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J Appl Physiol 91: 343-350, 2001;
8750-7587/01 $5.00
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Vol. 91, Issue 1, 343-350, July 2001

Adenosine A1 and A2A receptors modulate sleep state and breathing in fetal sheep

Brian J. Koos, Takatsugu Maeda, and Calvin Jan

Nicholas S. Assali Perinatal Research Laboratory, Department of Obstetrics and Gynecology and the Brain Research Institute, University of California at Los Angeles School of Medicine, Los Angeles, California 90095-1740


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study was designed to determine the adenosine (Ado) receptor subtype that mediates the depressant effects of Ado on fetal breathing and rapid eye movements (REM). In chronically catheterized fetal sheep (>0.8 term), intra-arterial infusion of N6-cyclopentyladenosine (CPA), an Ado A1-receptor agonist, increased the incidence of high-voltage electrocortical (ECoG) activity while virtually abolishing low-voltage activity, REM, and breathing. These effects were blocked by 9-cyclopentyl-1,3-dipropylxanthine (DPCPX), an Ado A1-receptor antagonist. Infusion of DPCPX alone increased breath amplitude but had no significant effect on inspiratory duration, breath interval, incidence of REM, or incidence of low-voltage activity. Ado A2A-receptor blockade with ZM-241385 increased the incidence of low-voltage ECoG activity, REM, and breathing but had no effect on breath amplitude or respiratory cycle. Both DPCPX and ZM-241385 eliminated the inhibitory effects of Ado on REM and breathing. We conclude that 1) Ado A1 receptors tonically inhibit fetal respiratory drive, 2) Ado A2A receptors tonically inhibit REM-like behavioral state, and 3) both Ado A1 and A2A receptors mediate the depressant effects of Ado on REM and breathing.

behavioral state; brain; respiration; rapid eye movements


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BREATHING IN FETAL SHEEP (>0.8 term) consists of rapid, irregular movements produced by contractions of the diaphragm (7). In sheep, fetal breathing (>0.8 term), which is present ~30-50% of the time in normal fetuses, occurs in episodes associated with low-voltage electrocortical (ECoG) activity and rapid eye movements (REM), a behavioral state that resembles REM sleep (7, 37). Fetal respiratory efforts are typically absent during high-voltage ECoG activity, a state similar to quiet or non-REM (NREM) sleep. The apnea of NREM states does not result from a lack of stimulation because hypercapnia, which increases the incidence and amplitude of fetal breathing in REM sleep, does not induce respiratory movements during NREM episodes. This inhibition of breathing appears to be linked directly or indirectly to medullary prostaglandins because systemic or central administration of cyclooxygenase inhibitors induces breathing in NREM sleep (7). The coincidence of breathing and REM suggests that the REM state may provide critical respiratory drive in fetal sheep (17, 22).

In adult mammals, adenosine (Ado) and Ado receptor agonists enhance sleep, whereas Ado receptor antagonists increase wakefulness, implicating Ado as an endogenous sleep-promoting agent (30). Ado production by the central nervous system is related to metabolic activity of neurons and glia (30). Compared with slow-wave sleep, extracellular levels of Ado in the brains of rats and cats tend to be greater during wakefulness, when the brain has a higher metabolic rate, with the magnitude of the change depending on the site of sampling (14, 30). Extracellular brain Ado concentrations in the basal forebrain increase steadily during sleep deprivation and slowly fall during rebound sleep (30). The modulation of sleep by Ado may involve a decrease in the activity of cholinergic neurons involved in behavioral arousal that reside in the basal forebrain (29) and mesopontine tegmentum (34).

In fetal sheep, intravascular infusion of Ado virtually eliminates REM and breathing without significantly altering the incidence of low-voltage ECoG activity; higher rates of infusion (>0.25 mg · min-1 · kg-1) can also reduce the incidence of low-voltage activity (21). These results have led to the hypothesis that the depressant effects of Ado on fetal breathing result from an Ado-induced reduction in REM sleep. Intravascular administration of 8-phenyltheophylline, a potent nonselective Ado-receptor antagonist, to fetal sheep increases the incidence of low-voltage activity, REM, and breathing (6). These effects of Ado have relevance to the fetus because central Ado concentrations are increased during hypoxia (20) and may fluctuate with behavioral state during normoxia.

