INTRODUCTION

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ADENOSINE IS A PURINE nucleoside produced by all cells that plays important roles in cellular metabolism and functions as an extracellular physiological regulator (24, 26, 32). As a metabolite of ATP, adenosine is uniquely suited to modulate the physiological responses to increased metabolic activity or decreased oxygen delivery. During conditions of increased ATP breakdown, such as hypoxia, intracellular adenosine levels increase (26, 32). Intracellular adenosine is then transported into the local extracellular space where it activates P₁ purinergic receptors, which include A₁ and A₃ receptors that couple with G_i and G_o and the A₂a and A₂b receptors that couple to G_s (24).

During fetal life, adenosine may be especially important. Fetal plasma adenosine levels are nearly fourfold greater than adenosine levels in the maternal circulation (30). With mild hypoxia, fetal plasma adenosine levels more than double (16, 35). Because adenosine is rapidly broken down in the circulation (20), changes in local adenosine levels will be much greater than those seen in the circulation.

Recently, A₁ adenosine receptors (A₁AR) expression has been found in rat hearts as early as postconception (PC) day 7.5, when the developing heart is a primitive cardiac tube and has not yet begun beating (4, 28). A₁ARs are thus among the earliest expressed G protein-coupled receptors in the mammalian heart, leading us to hypothesize that adenosine acts via A₁ARs to influence mammalian cardiac activity at embryonic stages.

During development, cardiac output is highly dependent on fetal heart rates (14). In mature hearts, A₁AR activation has been shown to slow atrioventricular conduction and alter the pacemaker current (26). Thus, to test the affects of adenosine on fetal cardiac function, we have examined the influence of adenosine on fetal heart rates beginning at the inception of spontaneous cardiac contractility. In addition, we have characterized the receptor subtypes through which adenosine acts to influence fetal cardiac activity and examined potential cellular effector systems that mediate adenosine action. We have used mice for these studies, since the ontogeny of A₁AR expression in rodents has been characterized, a considerable amount of information is known about murine cardiac development, and it is possible to culture murine embryos (4, 28, 29, 31).

	MATERIALS AND METHODS

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Fetal culture conditions. C57BL6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Timed-pregnant mice from 2-h matings (6 to 8 AM) were used. Embryos were dissected from the dams as previously described (21). Dams were killed by decapitation, and the uteri were then removed and cut into segments containing embryos. Uterine segments were placed in culture media [Dulbecco's modified Eagle's medium (DMEM)] with 10% fetal bovine serum (Fetal Clone II; Hyclone Laboratories, Logan, UT), penicillin (100 µg/ml), and streptomycin (100 mg/ml).

Embryos were dissected from the uterus under microscopic observation by sequentially removing the muscle layer, deciduum, Reichert's membrane, and the yolk sac. Intact embryos were then cultured, or whole hearts were dissected and cultured. All cultures were maintained at 37°C in 5% CO₂. After cultures were established, spontaneous cardiac contractility began within 2 h. To maintain uniform treatment conditions, cultures were generally studied between 8 and 16 h after dissection. Baseline heart rates were between 120 and 220 beats/min. All studies involving the use of mice were approved by the Indiana University Subcommittee for Animal Care and Use.

Heart rate assessment. Cultures were maintained at 37°C during all experiments using a thermostatically controlled, heated stage (Lab-Line). Each preparation was microscopically examined, and heart rates were manually counted over 30-s intervals. Heart rate counts were performed in duplicate or triplicate at baseline and at each drug concentration. Mean heart rate values were then determined.

Statistical analysis. To standardize heart rates across all studies, heart rates were expressed as a percentage of the basal (pretreatment) heart rates defined as 100%. The maximal decrease in heart rate from basal levels was defined as the E_max. The drug concentration that caused heart rates to decline from basal levels to 50% of the E_max value was defined as the EC₅₀. Statistical comparisons were performed using analysis of variance (ANOVA) (GraphPad PRISM version 2; San Diego, CA). A significant difference was defined as a P < 0.05. Values are expressed as means ± SE.

