J Physiol Volume 507, Number 3, 857-867, March 15, 1998
The Journal of Physiology (1998), 507.3, pp. 857-867
© Copyright 1998 The Physiological Society
Angiotensin II and cardiovascular chemoreflex responses to acute hypoxia in late gestation fetal sheep
L. R. Green, H. H. G. McGarrigle, L. Bennet and M. A. Hanson
Departments of Obstetrics & Gynaecology and Physiology, University College, London WC1E 6HX, UK
Received 3 March 1997; accepted after revision 19 November 1997.
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ABSTRACT |
- In six intact and nine carotid sinus denervated (CSD) fetal sheep (125-128 days gestation) we measured heart rate (FHR), mean systemic arterial blood pressure (MAP), femoral and carotid blood flows (FBF and CBF), and femoral and carotid vascular resistances (FVR and CVR). Three experiments were conducted on successive days: normoxia followed by acute isocapnic hypoxia (Pa,O2 to ca 12 mmHg) with infusion of vehicle (HV experiment), the same protocol but with infusion of the angiotensin converting enzyme (ACE) inhibitor, captopril (HC experiment), and normoxia alone with captopril infusion (NC experiment). Plasma angiotensin II concentration ([AII]) was measured in these fetuses, and in a separate group of fetuses (n = 5) that were infused with the nitric oxide (NO) synthesis inhibitor NŒG-nitro-L-arginine methyl ester (L-NAME) or saline vehicle.
- During normoxia, cardiovascular parameters and plasma [AII] were unaltered by captopril infusion, apart from a fall in MAP (NC experiment only, P < 0·05) and FHR (HC experiment only, P < 0·05) in intact and CSD fetuses, respectively. No differences were observed between intact and CSD groups.
- At the onset of hypoxia the rapid initial fall in FHR and rise in FVR was attenuated in CSD fetuses. In all fetuses FHR returned towards prehypoxic levels as hypoxia continued. In contrast, during hypoxia with vehicle infusion (HV experiment) plasma [AII] rose to a similar level in intact and CSD fetuses.
- In both intact and CSD fetuses, the rise in [AII] during hypoxia was blocked by captopril or L-NAME infusion. In CSD, but not intact, fetuses infused with captopril the rise in MAP was absent, and the fall in FBF and rise in FVR did not reach significance during hypoxia.
- Thus, during normoxia CSD alone, or combined with ACE inhibition, does not consistently alter basal cardiovascular control in the late gestation fetus. The rise in [AII] during hypoxia is not mediated by carotid reflexes but may involve NO-dependent mechanisms. In intact fetuses, AII does not appear to be pivotal in cardiovascular control during hypoxia. It is only when carotid reflex mechanisms are removed that a role for AII in the regulation of MAP and peripheral blood flow during hypoxia becomes apparent. These findings lend weight to the idea of multiple mechanisms of fetal cardiovascular control during hypoxia.
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INTRODUCTION |
The ability of the fetus to redistribute its blood flow appropriately during episodes of reduced oxygen supply is of great importance in its survival and development. We have investigated a possible role for angiotensin II (AII) in mediating this redistribution in the late gestation fetal sheep.
Fetal blood flow redistribution during hypoxia consists of cardiovascular responses with two temporal components. Initial responses, which occur in the first 15 min, are characterized by a rapid fall in fetal heart rate (FHR) and femoral blood flow (FBF) and are initiated by carotid rather than aortic chemoreflexes (Bartelds, Van Bel, Teitel & Rudolph, 1993; Giussani, Spencer, Moore, Bennet & Hanson, 1993). The efferent limb of the FBF and FHR reflex responses are provided by 
-adrenergic and vagal mechanisms, respectively. However, it seems likely that other, as yet unidentified, reflexly released mediators may complement the -adrenergic limb since while intact fetuses survive -adrenergic blockade alone, -adrenergic blockade combined with carotid sinus denervation (CSD) is deleterious to fetal survival during hypoxia (Giussani et al. 1993). ACTH and cortisol (Giussani, McGarrigle, Moore, Bennet, Spencer & Hanson, 1994a) and arginine vasopressin (AVP; Giussani, McGarrigle, Spencer, Moore, Bennet & Hanson, 1994b) are not likely to fulfil this role since their rise during hypoxia is not altered by CSD. An alternative candidate is the potent vasoconstrictor AII. Fetal plasma [AII] is known to rise during hypoxia (Broughton Pipkin, Lumbers & Mott, 1974), but there is no evidence as to whether this is reflexly mediated.