Four subtypes of Ado receptors (A1, A2A, A2B, and A3) have been identified based on agonist and antagonist binding affinities and cloning (10). The receptor subtypes that mediate the effects of Ado on fetal sleep state and breathing have not been clearly established. This study was designed to determine the role of Ado A1 and A2A receptors in mediating fetal behavior.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The following surgical procedures and experiments, which were approved by the Chancellor's Animal Research Committee, were performed in accordance with the guiding principles in the care and use of animals as endorsed by the American Physiological Society. Twenty-nine pregnant ewes (Rambouillet-Columbia cross) were operated on at ~120 days of gestation (~0.8 term). With the ewes under halothane anesthesia, polyvinyl catheters were inserted in the amniotic sac and in the right brachial and carotid arteries, external jugular vein, and trachea of the fetus (22). Bipolar stainless steel electrodes (Cooner Wire, Chatsworth, CA) were sutured to the lateral and medial canthus of an eye to record eye movements, and a second set of electrodes was placed on parietal dura to record the electrocorticogram. Stainless steel wire, which was sutured to the scalp, served as the common electrode.

Postoperatively, the pregnant ewes were kept in metabolic carts to permit long-term fetal recordings. Pressure transducers (Argon Medical, Dallas, TX) were used to measure amniotic, tracheal, and arterial pressures. Fetal tracheal and arterial pressures (both referenced to amniotic fluid pressure), heart rate, electrooculogram (EOG), and ECoG were recorded on a Grass Instruments chart recorder. Tracheal pressure was sampled at 100 Hz by microcomputer, and minute averages of breathing measurements (number of breaths, inspiratory time, breath interval, and breath amplitude) were stored on disk. Arterial blood gases and pH were measured with blood gas electrodes (model 1304, Instrumentation Laboratories) with values corrected to 39.5°C.

Fetal responses to adenosine receptor agonists and antagonists were determined in the unanesthestized state at least 4 days after surgery.

Ado A1-Receptor Agonist

N6-cyclopentyladenosine (CPA), which has a high degree of selectivity for Ado A1 receptors, was dissolved in 0.2 N HCl and 0.9% saline (50:50, vol/vol) and infused (0.008 mg · min-1 · kg-1) for 1 h into the external jugular vein. This dose of CPA produces an Ado A1-receptor-mediated bradycardia (19).

Ado A1-Receptor Antagonist

9-Cyclopentyl-1,3-dipropylxanthine (DPCPX) was dissolved in 0.04 M 2-hydroxypropyl-beta -cyclodextrin and 0.2 N NaOH (50:50, vol/vol). DPCPX (2.5 mg/ml) was infused into the right brachiocephalic trunk at 1.2 mg · min-1 · kg fetal wt-1 for 10 min and 0.24 mg · min-1 · kg-1 for 50 min. The DPCPX dose was determined from the rate of infusion that blocked the bradycardia induced by CPA (19).

Ado A2A-Receptor Antagonist

4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-alpha ][1,3,5]triazin- 5-yl amino]ethyl)phenol (ZM-241385, Zeneca Pharmaceuticals), which has a high degree of selectivity for the Ado A2A receptor, was dissolved in polyethylene glycol 400 and 0.1 N NaOH (50:50, vol/vol) and diluted to 30 ml with saline. ZM-241385 (0.33 mg/ml) was infused into the right brachiocephalic artery at 1.3 mg · min-1 · kg-1 for 5 min and 0.056 mg · min-1 · kg-1 for 55 min. The rate of infusion was determined by the dose that blocked the tachycardia (an A2A-mediated response) induced by Ado (19).

Ado

Ado (3.6 mg/ml saline) was infused (14 mg · min-1 · kg-1) into the external jugular vein of the fetus for 1 h. This dose of Ado inhibits REM and breathing (19, 21).

Ado and A1-Receptor Blockade

DPCPX was infused simultaneously with Ado to determine the role of Ado A1 receptors in mediating the effects of Ado on fetal sleep state and breathing.

Ado and A2A-Receptor Blockade

ZM-241385 was administered during 1 h of Ado infusion to determine the role of Ado A2A receptors in mediating the effects of Ado on fetal sleep state and breathing.

Control experiments were also performed in which only the vehicle for the Ado receptor agonist or antagonist was infused; our previous work has shown that slow infusions of saline do not alter fetal sleep state or breathing (21). The experiments were performed on separate days to minimize enduring effects of the drugs, and the order of the experiments was varied.

