Adenosine A_2a receptors and O₂ sensing in development

Abstract

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O₂ sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5′-nucleotidase and the resulting activation of adenosine A_2A receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A_2A receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A_2A receptors mediate hypoxic inhibition of breathing and rapid eye movements. A_2A receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A_2A receptors play virtually no role in O₂ sensing by the carotid bodies, but brain A_2A receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A_2A receptors have been implicated in O₂ sensing by carotid glomus cells, while central A_2A receptors likely blunt hypoxic hyperventilation. In conclusion, A_2A receptors are crucially involved in the transduction mechanisms of O₂ sensing in fetal carotid bodies and brains. Postnatally, central A_2A receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O₂ sensing in carotid chemoreceptors, particularly in developing lambs.

mammalian life critically depends upon oxygen, which provides cellular energy [adenosine triphosphate (ATP)] by accepting electrons at complex IV of the mitochondrial respiratory chain (Fig. 1). A continuous production of ATP is crucial for sustaining electrochemical gradients of cellular membranes, cell metabolism, organ function, and growth. Restrictions in mitochondrial oxygen supply, which can occur continuously or intermittently, trigger adaptive responses to help restore mitochondrial oxygen sufficiency and ATP synthesis.

Fig. 1.

Schematic diagram of electron and proton flow along the respiratory chain complexes (CI-CIV) of the inner mitochondrial membrane. Electrons (dotted line) from NADH (CI) and FADH₂ (CII) flow along the respiratory chain to O₂ (CIV), which is linked to a proton flux from the mitochondrial matrix to the intermembranous space. Passage of protons back to the matrix through the ATPase complex drives ATP synthesis. ATP passes through the inner and outer mitochondrial membranes via the adenine nucleotide translocator (ANT) into cytosol. Under normal conditions, 1–2% of electron flow through the respiratory chain reduces O₂ to superoxide anion radical (O₂^·−) at complexes I and III. Hypoxia diverts more of the electron flow to O₂^·− production, with O₂^·−'s generated at complex III a major source of mitochondria-derived reactive oxygen species (ROS) in cytosol. The dashed lines show the reversal of membrane transport for protons and ATP in O₂ deficiency. ATP generated by cytosolic glycolysis is carried into mitochondria, where it is hydrolyzed by ATPase. This supplies energy for the extrusion of protons from the mitochondrial matrix to the intermembranous space to maintain the mitochondrial membrane potential. [Adapted from Solaini et al. (317).]

Fetal hypoxia can arise from numerous maternal, placental, and fetal pathophysiologic states that reduce placental O₂ transport (Table 1). Normal fetuses maintain O₂ consumption, and thus placental O₂ transfer, with acute reductions up to ∼50% in uteroplacental blood flow, umbilical blood flow, or oxygen-carrying capacity of fetal or maternal blood. Under these conditions, oxygen sufficiency is sustained by an increase in O₂ extraction by fetal tissues and other mechanisms, although longer-term reductions can result in reduced fetal O₂ consumption and growth (42, 110, 157, 231). Clinically, insufficient fetal O₂ delivery can lead to significant perinatal morbidity and mortality (Table 2).

Table 1.

Causes of fetal hypoxia

Mother

↓ Arterial O2 content

High altitude

Asthma

Pneumonia

Cystic fibrosis

Cyanotic heart disease

Severe anemia

Malaria and sickle cell crisis

Seizures

Sleep apnea

↓ Uteroplacental blood flow

Hypotension*

Congenital or acquired heart disease

Chronic hypertension

Preeclampsia

Diabetes mellitus

Thrombosis

Long-term high altitude

Uterine contractions

Smoking

Illicit stimulants

Vasoconstrictors

Antiphospholipid antibody disorders

Placenta

↑ Intervillous fibrin deposition

↓ Villous capillary size and/or number

Villous edema

Thrombosis/infarction†

Abruptio placentae

Umbilical Circulation

Vasoconstrictors

Preeclampsia

Umbilical cord compression

Umbilical cord hypercoiling/knots

Polyhydramnios

Meconium

Fetus

↓ Systemic arterial pressure

Acute hemorrhage

Cardiac failure

↓ Arterial O2 content

Anemia

Smoking (carbon monoxide)

^*Due to septic shock, acute hemorrhage, supine hypotension, antihypertensive agents, regional anesthesia;

^†includes malaria and sickle cell crisis.

Table 2.

Consequences of fetal hypoxia

Intrauterine growth restriction

Prematurity

Hypoxic-ischemic encephalopathy

Multiorgan failure

Stillbirth

Neonatal demise

Meconium aspiration syndrome

Seizures

Cerebral palsy

Mental retardation

A major cause of neonatal O₂ deprivation includes the failure to establish effective pulmonary gas exchange at birth due to respiratory distress syndrome, meconium aspiration, pulmonary hypoplasia, fetal hydrops, diaphragmatic hernia, congenital cystic adenomatoid malformation of the lung, birth asphyxia, and central nervous system depressants. Postnatal hypoxia also occurs in pneumonia, apnea of prematurity, and airway obstruction in sleep.

Compensatory responses to these hypoxic challenges are essential to maintain metabolic homeostasis in the fetus and newborn. A crucial first-line defense involves O₂ sensors that respond almost instantaneously to reductions in Pa_O2 through mechanisms independent of metabolic compromise or gene transcription. Upon activation, these chemoreceptors initiate acute physiologic compensations that support oxygen homeostasis for critical organs. Examples include carotid bodies, chromaffin cells of the adrenal medulla in the fetus or neonate, neuroepithelial bodies in the airways, the central nervous system, and the smooth muscle of pulmonary and systemic vasculature.

Various triggers have been proposed to account for the hypoxia-induced chemosensory cascade (44, 116, 123, 163, 262, 349, 353). These include an O₂-limitation in mitochondrial oxidative phosphorylation at complex IV (Fig. 1), increased production of reactive oxygen species (ROS) at mitochondrial complex III and/or cell membrane NADPH oxidase, and inhibition of cell membrane potassium channels that are inhibited directly or indirectly by falls in Po₂. The latter include conformational changes in associated plasma membrane hemoproteins (e.g., NADPH oxidase) and enzymatic release of CO via hemeoxygenase-2. Hypoxia-inducible factor-1, a major regulator of oxygen-dependent gene expression instantaneously stabilized by hypoxia, activates hypoxic signaling pathways within the first 2 min of a fall in tissue Po₂, which inactivates anabolism, stimulates anaerobic glycolysis, and inhibits mitochondrial aerobic metabolism (151, 164, 310, 317, 325, 351). Although the relative roles of specific mechanisms are disputed, it is generally accepted that the transduction cascade in O₂ sensors involves inhibition of potassium currents, depolarization of cell membranes, and elevation in intracellular [Ca²⁺].

Progress has been made surrounding controversies of O₂ chemoreception and establishing the relative role of O₂ sensing processes in development. This information has important implications for acute respiratory, cardiovascular, metabolic, and endocrine responses of the fetus and newborn to hypoxia as well as for the emergence of chronic pathophysiologic disorders in children and adults. Thus, advances in this field have relevance to fetal, newborn, and adult medicine.

This essay reviews putative regulation by the mitochondrial respiratory chain of adenosine formation in development and the resulting autocrine, paracrine, and endocrine compensations that buffer against acute O₂ deprivation. A particular focus is the role of adenosine A_2A receptors in O₂ sensory transduction in the carotid body and the brain, as determined by cardiorespiratory responses to acute hypoxia. Potential A_2A receptor involvement in other major O₂ sensing tissues in perinatal life is also summarized.

ADENOSINE: AN OVERVIEW

Adenosine Production and Metabolism

Adenosine, a purine nucleoside, is primarily derived in normoxia from intracellular S-adenosyl homocysteine (Fig. 2). Cytosolic adenosine is phosphorylated via adenosine kinase to AMP, but conversion to inosine by adenosine deaminse can predominate at high levels of adenosine. Intracellular adenosine diffuses down a concentration gradient into the extracellular space via equilibrative membrane transporters. Adenosine can also be produced extracellularly from rapid hydrolysis (via membrane-bound ecto-5′-nucleotidase) of AMP that is derived from ATP or cAMP. Adenosine is not stored in vesicles, although synaptic exocytosis has been proposed for discrete brain sectors (347). Extracellular adenosine can be transported intracellularly via equilibration, as well as by sodium-dependent, concentrating transporters, which lower extracellular concentrations (93).

Fig. 2.

Cellular production of adenosine. CD39, nucleoside triphosphate diphosphohydrolase; CD73, ecto-5′-nucleotidase; 5NT, endo-5′-nucleotidase; AK, adenosine kinase; SAH, S-adenosyl homocysteine; cylinder, exocytosis exportation; circle, equilibrative membrane transporter. SAH hydrolase reversibly catalyses the degradation of SAH to adenosine and homocysteine. [Modified from Fredholm et al. (93).]

Hypoxic reductions in mitochondrial oxidative phosphorylation have been proposed to raise the cytosolic ADP/ATP ratio, which, via activation of adenylate kinase, converts two molecules of ADP into one molecule each of ATP and AMP (10, 85, 86, 262, 360). This reaction has two effects: 1) it blunts the fall in ATP, and 2) relative to the rise in the ADP/ATP ratio, it exponentially increases the cytosolic AMP/ATP ratio. The latter elicits adaptations that tend to restore and stabilize cellular ATP levels. Thus, negligible changes in cytosolic ATP can sufficiently increase the AMP/ATP ratio to activate AMP-activated protein kinase (AMPK), 5′-nucleotidase and other enzymes involved in chemoreceptor-signaling pathways (85, 86, 130, 142, 202, 262. 360). Compared with other tissues, the enhanced O₂ sensitivity of chemoreceptors has been attributed to higher levels of key enzymes linking changes in cellular AMP/ATP ratio to membrane depolarization, greater ATP dependency on mitochondrial oxidative phosphorylation, and lower resting energy state (i.e., ↑ADP/ATP ratio).

