Adenosine mediates metabolic and cardiovascular responses to hypoxia in fetal sheep

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1 Journal of Physiology (1995), 488.3, pp Adenosine mediates metabolic and cardiovascular responses to hypoxia in fetal sheep Brian J. Koos, Andrew Chau and Dotun Ogunyemi Nicholas S. Assali Perinatal Research Laboratory, Department of Obstetrics and Gynecology, Brain Research Institute, UCLA School of Medicine, Los Angeles, CA 90024, USA 1. In seven unanaesthetized fetal sheep ( > 80% term), isocapnic hypoxia (arterial partial pressure of 02, Pla,2, 15 mmhg) was induced for 1 h by lowering maternal inspired Po2. Fetal hypoxia was also produced during intra-arterial administration of the adenosine receptor antagonist 8-(p-sulphophenyl)-theophylline (8-SPT). The fetal 8-SPT infusion was begun just prior to hypoxia and was stopped when fetal Pa0 2 was returned to normal. 2. Hypoxia induced a progressive fetal acidosis, a rise in mean arterial pressure, a transient fall in heart rate and a decrease in breathing movements. 8-SPT significantly reduced the metabolic acidosis and abolished the hypertension and bradycardia without altering hypoxic inhibition of fetal breathing. Administration of the vehicle for 8-SPT during hypoxia did not significantly affect the normal fetal metabolic and cardiovascular responses to acute 02 deprivation. 3. It is concluded that adenosine mediates the fetal bradycardia and hypertension produced by hypoxia, indicating that adenosine modulates fetal autonomic responses to acute oxygen deficiency. Secondly, adenosine contributes to fetal metabolic acidaemia, suggesting that adenosine also modulates fetal glycolytic responses to hypoxia. In fetal sheep ( >80% term), acute reductions in arterial partial pressure of 02, Pa.,2, produce a transient fetal bradyeardia. For example, decreasing the mean fetal arterial 02 tension from 25 to 15 mmhg will usually lower fetal heart rate by beats min-' with an increase towards the control value within 30 min of sustained oxygen deficiency (Boddy, Dawes, Fisher, Pinter & Robinson, 1974; Koos, Sameshima & Power, 1987). This bradycardia is abolished by muscarinic blockade, vagotomy, bilateral division of the carotid sinus and aortic nerves (see Hanson, 1988), and bilateral denervation of the carotid bodies (Giussani, Spencer, Moore, Bennet & Hanson, 1993). Hypoxia also increases mean arterial pressure, a response which is largely caused by constriction of the femoral arteries (see Hanson, 1988). The hypoxia-induced fall in heart rate is undiminished in fetuses in which the rise in arterial pressure is eliminated by autonomic blockade (Lewis, Donovan & Platzker, 1980) or ablation of lumbar sympathetic efferents (see Hanson, 1988); therefore, the bradycardia appears to be a chemoreflex triggered by hypoxic stimulation of the carotid bodies. Although resulting from increased vagal tone, the bradycardia is attenuated over time by the rise in circulating catecholamines and probably other factors released by hypoxia (see Hanson, 1988). Such factors may include adenosine, a purine nucleoside which increases in the fetus during hypoxia (Koos & Doany, 1991). Because intravascular infusion of adenosine increases fetal heart rate through direct and indirect effects on the myocardium (Koos, Mason & Ducsay, 1993), this study determined whether adenosine modulates fetal heart rate during hypoxia. METHODS Nine pregnant ewes (Rambouillet-Columbia breed) were operated on under halothane anaesthesia (2-5 % halothane in 02) at about 120 days gestation (80% term). A polyvinyl catheter was inserted in the right brachial artery of the fetus and advanced into the brachiocephalic trunk, and a second catheter was inserted into the right carotid artery with the catheter tip advanced 7 cm toward the aortic arch. Other fetal catheters were placed in the right external jugular vein, trachea and the amniotic sac (Koos et al. 1993). Antibiotics were administered intramuscularly to the ewe before and immediately after surgery (Koos et al. 1993). Fetal arterial, tracheal and amniotic fluid pressures were measured using pressure transducers (Cobe Laboratories, Lakewood, CO, USA), and fetal tracheal and arterial pressures were corrected by subtracting amniotic fluid pressure. Fetal heart rate was determined from the arterial pulse pressure using a cardiotachometer. Fetal heart rate, arterial pressure, and tracheal pressure were recorded on a Grass polygraph (model 7E), and these signals were sampled at 0-01 s by an IBM-AT-compatible microcomputer using data-acquisition software with minute averages stored on disk.