Data Analysis

In fetal sheep (>0.8 term), the ECoG is differentiated into high- and low-voltage ECoG activity; low-voltage ECoG states are associated with episodes of REM and breathing. The incidence of REM and low- and high-voltage ECoG activity was determined by visual analysis of chart recordings, as previously described (15). Breathing, REM, and low- and high-voltage ECoG states were judged present if the activity occurred during at least 20 s of each 1-min epoch (21).

Fetal breathing was identified from the characteristic negative changes in tracheal pressure that exceeded 1 mmHg in amplitude. Inspiratory time and breath interval were determined from on-line computer analysis with minute averages stored on disk. Only breaths with intervals of <= 3 s were considered valid for this analysis; that is, isolated breaths or gasps were excluded.

Statistical Analysis

Statistical significance for serial measurements was determined by repeated measures ANOVA with post hoc comparison by Tukey's least significant difference criterion. Single comparisons between control and experimental measurements were performed using Student's t-test. Differences were significant at P < 0.05. Values are means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ado A1 Receptors

Ado A1-receptor agonist. CPA was infused to seven fetuses. CPA did not significantly alter arterial PO2 (PaO2; control = 24.3 ± 1.5, infusion = 25.1 ± 1.6 Torr), arterial partial pressure of CO2 (PaCO2; control = 48.5 ± 1.9, infusion = 48.8 ± 1.4 Torr), or pH (control = 7.341 ± 0.006, infusion = 7.328 ± 0.009). CPA more than doubled the incidence of high-voltage ECoG activity and virtually eliminated low-voltage states, REM, and breathing (Fig. 1). CPA did not significantly alter inspiratory duration (control = 0.49 ± 0.04; infusion = 0.46 ± 0.06 s), breath interval (control = 1.41 ± 0.17; infusion = 1.48 ± 0.35 s), and breath amplitude (control = 2.6 ± 0.1; infusion = 2.8 ± 0.3 mmHg). Administration of the vehicle alone did not significantly affect arterial blood gases, pH, and the incidence of high-voltage ECoG, low-voltage ECoG, REM, and breathing. The respiratory cycle and breath amplitude also were not significantly altered.


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  		Fig. 1.   The effects of intravascular infusion of an adenosine (Ado) receptor agonist N6-cyclopentyladenosine (CPA) on the incidence of high-voltage (HV) and low-voltage (LV) electrocorticogram (ECoG), rapid eye movements by electrooculogram (EOG), and breathing in fetal sheep (solid line). The broken line shows the effects of simultaneous infusion of CPA with an Ado A1-receptor antagonist 9-cyclopentyl-1,3-dipropylzanthine (DPCPX). The vertical bar shows the hour of infusion. *P < 0.05 compared with control. dagger P < 0.05 compared with CPA value at same time.

Ado A1-receptor agonist and antagonist. CPA and DPCPX were infused simultaneously in seven fetuses to determine whether DPCPX, the Ado A1-receptor antagonist, would abolish the behavioral effects of CPA. DPCPX and CPA administration was associated with a slight fall (P < 0.05) in PaO2 of 2-4 Torr compared with the control of 25.9 ± 2.4 without altering PaCO2 or arterial pH (pHa). DPCPX blocked the depressant effects of CPA on low-voltage ECoG, REM, and breathing (Fig. 1). Compared with preinfusion values, the Ado A1-receptor antagonist actually increased the incidence of REM by 30% and the incidence of breathing by nearly twofold. Simultaneous infusion of CPA and DPCPX significantly reduced inspiratory time (control = 0.49 ± 0.02, infusion = 0.46 ± 0.03 s) and breath interval (control = 1.66 ± 0.06, infusion = 1.20 ± 0.07 s) but not breath amplitude (control = 2.8 ± 0.1, infusion = 2.9 ± 0.1 mmHg). Thus DPCPX antagonized the inhibitory effects of CPA on low-voltage activity, REM, and breathing, which indicated that the rate of DPCPX administration was sufficient to block Ado A1 receptors.

