John L. Carroll

Determinants of growth in children with the obstructive sleep apnea syndrome.

Failure to thrive is a common complication of childhood obstructive sleep apnea syndrome (OSAS). To further evaluate its cause, we obtained 3-day dietary records, anthropometric measurements, polysomnography, and measurements of energy expenditure during sleep (SEE) in children with OSAS before and after tonsillectomy and adenoidectomy. Fourteen children were studied (mean age, 4 +/- 1 (SD) years). During initial polysomnography, patients had 6 +/- 3 episodes of obstructive apnea/hr, an arterial oxygen saturation nadir of 85% +/- 8%, and peak end-tidal carbon dioxide tension of 52 +/- 6 mm Hg. After surgery, OSAS resolved in all patients. The standard deviation score (z score) for weight increased from -0.30 +/- 1.47 to 0.04 +/- 1.34 (p < 0.005), despite unaltered caloric intake (91 +/- 30 vs 90 +/- 27 kcal/kg per day; not significant). The initial SEE (averaged over all sleep states) was 51 +/- 6 kcal/kg per day; postoperatively, it decreased to 46 +/- 7 kcal/kg per day (p < 0.005). Although SEE decreased during all sleep stages, the greatest decrease occurred during rapid eye movement sleep. The patients with the highest SEE on initial study had the lowest z scores (r = -0.62; p < 0.05). We conclude that SEE decreases and weight improves after resolution of OSAS. We speculate that the poor growth seen in some children with OSAS is secondary to increased caloric expenditure caused by increased work of breathing during sleep.

Sperm-triggered [Ca2+] oscillations and Ca2+ homeostasis in the mouse egg have an absolute requirement for mitochondrial ATP production.

At fertilisation, repetitive increases in the intracellular Ca2+ concentration, [Ca2+]i, drive the completion of meiosis and initiate the development of the quiescent egg into an embryo. Although the requirement for an ATP supply is evident, the relative roles of potential ATP sources remains unclear in the mammalian egg, and the specific role of mitochondria in [Ca2+]i regulation as well as in the sperm-triggered [Ca2+] oscillations is unknown. We have used fluorescence and luminescence imaging to investigate mitochondrial activity in single mouse eggs. Simultaneous imaging of mitochondrial redox state (NADH and flavoprotein autofluorescence) and [Ca2+]i revealed that sperm-triggered [Ca2+] oscillations are transmitted to the mitochondria where they directly stimulate mitochondrial activity. Inhibition of mitochondrial oxidative phosphorylation caused release of Ca2+ from the endoplasmic reticulum because of local ATP depletion. Mitochondrial ATP production is an absolute requirement for maintaining a low resting [Ca2+]i and for sustaining sperm-triggered [Ca2+] oscillations. Luminescence measurements of intracellular [ATP] from single eggs confirmed that mitochondrial oxidative phosphorylation is the major source of ATP synthesis in the dormant unfertilised egg. These observations show that a high local ATP consumption is balanced by mitochondrial ATP production, and that balance is critically poised. Mitochondrial ATP supply and demand are thus closely coupled in mouse eggs. As mitochondrial ATP generation is essential to sustain the [Ca2+] signals that are crucial to initiate development, mitochondrial integrity is clearly fundamental in sustaining fertility in mammalian eggs.

Heteromeric TASK-1/TASK-3 is the major oxygen-sensitive background K+ channel in rat carotid body glomus cells

Carotid body (CB) glomus cells from rat express a TASK‐like background K+ channel that is believed to play a critical role in the regulation of excitability and hypoxia‐induced increase in respiration. Here we studied the kinetic behaviour of single channel openings from rat CB cells to determine the molecular identity of the ‘TASK‐like’ K+ channels. In outside‐out patches, the TASK‐like background K+ channel in CB cells was inhibited >90% by a reduction of pHo from 7.3 to 5.8. In cell‐attached patches with 140 mm KCl and 1 mm Mg2+ in the bath and pipette solutions, two main open levels with conductance levels of ∼14 pS and ∼32 pS were recorded at a membrane potential of −60 mV. The K+ channels showed kinetic properties similar to TASK‐1 (∼14 pS), TASK‐3 (∼32 pS) and TASK‐1/3 heteromer (∼32 pS). The presence of three TASK isoforms was tested by reducing [Mg2+]o to ∼0 mm, which had no effect on the conductance of TASK‐1, but increased those of TASK‐1/3 and TASK‐3 to 42 pS and 74 pS, respectively. In CB cells, the reduction of [Mg2+]o to ∼0 mm also caused the appearance of ∼42 pS (TASK‐1/3‐like) and ∼74 pS (TASK‐3‐like) channels, in addition to the ∼14 pS (TASK‐1‐like) channel. The 42 pS channel was the most abundant, contributing ∼75% of the current produced by TASK‐like channels. Ruthenium red (5 μm) had no effect on TASK‐1 and TASK‐1/3, but inhibited TASK‐3 by 87%. In CB cells, ruthenium red caused ∼12% inhibition of TASK‐like activity. Methanandamide reduced the activity of all three TASKs by 80–90%, and that of TASK‐like channels in CB cell also by ∼80%. In CB cells, hypoxia caused inhibition of TASK‐like channels, including TASK‐1/3‐like channels. These results show that TASK‐1, TASK‐1/3 and TASK‐3 are all functionally expressed in isolated CB cells, and that the TASK‐1/3 heteromer provides the major part of the oxygen‐sensitive TASK‐like background K+ conductance.