When hypoxia is maintained for 1 h, the second, slower component of the cardiovascular responses to hypoxia is characterized by maintained peripheral vasoconstriction, e.g. in the femoral bed (Giussani et al. 1993), and vasodilatation of cerebral, myocardial and adrenal beds (Cohn, Sacks, Heymann & Rudolph, 1974), which redistribute combined ventricular output towards these latter organs. These changes are not primarily mediated by carotid chemoreflexes. A rise in plasma catecholamines, due in part to a direct action of hypoxia on the adrenal glands, and in plasma AVP (Giussani et al. 1994b), ACTH and cortisol (Giussani et al. 1994a) is likely to contribute, but cannot fully account for, the maintained vasoconstriction. A rise in AII, following increased renin angiotensin system (RAS) activity, would provide another candidate mechanism. Angiotensin II receptor blockade using saralasin has implicated AII in the maintainance of basal regional blood flow in the fetus (Iwamoto & Rudolph, 1979), but to date the role played by AII in cardiovascular control during acute hypoxia has not been fully elucidated.
In the fetus, as in the adult, the vascular endothelium may act as a hypoxic sensor and effector system, since endothelial cells release a variety of vasoactive substances including nitric oxide (NO; Vane, Anggard & Botting, 1990). Recently, we have demonstrated that NO synthesis inhibition with N G-nitro-L-arginine methyl ester (L-NAME) during normoxia decreases carotid blood flow (CBF) and FHR, and increases mean arterial pressure (MAP), femoral vascular resistance (FVR) and carotid vascular resistance (CVR) in the late gestation fetus during normoxia. Then, at the onset of hypoxia, the rapid initial chemoreflex-mediated fall in FHR and FBF is simply superimposed upon the altered baseline levels. In contrast, NO appears to play a key role in the rise in CBF during hypoxia (Green, Bennet & Hanson, 1996). In addition, endothelial (Lilly et al. 1985) and smooth muscle (Re, Fallon, Dzau, Quay & Haber, 1982) cells synthesize renin, and therefore locally generated AII may act in a paracrine and autocrine fashion on vascular smooth muscle. There is evidence of an inverse relationship between local vascular angiotensin converting enzyme (ACE) activity and NO synthase expression (Holtz & Goetz, 1994). Moreover, in studies on adult rabbits, blockade of NO synthesis decreases AII synthesis (Goyer, Bui, Chou, Evans, Keil & Reid, 1994). It is not appropriate therefore to consider changes in AII synthesis in isolation from the effects of NO synthesis.
Thus, in this study we have measured circulating AII in intact and CSD fetuses to investigate whether the rise in [AII] during hypoxia is controlled by carotid chemoreflex mechanisms. In addition we have monitored FHR, MAP, FBF and CBF in these fetuses and have used the ACE inhibitor, captopril, to determine the contribution of AII to the cardiovascular changes that occur in the fetus during hypoxia. In the light of the potential interaction between AII and NO, we also report plasma AII measurements made in a separate group of fetuses (Green et al. 1996) during the inhibition of NO synthesis by L-NAME.
