Almitrine inhibits breathing movements in fetal sheep in utero

Whilst hypoxia stimulates fetal peripheral chemoreceptors, fetal breathing movements do not increase as hypoxia also has central effects. We wondered whether specific stimulation of the arterial chemoreceptors by almitrine would produce a stimulation of fetal breathing movements. When almitrine was given to 5 intact and 3 peripherally-chemodenervated fetal sheep in utero, fetal breathing movements rapidly ceased for 1-12 h. There was also a decrease in the amount of time spent in low voltage electrocortical activity. The effects of almitrine are therefore similar to those of hypoxia, and are independent of the peripheral chemoreceptors. Thus it may be a valuable tool in the study of the control of fetal breathing.     

Fetal breathing and development of control of breathing

Technical advances during the last several decades have greatly facilitated research into fetal physiology and behavior, specifically fetal breathing (FB). Breathing movements have been demonstrated in the fetuses of every mammalian species investigated and appear to be part of normal fetal development. In this review we focus on the methods of measuring FB and on some of the problems associated with these measurements and their interpretation. We also review fetal behavior, the role of the peripheral and central chemoreceptors in spontaneous FB, the fetal respiratory response to hypercapnia and hypoxia, and the transition to continuous breathing at birth. It is clear that in many ways the control of breathing movements in utero differs from that after birth. In particular, inhibitory influences are much more prominent before than after birth. Possibly this is due to the unique fetal situation, in which conservation of energy may be more important than any advantage breathing activity imparts to the fetus.     

The response of few-fiber carotid chemoreceptor preparations to almitrine in the dog

Almitrine, a long-lasting peripheral chemoreceptor stimulant, was given to nine dogs via intracarotid injection. Carotid chemoreceptor activity was recorded from single or few-fiber afferent nerve preparations. Doses of 10-20 microgram/kg were generally sufficient to produce a brisk stimulatory response of less than 30 min duration. In four dogs decreasing arterial PO2 was found to allow a greater than additive response to almitrine. Infusions of NaHCO3 appeared to depress the response to almitrine whereas changing arterial PCO2 had little effect on the carotid chemoreceptor response to almitrine. Neither dopamine infusion nor dopamine receptor blockade altered the responsiveness of the carotid chemoreceptors to almitrine.     

Pulmonary vascular responses to surgical chemodenervation and chemical sympathectomy in dogs

We investigated the effects of surgical peripheral chemoreceptor denervation, chemical sympathectomy with 6-hydroxydopamine (6-OHDA), and the peripheral chemoreceptor stimulant almitrine on multipoint pulmonary arterial pressure-cardiac index (PAP/Q) plots in 30 pentobarbital sodium-anesthetized dogs ventilated alternatively in hyperoxia [fraction of inspired O2, (FIO2) = 0.4] and hypoxia (FIO2 = 0.1). A hypoxic pulmonary vasoconstriction (HPV), i.e., a hypoxia-induced increase in PAP over the entire range of Q studied, from 2 to 5 l.min-1.m-2, was elicited in all the animals. Surgical denervation of the carotid and aortic chemoreceptors in a first group of nine dogs increased PAP at the lowest Q of 2 and 3 l.min-1.min-2 in hyperoxia and increased PAP at all levels of Q in hypoxia, so that HPV was enhanced. Chemical sympathectomy in a second group of eight dogs increased PAP at all levels of Q to a comparable extent in hyperoxia and hypoxia so that HPV remained unchanged. Almitrine (8 micrograms.kg-1.min-1 iv) in a third group of eight dogs increased PAP at all levels of Q in hyperoxia but had no effect on PAP/Q plots in hypoxia, so that HPV was inhibited. Almitrine had these same pulmonary vascular effects when administered to the chemodenervated and the sympathectomized dogs. Sham operation and a 2-h delay in a final group of five dogs had no effect on hyperoxic or hypoxic PAP/Q plots. We conclude that in intact dogs 1) the sympathetic nervous system reduces both hyperoxic and hypoxic pulmonary vascular tone, 2) stimulation of the peripheral chemoreceptors inhibits HPV, and 3) almitrine has direct pulmonary vasoconstricting effects in hyperoxia but not hypoxia.     