The rise in cytosolic AMP/ATP ratio in acute hypoxia elevates adenosine levels by stimulating cytosolic and extracellular 5′-nucleotidases (131, 142, 185, 202), although other enzymes (e.g., ↓ adenosine kinase activity) can be involved (69). Because AMP levels regulate adenosine formation, the relative rates of ATP synthesis and degradation largely determine net adenosine production, which, in turn, is controlled by the ATP utilization rate and the availability of substrate (e.g., glucose, O₂) that is involved in ATP synthesis. Adenosine formation from extracellular AMP normally predominates under these conditions (115), but its relative contribution likely depends on the tissue and the extent of O₂ deprivation. Increased metabolic rate alone can reduce the adenylate energy state and activate 5′-nucleotidase; thus, adenosine levels can increase without a restriction in O₂ supply per se (22, 129, 305).

Adenosine levels can be measured in interstitial fluid dialysate or plasma by high-performance liquid chromatography (182, 185, 191, 306), and real-time changes in adenosine concentration can be tracked with enzyme-based probes (347). Circulating levels of adenosine in fetal sheep, which are 2–4 times those in adult ewes, derive from the low fetal Pa_O2 and possibly from specialized features of the umbilical circulation (182, 306).

Adenosine Receptors

Adenosine binds to four extracellular G protein liganded receptor subtypes (A₁, A_2A, A_2B, and A₃), which are distinguished by differential affinities for adenosine analogs and methylxanthanine antagonists, tissue distribution, and effector-coupling mechanisms (94, 146). Adenosine has the greatest affinity for A₁ receptors with less attraction for A_2A, A_2B, and A₃ receptors. Both the A₁ and A₃ receptors inhibit adenylate cyclase via pertussis-sensitive G proteins (G_i/o), which can stimulate K⁺ and K_ATP currents and inhibit Q-, P-, N-type Ca²⁺ channels. The importance of A₁ receptors in fetuses and newborns has been reviewed (290, 355).

A_2A and A_2B receptors primarily couple to G_s (G_olf for A_2A in striatum), which stimulates adenylyl cyclase, increases cAMP concentrations, and activates cAMP-dependent protein kinase A (PKA). PKA stimulates L-type calcium channels, as well as a number of other signaling pathways (Table 3). A_2A receptors have been implicated in inositol phosphate formation, activation of protein kinase C, and elevation of intracellular Ca²⁺ levels that result in contraction of smooth muscle or neurotransmitter release. A_2A receptor signaling also can occur independently of G proteins (93, 146, 382). A_2B receptors can couple to G_q, which activates phospholipase C. Specific tissue responses are determined by the number and relative expression of adenosine receptor subtypes, interaction with other receptors (including heteromeres), and extracellular concentrations of adenosine (93).

Table 3.

Adenosine A_2A signal transduction involving cAMP-dependent kinase^*

↑ L-type Ca2+ channel

↑ aPKC

↑ CREB→↓NFkB

↑ DARPP-32

↑ Rac1/Cdc42 → →→p38

↑ Src → Ras →Raf-1→MEK → ERK1/2

^*Via G protein activation of adenylyl cyclase and increase in intracellular cAMP.

aPKC, atypical protein kinase C; CREB, cAMP responsive element binding protein; NFkB, nuclear factor-кB; DARPP-32, dopamine- and cAMP-regulated neuronal phosphoprotein; p38, p38 mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinases 1 and 2. [lsqb]Modified from Fredholm et al. (93).[rsqb]

A_2A receptors, which have been localized to chromosome 22q11.23 in humans and chromosome 10 in mice (364), are widely expressed in tissues, including the immune system, platelets, myocardium, lungs, vasculature, carotid body, and central nervous system (93, 94, 146, 350). Thus, A_2A receptors are involved in diverse physiologic processes, such as respiration, sleep, vascular tone, angiogenesis, inflammation, and defenses against oxidative stress. The following summarizes the developmental role of these receptors in O₂ sensory transduction in the carotid body and the brain as well as the involvement of these receptors in metabolic, endocrine, and vascular responses to acute O₂ deprivation.

ADENOSINE AND O₂ SENSING

Carotid Body

In newborns and adults, hypoxic excitation of the carotid bodies triggers arousal from sleep, hyperventilation, and sympathetically driven cardiovascular responses that rapidly defend against acute oxygen deprivation. A detector of Po₂ in arterial blood supplying the brain, the carotid body also independently senses arterial Pco₂, pH, temperature, osmolality, and glucose (198).

General O₂ sensory mechanisms.

Oxygen chemoreception by the carotid body likely involves the functional interaction of multiple sensors with differing thresholds of Po₂ activation that enable chemosensory responses over a wide range of O₂ tensions (1, 117, 201, 274). The generally accepted paradigm of O₂ sensing by the carotid body involves depolarization of glomus cell membranes by hypoxic inhibition of K⁺ channels, a rise in intracellular Ca²⁺ levels, and subsequent release of neurotransmitters that stimulates adjacent fibers of the carotid sinus nerve.

While the mechanism by which hypoxia depresses K⁺ currents is not fully understood, it most likely involves an imbalance in the localized rates of ATP production/consumption within glomus cells (85, 86, 87, 360). The resulting elevation in the ADP/ATP ratio triggers, via adenylate kinase, a rise in the AMP/ATP ratio, which activates AMPK by allosteric stimulation of phosphorylation and by inhibition of dephosphorylation (127, 303). Activated AMPK, in turn, inhibits large-conductance, calcium-activated potassium channels (BK_Ca) and background potassium channels (e.g., TASK-like potassium currents, TREK channels), resulting in membrane depolarization, voltage-gated Ca²⁺ entry, and the release of an array of neurotransmitters and neuromodulators, including acetylcholine, catecholamines, peptides, amino acids, ATP, and adenosine (32, 85, 117, 168, 197, 201, 211, 262). ATP, acetylcholine, adenosine, and other transmitters/modulators have been implicated in chemoexcitation of the carotid sinus nerve (104, 200, 274, 288, 289, 380). This general schema of carotid O₂ sensing appears generally applicable to humans, because human carotid body expresses TASK and BK_Ca channels, as well as receptors for γ-aminobutryic acid, acetylcholine, and dopamine (88).

The duration and frequency of hypoxia modulate O₂ chemoreception. For example, chronic, intermittent hypoxia enhances carotid body sensitivity under basal and acute hypoxic conditions, a chemosensory plasticity that critically depends on ROS generation by mitochondria and NADPH oxidase (215, 275, 378).

A_2A receptors and O₂ sensing by adult carotid bodies.

Adenosine release by the carotid bodies is extremely sensitive to small reductions in Pa_O2, with the rise in interstitial levels due to catabolism of intracellular and extracellular ATP (38, 52). Exogenous adenosine, via A₂ receptors, increases chemosensory discharges in the carotid sinus nerve in rats and cats and stimulates respiration in normoxia and hypoxia (221, 222, 229, 308); thus, increased extracellular levels of adenosine have physiological relevance. Low doses of an A_2A receptor agonist (CGS-21680) stimulate respiration in rats, suggesting that these receptors mediate excitatory effects of adenosine (53, 308). Endogenously produced adenosine has been implicated in the stimulation of the carotid chemoreceptors in anesthetized rats (222, 230) and in hypoxic excitation of the carotid bodies in unanesthetized rhesus monkeys and human subjects (76, 138, 366).

A_2A and A_2B are the primary adenosine receptor subtypes in carotid glomus cells. Activation of A₂ receptors stimulates these O₂ sensors in vitro and in vivo (55, 90, 91, 172, 210, 229, 300, 308, 342, 350). Proposed transduction effects of adenosine A_2A receptor stimulation include activation of adenylate cyclase and PKA pathways to inhibit TASK-1 K⁺ channels, which results in membrane depolarization, Ca²⁺ entry through voltage-gated calcium channels, and release of neurotransmitters (47, 90, 91, 211, 342, 362), as depicted in Fig. 3. But the post-A_2A receptor cascade is likely to be more complex (51, 54, 172), and it may vary according to species, strains within species, and stage of development (11, 53, 55, 90, 172, 308).

Fig. 3.

Adenosine A_2A receptor transduction cascade proposed for carotid body activation. O₂-limitation of oxidative phosphorylation in mitochondria activates adenylate kinase (AK), which converts two molecules of ADP into ATP and AMP. The resulting increase in the AMP/ATP ratio promotes adenosine formation from AMP in cytosol and extracellular space. Intracellular adenosine is transported extracellularly via an equilibrative nucleoside transporter. Activation of extracellular A_2A receptors stimulates cAMP formation by adenylyl cyclase (AC), which, in turn, promotes protein kinase A (PKA) inhibition of potassium currents. The resulting fall in membrane potential (ΔV_m) activates voltage-gated Ca²⁺ channels, and the rise in intracellular Ca²⁺ leads to secretion of neurotransmitters that stimulate the carotid sinus nerve.

O₂ sensing by fetal carotid bodies.