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METHODS |
Surgical preparation
Twenty pregnant Suffolk and Blue-faced Leicester Cross ewes were instrumented at 113-121 days gestation (term is 147 days) under general anaesthesia (1 g thiopentone in solution I.V. for induction followed by 2 % halothane in O2 for maintenance). An incision was made in the mid-line of the lower abdominal wall and the uterus was palpated to determine the fetal number and position. A fetus was partially exteriorized through an incision in the uterine wall. Nine fetuses had the carotid sinus nerves (CSN) cut bilaterally (Giussani et al. 1993). On each side, below the angle of the jaw, the tissue surrounding the carotid body was carefully dissected using × 4 optical magnification. The CSN was identified within the triangle formed by the common carotid artery, occipital artery and glossopharyngeal nerve and dissected free of connective tissue. A 2-3 mm portion of the CSN was removed, the severed ends of the nerve were identified and the denervation was evidenced by the attenuation of the rapid bradycardia at the onset of hypoxia. Catheters filled with heparinized saline (i.d. = 1·0 mm, o.d. = 2·0 mm, translucent vinyl tubing, Portex Ltd) were placed in a fetal carotid artery, a jugular vein, and in the amniotic cavity. In addition, in five CSD fetuses, a catheter was placed in a brachial vein for drug administration. An ultrasonic blood flow probe (Transonic Systems Inc., Ithaca, NY, USA) was placed around the uncatheterized carotid artery, as this provides a good estimate of cerebral blood flow in the fetus (van Bel, Roman, Klautz, Teitel & Rudolph, 1994), and around a femoral artery, which gives an indication of carcass blood flow. Stainless-steel electrodes were sewn subcutaneously onto the chest and hindlimb to record ECG. Catheters and electrodes were exteriorized through the maternal flank and secured to the ewe's back in a plastic bag. A heparinized catheter was placed in a maternal pedal vein. Following surgery, antibiotics were administered to the ewe (4 ml Streptopen I.M.) and amniotic cavity (600 mg Crystapen and 80 mg Gentamicin). A period of at least 5 days post-operative recovery was allowed prior to experimentation during which daily antibiotic treatment was given to the ewe (300 mg Crystapen I.V.), fetus (150 mg Crystapen I.V.) and into the amniotic cavity (150 mg Crystapen). Gentamicin was administered into the amniotic cavity (40 mg) and to the ewe (40 mg, I.V.) on post-operative days 1 and 2 only. Patency of catheters was maintained by a continuous infusion of heparinized saline (50 i.u. ml-1 at 0·125 ml h-1) and fetal arterial blood was collected daily for blood gas analysis. At the end of the study, ewes were killed by an overdose of barbiturate (30-40 ml I.V., 200 mg ml-1 pentobarbitone sodium BP; Rhône Mérieux, UK).
Experimental procedures
Induction of hypoxia. Fetal oxygenation was manipulated by placing a loosely tied, transparent polyethylene bag over the ewe's head into which a controlled mixture of air, N2 and CO2 were passed at ca 44 l min-1. Normoxia was maintained by the administration of air only, and fetal isocapnic hypoxia (Pa,O2 to ca 12 mmHg) was induced by reducing the maternal inspired O2 fraction (FI,O2) (inspirate, 14-18 l min-1 air; 22 l min-1 N2; 1·2 l min-1 CO2).
L-NAME protocol. As previously described (Green et al. 1996), in five intact fetuses (123-129 days gestation) two experiments were conducted on successive days. An initial 1 h normoxic period was established, followed by the induction of 1 h fetal hypoxia, during the infusion of vehicle (4 ml h-1; five drops 150 mM NaOH per 10 ml saline). On a subsequent day the protocol was repeated during infusion of L-NAME (Sigma; 300 mg in 2 ml bolus I.V. followed by 3 ml h-1 infusion of 33 mg ml-1 in saline). Fetal arterial blood (2-3 ml) was collected just prior to the onset of infusion (time 0), during normoxia (45 min), hypoxia (75 and 105 min) and the subsequent recovery period (135 and 165 min). Blood was transferred immediately to chilled EDTA tubes and spun at 4°C (3000 r.p.m.) for 10 min. Plasma was decanted into tubes and stored at -20°C for subsequent hormonal analysis. A further 0·6 ml arterial blood was collected at these times and at 90 min (30 min into the hypoxic period) for blood gas and electrolyte analysis. Cardiovascular parameters were monitored continuously throughout the protocol and are reported elsewhere (Green et al. 1996).
Captopril protocol. In six intact and nine CSD fetuses (119-128 days gestation), three experiments were conducted on successive days. In the first experiment, 5 min after the onset of saline vehicle infusion (3 ml h-1 infusion, I.V. ) an initial 1 h period of normoxia was established followed by the induction of 1 h fetal hypoxia (Hypoxia with vehicle, HV). For the second experiment, the protocol was repeated with the I.V. infusion of captopril (Hypoxia with captopril, HC; Sigma; 1 mg in 1 ml bolus over 5 min, followed by 3 ml h-1 continuous infusion of 1 mg ml-1 captopril in saline). In the third experiment, the same dose regime of captopril was administered, but during 3 h normoxia (Normoxia with captopril, NC). In one CSD fetus the order of the first and second experiments was reversed, and in one intact fetus the order of the second and third experiments was reversed.