Ado A1-receptor antagonist. In 10 fetuses, DPCPX was administered to determine the effects of Ado receptor blockade. PaO2 was slightly reduced after 10 min of DPCPX administration (control = 24.8 ± 1.6, infusion = 21.5 ± 1.6 Torr; P < 0.05), but other measurements did not differ significantly from control. DPCPX had no significant effect on fetal pH or PaCO2. Ado A1-receptor blockade did not significantly affect the incidence of high-voltage ECoG activity (control = 23 ± 2.3, infusion = 20 ± 2.7 min/h), low-voltage activity (control = 31 ± 2.3; infusion = 31 ± 2.3 min/h), REM (control = 30 ± 1.3; infusion = 31 ± 2.7 min/h), or breathing (control = 27 ± 2.5, infusion = 30 ± 3.7 min/h). DPCPX increased breath amplitude (Fig. 2) but did not significantly affect inspiratory duration (control = 57 ± 0.03, infusion = 0.56 ± 0.04 s) or breath interval (control = 1.79 ± 0.17, infusion = 1.70 ± 0.19 s). Infusion of the vehicle did not alter fetal arterial blood gases, sleep state, or breathing.


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  		Fig. 2.   Mean tracheal pressure amplitude during infusion of DPCPX and ZM-241385. *P < 0.05 compared with control.

Ado and Ado A1-receptor antagonist. Ado and DPCPX vehicle were infused simultaneously to seven fetal sheep with normal arterial blood gases and pHa. Arterial blood gases were not significantly affected, but pHa declined significantly by 0.023 units (control = 7.335 ± 0.009). Ado significantly reduced the incidence of REM and breathing (Fig. 3) but did not significantly affect the inspiratory time, breath interval, or mean breath amplitude. The depressant effects of Ado were blocked by DPCPX.


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  		Fig. 3.   Effects of Ado plus the vehicle for DPCPX on the incidence of HV and LV ECoG, EOG, and breathing (broken line) in fetal sheep. The solid line shows fetal responses to simultaneous infusion of Ado and DPCPX, the Ado A1-receptor blocker. *P < 0.05 compared with control. dagger P < 0.05 compared with vehicle value at same time.

Ado A2A Receptors

Ado A2A-receptor antagonist. ZM-241385, which was administered to 10 fetuses, did not significantly affect mean PaO2, PaCO2, or pHa, but it did reduce the incidence of high-voltage ECoG by 56% and increase the incidence of low-voltage ECoG by virtually the same amount (Fig. 4). Ado A2A-receptor blockade increased the incidence of REM by about 70% and the incidence of breathing by more than 200% (Fig. 4); it did not significantly alter inspiratory time (control = 0.55 ± 0.04, infusion = 0.57 ± 0.05 s), breath interval (control = 1.72 ± 0.18, infusion = 1.55 ± 0.19 s), or breath amplitude (Fig. 2). No significant effects were observed with infusion of the vehicle.


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  		Fig. 4.   Effects of intravascular infusion (vertical bar) of ZM-241385 (solid line), an Ado A2A-receptor antagonist, on the incidence of HV and LV ECoG, EOG, and breathing. The effects of the vehicle for ZM-241385 are shown by the broken line. *P < 0.05 compared with control. dagger P < 0.05 compared with vehicle value at same time.

Ado and Ado A2A-receptor antagonist. Simultaneous infusion of Ado and the vehicle for ZM-241385 to eight fetuses was associated with arterial blood gases and pH effects similar to those for infusion of Ado with the vehicle for DPCPX. Ado decreased the incidence of REM and breathing but had no significant effect on the incidence of high- or low-voltage ECoG activity, inspiratory duration, breath interval, or breath amplitude (Fig. 5).


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  		Fig. 5.   Effects of intravascular administration of Ado plus ZM-241385 (solid line) or the ZM-241385 vehicle (broken line) on the incidence of HV and LV ECoG, EOG, and breathing. *P < 0.05 compared with control. dagger P < 0.05 compared with Ado + ZM-241385 vehicle value at same time.

Ado infusion during ZM-241385 administration slightly reduced fetal PaO2 (control = 24.2 ± 1.1, infusion = 22.1 ± 1.8 Torr) after 10 min; other PaO2 values did not significantly differ from control. Fetal PaCO2 and pHa were not significantly affected. Ado in conjunction with blockade of Ado A2A receptors was not associated with significant effects, relative to the preinfusion control, on the incidence of high- or low-voltage ECoG activity, REM, or breathing (Fig. 5). Compared with measurements during Ado and ZM vehicle infusion, ZM-241385 decreased the incidence of high-voltage ECoG and significantly increased the incidence of low-voltage ECoG, REM, and breathing. However, the incidence of low-voltage ECoG, REM, and breathing during infusion of Ado and ZM-241385 was significantly less than during administration of the Ado A2A blocker alone (Fig. 4).