In the sheep fetus, carotid chemoreceptors respond to changes in Po₂ and Pco₂ from at least 0.6 of term (24). The extremely low fetal Pa_O2 (25–30 Torr) greatly reduces the Po₂ setpoint of carotid glomus cells. As in the adult, the fetal carotid bodies respond selectively to changes in arterial Po₂ rather than in O₂ content (196). The carotid bodies of fetal rats express adenosine A_2A receptor mRNA (Table 4), which is consistent with a role for this receptor subtype in O₂ sensing in early development. It is reasonable to assume that fetal expression of transduction elements in carotid glomus cells differ from those in adults due to the much lower Pa_O2 as well as developmental immaturity. The responsiveness of the fetal carotid body to other stimuli (e.g., H⁺, hypoglycemia, hyperkalemia, and hyperosmolality) has not been specifically tested, although sensitivity to acidosis and glucose appears likely (35, 336).

Table 4.

Semiquantitative adenosine A_2A receptor gene expression in rat development revealed by in situ hybridization

	G16	G20	P0	P7	Adult
Caudate-putamen	2–3	3	2–3	3	3
Accumbens, n	1–2	3	2–3	3	3
Olfactory tubercle	2	3	2–3	3	3
Cortex	0–1	0–2	0	0	0
Parafascicular, n		0	0	2	0
Area postrema	0	0–2	0–2	0–2	0
Carotid body	2–3	2–3			2
Pituitary
Intermediate lobe	1–2	2–3	3	1–2	2
Anterior lobe	1–2	1	0	0	0

G, gestational days; P, postnatal days. [Data from Weaver (350).]

O₂ CHEMOREFLEXES.

In fetal sheep (>0.8 term), hypoxic excitation of the carotid bodies elicits autonomic reflexes that lower heart rate through increased vagal activity and constrict blood vessels via increased sympathetic tone (15, 111, 119, 125, 145, 170, 233). Sympathetic vasoconstriction, along with other factors, lower vascular conductance to nonessential tissues (e.g., kidneys, gastrointestinal tract, liver, and lungs), which support or raise systemic arterial pressure in the face of increased vascular conductance to the myocardium, brain, and adrenal glands (263). Similar cardiovascular adaptations have been identified in human fetuses (8, 16, 17, 39, 120, 132, 352). Thus, arterial O₂ chemoreflexes are involved in the redistribution of cardiac output and O₂ delivery to critical organs in O₂ deficient states. An important distinction in the fetus is that hypoxic excitation of the sinus nerve does not induce arousal or hyperpnea, as occurs postnatally, and fetal breathing differs in other respects as well (176).

Interestingly, acute and chronic hypoglycemia alters organ distribution of fetal combined ventricular output in a similar manner to that caused by hypoxia (34, 225, 322). Nutrient-deprived ovine fetuses have reduced liver blood flow and growth, which are abrogated by bilateral denervation of fetal carotid bodies (35). Thus, fetal carotid chemoreceptors have a role in redistribution of fetal cardiac output in both hypoxia and hypoglycemia, which can contribute to altered organ growth in oxygen- and nutrient-deprived fetuses (35, 110, 136).

The fetal O₂ deficiency of anemia differs in at least three ways from that of hypoxia: 1) lowered oxygen carrying capacity (i.e., hemoglobin concentration), 2) increased spacing of red cells in capillaries (a significant cause of O₂ deprivation in tissues), and 3) a minimal fall in Pa_O2. Because of the latter, fetal anemia has little effect on carotid O₂ chemoreflexes (196). Hemodynamic differences of anemia include elevated cardiac output and blood flow to most tissues (66).

A_2A RECEPTORS AND O₂ SENSING.

As in the adult, excitation of the fetal carotid bodies can be elicited by metabolic consequences of reduced ATP production in mitochondria. When central inhibition of breathing has been abolished by disruptions of the brain stem in fetal sheep, intra-arterial administration of oligomycin (a mitochondrial ATPase inhibitor, see Fig. 1), adenosine, or an adenosine A_2A receptor agonist rapidly increases the rate and amplitude of breathing (178, 179). This respiratory stimulation is eliminated by peripheral chemodenervation, which is consistent with a crucial role for A_2A receptors in exciting fetal carotid chemoreceptors.

Intravascular infusion of a potent, nonselective adenosine receptor antagonist with actions restricted to peripheral tissues [8-(p-sulphophenyl)-theophylline] abolishes the bradycardia and hypertension induced by hypoxic excitation of carotid chemoreflexes (108, 181), an effect that is replicated by highly selective A_2A receptor blockade (186), as shown in Fig. 4. These observations are consistent with the hypothesis that adenosine A_2A receptors associated with carotid bodies are critically involved in triggering hypoxic chemoreflexes that reduce heart rate (and thus myocardial O₂ consumption) and support O₂ delivery to critical organs. That adenosine A_2A receptors mediate the full expression of these cardiovascular chemoreflexes suggests that O₂ sensing in carotid bodies is critically linked to activation of 5′-nucleotidase.

Fig. 4.

Heart rate and mean arterial pressure responses in fetal sheep to isocapnic hypoxia during intravascular infusion of an adenosine A_2A receptor antagonist (ZM-241385) or vehicle. Vertical bars depict ± 1 SE. Horizontal bar shows time of hypoxia and infusion. *P < 0.05 compared with control at time 0; ^†P < 0.05 compared with vehicle at same time. During hypoxia, mean fetal Pa_O2 was ∼15 Torr (ΔPa_O2, about −9 Torr compared with normoxia). [Modified from Koos and Maeda (186).]

O₂ sensing by carotid bodies in postnatal development.

The control of breathing changes significantly at birth when respiratory gas exchange shifts from the placenta to the lungs.

CHANGES AT BIRTH.

The rise in Pa_O2 (from ∼25 to 50–70 Torr) at birth in sheep silences spontaneous carotid sinus nerve activity in the first 2–3 postnatal days, with the emerging discharge rate increasing in frequency in older lambs (24, 25, 79, 321). Although basal sinus nerve activity is depressed, isocapnic hypoxia stimulates respiration in newborn lambs and other neonates, an augmentation that is eliminated by carotid body denervation (23, 330). Thus, excitatory afferent discharges in the carotid sinus nerve are now functionally integrated into respiratory drive. The respiratory response becomes progressively more robust with advancing age, with the discharge rate increasing for a given Pa_O2 as well as for a change Pa_O2 (24, 126). This postnatal resetting of the carotid chemoreceptors, which can take days or weeks, potentially involves increases in the number of type I cells as well as changes in expression of receptors and ion channels, synaptic interactions, and afferent responsiveness (41, 74, 100, 330, 348). This adaptation does not include changes in carotid body perfusion (232).

PROLONGED OR RECURRENT PERINATAL HYPOXIA.

Chronic in utero exposure to sustained or intermittent hypoxia (112, 118, 267, 268) can result postnatally in resting hyperventilation with either a depressed or augmented ventilatory response to acute hypoxia in rats up to 3 wk of age (112, 267, 268). Naturally occurring growth restriction in fetal sheep (presumably chronically hypoxic) arrests the progressive increase in the hypoxic ventilatory response in lambs that normally occurs after the first week of age (239).

In several mammalian species, prolonged hypoxia initiated at birth induces long-term perturbations in respiratory control that include normoxic hyperventilation and/or blunting of the acute hypoxic respiratory response (79, 126, 251, 281, 282, 283, 314). The latter has been attributed to impairment of carotid body O₂ sensing by low Po₂ (79, 126, 321, 361).

Chronic intermittent hypoxia in the postnatal period in rats augments acute hypoxic respiratory hypernea, which persists even in adults (18, 153, 259, 260, 265, 275, 283). The exaggeration of acute carotid body O₂ sensing by chronic intermittent hypoxia, which is mediated largely through the release of ROS, is greater in neonatal than adult rats and is associated with glomus cell hyperplasia in only neonates (260, 265, 266, 275, 276). Intermittent hypoxia increases ROS levels by activating NADPH oxidases, inhibiting mitochondrial complex III activity (Fig. 1), and downregulating anti-oxidant enzymes via hypoxia-inducible factors 1 and 2 (275).

In mature mammals, respiratory acclimatization to low Pa_O2 reverses rapidly upon the restoration of normoxia (18). Thus, the development of persistent changes in respiratory control that occur in the fetus and newborn are largely restricted to the perinatal period.

HYPEROXIA.

Sustained postnatal hyperoxia also modulates respiratory control (78). For example, prolonged hyperoxia blunts early stimulatory effects of acute hypoxia on ventilation in adult rats, apparently by altering carotid body development (19, 20).

A_2A RECEPTORS AND POSTNATAL O₂ SENSING.

Postnatal changes in carotid body O₂ sensitivity, which are qualitatively similar in rats and sheep, may involve altered expression of A_2A receptors (101, 103, 350). In rats, A_2A receptor mRNA expression in the carotid body declines postnatally (103, 350); nevertheless, A_2A receptors still account for ∼50% of the hypoxia-induced enhancement of sinus nerve activity in adults (55).

Hypoxic hyperventilation in newborn lambs is almost entirely mediated by stimulation of the carotid chemoreceptors, with negligible contribution from the aortic bodies (357). In developing lambs (1–2 wk of age), hypoxia rapidly increases the rate and depth of respiration, with maximum ventilation achieved within 1–2 min. This hyperpnea coincides with a sharp rise in arterial pressure and heart rate. Contrary to the response in the fetus, systemic antagonism of adenosine A_2A receptors in developing lambs does not blunt the hypertension or the maximum ventilatory response (Fig. 5). Thus, adenosine A_2A receptors have little involvement in carotid O₂ chemoreflexes of the developing lamb (184). The effects of antagonism of the A_2A receptors on hypoxic chemoreflexes in older sheep require investigation.