2 762 B. K Koos, A. Chau and D. Ogunyemi J Physiol Haemoglobin concentration and 02 saturation were determined using an OSM Hemoximeter (Radiometer, Copenhagen, Denmark). Arterial blood gases and ph were measured using blood gas electrodes (model 1304, Instrumentation Laboratories, Lexington, MA, USA), with values corrected to 39 5 'C. Experiments were performed at least 4 days after surgery under chronic experimental conditions. After a 1 h control period, isocapnic hypoxia was induced in eight fetuses by having the ewe breathe for 1 h from a plastic bag receiving a continuous supply (30 1 min-') of a hypoxic gas mixture (8% 02, 3% C02, 89% N2). Isocapnic hypoxia was also induced in these fetuses while administering 8-(p-sulphophenyl)-theophylline (8-SPT; Research Biochemicals), a water-soluble, non-selective adenosine receptor antagonist. 8- SPT (8 mg ml-) in Tris, ph 7 5, was injected (17 mg kg-) into the fetal brachiocephalic trunk 4 min before lowering fetal Pa 2 and the adenosine receptor antagonist was infused intra-arterially at 0 9 mg min-' kg' during the 1 h of hypoxia (Koos, Mason & Ervin, Hypoxia experiments and hypoxia with 8-SPT were carried out in each fetus on separate days in varying order. This interval between studies helped minimize carry-over effects of hypoxia and 8-SPT from previous experiments. Hypoxia was also produced for 1 h in four fetuses (2 used in the above studies plus 2 others) in which Tris alone was administered as for 8-SPT experiments. These control experiments determined whether the vehicle for 8-SPT administration altered fetal responses to hypoxia. Because the purpose of this study was to determine whether adenosine receptor blockade would alter the hypoxia-induced bradycardia, only fetuses in which hypoxia reduced the fetal heart rate by at least 20 beats min-' were evaluated; this is the normal fetal response under these conditions (Koos et al. 1987). Using this criterion, one fetus was excluded from the analysis. Fetuses in this study included four from previous work having a different focus (Koos et al Fetal breathing incidence was determined by visual inspection of the polygraph tracings. Breathing movements were identified by characteristic negative deflections in tracheal pressure and were judged to be present if at least 20 s of each minute epoch contained breathing (Koos & Matsuda, 1990). Two-way analysis of variance with Duncan's multiple range test was used to determine significant differences (P < 0 05) for repeated measures, and ANOVA was used to determine significant differences in responses between experimental groups. The data are expressed as means + S.E.M. RESULTS Hypoxia Fetal responses to hypoxia were determined in seven fetuses in which the mean Pa,O2 was decreased by 8-10 mmhg from the control value of P2 mmhg (Table 1). This isocapnic fall in Pa 2 was associated with a significant reduction in arterial ph and base excess (Fig. 1). The effects of hypoxia on mean arterial pressure (MAP) are shown in Fig. 2. Compared with the control value of about 42 mmhg, MAP increased by 8 mmhg within 10 min of the onset of hypoxia and reached maximum levels of about 11 mmhg greater than the control mean after 60 min of 02 deprivation. MAP fell towards control values after restoring fetal oxygen supply. Fetal heart rate averaged about 170 beats min-1 during the control