Ado with ZM-241385 administration was associated with an increase in breath amplitude (control = 3.0 ± 0.3, infusion = 3.9 ± 0.4 mmHg), but inspiratory time (control = 0.56 ± 0.04, infusion = 0.57 ± 0.04 s) and breath interval (control = 1.63 ± 0.12, infusion = 1.47 ± 0.16 s) were not significantly affected. Infusion of Ado with the vehicle for ZM-241385 did not alter the depth of breathing or the respiratory cycle.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows that the depressant effects of Ado on REM and breathing are abolished by blockade of either A1 or A2A receptors, which indicates that both receptor subtypes are involved in the inhibition. Selective Ado receptor blockade provides additional information on the receptor subtypes involved in the regulation of fetal behavior and respiration under normal conditions.

Behavioral State

Ado A1 receptors. The Ado A1-receptor agonist CPA increased the incidence of high-voltage ECoG activity and reduced the incidence of low-voltage states and REM. These responses confirm previous studies in fetal sheep with systemic administration of a stable Ado analog [L-N6-(2-phenylisopropyl)-adenosine (L-PIA)] with a high selectivity for Ado A1 receptors (44, 46) and in adult rats with intraperitoneal or intracerebroventricular injection of L-PIA or CPA (2, 33). The enhancement of synchronized neuronal activity by stimulation of central Ado A1 receptors likely results from increased inward-rectifying potassium currents and decreased depolarizing inputs in the cerebral cortex and thalamus (2).

The new finding is that blockade of fetal Ado A1 receptors by DPCPX did not alter the incidence of low-voltage ECoG activity or REM, indicating that Ado A1-receptor activation does not have a tonic effect on fetal behavioral state under basal conditions. Thus A1 receptors do not mediate the enhancement of fetal REM state induced by nonselective Ado receptor antagonism (6).

Ado A2A receptors. Intravascular administration of an Ado A2A-receptor agonist (CGS-21680) depresses low-voltage ECoG activity and REM, indicating that activation of central A2A receptors inhibits REM-like behavioral state (16). In the present study, Ado A2A-receptor antagonism by ZM-241385 decreased the incidence of high-voltage ECoG activity, whereas it increased the incidence of low-voltage states and REM. These effects of systemic administration of ZM-241385, which are similar to those of nonselective Ado receptor blockade (4, 6, 21), indicate that central Ado A2A receptors exert tonic inhibitory effects on REM sleep in fetal sheep.

Infusion of ZM-241385 alone promoted the REM state to a significantly greater extent than during simultaneous infusion of ZM-241385 and Ado. These results are consistent with residual REM depression produced by Ado stimulation of central A1 receptors.

The effects of Ado A2A-receptor blockade on behavioral state likely depend on maturity and species. For example, intraperitoneal injection of an antagonist of Ado A2A receptors (SCH-58261) in adult rats reduces REM sleep and promotes wakefulness (3).

Brain Ado receptor network. Ado, which is the likely common pathway for prostaglandin D2 and other sleep-promoting substances (45), binds to several receptor subtypes in different brain sectors in adult mammals (26). For example, stimulation of Ado A1 receptors within the basal forebrain, preoptic and anterior hypothalamus, and the mesopontine tegmentum promotes sleep in rodents and cats (1, 24, 25, 28, 30, 31, 34, 40, 42, 43, 45, 48). Activation of Ado A2A receptors in the tuberculum olfactorium and ventral nucleus accumbens of rats enhances NREM and REM sleep (38, 39, 40). The neuronal network and Ado receptors that mediate behavioral states in fetal sheep have not been established.