Fig. 5.

Respiratory responses to isocapnic hypoxia in lambs at 7–16 days of age. Depicted are the effects of ZM-241385 or vehicle on maximum hypoxic stimulation (Phase 1) and on ventilation 10 min (Roll-off₁₀) and 15 min (Roll-off₁₅) after the start of hypoxia compared with normoxia (control). *P < 0.05 compared with control; ^†P < 0.05 compared with vehicle; ^□P < 0.05 compared with phase 1. During hypoxia, mean Pa_O2 was ∼31 Torr (ΔPa_O2, about −52 Torr compared with normoxia). [From Koos et al. (184).]

RESPIRATORY EFFECTS ON POSTNATAL HEART RATE.

The onset of pulmonary ventilation at birth profoundly affects the modulation of heart rate by the autonomic nervous system. As previously noted, acute hypoxia decreases fetal heart rate as a result of a carotid chemoreflex that increases vagal tone. In contrast, hypoxic stimulation of the carotid bodies in the newborn or adult elicits tachycardia. Postnatally, hypoxic hyperpnea increases heart rate via central autonomic effects of increased afferent traffic from pulmonary stretch receptors (60). In addition to being episodic, fetal breathing differs in that: 1) inspiration is associated with minimal changes in lung volume, which are insufficient to activate pulmonary stretch receptors, and 2) hypoxia inhibits breathing activity (176); hence the differences in heart rate response to hypoxia in fetal and postnatal life.

Brain

O₂ deprivation generally increases potassium conductance and thereby hyperpolarizes neurons in the central nervous system. But in some brain sectors, acute hypoxia depolarizes neurons with effects that are consistent with those of an O₂ sensor (330).

The rostral ventrolateral medulla, nucleus tractus solitarius, pre-Bötzinger complex, and posterior hypothalamus have neurons intrinsically sensitive to hypoxia that, when activated, stimulate sympathetic and/or respiratory activity in adult mammals (245, 271, 330). With respect to respiration, hypoxic excitation of the posterior hypothalamus induces tachypnea in awake cats with carotid chemodenervation (330). The neuronal O₂ sensing mechanisms likely involve O₂-sensitive ion channels, hemeoxygenase-2, ROS, and erythropoietin as well as other transduction pathways (2, 59, 99, 106, 271, 330). ROS activation of TASK-2 channels in cells of the retrotrapezoid region of the ventral medulla has been implicated in hypoxic respiratory depression (106).

Present in glia and presynaptic and postsynaptic neurons, adenosine A_2A receptors are concentrated in striatum, nucleus accumbens, olfactory tubercle, and pineal gland with lower levels widely distributed more generally in gray matter (30, 89, 309, 367, 379). Adenosine A_2A receptors can affect neuronal activity directly as well as indirectly by interacting with A₁ receptors and receptors for other neuromodulators or neurotransmitters (308, 309). A_2A receptors modulate the release of several neurotransmitters, including acetylcholine, GABA, and dopamine. A classic example is the striatum, where stimulation of A_2A receptors reduces the affinity of dopamine D₂ receptors.

Brain A_2A receptors modulate sleep, temperature, and many other central functions, including tonic depression of motor activity (308, 369). Acute intermittent hypoxia induces long-term facilitation of phrenic motor activity by ROS generated by NADPH oxidase in the spinal cord, which probably involves activation of A_2A receptors (133, 215).

Adenosine and fetal behavior.

A_2A receptor mRNA is expressed widely in embryonic/fetal rat brains, including the striatum, cortex, hippocampus, and thalamus (Table 4). In near-term fetal sheep, a high density of A_2A receptors is present in striatum and the pineal gland, with moderate levels in the thalamus, including the parafascicular nuclear complex (Pf) (367).

In the ovine fetus (>0.8 term), virtually all behavior is consistent with quiet sleep (high-voltage electrocortical activity, absence of rapid eye movements, and breathing) alternating with rapid eye movement sleep (low-voltage electrocortical activity coincident with rapid eye movements and breathing activity). Blockade of central adenosine A_2A receptors increases the incidence of rapid eye movement states, indicating that endogenous adenosine levels are involved in the regulation of sleep in normoxic fetuses (178, 187).

BRAIN HYPOXIA AND FETAL BREATHING AND RAPID EYE MOVEMENTS.

Hypoxia inhibits fetal movements through direct effects on the central nervous system (68, 193). With respect to breathing and rapid eye activity, isocapnic hypoxic depression is dose-dependent (195), beginning when fetal Pa_O2 acutely falls more than ∼6 Torr [Δarterial O₂ content (Ca_O2) about −2.00 ml/dl; control Ca_O2 = 7.18 ml/dl], with virtual absence of these movements when Pa_O2 decreases by 9–10 Torr (ΔCa_O2, about −3.81 ml/dl), as shown in Fig. 6. The amplitude and frequency of residual breathing are unaltered by isocapnic hypoxia (176).

Fig. 6.

Incidence of breathing and rapid eye movements in fetal sheep is shown in relation to the calculated mean end-capillary Po₂ of the fetal brain. The incidence of breathing and rapid eye movements declines in dose-dependent manner once the estimated end-capillary Po₂ falls below ∼14 Torr. The incidence of low-voltage electrocortical activity was not significantly reduced over the studied range of fetal Pa_O2. *P < 0.05 compared with normoxia with calculated mean end-capillary Po₂ ∼18 Torr. The corresponding measurements of mean fetal Pa_O2 were ∼25 Torr for normoxia and ∼20, 18, 17 Torr, respectively, for hypoxia. [Modified from Koos et al. (195).]

Denervation of the carotid and aortic chemoreceptors delays the onset, but not the extent, of the inhibition (193). The depressant effects of moderate hypoxia (without a continued decline in pHa) are transient with the incidence of breathing and rapid eye movements increasing toward normal within 3–4 h of sustained O₂ deficiency (176). This return of activity is consistent with actions of a central O₂ sensor that rapidly adapts to changes in Pa_O2.

Supramedullary disruptions of the brainstem abolish hypoxic inhibition of breathing; thus, this respiratory arrest does not arise from the intrinsic effects of hypoxia on medullary respiratory motoneurons (68, 114). More discrete neuronal lesions have identified a sector of the posteromedial thalamus encompassing the Pf as the most rostral locus of the neuronal network mediating hypoxic inhibition of breathing and rapid eye movements (179), as shown in Fig. 7. This conclusion is supported by the induction of apneas by electrical stimulation within or immediately adjacent to this locus (183). The crucial role of Pf, which integrates sensorimotor function and regulates behavioral state, supports the notion that a hypoxia-induced change in behavioral state (e.g., reduction in rapid eye movement sleep) underlies the depressant effects on fetal breathing (179, 195).

Fig. 7.

Effects of normoxia and isocapnic hypoxia on the incidence of breathing and rapid eye movements in normal fetal sheep and in fetuses with neuronal lesions that encompassed the parafascicular nuclear complex (Pf) of the posteromedial thalamus. In both groups, isocapnic hypoxia did not alter the amplitude or frequency of breathing. *P < 0.05 compared with normoxia. During hypoxia, Pa_O2 was ∼14 Torr (ΔPa_O2, about −10 Torr compared with normoxia). [Modified from Koos et al. (179).]

A_2A RECEPTORS AND FETAL BRAIN O₂ SENSING.

As in the carotid body (178, 192, 241) reduced mitochondrial ATP production has been implicated in the central O₂ sensing mechanism that triggers hypoxic inhibition of fetal breathing and rapid eye movements (194). This metabolic hypothesis of behavioral regulation has been bolstered by observations that 1) hypoxia increases fetal levels of adenosine in plasma and the brain (182, 191); 2) intravascular infusion of exogenous adenosine mimics the inhibitory effects of hypoxia on fetal breathing and rapid eye movements (192); and 3) hypoxic depression is abolished by blockade of central adenosine A_2A receptors (188), as depicted in Fig. 8. Thus, A_2A receptors are critically involved in central O₂-sensing mechanisms in the fetus.

Fig. 8.

Incidence of rapid eye movements and breathing during isocapnic hypoxia in normal fetal sheep with intravascular infusion of vehicle or ZM-241385. ZM-241385 blocked the inhibitory effects of hypoxia on breathing and rapid eye movements. During hypoxia, mean Pa_O2 was ∼14 Torr (ΔPa_O2, about −10 Torr compared with normoxia). *P < 0.05 compared with normoxia. [Modified from Koos et al. (188).]

Intracarotid infusion of a potent and highly selective A_2A receptor agonist (CGS-21680) has apposing effects on fetal breathing: stimulation (via carotid chemoreflexes) followed by inhibition due to activation of central A_2A receptors as the agonist accumulates in the brain (178). These breathing responses in the fetus are remarkably similar qualitatively to the biphasic respiratory response to hypoxia that is observed postnatally (see next section), and they are consistent with a crucial dual role of A_2A receptors in exciting fetal carotid chemoreflexes and triggering central hypoxic inhibition of fetal breathing.

Neuronal lesions in the diencephalon abolish inhibitory effects of adenosine on fetal breathing (180, 367). The locus of A_2A receptors that arrest breathing has yet to be established, but the immediate proximity of thalamic A_2A receptors to the rostral neuronal substrate (P_f) involved in hypoxic inhibition suggests a functional relationship.