period but declined by almost 50 beats min-' within 5 min of the onset of fetal hypoxia (Fig. 3). Mean fetal heart rate rose towards control values after 15 min of 02 deficiency, and increased further to a tachyeardia during the recovery period. A 7.4.m:l:::::::: 1:::.. ::.:::,:::::.:::.:::::::.....: :: :::::"...: :::,::: :'....:'..... ':,.. :':'H :: :-, -:1 ::::::-:'::::::::':::"::"":":'- i:i:..:: '' i:i:i:i:il:iij..:: :::.::::::::.::::.: !:::!!;,:;!:I :...::.:... :::: I Z a B L *t -1 *t Figure 1. Effects of hypoxia on arterial ph and base excess Fetal arterial ph and base excess during hypoxia (0) and 90 hypoxia with adenosine receptor blockade (0). Infusion of the adenosine receptor antagonist started 4 min before hypoxia and continued until the end of fetal 02 deprivation. Values are means + S.E.M. *P < 0 05 compared to control mean. t P < 0 05 compared with value at same time during adenosine receptor blockade a.) x a m '- L 0 I L_ Time (min) 90

3 J. Physiol Adenosine and fetal heart rate 763 Table 1. Fetal preductal arterial blood gases during hypoxia and hypoxia with adenosine receptor blockade Hypoxia Hypoxia and AR blockade Time (min) P02 Pc02 P02 Pc P P * P * P * P * * * t t AR, adenosine receptor. *P < 0 05 compared to control value. t Post-hypoxia recovery period. The incidence of fetal breathing averaged min h-' during the hour before hypoxia. During the hour of 02 deprivation, the incidence significantly decreased to min h-'. Hypoxia and Tris In four fetuses, the effects of isocapnic hypoxia were determined while infusing the vehicle for 8-SPT. Reducing fetal mean Pa02 by -IO mmhg from a control value of mmHg resulted in changes in fetal arterial ph, MAP and heart rate which were not significantly different from those described previously for hypoxia alone. These results indicated that the vehicle for 8-SPT did not alter fetal metabolic or cardiovascular responses to hypoxia. Hypoxia and adenosine receptor blockade In experiments in which hypoxia was induced while infusing 8-SPT, fetal mean PaO2 fell by 9-11 mmhg, a reduction comparable to the decrease during hypoxia alone (Table 1). Administration of 8-SPT blunted the fall in arterial ph normally observed during hypoxia (Fig. 1). Although a significant acidaemia was present 10 min after restoring fetal Pa 2 to normal, the ph values during the recovery period were significantly higher than those measured in experiments without adenosine receptor blockade. Compared with measurements during hypoxia alone, the base excess in fetuses receiving 8-SPT was significantly greater during the last 30 min of hypoxia and during the recovery period (Fig. 1). - Hypoxia 55 - *t I EE U) U) H ( I Time (min) Figure 2. Effects of hypoxia on fetal arterial pressure Fetal mean arterial pressure during hypoxia (0) and hypoxia *P < 005 compared to control mean. t P < 005 compared to receptor blockade. with adenosine receptor blockade (0). value at same time during adenosine