Drugs. Pertussis toxin and forskolin were obtained from Sigma Chemical (St. Louis, MO). All other drugs were obtained from Research Biochemicals (Natick, MA). All solutions were generally prepared on the day of the study. Drugs were initially dissolved in dimethyl sulfoxide and diluted in DMEM. Vehicle preparations contained the same concentration of dimethyl sulfoxide as solutions containing drugs. Drugs were directly added to cultures by pipetting 10 or 100× stock solutions prepared with culture media.

	RESULTS

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Influences of A₁AR agonists on heart rates. A₁AR expression has been recently detected in fetal hearts at very early stages of development (4, 28). Thus, to assess whether A₁AR activation influences fetal cardiac activity during embryogenesis, we tested whether A₁ARs regulated heart rates in whole embryos from PC days 9 through 12. These ages were selected since spontaneous cardiac contractions begin shortly before PC day 9, and cardiac structural differentiation occurs over this age range (29).

When the effects of the A₁AR agonist N⁶-cyclopentyladenosine (CPA) were examined from PC days 9 to 12, dose-dependent slowing of heart rates was observed (EC₅₀ = 1.8 × 10⁸ M). At the most effective CPA concentration, heart rates declined to 17.0 ± 5.6% of baseline values (E_max), with complete cessation of spontaneous contractility seen in 70% of preparations. No differences (ANOVA) in CPA effectiveness were observed among the different ages [PC day 9 (EC₅₀ = 2.1 × 10⁸ M, E_max = 22.0 ± 6.1%, n = 7), PC day 10 (EC₅₀ = 1.1 × 10⁸ M, E_max = 13.4 ± 6.2%, n = 15), PC day 11 (EC₅₀ = 9.8 × 10⁹ M, E_max = 12.1 ± 5.1%, n = 15), and PC day 12 (EC₅₀ = 2.3 × 10⁸ M, E_max = 11.4 ± 6.1%, n = 15)].

Next, we assessed whether the effects of CPA were due to direct action on the heart or were due to action at noncardiac sites. CPA dose-response studies were therefore performed on isolated whole heart preparations. Because it was not possible to isolate hearts at PC day 9 due to the extremely small sizes of the specimens, whole heart cultures from PC days 10 to 12 were examined. As in the whole embryos, CPA caused dose-dependent declines in heart rates. After CPA treatment, dose-dependent slowing of heart rates was observed (EC₅₀ = 3.6 × 10⁸ M, n = 7-10 separate studies at each age). Heart rates declined to 14.4 ± 4.6% of baseline values (E_max), with complete cessation of spontaneous contractility seen in 63% of preparations. Showing that CPA action was A₁AR mediated, the A₁AR antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX) reversed the inhibitory effects of CPA (1 × 10⁵ M) on heart rates in a dose-dependent manner (EC₅₀ = 9.3 × 10⁷ M, n = 5 separate studies).

We also assessed whether responsiveness to A₁AR activation was developmentally regulated. Thus responses to CPA were also examined in whole hearts isolated from fetuses at PC days 14, 16, and 20. At each age, CPA was as effective as at younger ages [PC day 14 (EC₅₀ = 9.2 × 10⁹ M, E_max = 11.0 ± 5.2%, n = 8), PC day 16 (EC₅₀ = 1.1 × 10⁸ M, E_max = 10.3 ± 6.0%, n = 6), and PC day 20 (EC₅₀ = 1.2 × 10⁸ M, E_max = 9.0 ± 4.5%, n = 6)].

Responses to A_2a, A_2b, and A₃AR agonists. Besides acting via A₁ARs, adenosine acts via A_2a, A_2b, and A₃ARs (24, 32). Thus, after A₁AR ligand studies, we tested whether other adenosine subtypes regulated fetal cardiac function using whole hearts from PC days 10 to 12.

To test for functional A₂aARs, dose-response studies were performed using the A₂aAR-selective agonist CGS-21680. However, even at high doses of CGS-21680 (1 × 10⁵ M, n = 5), no effects on heart rates were seen (Fig. 1).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Influences of adenosinergic compounds on contractility rates of cultured whole hearts. For some studies, cultures were preincubated with 1,3-dipropyl-8-cyclopentylxanthine (CPX, 1 uM) before drugs were added. Drugs used were 5'-(N-ethylcarboxamido)-adenosine (NECA) and CPX (), CGS-21680 (), N6-cyclopentyladenosine (CPA, ), and N6-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IBMECA, ). Data are mean values from at least 5 separate dose-response studies/drug. Data points are means ± SE. Please note that the A1 adenosine receptor (A1AR) agonist CPA most potently slowed heart rates.