Blockade of central α₂-adrenergic receptors increases the incidence, rate, and amplitude of breathing in normoxic fetal lambs. In hypoxic fetuses, breathing persists, although with lower incidence, rate, and amplitude compared with normoxic responses (13, 109). These studies implicate α₂-adrenergic receptors in a dopaminergic pathway that inhibits breathing in hypoxia.

GABA does not have a demonstrable role in hypoxic depression of breathing in unanesthetized fetal sheep (176). Thus, A_2A-GABA receptor interactions do not significantly contribute to hypoxic inhibition in the fetus.

Adenosine and brain O₂ sensing in postnatal development.

Adenosine is involved in central O₂ sensing as illustrated by acute respiratory responses to hypoxia in newborn and developing mammals.

ROLL-OFF RESPIRATORY RESPONSE TO HYPOXIA.

The hypoxic ventilatory response in many mammals is biphasic: an initial stimulation followed by a decline or roll-off. The second phase fall in ventilation, which is more prominent in immature newborns, has been attributed to several factors, including reduced metabolic rate, central effects of hypoxia on the respiratory neuronal network, and reduced carotid sinus nerve activity and/or its central integration into respiratory drive (23, 330). The roll-off becomes dampened with age as the phase-I stimulatory response becomes more robust.

A number of other factors influence the ventilatory decline, including sex, arousal state, anesthesia, Pa_O2, and O₂ ventilatory plasticity (330). For example, prenatal hypoxia exposure increases basal respiration in normoxia in neonatal rat pups, although this does not persist beyond postnatal day (P) 30 (112, 267, 268). In young male rats, continuous intrauterine hypoxia (embryonic days, E5–E20) abolishes the phase-2 ventilatory decline in hypoxia (268), while chronic intermittent hypoxia in utero accentuates the phase-2 depression in rat pups postnatally (118). Interestingly, perinatal exposure to alcohol changes the effects of episodic hypoxia in young rats from respiratory facilitation to depression (165), which is another example of in utero exposures disrupting postnatal respiratory regulation.

BRAIN SECTORS AND THE RESPIRATORY ROLL-OFF.

The inhibitory neuronal network involved in the roll-off includes neuronal substrate within or rostral to the pons in unanethetized young rabbits, as in fetal sheep (68, 217). Subsequent localization studies have demonstrated sectors proximate to the red nucleus in the midbrain of anesthetized young rabbits (3, 345) and to the locus coeruleus in the pons of anesthetized neonatal sheep (29, 234). Whether Pf of the posteromedial thalamus is critically involved in the ventilatory decline in developing lambs has yet to be established; thus, the developmental aspects of central hypoxic inhibition deserve further scrutiny.

A_2A RECEPTORS AND THE ROLL-OFF.

The roll-off respiratory response to hypoxia in newborns and adults is attenuated by nonselective adenosine receptor blockade with methylxanthines (63, 105, 208, 210). Studies in lambs with potent and highly selective antagonists have shown that the ventilatory depression critically depends on activation of adenosine A_2A receptors (184), as shown in Fig. 5. Thus, adenosine A_2A receptors mediate the depressant effects of hypoxia on respiration in both fetal sheep and developing lambs, indicating that a central adenosinergic inhibitory pathway persists in postnatal life. As in fetal sheep, the thalamus has a moderate density of A_2A in lambs and adult sheep, but it remains to be determined whether this locus of receptors is involved in hypoxic ventilatory depression.

Whether activation of adenosine A_2A receptors is indispensible for hypoxic respiratory depression has not been established. So far, respiratory responses to hypoxia have not been reported in neonatal mice with knockouts of the A_2A receptor. Other neurotranmitters or modulators [e.g., opioids, nitric oxide, substance P, serotonin, dopamine, GABA (23)] may assume more prominent roles in central respiratory depression in the absence of adenosine A_2A receptor activation.

Stimulation of medullary A_2A receptors activates inhibitory GABAergic inputs onto brainstem respiratory neurons in immature rats and piglets (219). These findings implicate A_2A receptors in GABAergic influences on respiratory timing and respiratory drive that are involved in apnea of prematurity (219, 358).

Endocrine Systems

Adenosine modulates hormone secretion by a number of tissues, including adrenal glands, pituitary, and myocardium.

A_2A receptors and fetal adrenal glands.

Adenosine is potentially involved in hormone release by the adrenal medulla and cortex.

ADRENAL MEDULLA.

In immature mammals, adrenomedullary chromaffin cells are directly sensitive to hypoxia, and the resulting rise in plasma catecholamine concentrations can be critical for survival (315). For example, in the fetus and newborn, elevated plasma catecholamine levels support cardiac contractility (via α-adrenergic receptors) and heart rate (via β₁-adrenergic receptors), constrict peripheral vasculature (via α-adrenergic receptors), and signal metabolic adaptations to acute oxygen deficiency. Fetal hypoxia or asphyxia in labor can contribute to the catecholamine surge at birth, which is involved in absorption of lung fluid and secretion of surfactant by stimulating pulmonary β₂-adenergic receptors.

The O₂ chemosensory cascade of chromaffin cells involves inhibition of K⁺ channels, membrane depolarization, and calcium entry via T-type voltage-gated Ca²⁺ channels (27, 160, 161, 205). Mitochondrial regulation of intracellular redox levels appears to modulate O₂-sensitive K⁺ currents in mature ovine adrenal medullary cells (160), which is consistent with other studies implicating mitochondria-derived ROS in hypoxic sensitivity in these chromaffin cells (248). Gene deficiency studies in mice have shown that NADPH oxidase (324) and hemeoxygenase-2 (253) are not essential components of the O₂-sensing mechanism.

Adrenomedullary chromaffin cells generally lose their direct O₂-sensing ability when splanchnic nerve innervation is completed, which occurs at approximately P10 in rats. In fetal sheep, sympathetic innervation of ovine adrenal occurs by 130 days of gestation (∼0.9 term), although ovine chromaffin cells in vitro retain ability to secrete catecholamines by a nonneurogenic mechanism (5, 48). Changes in the expression of T-type Ca²⁺ channels in chromaffin cells contribute to maturity-related loss of intrinsic O₂ sensitivity (161, 205).

In adult and neonatal rodents, intermittent hypoxia augments the response of adrenomedullary chromaffin cells to acute hypoxia via ROS generated by NADPH oxidase and mitochondria, as well as by downregulation of endogenous antioxidants. Both HIF-1α and HIF-2α are involved in the augmentation of ROS activity under these conditions (275). While reversible in adult rats, facilitation of catecholamine secretion from chromaffin cells by intermittent hypoxia in rat pups (P0-P5) persists after >30 days of subsequent normoxia. In contrast, continuous hypobaric hypoxia (P0-P5) blunts catecholamine secretion (305).

Adenosine A_2A and A_2B receptors have been identified on rat pheochromocytoma cells (PC12), while only A_2B receptors have been detected on plasma membranes of bovine adrenal chromaffin cells (43, 173). Whether A_2A and/or A_2B receptors are expressed in sheep adrenomedullary chromaffin cells has not been reported; however, the predominant plasma norepinephrine response to systemic administration of an A_2A receptor agonist (CGS21680) suggests that A_2A receptor activation is not directly involved in catecholamine release by the adrenal medulla in near-term fetal sheep (189). Epinephrine and norepinephrine responses have not been studied in younger fetuses when chromaffin responses to hypoxia more clearly result from a direct, nonneurogenic mechanism.

ADRENAL CORTEX.

Adenosine modulates cortisol production by the adrenal cortex. In fetal sheep, adenosine dampens the cortisol response to hypoxia by blunting corticotrophin release and by direct effects on the adrenal cortex (45). Depressant effects on adrenal secretion of cortisol are mediate by A₁ receptors (150).

A_2A receptors and fetal pituitary.

In the ovine fetus, acute hypoxia (preductal Pa_O2 ∼15 Torr) elicits a rapid rise in plasma arginine vasopressin (AVP) concentrations, with peak levels ∼24 times normoxic values (190). This AVP response to hypoxia is mimicked by intravascular administration of a selective A_2A receptor agonist (CGS21680) in normoxic fetuses, which is unaltered by bilateral denervation of the carotid bodies and section of the cervical vagi. Nonselective blockade of adenosine receptors abolishes ∼75% of the hypoxia-induced rise in AVP (190). These results are consistent with a critical role of A_2A receptor activation in the fetal AVP response to acute O₂ deficiency.

A_2A receptors and fetal myocardium.

Acute hypoxia (ΔPa_O2, about −10 Torr) reduces fetal plasma volume by ∼9% through mechanisms that include increased sympathetic activity and a rise in plasma concentrations of atrial natriuretic peptide (49). About 50% of the hypoxia-induced elevations in plasma atrial natriuretic peptide likely involve A_2A receptor enhancement of sympathetic activity and AVP release as well as direct stimulation of atrial A₁ receptors (250).

Metabolism

Adenosine and AMPK are potentially involved in hypoxic metabolic perturbations, either directly through effects on the fetus or indirectly through actions on the placenta.

A_2A receptors and fetal glucose metabolism.

Hypoxia increases fetal plasma glucose levels by reducing glucose consumption and increasing circulating catecholamine levels, which inhibit insulin release and promote hepatic glycogenolysis. Selective blockade of either adenosine A₁ or A_2A receptors blunts the hyperglycemia, indicating that both receptor subtypes are involved in these metabolic perturbations (216).