4 764 B. J Koos, A. Chau and D. Ogunyemi J Physiol The adenosine receptor antagonist prevented the fetal hypertension and bradycardia normally produced by hypoxia (Figs 2 and 3). As in experiments without 8-SPT infusion, a tachycardia developed during the recovery period. During hypoxia the incidence of fetal breathing was min h-', a value significantly less than the control mean of min h-'. Thus adenosine receptor blockade did not significantly alter the inhibitory effects of hypoxia on fetal breathing activity. DISCUSSION Although it causes a tachycardia in young fetuses at 65-75% term, hypoxia typically produces a transient bradycardia in older fetal sheep (Boddy et al. 1974), with the fall in heart rate depending on the extent of oxygen deficiency (Koos et al. 1987). After about min of hypoxia, fetal heart rate rises as a result of increased B6-adrenergic stimulation from the rise in circulating catecholamine concentrations and possibly other factors (see Hanson, 1988). The bradyeardia produced by hypoxia in the mature fetus contrasts with the tachycardia normally observed in the newborn or adult. This rise in heart rate in the adult depends on increased pulmonary ventilation because a bradycardia occurs if ventilation is controlled (see Hanson, 1988). Thus the mechanisms regulating heart rate after birth appear to be similar to those in the fetus whose breathing is inhibited by hypoxia. In the present study, adenosine receptor blockade maintained fetal heart rate during hypoxia, suggesting that adenosine mediates the fall in heart rate caused by acute reductions in fetal Pa 2. Because 8-SPT administration to normoxic fetuses does not significantly affect fetal heart rate or MAP (B. J. Koos, personal observations), the stabilizing effect of 8-SPT on heart rate during hypoxia results from an inhibition of the bradycardic reflex rather than from a drug-induced tachycardia which counterbalances the normal fall in heart rate. The bradycardia elicited by adenosine appears to be produced by indirect effects on the heart because similar levels of 02 deprivation do not lower heart rate in fetuses with denervated peripheral arterial chemoreceptors (see Hanson, 1988; Giussani et al. 1993). Because 8-SPT poorly penetrates the central nervous system (Baumgold, Nikodijevic & Jacobson, 1992), the heart rate responses observed in these mature fetuses with an established blood-brain barrier (Evans, Reynolds, Reynolds, Saunders & Segal, 1974) are probably caused by peripheral effects of the purine nucleoside. The effects of adenosine on heart rate would appear to be mediated by the carotid bodies because the bradycardia is triggered by stimulation of this chemoreceptive tissue (Giussani et al. 1993) and because adenosine excites the peripheral arterial chemoreceptors in fetal sheep (Koos, Chau & Doany, 1992). However, intra-arterial infusion of adenosine in normoxic fetuses produces a tachycardia rather than a bradycardia (Koos & Matsuda, 1990), suggesting that the effects of adenosine on heart rate during hypoxia may differ from those during intravascular infusion under normoxic conditions. Thus, adenosine may modulate heart rate by acting at sites other than or in addition to the peripheral arterial chemoreceptors. For example, the inhibitory effects of 8-SPT on hypoxiainduced bradycardia may involve the antagonism of adenosine receptors within circumventricular organs (Gross & Weindl, 1987), such as the area postrema. Because it 220 r.- Hypoa ::. f--.0 a, Q I 180 F- 140 F *t Time (min) Figure 3. Effects of hypoxia on fetal heart rate Fetal heart rate during hypoxia (0) and hypoxia with adenosine receptor blockade (0). *P< 0-05 compared to control mean. t P < 0 05 compared to value at same time with adenosine receptor blockade.