The incidence of breathing movements of fetal sheep in normoxia and hypoxia after peripheral chemodenervation and brain-stem transection

The incidence of fetal breathing movements and low voltage electrocortical activity was measured in three groups of fetal sheep, at 123-137 days gestation. The first group (transected & denervated) had the brainstem transected at the level of the colliculi and also had peripheral arterial chemodenervation. The second group (denervated) had a sham brain-stem transection and peripheral arterial chemodenervation. The third group (sham-operated) had sham brain-stem transection and sham peripheral chemodenervation. No differences were observed in the incidence of fetal breathing movements or low voltage electrocortical activity between the sham-operated and the denervated groups in normoxia, or in hypoxia when all these fetuses became apnoeic. There were however differences between these 2 groups and the transected & denervated group, in which fetal breathing movements where dissociated from electrocortical activity and which in some fetuses were continuous. During isocapnic hypoxia 3 of 8 transected & denervated fetuses made fetal breathing movements. We discuss the problems of interpreting data from brain-stem transected fetuses, but conclude that the evidence reveals no tonic influence of the peripheral arterial chemoreceptors on fetal breathing movements.     

Adenosine stimulates breathing in fetal sheep with brain stem section.

Breathing responses to adenosine were determined in 12 chronically catheterized fetal sheep (greater than 0.8 term) in which hypoxic inhibition of breathing had been eliminated by brain stem section. The caudal extent of transection varied from the rostral midbrain to the pontomedullary junction. Isocapnic hypoxia [delta arterial PO2 (PaO2) of -12 Torr] doubled the incidence and depth of breathing activity and increased the incidence of eye movements. Intra-arterial infusion of adenosine (0.30 +/- 0.03 mg.min-1.kg fetal wt-1) increased the incidence and amplitude of breathing without affecting blood gases. Adenosine did not significantly alter the incidence of eye activity. Intra-arterial injection of oligomycin (120 +/- 26 micrograms/kg fetal wt), an inhibitor of mitochondrial oxidative phosphorylation, also stimulated breathing activity. In four fetuses with brain stem section, peripheral arterial chemodenervation blunted the stimulatory effects of hypoxia on breathing activity and abolished altogether the excitatory effects of adenosine. It is concluded that 1) hypoxia and adenosine likely inhibit breathing in normal fetuses by affecting similar areas of the brain stem and 2) in fetuses with brain section, hypoxic hyperpnea depends on peripheral and central mechanisms, whereas adenosine stimulates breathing via the peripheral arterial chemoreceptors.     

Denervation of peripheral chemoreceptors decreases breathing movements in fetal sheep

The role of the peripheral chemoreceptors in the control of fetal breathing movements has not been fully defined. To determine whether denervation of the peripheral chemoreceptors affects fetal breathing movements, we studied 14 chronically catheterized fetal sheep from 120 to 138 days of gestation. In seven fetuses the chemoreceptors were denervated by bilateral section of the vagus and carotid sinus nerves; in seven others, sham operations were performed. We compared several variables during two study periods: 0-5 and 6-13 days after operation. In the denervated fetuses there were significant decreases in the incidence and amplitude of fetal breathing movements during both study periods. There were no differences between the two groups in incidence of low-voltage electrocortical activity, arterial pH and blood gas tensions, fetal heart rate, mean arterial blood pressure, or duration of survival after operation or birth weight. We conclude that denervation of the peripheral chemoreceptors decreases fetal breathing movements. These results indicate that the peripheral chemoreceptors are active during fetal life and participate in the control of fetal breathing movements.     