To test for functional A₃ARs, dose-response studies were performed using the A₃AR-selective agonist, N⁶-(3-iodobenzyl)-adenosine-5'-N-methylcarboxamide (IB-MECA). In addition, we tested the A₁AR/A₃AR agonist N⁶-2-(4-aminophenyl)-ethyladenosine (APNEA) after cultures were pretreated with CPX (1 × 10⁶ M) to block A₁ARs. In both the IB-MECA- and APNEA/CPX-treated cultures, heart rates decreased in a dose-dependent manner to 50% of baseline levels (IB-MECA, EC₅₀ = 7.2 × 10¹¹ M, E_max = 52.9 ± 15.2%, n = 7; APNEA/CPX, EC₅₀ = 6.7 × 10⁹ M, E_max = 50.8 ± 6.6%, n = 6; Fig. 1). When compared with CPA, the declines in heart rates following addition of APNEA/CPX or IB-MECA were significantly less (P < 0.01; ANOVA, Fig. 1).

Finally, dose-response studies were performed using 5'-(N-ethylcarboxamido)-adenosine (NECA), which activates A₁, A₂a, A₂b, and A₃ARs. With NECA treatment alone, heart rates declined in a dose-dependent manner consistent with activation of A₁ARs (EC₅₀ = 1.3 × 10⁷ M, E_max = 37.2 ± 10.0, n = 6). However, when cultures were preincubated with CPX (1 × 10⁶ M), only modest declines in heart rates were seen at high NECA concentrations, consistent with activation of A₃ARs (EC₅₀ = >10 × 10⁶ M, E_max = 71.0 ± 9.8%, n = 7).

P₂ purinergic receptor ligand studies. ATP is a precursor of adenosine that can act to directly affect cell function via P₂ purinergic receptors (24). Thus, to assess whether ATP also can influence fetal heart rate, we examined fetal heart rates after treating whole heart cultures (PC days 10-12) with P₂ receptor agonists and antagonists.

In contrast to P₁ agonist studies, the P₂ agonist 2-methylthio-ADP (1 × 10⁶ M) had no effect on fetal heart rates (162.5 ± 33.5 beats/min at baseline vs. 160.8 ± 26.6 beats/min with 2-methylthio-ADP, n = 8, P > 0.05). Similarly, we saw no effects on heart rates with the addition of 2-methylthio-ATP (1 × 10⁵ M; 131.0 ± 34.4 beats/min at baseline vs. 122.5 ± 27.7 beats/min with 2-methylthio-ATP, n = 6, P > 0.05). Addition of ATP (1 × 10⁵ M) caused dose-dependent decreases in heart rates (121.0 ± 11.8 beats/min at baseline vs. 94.0 ± 16.4 beats/min with ATP, n = 6, P < 0.01). This effect was blocked by 1 × 10⁶ M CPX (119.7 ± 18.8 beats/min with CPX and ATP, n = 6), suggesting that ATP action was mediated by A₁ARs.

We also tested for the presence of endogenously activated P₂ receptors by incubating heart preparations with the nonspecific P₂ receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2',4'-disufonic acid tetrasodium (PPADS). Suramin (1 × 10⁵ M) had no significant effects on heart rates (155.0 ± 14.4 beats/min at baseline vs. 144.5 ± 17.8 beats/min with suramin, n = 4, P > 0.05). PPADS (1 × 10⁵ M) similarly had no significant effects on heart rates (141 ± 37.1 beats/min at baseline vs. 131 ± 34.4 beats/min with PPADS, n = 4, P > 0.05).