Oxygen deprivation shifts ATP production from mitochondria to anaerobic glycolysis in cytosol, a metabolic adaptation that is mediated in adult tissues by HIF-1 (310). Although stimulation of A_2A receptors has been linked to HIF-1 induction (7, 71), this adenosine receptor subtype does not modulate the rise in plasma lactate levels in acutely hypoxic, near-term fetal sheep (216).

AMPK and fetal metabolism.

Besides redistributing fetal cardiac output, hypoxia and/or nutritional deficiency may impair growth by upregulating AMPK activity (via activation of adenosyl kinase and the ↑AMP/ATP ratio) in fetal tissues. In adult mammals, AMPK activation inhibits synthesis of fatty-acids, glycogen, and protein, while promoting glycolysis, glucose uptake, and other catabolic effects (127, 340). At least part of these effects of AMPK is mediated through inhibition of mammalian target of rapamycin, a kinase that promotes the efficiency of translation initiation (312). AMPK activity is inversely related to adipogenesis in fetal sheep muscle (339), which is consistent with a role in fetal metabolic homeostasis. Adverse intrauterine conditions may result in dysregulation of AMPK activity postnatally, predisposing in later life to obesity, diabetes, and the metabolic syndrome (297).

Placenta and fetal metabolism.

Oxygen defficiency, via HIF-1, may also reduce fetal growth by restricting placental transfer of substrates to the fetus. One proposed mechanism involves HIF-1-mediated fall in placental mitochondrial O₂ consumption and increase in anaerobic glucose consumption (140). This metabolic reprogramming of the placenta would increase fetal O₂ delivery but at the expense of lowering glucose transfer and thus fetal growth. Other alterations in placental transport and metabolism can be involved in fetal undernutrition (57).

Vasculature

In the fetus and newborn, inherent vascular responses to changes in arterial Po₂ are particularly notable for the brain, heart, and lungs, and the ductus arteriosus. These direct effects of O₂ are involved primarily in circulatory adjustments to hypoxia in the fetus (148, 263) as well as to the rise in Pa_O2 at birth. Adenosine A_2A receptors, which are widely distributed in vasculature, are potentially involved in autocrine/paracrine and hormonal vascular responses to hypoxia, including hypoxia-induced angiogenesis (4, 324).

A_2A receptors and the brain.

Hypoxia increases cerebral vascular conductance in the ovine fetus, a response that is augmented with gestational age and is generally independent of endothelium (128, 261). Stimulation of A₂ receptors accounts for ∼50% of the cerebrovascular response to acute hypoxia in older fetuses (>0.8 term). As in adult rats (56), the A_2A receptor subtype likely mediates the vasodilatation, although a contribution by A_2B receptors cannot be excluded (26, 199). Besides direct vascular actions, the relaxant effects of adenosine on the cerebral vasculature may involve indirect effects via A_2A-mediated release of arginine vasopressin, a vasodilator of the fetal cerebral circulation (190).

A_2A receptors and the heart.

Adenosine is a potent dilator of coronary arteries in fetal sheep (336). A_2A receptors likely mediate the vasodilation, as in most mature mammals (242, 341), although this action has not been established. The maximum coronary flow induced by exogenous adenosine in the fetus is less than the response to hypoxia, indicating that nitric oxide and other factors are also involved in hypoxia-induced coronary vasodilatation (128, 336).

A_2A receptors and the lungs.

The fetal pulmonary circulation has a very high vascular resistance due to lack of gaseous alveolar expansion as well as the low fetal Pa_O2 (67, 107). Local factors primarily regulate pulmonary vascular resistance, although carotid body chemoreflexes enhance pulmonary vasosconstriction in acute hypoxia (233). As in postnatal mammals, the direct constrictor effects of hypoxia on pulmonary artery smooth muscle cells is endothelium independent and likely involves ROS generated principally from mitochondria, inhibition of O₂-sensitive K⁺ channels, and a rise in [Ca²⁺]_i (9, 353, 354).

A_2A RECEPTORS AND FETAL PULMONARY ARTERY.

In fetal sheep, exogenous adenosine increases pulmonary vascular conductance through activation of A_2A receptors and release of nitric oxide (174, 175, 323). The effects of selective A_2A receptor blockade on pulmonary artery conductance have not been reported; therefore, the extent to which endogenous adenosine modulates pulmonary vascular resistance in the fetus remains to be established. Postnatally, A_2A receptors have a key role in modulating pulmonary vascular resistance because genetic inactivation of murine A_2A receptors confers pulmonary hypertension (363).

A_2A RECEPTORS AND POSTNATAL PULMONARY ARTERY.

Gaseous expansion of the lungs and the abrupt rise in Pa_O2 at birth substantially reduce pulmonary vascular resistance, which is associated with ∼10-fold increase in pulmonary artery blood flow (329). Whether A_2A receptors have a significant role in modulating pulmonary vascular conductance in the newborn has yet to be determined. Recruitment and distention in the pulmonary vasculature as well as arterial remodeling further reduce pulmonary vascular resistance over the following 2 wk in human newborns (107). As a result of the fall in pulmonary vascular resistance, ductal flow is reversed (aorta to pulmonary artery) within 60 min after birth. In contrast, right-to-left shunting of blood persists in pulmonary hypertension of the newborn (107).

A_2A receptors and the ductus arteriosus.

The high vascular resistance in the fetal pulmonary circulation necessarily results in 80–90% of right ventricular output being shunted, via the ductus arteriosus, to the descending aorta (224, 298).

A_2A RECEPTORS AND DUCTAL PATENCY.

In sheep and baboons, prostaglandin E₂ (via EP2, EP3, EP4 receptors) and other vasoactive modulators maintain the patency of the ductus arteriosus at the normally low fetal Pa_O2 via activation of adenylyl cyclase, K_ATP channels, and probably protein kinase A-regulated pathways (211). In the ovine fetus, cytosolic Ca²⁺ efflux, via the forward mode of Na⁺/Ca²⁺ exchanger, also appears to be involved in maintaining reduced [Ca²⁺]_i in smooth muscle cells of the ductus arteriosus (134).

Adenosine, which stimulates cAMP-dependent pathways via A_2A receptors, has been implicated in patency of the ductus arteriosus in utero (223). But a significant role for adenosine is unlikely because caffeine lacks in vitro effects on ductal tension (50), and intravascular administration of a potent, nonselective adenosine receptor antagonist (8-phenyltheophylline) does not reduce ductal blood flow in vivo in normoxic fetal sheep (B. J. Koos, unpublished observations).

MODULATORS OF DUCTAL CLOSURE.

The acute rise in Pa_O2 at birth is associated with constriction of ductal smooth muscle, a functional closure that involves inhibition of O₂-sensitive K⁺ channels, activation of L-type Ca²⁺ channels, release of Ca²⁺ from sarcoplasmic reticulum, and increased Ca²⁺ sensitization of actin-myosin filaments (135, 156). The constricted lumen subsequently undergoes morphological remodeling and anatomical closure.

Administration of potent nonsteroid anti-inflammatory agents to pregnant women is a particular concern because these inhibitors of prostaglandin synthesis can constrict the ductus arteriosus, particularly when administered after 32 wk of gestation. Postnatally, these drugs are therapeutically useful in premature newborns with persistent ductal patency that results from immaturity of the oxygen-dependent constriction cascade (156). On the other hand, prostaglandin E therapy can be critical to maintain ductal patency in neonates with congenital heart disease and ductal-dependent pulmonary blood flow.

A_2A receptors and fetoplacental vasculature.

Clear fluid fills the intervillous space until the intervillous circulation becomes established by ∼11 wk of gestation when trophoblastic occlusion of the spiral arteries regresses (37, 256). As a result, early human development occurs in a hypoxic environment. A critical regulator of cell proliferation, angiogenesis, and probably extravillous trophoblast invasion, this low Po₂ modulates organogenesis in the embryo and may protect against ROS-induced teratogenesis (36, 256, 278, 310, 312). The onset of uteroplacental blood flow greatly increases oxygen delivery, supporting increased placental metabolism and fetal growth. All four adenosine receptor subtypes have been identified in syncytiotrophoblast, endothelial cells, and myo/fibroblasts of the human placenta, which implicates adenosine in the regulation of placental angiogenesis, vascular tone, and transfer of nutrients and oxygen (84, 286, 307, 316, 343).

HYPOXIA AND UMBILICAL ARTERY CONDUCTANCE.

The umbilical vasculature, which lacks autonomic innervation, is regulated by hormonal and autocrine/paracrine factors (243, 270). In fetal lambs, the umbilical arteries and placental vasculature contribute ∼80% of the total resistance to umbilical blood flow (257). Acute hypoxia (postductal Pa_O2, ∼13 Torr) transiently reduces umbilicoplacental vascular conductance, but greater O₂ deficiency augments conductance (98, 257, 332). In both cases, umbilical blood flow increases primarily from the rise in perfusion pressure, with greater involvement of local and/or circulating vasodilators at lower O₂ tensions. Increased villous capillary blood flow in hypoxia would be expected to enhance placental O₂ transfer under these conditions (209).

A_2A RECEPTORS AND UMBILICAL CONDUCTANCE.

In sheep, the fetoplacental vasculature exhibits a biphasic response to systemic infusion of adenosine/transient vasoconstriction followed by prolonged vasodilatation (285). In vitro studies have implicated adenosine A_2B receptors in vasoconstriction (75) and A₂ receptors in vasodilatation (280) of human chorionic vessels. Although not specifically studied, the vasodilatory response is likely mediated by A_2A receptors. Thus, adenosine (via A_2A receptors), atrial natriuretic peptide, calcitonin gene-related peptide, prostaglandins, and probably other factors appear to be involved in the enhancement of umbilical vascular conductance in hypoxia (98, 250, 280, 285, 332).