Effects of almitrine on hypoglossal and phrenic electroneurograms

Almitrine increases breathing by stimulating peripheral chemoreceptors. Previous studies suggest clinical usefulness in the adult with chronic obstructive pulmonary disease, but little data are available to decide whether almitrine would be helpful in diseases involving pharyngeal airway obstruction, such as apnea of prematurity or obstructive sleep apnea. We investigated the effect of intravenous almitrine on hypoglossal (HG), an upper airway nerve, and phrenic (PHR) neural activity in eight alpha-chloralose-urethan anesthetized, paralyzed, vagotomized, and artificially ventilated cats. Recordings were made of raw and integrated HG and PHR electroneurograms (ENGs), alveolar PCO2, arterial PO2, arterial blood pressure, and rectal temperature. A dose-response study of cumulative almitrine doses ranging from 0.1 to 4.0 mg/kg was performed in three cats. The interactive effects of almitrine and hypoxic stimulation were investigated in four cats. The interactive effects of almitrine and hypercapnic stimulation were investigated in five cats. The interactive effects of almitrine and ventilatory timing were investigated in six cats. We found that 1) almitrine doses as low as 0.1 mg/kg iv increased both HG and PHR ENG activity, with a maximum effect at approximately 1.0 mg/kg; 2) almitrine markedly increased HG and PHR ENG activity at all arterial PO2 values from 35-175 Torr; 3) almitrine increased HG and PHR ENG activity at all arterial PCO2 values from 30-70 Torr; and 4) almitrine increased the ratio of tidal volume to inspiratory time and decreased the inspiratory muscle duty cycle at normoxia and eucapnia.     

Ventilatory, circulatory, endocrine, and renal effects of almitrine infusion in man: a contribution to high altitude physiology

Diuresis at altitude was thought to be the result of chemoreceptor stimulation leading to a reduction of cardiac volume overload. This hypothesis was tested in ten young, healthy subjects by infusion of almitrine (0.5 mg.kg-1 body mass within 30 min) assuming analogous sites of action, i.e. arterial chemoreceptors and pulmonary vessels, for almitrine as for hypoxic hypoxia. The results show that almitrine increases ventilation, heart rate, systolic blood pressure, central venous pressure and natriuresis, but fails to increase significantly atrial natriuretic peptide plasma concentration and diuresis. It is concluded: (1) that almitrine has similar sites of action as hypoxic hypoxia at about 5000 m, (2) that natriuresis during arterial chemoreceptor stimulation might reduce cardiac volume overload, (3) that the volume excretion hypothesis, in particular the pathways from the cardiac volume overload to the water diuresis, need, for an understanding of the hypoxia-induced diuresis, further direct investigations at altitude.     

Effects of prostaglandins on fetal breathing do not involve peripheral chemoreceptors

In sheep, prostaglandin (PG) E2 inhibits fetal breathing movements and meclofenamate, a PG synthetase inhibitor, causes a marked stimulation of fetal breathing movements; the site of action of these agents is not known. To determine whether these effects are mediated through the peripheral chemoreceptors, we studied 13 fetal sheep at gestational ages of 127 to 138 days. Seven fetuses had bilateral section of the carotid sinus and vagus nerves (denervated); six had sham operations. Beginning at least 6 days after the operation, we infused PGE2 (0.6 microgram X kg-1 X min-1) into five denervated and five sham-operated fetuses and meclofenamate (0.4 mg X kg-1 X h-1) into six denervated and four sham-operated fetuses. Infusions averaged 20 h in duration. During preinfusion control periods, the incidence of fetal breathing movements (% of time) was lower in denervated than in sham-operated fetuses (18.9% vs. 31.5%; P less than 0.005). In both groups, the incidence of fetal breathing movements was decreased by PGE2 and was increased by meclofenamate; when expressed as absolute values, the magnitude of the changes with both agents was greater in sham-operated fetuses than denervated fetuses. However, the effects were similar in both groups when the changes were expressed as a percent of the respective control values. The incidence of fetal breathing movements (% of control) was decreased by PGE2 to 25.4% in denervated and to 28.2% in sham-operated fetuses and was increased by meclofenamate to 297.3% in denervated and to 304.0% in sham-operated fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha 2 adrenergic control of ventilation in the rat]