Adenosine influences on heart rates. The above pharmacology studies strongly suggest that of the known purinergic receptor subtypes, A₁ARs most potently influence fetal heart rates. Because the above experiments involved the use of adenosine analogs, we next tested whether adenosine itself acts via A₁ARs to influence fetal heart rates. To test the ability of endogenous adenosine to tonically suppresses heart rates, whole hearts (PC days 10-12) were incubated with the A₁AR antagonist CPX. These studies showed that CPX caused dose-dependent increases in heart rates (E_max = 115.6 ± 23.1%, EC₅₀ = 3.8 × 10⁹; n = 5; Fig. 2).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Influences of adenosine and A1AR blockade on contractility rates of cultured whole hearts. For some studies, cultures were preincubated with CPX (1 µM) before drugs were added. Data are mean values from at least 5 separate dose-response studies/drug. Data points are means ± SE. Please note that adenosine (ADO) and dipyridamole (DPY) slowed heart rates, whereas A1AR antagonist CPX treatment increased heart rates above baseline.

Next, we tested whether alterations in endogenous adenosine levels altered heart rates. For these studies, cultures were treated with the adenosine reuptake blocker dipyridamole. To show that increasing local adenosine levels influences heart rates, dipyridamole produced dose-dependent decreases in heart rates (E_max = 36.5 ± 9.9%, EC₅₀ = 7.1 × 10⁷; Fig. 2).

Finally, dose-response curves to exogenous adenosine were examined. These studies demonstrated decreases in heart rates with increasing adenosine concentrations (EC₅₀ = 2.2 × 10⁶ M, E_max = 28 ± 11.2%; Fig. 2). When adenosine was added to cultures in the presence of CPX, the effects of adenosine were markedly attenuated (Fig. 2).

Influences of G_i/G_o and cAMP on A₁AR action. After defining the purinergic receptor subtypes that mediate adenosine action on fetal heart rates, we next examined the effector systems mediating A₁AR action in embryonic hearts. Because A₁ARs regulate cAMP levels via G proteins (24), we first tested whether fetal A₁AR action was G protein mediated. Cultures of whole hearts (PC days 10-12) were therefore preincubated with pertussis toxin to inhibit G_i and G_o (1 mg/ml; 30 min) (12). To confirm a requirement for G_i (or G_o), preincubation with pertussis toxin completely blocked CPA action (EC₅₀ = >10 × 10⁵ M, E_max = 99.9 ± 2.1%; Fig 3).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Influence of pertussis toxin (PTX, A) and forskolin (For) and 8-bromo-cAMP (8-BR) (B) on CPA-induced changes in heart rates. Solid lines, CPA dose-response curves in the presence of drugs. Dotted lines, dose-response curves generated using CPA alone. Data are mean values from at least 5 separate dose-response studies/drug. Data points are means ± SE. *** P < 0.001 vs. CPA alone for maximal decrease in heart rate from basal levels (Emax) values.

Because A₁ARs act to decrease cellular cAMP accumulation (24), and cellular cAMP levels influence heart rates (6), we then assessed whether altering intracellular cAMP levels modifies CPA action. Thus CPA-induced changes in fetal heart rates were observed in the presence of forskolin, which stimulates adenylate cyclase activity, or with the nonhydrolyzable cAMP analog 8-bromo-cAMP.

Cultures were preincubated for 10 min with forskolin (10 nM), or with 8-bromo-cAMP (0.5 mg/ml). Dose-response studies were then performed with CPA.

Incubation with forskolin or 8-bromo-cAMP resulted in increased heart rates to 125.4 ± 9.9% (n = 9) and 133.8 ± 11.9% (n = 7) above baseline level, respectively. In the presence of forskolin and CPA, heart rates declined by 64% to an E_max of 44.4 ± 15.5% (Fig. 3). In the presence of 8-bromo-cAMP, heart rates declined by 63% to an E_max of 47.0 ± 20.6% (Fig. 3). These values differed from those observed with CPA treatment alone, in which heart rates declined by 85% to an E_max of 15 ± 4.4% (P < 0.01, Fig. 3). Forskolin and 8-bromo-cAMP thus attenuated both absolute and relative reductions in heart rates seen with CPA treatment.

Influences of ion channel blockade on A₁AR action. Although treatment of fetal specimens with forskolin or 8-bromo-cAMP attenuated CPA action, the responses to CPA were only partially blocked, suggesting that other effector systems mediate A₁AR action. In the mature myocardium, A₁AR activation slows atrioventricular nodal conduction via activation of adenosine- and acetylcholine-sensitive ligand-gated potassium channels (K_Adeno/ACh) (26). A₁AR stimulation also activates potassium ATP (K_ATP) channels (9) and alters the pacemaker current (I_f) (7, 26). Thus A₁AR action on embryonic heart rates may involve ion channel-mediated events. To examine this possibility, CPA-induced changes in heart rates were observed in the presence of specific and nonspecific ion channel inhibitors.