In human fetuses, the placental fraction of the combined ventricular output is less in fetuses with chronic O₂ deficiency and growth restriction from placental dysfunction, even though the combined ventricular output per kilogram estimated weight remains unchanged. Of particular interest is that the reduced umbilical blood flow, which is evident by 20–24 wk of gestation, precedes the detected impairment of fetal growth and abnormal Doppler velocimetry of the umbilical artery (292). Whether the elevated circulating adenosine concentrations in growth-restricted fetuses (377) modulate fetoplacental blood flow via A_2A receptors remains to be determined.

A_2A RECEPTORS AND PLACENTAL BLOOD-FLOW DISTRIBUTION.

Hypoxia more uniformly distributes fetal blood flow (and maternal flow) in the ovine placenta, improving the efficiency of respiratory gas exchange (273). Several mechanisms potentially contribute to the effects of hypoxia on placental blood flow distribution, and one of which is the direct vascular effect of Po₂. In human placental cotyledons in vitro, hypoxia reversibly contracts previllous arterioles in human cotyledons via inhibition of O₂-sensitive voltage-gated potassium channels and activation of voltage-gated calcium channels (124, 167). The role A_2A receptors in this in vitro vascular response warrants investigation. The significance of O₂-modulation of precapillary arteriolar resistance in the placenta in vivo remains controversial (98, 257, 332).

A_2A RECEPTORS AND UMBILICAL VENOUS BLOOD-FLOW DISTRIBUTION.

In sheep and humans, hypoxia increases the proportion of umbilical venous flow that passes through the ductus venosus at the expense of the liver. This redistribution enhances the O₂ content of blood flowing through the foramen ovale and subsequently to the heart and brain, while reducing O₂ delivery to the right lobe of the liver (21, 77, 166, 169, 258, 286, 326, 327, 328). The diversion of hepatic venous blood to the ductus venosus results, at least in part, from the vasoconstrictive effects of circulating catecholamines on hepatic veins (258); and thus activation of A_2A receptors could be involved indirectly via stimulation of the sympathetic nervous system.

A_2A RECEPTORS AND PREECLAMPSIA.

Placental hypoxia and/or abnormalities of O₂ sensing have been linked to preeclampsia (141, 279, 293, 318), and ROS from hypoxia-reoxygenation have been implicated in placental damage (139). Stable (31) or fluctuating elevations in spiral artery vascular resistance as well as increased heterogeneity of blood flow in the intervillous space are potentially involved in the placental pathophysiology of preeclampsia.

In preeclampsia, adenosine levels in the umbilical vein are elevated threefold in fetuses with abnormally high impedances in the umbilical artery with elevated adenosine concentrations present even in normoxic fetuses (374). Preeclampsia is also associated with elevated maternal plasma adenosine concentrations, which supports a more general systemic disruption in adenosine production, uptake, and/or metabolism (83, 154, 158, 311, 370–373, 375, 376).

In preeclampsia, A_2A and other adenosine receptor subtypes have enhanced expression in microvascular endothelium of the placenta. Although hypoxia increases A_2A receptor expression in normal term placenta in vitro, low Po₂ in fetuses with growth restriction without preeclampsia does not affect placental adenosine receptor expression. Thus, factors other than O₂ deficiency per se are likely involved in the preeclampsia-associated alteration of placental adenosine receptor expression.

Limitations

Studies of unanesthetized, chronically catheterized sheep have many advantages, including measuring cardiorespiratory responses to hypoxia that are free from pharmacologic analgesia and surgical stress and that reflect integrated physiologic mechanisms. The involvement of peripheral and central O₂ chemoreceptors was inferred from the time course of cardiorespiratory responses, effects of polar agents that poorly cross the blood-brain barrier, peripheral arterial chemodenervation, and localized neuronal lesions. With respect to the latter, the relatively large size of the fetal sheep brain provides sufficient spatial resolution to identify posteromedial thalamic sectors involved in hypoxic inhibition. However, this approach has limitations in studying neuronal circuits or communication within or between cells, which can be more easily carried out in reduction preparations.

Sheep fetuses differ from human fetuses in several respects, including epitheliochorial placentation, lower hemoglobin concentrations, lack of a fetal zone in adrenal glands, reduced brain size relative to body weight, and greater neurologic maturity at birth. Nevertheless, ultrasound imaging and Doppler velocimetry studies in human fetuses have confirmed qualitatively the major cardiorespiratory measurements that have been observed in normoxic and hypoxic sheep fetuses.

The physiologic role of adenosine A_2A receptors in fetal sheep and developing lambs derives from studies with potent, highly specific agonists and antagonists to A₁ and A_2A receptors. While these pharmacologic agents have sufficient selectivity to distinguish between A₁, A_2A, and A₃ receptor activation in sheep, they cannot exclude the involvement of A_2B receptors (178, 184, 186, 187, 188).

Among mammals, the brain has similarities in the general sequence of brain growth and brain composition, although the timing of the brain-growth spurt relative to birth varies greatly among species (73). Thus, stages of brain development, rather than prenatal or postnatal ages, are often more useful for interspecies comparisons of central physiologic mechanisms. Based on brain growth spurts, the newborn rat, rabbit, or mouse roughly corresponds to an 18-wk human fetus, the neonatal piglet approximates a term human fetus, and the newborn lamb and rhesus monkey resemble a 15-mo-old human infant (73). Other models have been proposed to predict the timing of developmental events in metatherian and eutherian mammals (62).

Referencing maturation to the pattern of brain growth should be used cautiously because not all physiologic mechanisms develop in a parallel manner. For example, those that initiate labor are normally associated with pulmonary maturity, suckling, and gastrointestinal function even though brain development is delayed, as in altricial species (e.g., mice, rats, rabbits). The transition to postnatal life also involves an increase in Pa_O2 and the establishment of pulmonary ventilation, which change the function and central integration of O₂ sensing by the carotid bodies. Thus comparisons among species should be made in light of functional maturity of the involved physiologic mechanism as well as age. Although the neonatal sheep brain is relatively mature, the physiologic responses of the carotid body and brain to hypoxia are qualitatively similar to those of less neurologically mature newborn mammals.

SUMMARY

In fetal sheep, adenosine A_2A receptors are critically involved in acute O₂ sensing by both the carotid bodies and brain (Table 5) as well as in hypoxic regulation of endocrine systems, metabolism, and vascular conductance. Activation of A_2A receptors accounts for the complete expression of hypoxic carotid chemoreflexes that are involved in reducing myocardial oxygen consumption by lowering heart rate and in redistributing cardiac output (and O₂ delivery) to essential organs by supporting or increasing perfusion pressure. Stimulation of central A_2A receptors contributes to a regulated reduction in fetal O₂ consumption by inhibiting rapid eye movement sleep and breathing. Postnatally, A_2A receptors do not have a significant role in transduction processes of carotid O₂ sensing in developing lambs, whereas A_2A and A_2B receptors contribute to about half of hypoxic hyperventilation in adult rats. Activation of brain A_2A receptors mediates the second-phase ventilatory fall in hypoxia in developing lambs and likely other species as well. In the fetus, adenosine A_2A receptors in carotid bodies and the brain are crucially involved in triggering cardiobehavioral adaptations to acute hypoxia that support mitochondrial energy supply to critical organs. Postnatally, the importance of adenosine A_2A receptors in hypoxic carotid chemoreflexes likely depends on age as well as species, while activation of central A_2A receptors remains critically involved in hypoxic respiratory depression.

Table 5.

O₂ sensing in sheep

	Carotid Body	Brain*
Fetus (>0.8 term)
Modulator	Adenosine	Adenosine
Receptor	A2A	A2A
Action	↓ Heart rate	↓ Breathing
	↑ Arterial pressure	↓ REM
Lamb (1–2 wk of age)
Modulator	Not established	Adenosine
Receptor	Not established	A2A
Action	↑ Ventilation (phase 1)	↓ Ventilation (phase 2)
	↑ Heart rate**
	↑ Arterial pressure
	Arousal

REM, rapid-eye movements.

^*Functional brain O₂ sensor that inhibits breathing and REM;

^**secondary to increased ventilation.

CLINICAL PERSPECTIVE

The mitochondrial respiratory chain is involved in many physiologic adaptations and pathophysiologic disorders in development through mechanisms that include release of superoxide anion radicals and adenosine.

A_2A Receptors and Perinatal Hypoxia

Activation of A_2A receptors has particular relevance to hypoxia in the fetus and newborn.

Fetal assessment.

The clinical identification of hypoxic fetuses depends critically upon activation of adenosine A_2A receptors in the fetal carotid bodies and brain.

FETAL HEART RATE.

Fetal heart rate monitoring is commonly performed to detect fetal heart rate decelerations, which are chemoreflexes triggered by hypoxic activation of carotid body A_2A receptors. Acute reductions in fetal O₂ delivery associated with repetitive late or severe variable fetal heart rate decelerations release catecholamines, which progressively increase baseline heart rate. Adenosine likely contributes to the rise in heart rate by stimulating sympathetic activity via A_2A receptors and by activating A_2A receptors in myocardium (189). The decline in fetal heart rate that eventually develops in prolonged, severe hypoxia represents preterminal myocardial decompensation.

FETAL BREATHING.

Fetal breathing movements are one of the most sensitive components of the biophysical profile in the detection of fetal hypoxia. Falls in brain Po₂ inhibit breathing through activation of central A_2A receptors. The relevance of central A_2A receptors to limb and body movements has not been investigated.