In anaesthetized rats, ventilatory stimulation induced by phentolamine, an alpha sympatholytic agent, emphasizes the role of some adrenergic mechanisms in the control of the respiratory centres activity. Phentolamine (5 and 10 mg.kg-1, iv) stimulates ventilation after a 4 s latency, tidal volume and respiratory rate being both increased. A same response can also be provoked 10 min later, by a second identical iv administration, systemic blood pressure remaining then stable at its previous low level. Hyperventilation is also observed when phentolamine is injected in totally denervated rats, without any remaining baro- or chemosensitivity. Stimulation is thus due to a central activity in relation with the release of inhibitory influences. Phentolamine also causes hyperventilation after prazosin pretreatment indicating that the alpha 1 adrenergic blockade is not involved in the post-phentolamine stimulation. This is an alpha 2 adrenergic transmission dependent mechanism. Variation of the systemic blood pressure is not the main mechanism involved in the hyperventilation induced by phentolamine. Meanwhile, baroreceptor activity modulates the central response to the drug, as shown by the negative influence of the post-vasopressin arterial hypertension. Hyperoxia is also a modulating factor acting by two ways: an inhibition of the peripheral chemoreceptors activity is added to an arterial hypertension. On the other side, activation of these chemoreceptors by almitrine bismesilate increases the respiratory responses to phentolamine. As already shown by one of us (Lagneuax, 1986), phentolamine pretreated rats are more responsive to hypoxia and to almitrine. Moreover, these phentolamine pretreated rats are protected against cardiovascular collapses and against apnea, frequently observed during hypoxia without CO2 compensation.     

Effects of naloxone on the breathing, electrocortical, heart rate, glucose and cortisol responses to hypoxia in the sheep fetus

Continuous infusions of naloxone HC1 (0.5 mg/kg or 3.8 mg/kg) or saline were given intravenously to fetal sheep at 119 to 137 days of gestation during a one hour period of air administration and a one hour period of hypoxia induced by having ewes breathe 9% O2, 3% CO2 and 88% N2. Fetal carotid PaO2 fell to 13.0 +/- 0.5 mmHg during hypoxia with no change in pH. During hypoxia, plasma cortisol concentration increased significantly more in naloxone-infused fetuses than controls. Ewes, whose fetuses received naloxone, showed a significant increase in cortisol during hypoxia whereas no increase was observed in controls. There were no significant differences between saline and naloxone-infused fetuses during hypoxia in fetal breathing incidence, amplitude, frequency, number of deep inspiratory efforts per hour, heart rate, electrocortical activity or in the rise in plasma glucose caused by hypoxia. Results suggest that endogenous opiates may have a role in modulating cortisol production in the ewe and fetus during hypoxia but do not have a role in mediating the decrease in incidence of breathing activity or rise in plasma glucose. During air administration, naloxone significantly increased fetal breath amplitude, fetal and maternal plasma glucose, fetal heart rate, and the number of electrocortical changes per hour. This suggests endogenous opiates may have a more important role in the normoxic pregnant ewe and fetus.     

Effects of hypoxaemia and hypercapnia on breathing movements and sleep state in sinoaortic-denervated fetal sheep

The role of the systemic arterial chemoreceptors in regulating breathing movements was determined in 7 chronically catheterized fetal sheep with carotid denervation and vagal section. Fetal hypoxaemia (delta PaO2 = -11.4 +/- 0.6 mmHg) decreased significantly the incidence of rapid-eye-movements (control = 26 +/- 1.5 min/h; hypoxia = 12 +/- 2.6 min/h, P less than 0.001) and breathing activity (control = 18 +/- 1.0 min/h; hypoxia = 8 +/- 1.1 min/h, P less than 0.001). However, the lag in onset of inhibition (approximately 8 min) was significantly greater (P less than 0.05) than for normal fetuses. The incidence of low voltage electrocortical activity was not affected. Hypercapnia (delta PaCO2 = 9.5 +/- 1.1 mmHg) increased significantly the incidence of rapid-eye-movements and breathing activity. Hypercapnia also increased the mean amplitude of breathing activity and reduced the average breath interval. Rapid-eye-movements and breathing activity were depressed significantly by hypoxaemic hypercapnia. These observations suggest that hypoxic inhibition does not require afferent activity from the aortic or carotid bodies nor from other chemoreflexes mediated by the vagus. However, such peripheral input may be responsible for a more rapid onset of inhibition in normal fetuses.     