Effect of nicotine exposure on postnatal ventilatory responses to hypoxia and hypercapnia

The risk of SIDS is increased up to fourfold by maternal smoking, by an unknown mechanism. We tested the hypothesis that prenatal nicotine exposure can cause abnormal postnatal development of breathing control. Osmotic minipumps were implanted into pregnant rats to deliver either nicotine bitartrate (6 mg kg-1 day-1) (NIC) or saline (CON) throughout gestation and for 1 week postnatal. NIC and CON rat pups from 4 age groups (means 3, 8, 18 and 34 days) were studied. Ventilation was recorded at 30 degrees C in air and after 10 min at FIO2 = 0.1 and 0.15, and at FICO2 = 0.05. Ventilatory responses to FIO2 = 0.1 and FICO2 = 0.05 showed significant changes with age but were unaffected by NIC at all ages. The weak respiratory responses to FIO2 = 0.15 were unaffected by NIC or age. Oxygen consumption in normoxia and hypoxia, and hypoxic depression of oxygen consumption, declined with age but were not affected by NIC. We conclude that NIC exposure alone has no detectable effect on the postnatal development of respiratory responses to moderate levels of hypoxia or hypercapnia for short periods. However, effects of NIC on the responses to more severe or prolonged stimuli cannot be ruled out.

Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei

In mammals, the sperm triggers a series of cytosolic Ca2+ oscillations that continue for ∼4 hours, stopping close to the time of pronucleus formation. Ca2+ transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca2+ signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca2+ oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca2+ oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca2+ oscillations. A role for pronucleus formation in regulating Ca2+ signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca2+ oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin β that inhibits nuclear transport, also prevents pronucleus formation and causes Ca2+ oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca2+ oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca2+-releasing activity contributes to the temporal organization of Ca2+ transients in meiosis and mitosis in mice.

Resetting and postnatal maturation of oxygen chemosensitivity in rat carotid chemoreceptor cells

1 Carotid chemoreceptor sensitivity is minimal immediately after birth and increases with postnatal age. In the present study we have investigated the peri‐ and postnatal developmental time course of [Ca2+]i responses to hypoxia in clusters of type I cells isolated from near‐term fetal rats and rats that were 1, 3, 7, 11, 14 and 21 days old, using the Ca2+‐sensitive fluoroprobe fura‐2. 2 In type I cells from all age groups a graded increase in [Ca2+]i occurred in response to lowering the P  O 2 from 150 mmHg to 70, 35, 14, 7, 2 and 0 mmHg. The graded [Ca2+]i response to hypoxia was hyperbolic at all ages. 3 Type I cells from rats near‐term fetal to 1 day old exhibited small [Ca2+]i responses, mainly to the most severe levels of hypoxia. After day 1, an increase in the [Ca2+]i responses to submaximal hypoxia stimulation resulted in a rightward shift in the O2 response curve. Using the Δ[Ca2+]i between 35 and 2 mmHg P  O 2 as an index of O2 sensitivity, type I cell O2 sensitivity increased approximately 4‐ to 5‐fold between near‐term fetal to 1 day old and 11 to 14 days of age. 4 Exposure to elevated extracellular potassium (10, 20 and 40 mm K+) caused a dose‐dependent [Ca2+]i rise in type I cells from all age groups. There were no age‐related changes in [Ca2+]i responses to any level of K+ between near‐term fetal and 21 days. 5 We conclude that the maximal type I cell [Ca2+]i response to anoxia, as well as the sensitivity to submaximal hypoxic stimulation, of rats aged from near‐term fetal to 21 days depends on the level of postnatal maturity. The lack of an age‐related increase in the [Ca2+]i response to elevated K+ during the timeframe of maximal development of O2 sensitivity suggests that resetting involves maturation of O2 sensing, rather than non‐specific developmental changes in the [Ca2+]i rise resulting from depolarization.

Mitochondrial dysfunction in oocytes of obese mothers: transmission to offspring and reversal by pharmacological endoplasmic reticulum stress inhibitors