MIDDLE CEREBRAL ARTERY DOPPLER VELOCIMETRY.

Significant reductions in middle cerebral artery (MCA) impedance, which reflect increased vascular conductance, are used clinically to detect fetal hypoxia (70). A_2A receptors appear to have a major role in hypoxic cerebrovasodilation. In contrast, the hemodynamics of reduced blood viscosity, rather than oxygen deprivation per se, largely account for the elevated Doppler peak systolic velocities of fetal anemia (66, 277, 344).

UMBILICAL ARTERY DOPPLER VELOCIMETRY.

Doppler studies of the umbilical artery are used to assess the well being of growth-restricted fetuses, with elevated impedances generally reflecting reduced conductance in the umbilical circulation (6, 95, 218, 235). Absent or reversal of diastolic flow signifies further decompensation. Elevated impedance of the umbilical artery largely derives from remodeling of previllous resistance vessels due most likely to the release of oxygen radicals, nitric oxide derivatives, and other factors (92, 124, 147, 167, 281). Further work should be performed on the potential role of A_2A receptors in modulating fetoplacental vascular conductance in normal and pathologic states.

Fetuses can be hypoxic even though Doppler velocimetry of the umbilical artery is normal (46, 58, 313). In such cases, the placenta presumably lacks the typical vasculopathy of chronic hypoxia (170, 235–237, 240, 284, 326). Thus, the pulsatility indices of the MCA are generally a better indicator of the sufficiency of fetal oxygenation than those of the umbilical artery (291).

Hypoxic brain injury.

Activation of vascular A_2A receptors likely contributes substantially to the hypoxia-induced increase in cerebrovascular conductance, brain blood flow, and O₂ delivery thus buffering against neurologic injury due to fetal O₂ deficiency. ROS generated by NADPH oxidase contributes to vasodilatation, but excessive ROS production (via NADPH oxidase, xanthine oxidase or mitochondrial respiratory chain) in severe hypoxia or ischemia causes vasoconstriction. This fall in conductance further impairs brain O₂ delivery and exacerbates neuronal injury (via ROS, glutamate neurotoxicity, metalloproteinase activity, and inflammation), leading to hypoxic-ischemic encephalopathy, seizures, stroke, and long-term disabilities (121, 203, 249, 294, 331, 338).

Despite the rise in cerebral blood flow, O₂ delivery can be insufficient to maintain brain integrity over time even in moderate hypoxia. For example, reduced MCA impedance or a rise in brain-tissue perfusion in growth-restricted fetuses correlates with postasphyxial encephalopathy and impaired neurodevelopment (8, 58, 81, 152, 320). Loss of variable tone and a fall in conductance in the middle cerebral artery indicate cerebrovascular dysregulation and brain injury (301).

Hypoxic vasodilatation of the coronary arteries via A_2A receptors likely has an important role in maintaining fetal myocardial O₂ delivery and function. In severe hypoxia or ischemia, the vasoconstrictive effects of excessive ROS release counters hypoxic coronary vasodilatation, reducing myocardial O₂ delivery, ATP production, and contractility (97). Cardiac function is further impaired by increased afterload from systemic arterial vasoconstriction. These conditions create a feed-forward mechanism of myocardial decompensation, which, in turn, further compromises brain blood flow and oxygen delivery and exacerbates neurologic injury.

Placental dysfunction.

Abnormalities of placental growth and vascular function have been attributed, in part, to enhanced release of ROS and/or reduced production of enzymatic and nonenzymatic antioxidants. Such increased oxidative stress has been implicated in the pathophysiology of preterm birth, intrauterine growth restriction, and preeclampsia (28, 65, 158, 159, 370). Clinical trials lack consensus on benefits of antioxidant therapy in preeclampsia (122).

With respect to adenosine, it remains to be determined whether the preeclampsia-associated perturbations in circulating adenosine concentrations and in adenosine receptor expression in placental microvasculature arise as compensations to vascular dysregulation or as part of the pathophysiology of the disorder. It is also unknown whether adenosine is a modulating factor in the increased risk of stroke in adult offspring of preeclamptic pregnancies (155).

Fetal growth restriction.

Umbilical venous adenosine concentrations are elevated in human fetuses with growth restriction, with adenosine levels inversely proportional to Po₂ (374, 377). The increased adenosine levels likely contribute to metabolic and circulatory adaptations that protect the fetus against chronic O₂ deficiency, and the extent of A_2A receptor involvement in these compensations warrants further exploration.

Chronic hypoxia impairs fetal growth through several mechanisms that potentially include upregulation of fetal AMPK activity, increased placental anaerobic glucose metabolism, and other placental adaptations (57, 127, 312, 340). Elevated adenosine concentrations (via A₁ and A_2A receptors) may also modulate fetal metabolism and growth under these conditions, and this possibility should be explored. Besides perinatal complications (Table 2), poor fetal growth is associated with epigenetic changes in postnatal physiological regulation that predispose to major adult diseases, as summarized in Table 6 (14, 36, 80, 83, 220, 244, 278, 293, 335).

Table 6.

Adult diseases predisposed by poor fetal growth

Diabetes, type II

Hypertension

Stroke

Heart disease

Osteoporosis

Disorders of prematurity.

Premature infants, who have reduced antioxidant production, are predisposed to ROS injury due to the rise in postnatal Pa_O2 and the resulting increase in superoxide anion radical formation by the mitochondrial respiratory chain. Excessive oxidative stress has been implicated in several diseases of prematurity, including bronchopulmonary dysplasia, periventricular leukomalacia, and retinopathy (65).

Pulmonary hypertension.

In the human neonate, pulmonary hypertension most commonly arises from respiratory failure due to pneumonia, respiratory distress syndrome, meconium aspiration syndrome, or pulmonary hypoplasia (107). Pulmonary hypertension can occasionally be a significant cause of hypoxemia in growth-restricted, premature infants (61). Impairment of A_2A receptor function in the pulmonary vasculature may be involved in the development of pulmonary hypertension (363).

Disorders of cardiorespiratory control.

Given the prolonged, if not permanent, dampening of acute hypoxic respiratory responses reported in mammals by sustained hypoxic exposure in the perinatal period, there is concern that such exposures in early human development, as in growth-restricted fetuses, might blunt postnatal protective arousal and cardiorespiratory responses to obstructive sleep apneas or other forms of hypoxic stress.

Intermittent hypoxia in the perinatal period can result from repetitive compressions of the umbilical cord in the fetus and from recurrent central or obstructive sleep apneas in preterm infants. In addition to ROS, fluctuations in adenosine levels, caused by episodic arterial O₂ desaturations, potentially contribute to the enhancement of carotid body O₂ sensitivity by activating A_2A receptors that modulate gene transcription and translation (71, 76, 266, 295). Augmented carotid O₂ sensitivity can lead to respiratory instability, sympathetic overactivity, hypertension, and brain lesions (102, 113, 137, 144, 212, 213, 247). Furthermore, hypoxia-induced rises in brain adenosine levels, via activation of central A_2A receptors, potentially blunt hypoxic arousal and hyperventilation induced by afferent chemosensory discharges. Thus, sustained functional disruptions in peripheral and/or central O₂ sensors, or the central integration thereof, in fetuses or premature infants may depress arousal and respiratory responses to hypoxia and thus predispose susceptible sleeping infants and adults to sudden death (96, 102, 137, 365).

A_2A Receptors and Perinatal Caffeine Exposure

Long-term stimulant effects of caffeine appear to involve blockade of A₁ and A_2A receptors (368); and thus the question naturally arises as to whether maternal consumption of caffeine has adverse perinatal consequences.

Development disorders.

Exposure of pregnant mice to the equivalent of two cups of coffee per day from E8.5 to E10.5 substantially inhibits hypoxia-induced HIF-1α protein expression and cardiac ventricular development in embryos, resulting in long-term impairment of cardiac function in adult offspring (356). Modest maternal subcutaneous injections of caffeine (daily, E9.5 to E18.5) adversely affects embryonic cardiovascular function and growth, possibly through A_2A receptor blockade (226). Encouragingly, clinical studies have not found that caffeine increases the risk of developmental abnormalities, and they lack consensus on increased risk for spontaneous abortion.

Impairment of fetal hypoxic responses.

Another concern to be considered is whether blockade of A_2A receptors by caffeine would alter fetal breathing and heart rate responses, thereby rendering clinical assessment less reliable. In normal human fetuses, maternal coffee ingestion increases breathing incidence, presumably through antagonism of central A_2A receptors that modulate fetal behavioral state (187, 302). By antagonizing A_2A receptors, caffeine potentially blunts falls in heart rate and breathing incidence that would normally occur in hypoxic fetuses, but this is not likely a concern for normal fetuses whose mothers drink two or fewer cups of coffee (<200 mg caffeine) per day.

In sheep, caffeine enhances fetal oxygen consumption (via A₁ receptor blockade) and lowers brain Po₂ (337). Excessive maternal caffeine consumption in human pregnancy has been implicated in fetal growth restriction (40). These observations support the need for limited caffeine intake in pregnancy, with avoidance in gravidas with compromised fetuses.

Long-term regulatory changes.

In rats, perinatal exposure to caffeine induces long-term changes in sleep and cardiorespiratory regulation, which likely involve changes in the expression and/or interactions of A_2A receptors in the carotid body and brain (12, 227, 228). Whether this occurs in humans is unknown, but this possibility is particularly relevant given the widespread use of caffeine in treating apnea of prematurity.

GRANTS

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

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).