Before studies with CPA were performed, dose-response studies (n = 3 or more/agent) were performed with ion channel blockers to identify the maximal drug concentration that did not alter heart rates by >10% of pretreatment values. These concentrations were used in subsequent studies. Each of these concentrations corresponded to doses that have been shown to inhibit the activity of the target channels (2, 5, 8, 15, 17, 23, 25, 26, 33, 34). The drugs used were 4-aminopyridine (4AP; 5 × 10⁴ M; nonspecific K channel blocker), charybdotoxin (CTX, 1 × 10⁸ M; calcium-activated K channel blocker); glibenclamide (1 × 10⁸ M; K_ATP channel blocker); cesium chloride (CsCl, 2 mM; inhibitor of the I_f); procainamide (1 × 10⁵ M; Na channel blocker), tetrodotoxin (TTX; 1 × 10⁶ M; Na channel blocker); indanyloxyacetic acid 94 (IAA-94; 1 × 10⁶ M; chloride channel blocker); and verapamil (1 × 10⁸ M; L-type Ca channel blocker). Whole heart cultures were preincubated with these drugs for 10 min before dose-response studies with CPA were performed.

Results of these studies showed that glibenclamide, CsCl, verapamil, IAA-94, procainamide, and tetrodotoxin all significantly inhibited CPA action (P < 0.05 for CsCl, P < 0.01 for all other drugs) (Table 1, Fig. 4). In contrast, neither of the K_Adeno/ACh channel inhibitors (4AP, CTX) attenuated CPA action (Table 1, Fig. 4). Collectively, these observations suggest that Na, K_ATP, and Cl channels and I_f directly or indirectly mediate A₁AR action on fetal heart rate.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Influences of ion channel blockers on CPA-induced changes in heart rates. Solid lines, CPA dose-response curves generated in the presence of ion channel blockers. Dotted lines, dose-response curves generated using CPA alone. CTX, charybdotoxin; 4-AP, 4-aminopyridine; TTX, tetrodotoxin; Pro, procainamide; Ver, verapamil; Gli, glibenclamide; IAA, indanyloxyacetic acid 94. Data are mean values from at least 5 separate studies/drug. Data points are means ± SE. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. CPA alone for Emax values.

	DISCUSSION

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Our observations support the hypothesis that adenosine acts via A₁ARs to potently regulate cardiac pacemaker activity in hearts at very early stages of embryonic development. Shortly after the onset of spontaneous cardiac contractions, we found that the A₁AR agonist CPA potently slowed heart rates in embryos. When isolated whole hearts were examined, CPA was similarly effective, indicating that CPA acts directly on cardiac tissue.

Pharmacology studies showed that A₁ARs were by far the most potent regulators of fetal cardiac activity. A₂AR agonists did not have any effects on heart rates, and activation of A₃ARs produced only modest decreases in heart rates. In contrast to studies using P₁ receptor ligands, no effects of P₂ purine receptor analogues were seen on heart rates. When ATP alone was used, heart rates fell. However, since ATP is hydrolyzed to adenosine (26) and ATP action was blocked by CPX, our observations indicate that the effects of ATP were mediated by conversion to adenosine and activation of A₁ARs.

Our findings also showed that endogenous adenosine itself modulates fetal heart rates. Treatment of hearts with the A₁AR antagonist CPX increased baseline heart rates, whereas heart rates slowed following treatment with the adenosine reuptake blocker dipyridamole. To support the concept that adenosine itself activates A₁ARs to slow fetal heart rates under physiological conditions, changes in heart rates were seen at adenosine concentrations present physiologically (10, 16, 22, 35).

Our studies of possible effector systems that mediate A₁AR action on fetal heart rate suggests that several cellular mechanisms transduce the effects of adenosine on heart rates. When hearts were treated with pertussis toxin, A₁AR action was completely blocked, showing that A₁AR action was G_i/G_o dependent. Consistent with this observation, G_i proteins have been detected in murine fetuses as early as gestation day 6.5 (11).