Arterial blood pressure, FHR, CBF and FBF were recorded continuously throughout the protocol on a chart recorder (ES1000, Gould, France). Fetal arterial blood (2-3 ml) was collected prior to the onset of infusion (-20 min), during normoxia (45 min), hypoxia (75 and 105 min) and the subsequent recovery period (135 and 165 min). Blood was transferred immediately to chilled EDTA tubes and spun at 4°C (3000 r.p.m.) for 10 min. Plasma was decanted into tubes and stored at -20°C for subsequent hormonal analysis. A further 0·6 ml arterial blood was collected at these times and at 90 min (hypoxic period) for analysis of pH, blood gases, electrolytes (BG Electrolytes system, Instrumentation Laboratory, UK), haemoglobin (Hb), saturation of haemoglobin with oxygen (SaO2) and haematocrit (Hct; Co-oximeter, Instrumentation Laboratory, UK; calibrated for fetal haemoglobin), glucose and lactate (2300 Stat Plus, YSI Inc., Yellow Springs, OH, USA).
In four CSD fetuses the cardiovascular response to 5 µg AI I.V. (Sigma) was investigated on all experimental days prior to the onset of vehicle or captopril infusion, and at the end of the 3 h protocol.
Angiotensin II radioimmunoassay
Angiotensin II was measured in plasma samples from the captopril (n = 5) and L-NAME (n = 5) protocols by a sensitive and specific competitive protein-binding radioimmunoassay, following its separation from plasma proteins by methanol extraction and chromatography, using a kit supplied by Nichols Institute (Diagnostics B.V., Saffron Walden, Essex, UK).
Briefly, C18 chromatography columns (Sep Pak, Waters Associates, Millford, MA, USA) were mounted on a Super Separator-24 manifold, containing a vacuum facility for eluting the columns. The columns were washed with 10 ml methanol and 10 ml phosphate buffer. Aliquots of plasma (0·5 ml) were mixed with equal volumes of 10 mM phosphate buffer (pH 7·4) and added to the column. The columns were then washed with 10 ml phosphate buffer and 2·5 ml methanol was added to the columns to elute the AII. The methanol was evaporated with a jet of air (at 37°C) and the residue reconstituted with 0·84 ml of Tris buffer. Duplicate 0·4 ml aliquots of extract in Tris buffer were transferred to polstyrene tubes and 0·1 ml of anti-AII (rabbit) antiserum was added. The tubes were mixed, covered and incubated for 6 h at 2-8°C. Then 0·1 ml iodinated (125I) AII (Sigma) was added to the tubes, which were mixed and incubated for 18 h at 4°C. Finally, 0·1 ml anti-rabbit-antiserum (donkey) precipitant was added and the tubes were mixed and incubated at room temperature for 30 min. Deionized water (1 ml) was then added to each sample, and the tubes centrifuged at room temperature for 15 min (2000 g). The supernatant was decanted and the tubes containing the residue were transferred to a gamma counter to determine radioactive content.
Samples from the captopril and L-NAME protocols were assayed in separate batches. Recoveries averaged 83 % (range, 75-91 %). The assay sensitivity was 3·8 pg ml-1. The intra- and interassay coefficients of variation of the assay were 4 % for a value of 42 pg ml-1 and 5·1 and 9·3 % for values of 31 and 96 pg ml-1, respectively. The assay cross-reacted with Asp1-Ileu5-AII (100 %), Val5-AII (100 %), Asn1-Val5-AII (30 %) and Asp1-Ileu5-AI (0·1 %).
Data analysis
Six intact and nine CSD fetuses were originally included in the captopril protocol. For a given cardiovascular parameter, fetuses were excluded from group analysis when measurements from the HV experiment were missing.
Vascular resistances (mmHg min ml-1) were calculated using the formula: MAP/Flow. To avoid multiple comparison of data at individual time points, arterial blood measurements and cardiovascular data were reduced to summary measures to describe normoxic and hypoxic periods (Matthews, Altman, Campbell & Royston, 1990; Finney, 1990). Student's t test was then used to analyse summary measure values within (paired) and between (unpaired) intact and CSD groups. For cardiovascular measurements, the periods of time over which measurements were reduced to summary measures are indicated in Figs 4, 5 and 6 by horizontal filled bars. In addition, Student's t test was used to analyse rapid cardiovascular changes at the onset of hypoxia (60 vs. 65 min). Statistical significance was accepted when P < 0·05.