5 J Physiol Adenosine and fetal heart rate 765 modulates sympathetic activity and has a high density of receptors for a number of hormones in postnatal animals (Ferrario, Barnes, Diz, Block & Averill, 1987), the area postrema in the fetus may have adenosine receptors which modulate autonomic responses elicited by hypoxic stimulation of the carotid bodies. Adenosine also triggers the hypoxic release of arginine vasopressin through stimulation of receptors which also lie outside the blood-brain barrier (Koos et al Because 8-SPT blunts the hypoxic secretion of AVP, the effects of 8-SPT on heart rate during hypoxia may be modulated by arginine vasopressin or other hormones released by adenosine. Adenosine may also modulate heart rate through other secondary effects, such as those related to fetal metabolism. Previous work by Yoneyama & Power (1992) has shown that theophylline, a comparatively weak adenosine receptor antagonist, did not prevent the bradycardia or hypertension induced in the fetus by hypoxia. Besides having a greater affinity for adenosine receptors, the more polar antagonist 8-SPT poorly penetrates the blood-brain barrier and does not affect intracellular enzymes such as phosphodiesterase. These differences between theophylline and 8-SPT could account for these disparate results. Although arginine vasopressin (Koos et al and probably other vasoactive hormones (see Hanson, 1988) contribute to the rise in MAP, the hypoxia-induced hypertension depends principally upon femoral vasoconstriction caused by a-adrenergic stimulation (Lewis et al. 1980; Giussani et al. 1993). Because adenosine receptor blockade abolished the increase in MAP, adenosine apparently elicits sympathetic responses to acute 02 deficiency. This conclusion is consistent with our previous work suggesting that adenosine (through A2 receptors) excites the fetal autonomic nervous system (Koos et al. 1993), a response which may be mediated via vasomotor neurons in the ventrolateral medulla (Sun & Reis, 1994). Hypoxic stimulation of sympathetic activity supports cardiac output through activation of,-adrenergic receptors and helps redistribute cardiac output to important organs through stimulation of a-adrenergic receptors (Giussani et al. 1993). The present work indicates that adenosine has an important role in modulating these autonomic responses to oxygen deficiency. Besides stimulating sympathetic activity, adenosine helps mediate the redistribution of fetal cardiac output by triggering arginine vasopressin release (Koos et al and by increasing vascular conductance in vessels supplying the placenta (Read, Boura & Walters, 1993), heart (Reller, Morton, Giraud, Wu & Thornburg, 1992) and probably the brain (Koos & Doany, 1992; Koos, Mason, Punla & Adinolfi, 1994b). Thus, adenosine elicits several cardiovascular responses which help the fetus tolerate periods of oxygen deficiency. An interesting metabolic effect of adenosine receptor blockade is the reduced fall in arterial ph during hypoxia. Hypoxia normally produces an acidaemia resulting from increased lactic acid production. Because 8-SPT blunted the fall in ph and base excess, adenosine apparently contributed to this metabolic acidaemia, an effect which could be produced via adrenergic stimulation and/or by direct effects on glucose release, cellular uptake or metabolism. Although fetal adenosine concentrations were not measured in the present study, our previous work would indicate that fetal adenosine levels were elevated. For example, a similar degree of hypoxaemia increased fetal plasma (Koos & Doany, 1991) and brain (Koos et at. 1994b) adenosine concentrations by about twofold. Thus the metabolic and cardiovascular effects of adenosine were probably produced by the hypoxia-induced rise in fetal adenosine concentrations. Adenosine administration to the fetus inhibits breathing activity, a depression which is very similar to that produced by hypoxia (Koos & Matsuda, 1990). Theophylline significantly attentuates the inhibitory effects of hypoxia on breathing movements (Bissonnette, Hohimer, Chao, Knopp & Notoroberto, 1990; Koos & Matsuda, 1990), indicating that adenosine contributes to hypoxic inhibition of fetal breathing. Because 8-SPT poorly crosses the blood-brain barrier, the failure of 8-SPT to blunt hypoxic inhibition of breathing is