Over-nutrition in females causes altered fetal growth during pregnancy and permanently programs the metabolism of offspring; however, the temporal and mechanistic origins of these changes, and whether they are reversible, are unknown. We now show that, in obese female mice, cumulus-oocyte complexes exhibit endoplasmic reticulum (ER) stress, high levels of intracellular lipid, spindle abnormalities and reduced PTX3 extracellular matrix protein production. Ovulated oocytes from obese mice contain normal levels of mitochondrial (mt) DNA but have reduced mitochondrial membrane potential and high levels of autophagy compared with oocytes from lean mice. After in vitro fertilization, the oocytes of obese female mice demonstrate reduced developmental potential and form blastocysts with reduced levels of mtDNA. Blastocysts transferred to normal weight surrogates that were then analyzed at E14.5 showed that oocytes from obese mice gave rise to fetuses that were heavier than controls and had reduced liver and kidney mtDNA content per cell, indicating that maternal obesity before conception had altered the transmission of mitochondria to offspring. Treatment of the obese females with the ER stress inhibitor salubrinal or the chaperone inducer BGP-15 before ovulation increased the amount of the mitochondrial replication factors TFAM and DRP1, and mtDNA content in oocytes. Salubrinal and BGP-15 also completely restored oocyte quality, embryo development and the mtDNA content of fetal tissue to levels equivalent to those derived from lean mice. These results demonstrate that obesity before conception imparts a legacy of mitochondrial loss in offspring that is caused by ER stress and is reversible during the final stages of oocyte development and maturation. Highlighted article: In mice, obesity induces oocyte mitochondrial dysfunction that is transmitted to offspring but that can be reversed using interventions prior to conception.

Consequences of sleep-disordered breathing in childhood.

Obstructive sleep-disordered breathing (SDB) is a common problem in children that may lead to growth failure, neurocognitive and behavioral abnormalities, cor pulmonale, and death. Primary snoring, upper airway resistance syndrome, and obstructive sleep apnea syndrome represent a spectrum of clinical manifestations accompanying increasing degrees of upper airway obstruction. Clearly, children with severe SDB need to be identified and treated promptly. Appropriate management strategies for milder forms of SDB are less clear. Some snoring children, for example, may have an increased frequency of obstructive apnea during sleep, with or without mild hypoxemia, but have essentially no daytime symptoms or apparent clinical consequences. Should these children be treated? If untreated, will these children eventually develop more severe obstructive SDB? Development of management strategies is hampered by the lack of data on the natural history of childhood SDB and on the correlation of specific polysomnographic abnormalities to symptoms and complications. In this review, we highlight recent information about the consequences of obstructive SDB in children, with particular emphasis on areas in which more data are needed.

Dynamic ventilatory responses in rats: normal development and effects of prenatal nicotine exposure

Infants of smoking mothers are at increased risk of SIDS, one cause of which is thought to be due to impaired ventilatory responses. We tested the hypotheses that prenatal nicotine exposure impairs the development of dynamic carotid chemoreceptor-driven ventilatory responses, and reduces the ability to lower metabolic rate in hypoxia. Osmotic minipumps were implanted into 20 pregnant rats at day 3 of gestation to deliver nicotine (6 mg/kg per day free base) or saline for 4 weeks. Minute ventilation was recorded breath by breath in rat pups at 3, 8 and 18 days (n = 6, 8 and 6) postnatal in response to 5-sec challenges of 100% O2 (Dejours test) and 5% O2 + 5% CO2. Carotid sinus nerve (CSN) responses to hypoxia and CO2 were recorded from 22 control and 17 nicotine-exposed preparations at ages between 3-20 days. Oxygen consumption (V(O)2) was measured in groups of pups at 3 days (n = 7 each for nicotine and control) and 8 days (n = 5 each for nicotine and control) in room air and 10% O2. There was no detectable effect of nicotine exposure on the development of CSN responses. Ventilatory responses to 5% O2-5% CO2 increased with age but did not differ between nicotine and control groups. Ventilatory responses to 100% O2 were unaffected by nicotine exposure at 8 and 18 days. However, the 3-day nicotine group showed no significant response to 100% O2 whereas V(E) was significantly reduced in the control group by 100% O2. There was no significant effect of nicotine exposure on the ability to reduce oxygen consumption in hypoxia at 3 or 8 days, but at 3 days, baseline (room air) variability in oxygen consumption was greater in the nicotine group. We conclude that nicotine exposure appears to result in abnormal ventilatory responses to withdrawal of baseline peripheral chemoreceptor drive during a period of early postnatal life. We speculate that a transient abnormality could contribute to a period of instability and increased vulnerability to challenges.

Sleep-disordered breathing in children with achondroplasia

OBJECTIVE Our objective was to characterize sleep-disordered breathing in 88 children with achondroplasia aged 1 month to 12.6 years. RESULTS At the time of their initial polysomnography, five children had previously undergone tracheostomy, and seven children required supplemental oxygen. Initial polysomnography demonstrated a median obstructive apnea index of 0 (range, 0 to 19.2 apneas/hr). The median number of central apneas with desaturation per study was 0.5 (0 to 49), the median oxygen saturation nadir was 91% (50% to 99%), and the median peak end-tidal pCO2 was 47 mm Hg (36 to 87 mm Hg). Forty-two children (47.7%) had abnormal initial study results, usually caused by hypoxemia. Two children with severe obstructive sleep apnea eventually required continuous positive airway pressure therapy, and three additional children required tracheostomies. CONCLUSIONS (1) Children with achondroplasia often have sleep-related respiratory disturbances, primarily hypoxemia. (2) The majority do not have significant obstructive or central apnea; however, a substantial minority are severely affected. (3) Tonsillectomy and adenoidectomy decreases the degree of upper airway obstruction in most but not all children with achondroplasia and obstructive sleep apnea. (4) Restrictive lung disease can present at a young age in children with achondroplasia.