Initial analysis of plasma AII measurements was carried out using two-way analysis of variance (ANOVA) for repeated measures allowing for missing data to test the effect of time and treatment. A post hoc Student's t test was used to analyse the change in [AII] during hypoxia (75 and 105 min) from normoxia (45 min). The Bonferroni method of correction was used so that statistical signifiance was accepted when P < 0·025.
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RESULTS |
Response to exogenous angiotensin I
A 5 µg AI (I.V.) bolus administered to four CSD fetuses produced a rise in MAP on all days of study (see Fig. 1). This response was absent following captopril (HC and NC experiments), but not vehicle (HV experiment), infusion (Fig. 1).
Blood gases, electrolytes, glucose and lactate
During normoxia, arterial blood measurements were not altered by the onset of vehicle or captopril infusion (Table 1). In the HC experiment, normoxic glucose levels were greater in CSD than in intact fetuses, although there was no difference between the groups during the subsequent hypoxia and recovery periods (Table 1).
Table 1. Blood gas and electrolyte measurements during normoxia, hypoxia and recovery with vehicle and captopril infusion in intact and CSD fetuses
| | Intact | CSD |
| Expt | Pre-infusion | Normoxia | Hypox./Normox. | Recovery | Pre-infusion | Normoxia | Hypox./Normox. | Recovery |
| Pa,O2 | HV | 21·3 ± 0·7 (3) | 21·9 ± 0·8 | 11·5 ± 0·7** | 22·3 ± 1·2 | 22·0 ± 1·0 (8) | 21·3 ± 0·7 (8) | 12·2 ± 0·5** (8) | 22·6 ± 1·1 (8) |
| HC | 23·0 (2) | 22·9 ± 0·5 (5) | 11·4 ± 0·6** (5) | 23·2 ± 0·3 (5) | 23·7 ± 0·6 (7) | 23·3 ± 0·9 (7) | 12·6 ± 0·3** (7) | 22·2 ± 0·8* (7) |
| NC | 24·0 (1) | 24·3 ± 0·8 (5) | 23·4 ± 0·8 | 23·8 ± 0·7 (5) | 22·0 ± 0·7 (8) | 22·4 ± 1·0 (7) | 22·6 ± 0·8 (8) | 22·4 ± 0·6 (8) |
| SaO2 | HV | 76·2 ± 1·3 (3) | 77·1 ± 1·1 | 33·8 ± 2·7** | 74·0 ± 1·7* | 77·8 ± 2·3 (8) | 75·8 ± 1·6 (8) | 37·0 ± 2·1** (8) | 74·7 ± 2·0 (8) |
| HC | 79·4 (2) | 77·5 ± 1·9 | 31·7 ± 2·6** | 73·6 ± 3·2 | 80·7 ± 1·2 (7) | 78·8 ± 1·3 (7) | 33·9 ± 1·2**(7) | 72·8 ± 1·1** (7) |
| NC | 79·2 ± 1·5 (3) | 80·4 ± 2·0 | 78·3 ± 1·6** | 79·2 ± 0·8 | 78·7 ± 1·8 (7) | 76·4 ± 3·4 (6) | 76·7 ± 2·5 (7) | 75·6 ± 1·9 (7) |
| Pa,CO2 | HV | 50·7 ± 2·1 (3) | 47·4 ± 1·6 | 45·9 ± 1·5 | 45·9 ± 1·4 | 48·9 ± 1·0 (8) | 47·3 ± 0·6 (8) | 46·0 ± 1·2 (8) | 46·1 ± 0·8 (8) |