consistent with a central site of action of adenosine relative to hypoxic inhibition. In summary, 8-SPT significantly reduced the fetal acidaemia produced by hypoxia, suggesting that adenosine modulates fetal glycolytic responses to acute oxygen deficiency. Adenosine receptor blockade also abolished the bradycardia and hypertension which normally occur during hypoxia in fetal sheep. Together with other studies (Reller et al. 1992; Koos et al. 1993, 1994, these results suggest that adenosine triggers metabolic, hormonal, vascular, and autonomic responses which help the fetus survive acute 02 deficiency. BAUMGOLD, J., NIKODIJEVIC, 0. & JACOBSON, K. A. (1992). Penetration of adenosine antagonists into mouse brain as determined by ex vivo binding. Biochemical Pharmacology 43, BISSONNETTE, J. M., HOHIMER, A. R., CHAO, C. R., KNOPP, S. J. & NOTOROBERTO, N. F. (1990). Theophylline stimulates fetal breathing movements during hypoxia. Pediatric Research 28, BODDY, K., DAWES, G. S., FISHER, R., PINTER, S. & ROBINSON, J. S. (1974). Foetal respiratory movements, electrocortical and cardiovascular responses to hypoxia and hypercapnia in sheep. Journal of Physiology 243,

6 766 B. J Koos, A. Chau and D. Ogunyemi J Physiol EVANS, C. A. N., REYNOLDS, J. M., REYNOLDS, M. L., SAUNDERS, N. R. & SEGAL, M. B. (1974). The development of a blood-brain barrier mechanism in foetal sheep. Journal of Physiology 238, FERRARIO, C. M., BARNES, K. L., Diz, D. I., BLOCK, D. H. & AVERILL, D. B. (1987). Role of area postrema pressor mechanisms in the regulation of arterial pressure. Canadian Journal of Physiology and Pharmacology 65, GIUSSANI, D. A., SPENCER, J. A. D., MOORE, P. J., BENNET, L. & HANSON, M. A. (1993). Afferent and efferent components of the cardiovascular responses to acute hypoxia in term fetal sheep. Journal of Physiology 461, GROSS, P. M. & WEINDL, A. (1987). Peering through the windows of the brain. Journal of Cerebral Blood Flow and Metabolism 7, HANSON, M. A. (1988). The importance of baro- and chemoreflexes in the control of the fetal cardiovascular system. Journal of Developmental Physiology 10, Koos, B. J., CHAO, A. & DOANY, W. (1992). Adenosine stimulates breathing in fetal sheep with brain stem section. Journal of Applied Physiology 72, Koos, B. J. & DOANY, W. (1991). Role of plasma adenosine in responses to hypoxia in fetal sheep. Journal of Developmental Physiology 16, Koos, B. J., MASON, B. A. & DucsAY, C. A. (1993). Cardiovascular responses to adenosine in fetal sheep: autonomic blockade. American Journal of Physiology 264, H Koos, B. J., MASON, B. A. & ERVIN, M. G. (1994. Adenosine mediates hypoxic release of arginine vasopressin in fetal sheep. American Journal of Physiology 266, R Koos, B. J., MASON, B. A., PUNLA, 0. & ADINOLFI, A. M. (1994b). Hypoxic inhibition of breathing in fetal sheep: relationship to brain adenosine concentrations. Journal of Applied Physiology 77, Koos, B. J. & MATSUDA, K. (1990). Fetal breathing, sleep state, and cardiovascular responses to adenosine in sheep. Journal of Applied Physiology 68, Koos, B. J., SAMESHIMA, H. & POWER, G. G. (1987). Fetal breathing, sleep state, and cardiovascular responses to graded hypoxia in sheep. Journal of Applied Physiology 63, LEWIS, A. B., DONOVAN, M. & PLATZKER, A. C. G. (1980). Cardiovascular responses to autonomic blockade in hypoxemic fetal lambs. Biology of the Neonate 37, READ, M. A., BouRA, A. L. A. & WALTERS, W. A. W. (1993). Vascular actions of purines in the foetal circulation of the human placenta. British Journal of Pharmacology 110, RELLER, M. D., MORTON, M. J., GIRAUD, J. D., Wu, D. E. & THORNBURG, K. L. (1992). Maximal myocardial blood flow is enhanced by chronic hypoxemia in late gestation fetal sheep. American Journal of Physiology 263, H SUN, M.-K. & REIS, D. J. (1994). Hypoxia selectively excites vasomotor neurons of rostral ventrolateral medulla in rats. American Journal of Physiology 266, R YONEYAMA, Y. & POWER, G. G. (1992). Plasma adenosine and cardiovascular responses to dipyridamole in fetal sheep. Journal of Developmental Physiology 18, Acknowledgements We thank Oscar Punla for technical assistance and Karleen Wetherald for typing the manuscript. This study was supported in part by National Institute of Child Health and Human Development grant HD Received 21 November 1994; accepted 10 May 1995.