Hypoxia and electrocortical activity in the fetal lamb: effects of brainstem transection and chemoreceptor denervation

In order to investigate possible mechanisms for the effect of hypoxia on fetal electrocortical (ECoG) activity, the effects of 30 min of isocapnic hypoxia on ECoG were studied in three groups of unanaesthetized late-gestation fetal lambs in utero. One group was intact, in the second the brainstem was transected between the colliculi, and in the third the carotid sinus nerves and cervical vagosympathetic trunks were cut bilaterally to denervate the systemic arterial chemoreceptors. The incidence of high voltage (HV) ECoG activity was lower in brainstem-transected fetuses than in the other groups. All three groups showed an increased number of changes from low to high voltage and an increase in the incidence of HV activity at the onset of hypoxia, but the increases reached statistical significance only in the brainstem-transected group. It is concluded that the onset of hypoxia is often associated with an increase in HV ECoG activity, with the most consistent changes occurring after brainstem transection and similar but smaller increases in intact and denervated fetuses. Thus the response of fetal electrocortical activity to the onset of hypoxia does not depend on intact connections with the lower brainstem. However, the effect of hypoxia on fetal ECoG is minor and inconsistent and may be physiologically unimportant.     

Dopamine and ventilatory effects of hypoxia and almitrine in chronically hypoxic rats

We hypothesized that the temporary blunted ventilatory response to hypoxia seen in chronically hypoxic rats could be related to the increased amount of dopamine found in their carotid bodies. Rats, kept 2-3 wk in 10% O2, showed reduced nonisocapnic ventilatory responses to 21-12% inspiratory O2 fraction compared with control rats. Stimulus-response curves to almitrine, which simulates the action of hypoxia on the carotid body, were also depressed in chronically hypoxic rats. Responses to hypoxia and almitrine were significantly correlated in the two groups of rats. Dopamine depressed ventilation during normoxia, hypoxia, and almitrine stimulation in both groups, an action abolished by the dopamine-2 antagonist domperidone. Domperidone slightly increased responses to hypoxia and almitrine in control rats but had a greater enhancing effect in chronically hypoxic rats, such that there was no longer a difference between the responses of the two groups.     

Flux-dependent increase in the stoichiometry of charge translocation by mitochondrial ATPase/ATP synthase induced by almitrine

After studying the effects of almitrine, a new kind of ATPase/ATP synthase inhibitor, on two kinds of isolated mammalian mitochondrion, we have observed that: (1) Almitrine inhibits oligomycin-sensitive ATPase; it decreases the ATP/O value of oxidative phosphorylations without any change in the magnitude of delta mu H+. (2) Almitrine increases the mechanistic H+/ATP stoichiometry of ATPase as shown by measuring either (i) the extent of potassium acetate and of potassium phosphate accumulation sustained by ATP utilisation, or (ii) the electrical charge/ATP (K+/ATP) ratio at steady-state of ATPase activity. (3) Rat liver mitochondria are at least 10-times more sensitive to almitrine than beef heart mitochondria. (4) The change in H+/ATP stoichiometry induced by almitrine depends on the magnitude of the flux through ATPase. The inhibitory effect of almitrine on ATPase/ATP synthase complex, as a consequence of such an H+/ATP stoichiometry change, is discussed.     

Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life

Selected topics in the respiratory response to acute hypoxia in the fetus and newborn are reviewed. Peripheral chemoreceptors acting through ionotrophic glutamate receptors play an important role in affecting the initial augmentation phase. Whether fall off in peripheral chemoreceptor activity contributes to the secondary depressive phase remains controversial. A number of approaches including permanent electrolytic and reversible cooling lesions, Fos protein activation, and double-labeling immunohistochemistry has converged to show that an area in and around the locus ceruleus in the rostral pons affects the central depression. There is evidence that this is mediated by catecholamines acting at alpha(2)-adrenergic receptors. Tonic activity in early expiratory (postinspiratory) neurons may contribute to hypoxia-induced apneic episodes in the fetus and newborn. Desensitization of alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid receptors has been demonstrated in respiratory-related neurons both in vivo and in vitro. The role that this process might play in the depressive phase of the hypoxic ventilatory response has not been established. In vitro experiments with isolated brain stem-spinal cord preparations or transverse brain stem slices usually involve anoxia, whereas whole animal experiments use 8-15% O(2). Therefore, caution must be exercised in attempting to construct a unifying framework from these two approaches.     

