins migrating at regions corresponding to molecular masses of 38‐42, 50, 55, and 60 kDa, which were attenuated by pretreatment with the NMDA receptor channel blocker, MK‐801. As extracellular signal‐regulated kinase (Erk) and stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) phosphorylation may induce Fos and Jun gene transcription and activator protein‐1 (AP‐1) DNA binding, the activation of Erk1, Erk2, p38, and SAPK/JNK was examined in the nucleus tractus solitarii and neocortex during hypoxia and following administration of MK‐801. Hypoxia enhanced Erk1, Erk2, and p38 activity in the cortex, but not in the nucleus tractus solitarii. Increased phosphorylation of SEK1 and SAPK/JNK‐2 occurred in the nucleus tractus solitarii during hypoxia, whereas both SAPK/JNK‐1 and SAPK/JNK‐2 were recruited in cortex. MK‐801 attenuated hypoxia‐induced SEK1, SAPK/JNK‐2, and AP‐1 binding in the nucleus tractus solitarii, and the widespread activation of all MAP kinases in the cortex was also attenuated. We conclude that in conscious rats, a moderate hypoxic stimulus elicits NMDA‐dependent widespread mitogenactivated protein kinase activation in cortex, but selective SAPK/JNK‐2 and AP‐1 activation in the nucleus tractus solitarii, thereby suggesting a functional role for the SAPK/JNK‐2‐AP‐1 pathway.

Developmental patterns of NF-κB activation during acute hypoxia in the caudal brainstem of the rat

NF-kappaB, an ubiquitous transcription factor which plays a major role in the regulation of stress-related genes, is activated during environmental hypoxia in the dorsocaudal brainstem of adult rats. To examine the developmental pattern of NF-kappaB basal activity in the brainstem and the response to hypoxia, electromobility shift assays and immunohistochemical staining for the P65 subunit of NF-kappaB were performed in caudal brainstem samples of rats at 2, 5, 10, 15, and 60 days postnatal age, following normoxic or hypoxic (1 h in 10% O2) exposures. In addition, the expression of IkappaB-alpha, and IkappaB kinases (ikk)-alpha and -beta was also examined using Western blots. Basal NF-kappaB nuclear activity and nuclear P65 immunoreactivity increased with maturation. In contrast, hypoxia induced enhanced activation of NF-kappaB and nuclear translocation of P65 in youngest animals. Expression of both IkappaB-alpha and ikk-alpha was highest in the more immature rats, and decreased with postnatal age. In contrast, ikk-beta expression was unchanged over time. We conclude that NF-kappaB activity in caudal brainstem is developmentally regulated, and that hypoxia-induced NF-kappaB activation is more prominent in youngest rats. We postulate that postnatal regulation of NF-kappaB complex expression and function may underlie fundamental genomic processes mediating developmental changes in neuronal hypoxic tolerance.

PDGF-β Receptor Expression in the Dorsocaudal Brainstem Parallels Hypoxic Ventilatory Depression in the Developing Rat

The temporal trajectory of platelet-derived growth factor (PDGF)-β receptor activation within the dorsocaudal brainstem parallels that of the mild hypoxic ventilatory depression (HVD) seen in adult rats. We hypothesized that enhanced PDGF-β receptor activity may account for the particularly prominent HVD of developing mammals. To study this issue, 2-, 5-, 10-, and 20-d-old rats underwent hypoxic challenges (10% O2 for 30 min) after pretreatment with either vehicle (Veh) or the selective PDGF-β receptor antagonist CGP57148B (intraperitoneal 100 mg/kg). The developmental characteristics and magnitude of the peak hypoxic ventilatory response (HVR) were not modified by the PDGF-β receptor blocker. However, HVD was markedly attenuated by CGP57148B, and such effect, although still present, gradually abated with increasing postnatal age [p < 0.001, analysis of variance (ANOVA)]. Hypercapnic ventilatory responses were not affected by CGP57148B. The expression of PDGF-β receptor in the dorsocaudal brainstem was assessed by immunoblotting and confirmed progressively decreasing expression with maturation. We conclude that PDGF-β receptor activation during hypoxia is an important contributor to HVD at all postnatal ages but more particularly in the immature rat.

Hypoxia induces activation of a N-methyl-D-aspartate glutamate receptor-protein kinase C pathway in the dorsocaudal brainstem of the conscious rat.

To study in vivo phosphorylation of N-methyl-D-aspartate (NMDA) glutamate receptors and the recruitment of protein kinase C isoforms during acute hypoxia, dorsocaudal brainstem lysates were harvested from conscious rats exposed to either room air or hypoxia (10% O2 for 5 and 15 min). Increased phosphorylation of the NR-1 subunit at serine residue 896 occurred during hypoxia and was blocked by pre-treatment with MK-801. Immunoblots of soluble and particulate fractions revealed subcellular translocation for PKC-beta, -gamma, -delta, -epsilon, and -iota during hypoxia with no changes in PKC-alpha, -mu, and -zeta. Translocation of PKC-beta, -delta and -epsilon was selectively attenuated following MK-801. We demonstrate that hypoxia leads to PKC-mediated activation of NMDA receptors in the brainstem, and that PKC-beta, -delta and -epsilon are the most likely candidates for NR-1 phosphorylation.

Nocturnal Oximetry–based Evaluation of Habitually Snoring Children

Rationale: The vast majority of children around the world undergoing adenotonsillectomy for obstructive sleep apnea‐hypopnea syndrome (OSA) are not objectively diagnosed by nocturnal polysomnography because of access availability and cost issues. Automated analysis of nocturnal oximetry (nSpO2), which is readily and globally available, could potentially provide a reliable and convenient diagnostic approach for pediatric OSA. Methods: Deidentified nSpO2 recordings from a total of 4,191 children originating from 13 pediatric sleep laboratories around the world were prospectively evaluated after developing and validating an automated neural network algorithm using an initial set of single‐channel nSpO2 recordings from 589 patients referred for suspected OSA. Measurements and Main Results: The automatically estimated apnea‐hypopnea index (AHI) showed high agreement with AHI from conventional polysomnography (intraclass correlation coefficient, 0.785) when tested in 3,602 additional subjects. Further assessment on the widely used AHI cutoff points of 1, 5, and 10 events/h revealed an incremental diagnostic ability (75.2, 81.7, and 90.2% accuracy; 0.788, 0.854, and 0.913 area under the receiver operating characteristic curve, respectively). Conclusions: Neural network‐based automated analyses of nSpO2 recordings provide accurate identification of OSA severity among habitually snoring children with a high pretest probability of OSA. Thus, nocturnal oximetry may enable a simple and effective diagnostic alternative to nocturnal polysomnography, leading to more timely interventions and potentially improved outcomes.