Breathing

Adenosine A1 receptors. Ado A1-receptor blockade increased mean breath amplitude, which suggests that A1 receptors tonically inhibit fetal respiratory motoneurons. The incidence of breathing remained unchanged most likely because DPCPX did not enhance REM sleep. These results are consistent with previous work in which injection of L-PIA into the fourth ventricle of fetal sheep decreased breath amplitude, indicating that stimulation of brain stem A1 receptors has a direct inhibitory effect on respiratory neurons (5). Thus medullary motoneurons are the likely site of action of Ado relative to the modulation of breath amplitude. Neither the agonist (5) nor the antagonist of the A1 receptor altered inspiratory time or breath interval, indicating that Ado A1 receptors are not a significant modulator of respiratory rhythm under these conditions.

Intracerebroventricular administration of small doses of L-PIA in anesthetized rats reduces respiratory frequency and tidal volume, suggesting that Ado depresses respiratory drive in adult animals (11). Using an in vitro brain stem and spinal cord preparation, Dong and Feldman (8) showed that CPA produces a dose-dependent reduction in the transmission of inspiratory drive from bulbospinal neurons to phrenic motoneurons, an effect that was blocked by selective antagonism of Ado A1 receptors. Other investigators using this experimental model have shown that an Ado A1-receptor agonist depresses inspiratory-related neurons in the rostral ventrolateral medulla and decreases the respiratory burst rate in phrenic motoneurons (13, 14). An effect on respiratory drive has also been observed in intact animals. For example, systemic administration of DPCPX to anesthetized cats with denervation of the peripheral arterial chemoreceptors increased mean peak phrenic nerve activity (41). Ado A1 receptors probably modulate respiratory output under these conditions by decreasing synaptic transmission between respiratory neurons via presynaptic inhibition of neurotransmitter release and by inactivating medullary expiratory neurons via postsynaptic membrane hyperpolarization (8, 12, 41).

Ado A2A receptors. Blockade of Ado A2A receptors increased the incidence of breathing, indicating that central Ado A2A receptors have a tonic inhibitory effect on fetal breathing. ZM-241385 did not significantly affect breath amplitude, inspiratory time, or breath interval, which suggests that the mechanism does not involve a direct effect on brain stem respiratory neurons at basal concentrations of Ado. Thus the rise in breathing incidence likely resulted from the ZM-241385-induced increase in REM-like sleep.

In contrast, blockade of Ado A2A receptors during Ado administration increased breath amplitude, indicating that A2A receptors may modulate respiration-related neurons at higher levels of Ado. Further studies need to be performed to determine whether this respiratory stimulation involves activation of peripheral and/or central Ado A2A receptors.

These results extend our laboratory's previous work with intravascular infusion of CGS-21680, a highly selective Ado A2A-receptor agonist (16). CGS-21680 initially increased the incidence, frequency, and amplitude of fetal breathing, but this stimulation was followed by a prolonged depression. Sinoaortic denervation abolished the hyperpnea but not the inhibition. Ado A1-receptor blockade also had no effect on the depressant effects of CGS-21680 on breathing, indicating that A1-receptor activation by CGS-21680 was not the mechanism of depression (16). Thus the biphasic effects of the Ado A2A-receptor agonist on fetal breathing appear to result from stimulation of Ado A2A receptors in the carotid bodies resulting in hyperpnea, which is followed by activation of central A2A receptors that elicit apnea as the drug diffuses into the brain.

Systemic administration of Ado arrests fetal breathing, an inhibition that is abolished by discrete neuronal lesions of the posteromedial thalamus (18, 21). Thus the rostral brain sectors involved in controlling REM sleep are crucial to the depressant effects of Ado on fetal breathing (18). In the present experiments, ZM-241385 blocked the inhibitory effects of Ado on breathing, which suggests that Ado A2A receptors mediate this respiratory depression.

Ado A2A receptors are involved in peripheral arterial chemoreception in fetal (15, 16) and adult animals (27). Ado A2A receptors are heterogeneously distributed in the brain stem of rats with particularly high concentrations in the rostral nucleus tractus solitarius and ventrolateral medulla (47). Although these brain stem Ado A2A receptors have been implicated in cardiorespiratory regulation (47), their effects on respiratory control have not been established.

Physiological Significance

Fetal brain extracellular concentrations of Ado increase during hypoxia and mediate the inhibitory effects of acute O2 deprivation on REM and fetal breathing (4, 6, 20, 21). The mechanism of inhibition includes the parafascicular nuclear complex, a thalamic sector involved in sensorimotor integration and sleep-state regulation (17). The arrest of fetal movements by hypoxia appears to be an adaptive mechanism because it increases O2 availability for critical organs such as the heart, brain, and adrenals.

