Marie St-Hilaire

MxA, a member of the dynamin superfamily, interacts with the ankyrin-like repeat domain of TRPC

Mammalian transient receptor potential canonical channels have been proposed as the molecular entities associated with calcium entry activity in nonexcitable cells. Amino acid sequence analyses of TRPCs revealed the presence of ankyrin-like repeat domains, one of the most common protein-protein interaction motifs. Using a yeast two-hybrid interaction assay, we found that the second ankyrin-like repeat domain of TRPC6 interacted with MxA, a member of the dynamin superfamily. Using a GST pull-down and co-immunoprecipitation assay, we showed that MxA interacted with TRPC1, -3, -4, -5, -6, and -7. Overexpression of MxA in HEK293T cells slightly increased endogenous calcium entry subsequent to stimulation of Gq protein-coupled receptors or store depletion by thapsigargin. Co-expression of MxA with TRPC6 enhanced agonist-induced or OAG-induced calcium entry activity. GTP binding-defective MxA mutants had only a minor potentiating effect on OAG-induced TRPC6 activity. However, a MxA mutant that could bind GTP but that lacked GTPase activity produced the same effect as MxA on OAG-induced TRPC6 activity. These results indicated that MxA interacted specifically with the second ankyrin-like repeat domain of TRPCs and suggested that monomeric MxA regulated the activity of TRPC6 by a mechanism requiring GTP binding. Additional results showed that an increase in the endogenous expression of MxA, induced by a treatment with interferon α, regulated the activity of TRPC6. The study clearly identified MxA as a new regulatory protein involved in Ca2+ signaling.

Effects of postnatal smoke exposure on laryngeal chemoreflexes in newborn lambs.

Laryngeal chemoreflexes (LCR), which are elicited by the contact of liquids such as gastric refluxate with laryngeal mucosa, may trigger some cases of sudden infant death syndrome. Indeed, while LCR in mature mammals consist of protective responses, previous animal data have shown that LCR in immature newborns can include laryngospasm, apnea, bradycardia, and desaturation. The present study was aimed at testing the hypothesis that postnatal exposure to cigarette smoke is responsible for enhancing cardiorespiratory inhibition observed with LCR. Eight lambs were exposed to cigarette smoke (20 cigarettes/day) over 16 days and compared with seven control lambs. Urinary cotinine/creatinine ratio was measured at a level relevant to previously published levels in infants. On days 15 and 16, 0.5 ml of HCl (pH 2), milk, distilled water, or saline was injected onto the larynx via a chronic supraglottal catheter during sleep. Results showed that exposure to cigarette smoke enhanced respiratory inhibition (P < 0.05) and tended to enhance cardiac inhibition and decrease swallowing and arousal during LCR (P < 0.1). Overall, these results were observed independently of the state of alertness and the experimental solution tested. In conclusion, 16-day postnatal exposure to cigarette smoke increases cardiorespiratory inhibition and decreases protective mechanisms during LCR in nonsedated full-term lambs.

Laryngeal Stimulation by an Acid Solution in the Pre-term Lamb

In a mature organism, the contact between various liquids and the laryngeal mucosa triggers lower airway protective responses (cough, swallowing, arousal). These laryngeal chemoreflexes (LCR) are essential for preventing aspiration. In contrast, previous studies showed that LCR are responsible for apnea and bradycardia in the neonatal mammal. Consequently, LCR, especially when triggered by acid gastrolaryngeal reflux, are deemed responsible for some apneas of prematurity and many life-threatening events of infancy and, probably, for some cases of sudden infant death syndrome. Recently, we have revisited LCR in full-term lambs during quiet sleep. Our results showed that the LCR triggered by HCl (pH 2), mimicking the acid component of an acid gastro-oesophageal reflux, were consistently like the mature LCR reported in adult mammals, without significant apneas and bradycardias (St-Hilaire 2005). These results prompted us to question whether premature birth alters LCR. Results show that LCR triggered in pre-term lambs by both saline and HCl are much more marked and clinically relevant than the ones observed in full-term lambs. Indeed, life-threatening responses to HCl, including repetitive apneas for more than 90 seconds, severe desaturation and bradycardia, were observed in 2 lambs at postnatal day 7 (D7). In addition, LCR were significantly blunted at D14. In conclusion, HCl can trigger potentially dangerous LCR in pre-term lambs at D7, suggesting that LCR in response to acid gastrolaryngeal refluxes are likely involved in some apnea/bradycardia/desaturation in pre-term infants, before they reach a post-conceptional age equivalent to full gestation.

Validation of a new automatic smoking machine to study the effects of cigarette smoke in newborn lambs

The aim of this study was to describe the characteristics and validate the use of a new, custom-built automatic smoking machine (ASM), primarily designed to study the effects of an environmental tobacco smoke surrogate (ETS surrogate) exposure in animals of various sizes, including large animals. The equipment includes a programmable ASM coupled to a vented whole body chamber, where animals can be exposed to both mainstream and sidestream smoke. The user-friendly interface allows for full programming of puff volume (1–60 mL), time interval between two puffs (1–60 s) and between two cigarettes (1–60 min). Eight newborn lambs were exposed to either 10 (4 lambs, C10 group) or 20 (4 lambs, C20 group) cigarettes, 8 h per day for 15 days. Four additional control, lambs were exposed to air (C0 group). Weight gain was identical in all three groups of lambs. Urinary cotinine/creatinine ratio increased with the number of cigarettes smoked (C0: 11 ± 7 ng/mg; C10: 961 ± 539 ng/mg; C20: 1821 ± 312 ng/mg), with levels in the C10 and C20 groups in keeping with values published in infants exposed to ETS. Overall, results show that our new ASM is especially well suited for ETS surrogate exposure in non-restrained, non-anaesthetized large animals such as sheep.