| HC | 47·8 ± 1·1 (3) | 47·1 ± 2·2 | 47·2 ± 0·9 | 46·3 ± 0·4 | 49·1 ± 0·7 (7) | 46·7 ± 1·4 (7) | 48·4 ± 1·1 (7) | 47·1 ± 0·5 (7) |
| NC | 50·5 (2) | 47·9 ± 1·3 | 48·2 ± 0·8 | 47·9 ± 0·8 | 50·2 ± 0·9 (8) | 48·4 ± 1·6 (7) | 49·0 ± 1·2 (8) | 50·3 ± 0·6 (8) |
| pH | HV | 7·36 ± 0·01 (3) | 7·36 ± 0·01 | 7·29 ± 0·02* | 7·27 ± 0·02** | 7·36 ± 0·01 (8) | 7·36 ± 0·01 (8) | 7·31 ± 0·01** (8) | 7·29 ± 0·01** (8) |
| HC | 7·36 ± 0·01 (3) | 7·34 ± 0·01 | 7·27 ± 0·03 | 7·26 ± 0·04 | 7·36 ± 0·00 (7) | 7·35 ± 0·01 (7) | 7·29 ± 0·01** (7) | 7·30 ± 0·01** (7) |
| NC | 7·35 ± 0·01 (3) | 7·35 ± 0·00 | 7·35 ± 0·00 | 7·35 ± 0·00 | 7·35 ± 0·00 (8) | 7·35 ± 0·00 (7) | 7·35 ± 0·00 (8) | 7·35 ± 0·00 (8) |
| Na+ | HV | 140·3 ± 0·9 (3) | 140·8 ± 0·7 | 138·9 ± 0·8 | 138·1 ± 0·9** | 137·0 ± 3·3 (8) | 139·9 ± 1·2 (8) | 140·3 ± 1·4 (8) | 138·4 ± 0·9 (8) |
| HC | 140·5 (2) | 139·4 ± 1·2 (5) | 139·1 ± 1·8 | 138·9 ± 1·85 (5) | 139·3 ± 1·0 (6) | 140·2 ± 2·7 (6) | 137·8 ± 1·5 (6) | 136·8 ± 1·3 (7) |
| NC | 139·7 ± 1·9 (3) | 139·4 ± 2·5 (5) | 139·3 ± 1·6 (5) | 141·8 ± 2·6 (5) | 138·1 ± 1·1 (8) | 138·9 ± 1·0 (7) | 138·4 ± 1·2 (8) | 137·8 ± 1·4 (7) |
| K+ | HV | 3·7 ± 0·2 (3) | 3·6 ± 0·2 | 4·2 ± 0·2* | 3·8 ± 0·1 | 4·0 ± 0·3 (8) | 3·5 ± 0·1 (8) | 4·0 ± 0·2** (8) | 3·8 ± 0·1** (8) |
| HC | 3·6 ± 0·3 (3) | 3·5 ± 0·3 | 4·3 ± 0·2** | 4·0 ± 0·2* | 3·6 ± 0·2 (6) | 3·6 ± 0·2 (6) | 4·0 ± 0·1 (7) | 3·7 ± 0·2 (6) |
| NC | 3·7 ± 0·3 (3) | 3·7 ± 0·3 | 3·7 ± 0·2 | 3·8 ± 0·2 | 3·6 ± 0·2 (8) | 3·6 ± 0·2 (7) | 3·6 ± 0·2 (8) | 3·7 ± 0·3 (7) |
| Hct | HV | 29·3 ± 1·5 (3) | 27·1 ± 1·0 | 28·5 ± 1·2* | 27·6 ± 1·0 | 26·4 ± 1·1 (8) | 25·9 ± 1·0 (8) | 29·3 ± 1·1** (8) | 27·5 ± 0·8 (8) |
| HC | 28·3 ± 1·9 (3) | 25·7 ± 1·7 | 29·5 ± 0·9* | 28·3 ± 1·2 | 27·1 ± 1·3 (7) | 25·6 ± 1·3 (7) | 29·5 ± 1·1* (7) | 27·3 ± 0·8 (7) |
| NC | 29·0 ± 2·1 (3) | 27·3 ± 1·6 | 27·5 ± 1·6 | 28·8 ± 2·0 | 28·1 ± 1·0 (7) | 27·2 ± 1·4 (6) | 27·4 ± 1·1 (7) | 27·4 ± 0·8 (7) |
| Hb | HV | 7·9 ± 0·2 (3) | 7·4 ± 0·2 | 6·0 ± 0·2** | 7·4 ± 0·2 | 7·7 ± 0·2 (8) | 7·4 ± 0·3 (8) | 6·4 ± 0·2** (8) | 7·5 ± 0·2 (8) |
| HC | 8·0 (2) | 7·4 ± 0·2 | 6·0 ± 0·2** | 7·3 ± 0·3 | 7·9 ± 0·2 (7) | 7·6 ± 0·3 (7) | 6·0 ± 0·2** (7) | 7·0 ± 0·6 (7) |
| NC | 7·9 ± 0·3 (3) | 7·7 ± 0·4 | 7·8 ± 0·3 | 7·9 ± 0·3 | 7·7 ± 0·3 (7) | 7·3 ± 0·3 (6) | 7·4 ± 0·2 (7) | 7·5 ± 0·2 (7) |
| Glu. | HV | 0·70 ± 0·08 (3) | 0·77 ± 0·11 | 1·09 ± 0·07* | 1·00 ± 0·03 | 0·88 ± 0·10 (7) | 0·87 ± 0·10 (7) | 1·24 ± 0·12** (7) | 1·20 ± 0·14** (7) |