The enhancement of high-voltage ECoG states by Ado (21) has relevance to the increased incidence of high-voltage ECoG activity that occurs with severe fetal hypoxia (arterial O2 content ~1.5 mmol/l) (35). Fetal cerebral O2 consumption falls by ~17% when the ECoG changes from low- to high-voltage ECoG activity (36), which contributes to the decrease in cerebral O2 consumption when preductal arterial O2 contents are less than ~1 mmol/l (9). This decline in cerebral O2 consumption, which is mediated by an inhibition of excitatory amino acid release and postsynaptic hyperpolarization, protects the brain against acute O2 deprivation by reducing brain O2 requirement. Central Ado A1 and possibly A2A receptors are likely involved in promoting the high-voltage state. Ado also protects the brain against O2 deprivation by increasing brain blood flow (via A2A and A2B receptors), decreasing Ca2+ influx (via A1 receptors), and reducing free radical formation (via A2A and A3 receptors) (10).

Extracellular levels of Ado in the fetal brain have the potential to modulate sleep and breathing in the fetus. Fetal brain Ado concentrations are likely be to be higher during REM sleep than during slow-wave (NREM) sleep because the REM state has a greater brain metabolic rate (36). It is hypothesized that, as in postnatal animals, brain extracellular Ado would provide a negative feedback loop whereby the higher central Ado concentrations during REM would promote NREM sleep. Whether central Ado levels fluctuate with sleep state needs to be established before it can be determined whether Ado provides a critical link between brain metabolism and fetal behavior.

Receptor Blockade

In rodent brain tissue, DPCPX has been shown to be highly selective (>500-fold) for Ado A1 receptors relative to A2A and A3 receptors and moderately selective (50-fold) relative to A2B receptors (10). But this selectivity is species dependent because DPCPX is only moderately selective for A1 over A2A receptors in human tissues (23). ZM-241385 is highly selective for A2A relative to A1 and A3 receptors but only moderately selective (30- to 80-fold) relative to A2B receptors (29, 32).

Little information is available on the affinity and selectivity of DPCPX and ZM-241385 for Ado receptor subtypes in sheep. In our previous work (19), DPCPX prevented CPA-induced bradycardia, confirming that DPCPX blocked myocardial Ado A1 receptors. However, DPCPX also reduced by ~30% the tachycardia induced by Ado stimulation of A2A receptors, which indicates some A2A-receptor antagonism under the conditions of study. In the present study, DPCPX and ZM-241385 had distinctive effects on breath amplitude and behavioral state, which suggests at least a moderate degree of selectivity relative to Ado A1- and A2A-receptor subtypes in fetal sheep under normal conditions. These observations are consistent with the separate effects of DPCPX and ZM-241385 on fetal cardiovascular responses to hypoxia (19). Thus respiratory and behavioral effects of ZM-241385 were apparently mediated by antagonism of A2A receptors, although interaction with Ado A2B receptors cannot be excluded.

In summary, DPCPX increased breath amplitude but had no significant effects on the respiratory cycle, incidence of breathing, or behavioral state. These results indicate that brain stem Ado A1 receptors have a tonic depressant effect on fetal respiratory motoneurons but not sleep state. ZM-2413845 increased the incidence of REM-like state and breathing without altering breath amplitude or respiratory cycle. These results suggest that Ado A2A receptors have a tonic inhibitory effect on REM-like sleep, which, in turn, reduces the incidence of breathing. DPCPX and ZM-241385 eliminated the inhibitory effects of Ado on REM and breathing. Thus central Ado A1 and A2A receptors mediate the depressant effects of Ado on REM and breathing in fetal sheep.


    ACKNOWLEDGEMENTS

The authors thank Leland Patron and Fanor Bohorquez for technical assistance. S. M. Poucher of Zeneca Pharmaceuticals kindly supplied ZM-241385.


    FOOTNOTES

This work was supported in part by National Institute of Child Health and Human Development Grant HD-18478.

Address for reprint requests and other correspondence: B. J. Koos, Dept. of Obstetrics and Gynecology, 27-168 CHS, UCLA School of Medicine, Los Angeles, CA 90095-1740 (E-mail: bkoos{at}mednet.ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 December 2000; accepted in final form 2 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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