Postnatal autonomic activity in the preterm lamb

BACKGROUND Early postnatal abnormalities of autonomic nervous system (ANS) activity, including at baseline and following cardiorespiratory challenge, are involved in apneas-bradycardias of prematurity, apparent life-threatening events of infancy and sudden infant death syndrome. Literature data suggest that baseline ANS activity does not mature normally after premature birth. OBJECTIVES This study performed in preterm lambs was aimed at assessing ANS maturation at baseline and following laryngeal chemoreflexes (LCR), a group of reflexes triggered by the contact of liquids with the laryngeal mucosa. METHODS Heart rate variability (HRV) and baroreflex sensitivity (BRs) were measured at baseline and after LCR during polysomnographic recordings performed in five non-sedated lambs born 15 days prematurely. Laryngeal chemoreflexes were induced by distilled water or acid (pH 2) during sleep and wakefulness on postnatal days 7 (D7) and 14 (D14, full-term equivalence). RESULTS While the life-threatening cardiorespiratory events of the LCR observed at D7 were no longer present at D14, baseline and post-LCR HRV and BRs indices were significantly lower at D14 compared to D7 (up to p<0.001). These results suggest that an initial autonomic overactivity was present at D7 and normalized at D14. CONCLUSION The autonomic cardiac and baroreflex control appears to follow a specific evolution in the preterm compared to the full-term newborn lamb, with an important initial autonomic stress, which normalizes at an age equivalent to full-term. Potential relevance of these findings in relation to anomalies of cardiac control in the early postnatal period, such as apneas-bradycardias of prematurity, apparent life-threatening events of infancy and sudden infant death syndrome, awaits further studies.

Effects of postnatal environmental tobacco smoke on non-nutritive swallowing-breathing coordination in newborn lambs.

While prenatal environmental tobacco smoke (ETS) exposure is a well-known risk factor for sudden infant death syndrome, the effect of postnatal ETS exposure is less clear. The objective of this study was to investigate the effect of postnatal ETS exposure on non-nutritive swallowing (NNS) and NNS-breathing coordination, which are crucial to prevent aspiration related-cardiorespiratory events. Eighteen newborn lambs (6 per group) were randomly exposed to either 10 cigarettes/day, 20 cigarettes/day or room air for 15 days. Lambs were instrumented for recording states of alertness, swallowing, electrocardiogram and breathing; recordings were performed in non-sedated lambs at the end of ETS exposure. Urinary cotinine/creatinine ratio confirmed relevant real-life exposure. Postnatal ETS exposure had no effect on NNS frequency but tended to decrease inspiratory NNS (p=0.07) during quiet sleep. No effect on respiratory or heart rate (p>0.6), apnea index (p=0.2) or sleep states (p=0.3) was observed. In conclusion, postnatal ETS exposure in lambs had only mild effects on NNS-breathing coordination.

CPAP Inhibits Non-nutritive Swallowing Through Stimulation of Bronchopulmonary Receptors

While swallowing and respiratory problems are among the most frequent disorders encountered in neonates, the interrelationships between both functions are not completely understood. This is especially true for non-nutritive swallowing (NNS), which fulfills the important function of clearing upper airways from both local secretions and liquids refluxed from the stomach. Recently, we showed that nasal CPAP inhibits NNS during quiet sleep in the newborn lamb (Samson, St-Hilaire, Nsegbe, Reix, Moreau-Bussière and Praud 2005). The present study was aimed at testing the hypothesis that NNS inhibition is eliminated when CPAP is directly administered through a tracheostomy, thus eliminating reflexes originating from upper airway receptors. Results show that both nasal and tracheal CPAP 6cm H2O similarly inhibit total NNS during quiet sleep, thus suggesting that the inhibiting effect of nasal CPAP on NNS is mainly mediated through bronchopulmonary mechanical receptors with minimal participation of the upper airways.

Effects of Hypercapnia on Non-nutritive Swallowing in Newborn Lambs

The purpose of this study was to determine the effect of hypercapnia on non-nutritive swallowing (NNS) frequency and on NNS-breathing coordination in newborn lambs. Six lambs were chronically instrumented for recording electroencephalogram, eye movements, diaphragm and thyroarytenoid muscle activity, nasal airflow and electrocardiogram. Each lamb was placed in a Plexiglas chamber and exposed to a hypercapnic gas mixture (21% O2, 5% CO2). Polysomnographic recordings were conducted in non-sedated lambs using a custom-designed radiotelemetry system. Results show that hypercapnia increased NNS frequency in all three states of alertness (p < 0.0001 to 0.03), through a specific increase in ie-type NNS. Causal mechanisms and potential consequences of such observations on aspirations and apneas, as well as on swallowing maturation, will require further studies.