A₁AR action also appears to involve the adenylyl cyclase system, as measures aimed at increasing cellular cAMP levels attenuated CPA-induced declines in heart rates. Because of the extremely small sizes of the fetal cardiac specimens, it was not possible to directly measure changes in cAMP levels within specimens to confirm that A₁AR activation indeed alters cAMP levels. However, in other systems (27), A₁AR-mediated inhibition of forskolin-stimulated cAMP accumulation is observed at doses used in our studies.

Because the effects of CPA were only partially blocked by forskolin or 8-bromo-cAMP, our observations suggest that other effector systems mediate A₁AR action on heart rate. A₁AR-mediated activation of ion channels has been observed to influence atrioventricular nodal conduction and pacemaker activity in mature myocardium (26). Thus we assessed whether A₁AR action in the fetal heart was mediated by ion channels.

With glibenclamide, which inhibits the K_ATP channel that plays an important role in mediating adenosine action during conditions of hypoxia (26), we were able to greatly reduce CPA efficacy. Because the intrauterine environment is relatively hypoxic (PO₂ = 20 mmHg) (1), K_ATP channels may play important roles in mediating A₁AR action during fetal life.

Similar to observations in adult tissues (13), we found that L-type Ca channel blockade inhibited A₁AR action on heart rate. We also found that sodium and chloride channel inhibitors impaired CPA action, as did CsCl. Because CsCl inhibits the pacemaker current (5), our findings suggest that I_f is regulated by A₁AR activation. Please note that, although A₁ARs may directly regulate ion channel function, A₁AR channel coupling was not directly assessed in our studies. Thus it is also possible that ion channel blockade initiates other events that indirectly attenuate A₁AR action.

In adult cardiac tissue, a large body of evidence suggests that adenosine cardiac action involves the activation of the K_Adeno/ACh channel (26). However, in contrast to that observed in mature myocardium, CPA action was not blocked when hearts were preincubated with effective concentrations of the K_Adeno/Ach channel inhibitor 4-aminopyridine (2, 15, 23, 33, 34). We are currently unaware of any studies examining the ontogeny of this ion channel. Thus K_Adeno/ACh channels may not be expressed or may not mediate A₁AR action at the fetal stages examined. Future studies in which the ionic current regulated by A₁AR activation are directly assessed will thus be needed to address this issue.

Although our understanding of A₁AR action on embryos is at early stages, functional A₁ARs that have also been identified in heart cells of avian species (18, 19). In chick embryos, A₁AR receptor activation has been observed to reduce the rate and amplitude of spontaneous contractility on dispersed atrial myocytes (18, 19). However, in comparison with the 80-100% reductions in heart rates that we observed in mice (including in dispersed murine heart cells; data not shown), rates of spontaneous contractility decline by only 20% in chick atrial preparations (18). Our findings thus show that A₁AR action on pacemaker function is much more pronounced in mammalian than in avian hearts.

Our observations also suggest that the adenosinergic system may be an essential regulator of fetal cardiac function during early development. In contrast to the end of gestation and the newborn period, the autonomic nervous system is not functional at early embryonic stages (14). Because fetal cardiac output is integrally dependent on heart rate at the ages examined (14) and local adenosine levels can dynamically change, changes in heart rate in response to activation of A₁ARs is expected to alter fetal cardiac output. It is important to recognize, however, that since these studies were performed in vitro, additional studies will be needed to confirm these observation in utero.

Our findings also raise the possibility that activation of A₁ARs during early development may result in fetal death, as factors that adversely affect cardiac output will result in fetal demise (3). Available evidence indicates that fetal adenosine levels are dynamically regulated by the oxygenation state of the fetus, with adenosine levels increasing during fetal hypoxia or stress (16, 35). Intrauterine hypoxia or stress triggers a cascade of A₁AR-mediated events resulting in bradycardia and asystole at embryonic stages. Currently, we are unaware of other extracellular chemical signals that so potently influence heart rates in the fetus. As such, further studies will be needed to define the potential pathological influences of prenatal A₁AR activation on mammalian embryos and test whether A₁AR antagonism protects against fetal bradycardia in vivo.