Effects of almitrine on genioglossal and diaphragmatic electromyograms

We previously demonstrated dose-dependent increases in both hypoglossal and phrenic electroneurograms after almitrine in anesthetized, paralyzed, and vagotomized cats. We have now investigated the effect of this peripheral chemoreceptor stimulant on diaphragmatic and genioglossal (GG, an upper airway-maintaining muscle) electromyograms in five unanesthetized, chronically instrumented, spontaneously breathing adult cats during slow-wave sleep. In 12 studies almitrine doses of 1.0-6.0 mg/kg increased inspired minute ventilation (VI), frequency (f), and tidal volume (VT) and decreased expiratory time (TE). However, almitrine doses as high as 6.0 mg/kg failed to augment phasic inspiratory GG activity. To determine why almitrine induced phasic inspiratory upper airway activity in anesthetized, vagotomized cats but not in sleeping cats, additional studies were performed. In four dose-response studies in three pentobarbital-anesthetized cats, almitrine, 1.0-6.0 mg/kg, did not produce phasic inspiratory GG activity. Almitrine did induce phasic inspiratory GG activity in two of three studies in three vagotomized, tracheostomized, alpha-chloralose-urethan-anesthetized cats. These results suggest that almitrine would not be useful in obstructive sleep apnea, yet because almitrine markedly increased VI, f, and VT and decreased TE in unanesthetized sleeping cats the drug may be effective in patients who lack normal central neural respiratory drive, such as the preterm infant.     

The role of peripheral chemoreceptors in the rapid response of the pulmonary vasculature of the late gestation sheep fetus to changes in PaO2.

The effect of removing the input from the peripheral arterial chemoreceptors on pulmonary vascular responses to changes in PaO2 was examined in late gestation fetal sheep. Blood flow in the left pulmonary artery and driving pressure across the pulmonary vascular bed were monitored in chronically prepared fetal sheep at 126-129 days gestation. Five fetuses had carotid sinus and vagus nerves sectioned bilaterally and four were left intact. In normoxia (PaO2 ca. 23 mmHg) pulmonary vascular resistance was slightly greater and pulmonary blood flow reduced in the denervated group relative to the intact group but these differences were not significant. When made hypoxic (PaO2 ca. 14 mmHg), pulmonary blood flow fell and pulmonary vascular resistance increased in all fetuses. However, in the intact fetuses these changes were significantly more rapid. In all fetuses the vasoconstriction was prolonged after their return to normoxia. When made hyperoxic (PaO2 ca. 27 mmHg), pulmonary blood flow increased by a similar amount in all fetuses. We conclude that in the term fetus the peripheral chemoreceptors play no appreciable role in the maintenance of the high pulmonary vascular resistance in normoxia, or the fall in resistance produced by a rise in PaO2. The chemoreceptors do however initiate the rapid phase of pulmonary vasoconstriction in hypoxia.     

The essential role of carotid body chemoreceptors in sleep apnea

Sleep apnea is attributable, in part, to an unstable ventilatory control system and specifically to a narrowed "CO2 reserve" (i.e., the difference in P(a)CO2 between eupnea and the apneic threshold). Findings from sleeping animal preparations with denervated carotid chemoreceptors or vascularly isolated, perfused carotid chemoreceptors demonstrate the critical importance of peripheral chemoreceptors to the ventilatory responses to dynamic changes in P(a)CO2. Specifically, (i) carotid body denervation prevented the apnea and periodic breathing that normally follow transient ventilatory overshoots; (ii) the CO2 reserve for peripheral chemoreceptors was about one half that for brain chemoreceptors; and (iii) hypocapnia isolated to the carotid chemoreceptors caused hypoventilation that persisted over time despite a concomitant, progressive brain respiratory acidosis. Observations in both humans and animals are cited to demonstrate the marked plasticity of the CO2 reserve and, therefore, the propensity for apneas and periodic breathing, in response to changing background ventilatory stimuli.