| HC | 0·56 (2) | 0·63 ± 0·07 (5) | 1·10 ± 0·12* (5) | 1·14 ± 0·23 (5) | 1·01 ± 0·12 (6) | 1·05 ± 0·09 (6) | 1·31 ± 0·06** (6) | 1·21 ± 0·09** (6) |
| NC | 0·61 ± 0·12 (3) | 0·71 ± 0·10 | 0·75 ± 0·12 | 0·78 ± 0·11 | 0·95 ± 0·07 (8) | 0·85 ± 0·08 (7) | 1·92 ± 0·12 (8) | 1·10 ± 0·18 (8) |
| Lac. | HV | 0·86 ± 0·12 (3) | 0·85 ± 0·10 | 3·69 ± 0·56** | 5·16 ± 1·04* | 0·87 ± 0·04 (7) | 0·79 ± 0·04 (7) | 3·65 ± 0·36** (7) | 5·10 ± 0·59** (7) |
| HC | 0·74 ± 0·10 (3) | 0·86 ± 0·12 | 3·99 ± 0·59** | 5·58 ± 0·97** | 0·86 ± 0·09 (6) | 0·86 ± 0·02 (6) | 3·41 ± 0·13** (6) | 4·14 ± 0·27** (6) |
| NC | 0·81 ± 0·14 (3) | 0·81 ± 0·11 | 0·83 ± 0·11 | 0·86 ± 0·01 | 0·89 ± 0·05 (8) | 0·83 ± 0·05 (7) | 1·12 ± 0·25 (8) | 1·40 ± 0·52 (8) |
Values are means ± S.E.M. of arterial blood measurements in intact and CSD fetuses (n = 6 and n = 9, respectively, except where indicated by numbers in parentheses; values for different parameters are given in the following units: Pa,O2, mmHg; Sa,O2, %; Pa,CO2, mmHg; Na+, mmol l-1; K+, mmol l-1; Hct, %; Hb, g dl-1; glucose (Glu.), mmol l-1; lactate (Lac.), mmol l-1. * P < 0·05 and ** P < 0·01, significantly different from normoxia, by Student's paired t test. P < 0·01, significantly different from intact group by Student's unpaired t test.
During hypoxia (HV and HC experiments) Pa,O2 fell from ca 22 to ca 12 mmHg and SaO2 fell from ca 80 to ca 30 % in intact and CSD fetuses. pH fell during hypoxia and recovery in intact and CSD fetuses infused with vehicle (HV experiment), and in CSD, but not intact, fetuses infused with captopril (HC experiment). In intact and CSD groups, Pa,CO2 did not change throughout the protocol, but blood lactate rose during hypoxia and remained elevated during the recovery period (HV and HC experiments; Table 1). In both groups, arterial glucose rose during hypoxia (HV and HC experiments), but only remained elevated during the recovery period in CSD fetuses. Haematocrit increased and Hb fell during hypoxia in both groups (HV and HC experiments; Table 1). In all experiments, arterial Na+ was unaltered, except for a small fall during the recovery period in vehicle-infused intact fetuses (HV experiment; Table 1). During hypoxia, arterial K+ increased in intact fetuses (HV and HC experiments), and in vehicle-infused (HV experiment), but not captopril-infused (HC experiment) CSD fetuses.
Plasma angiotensin II concentration
In vehicle-infused intact fetuses, plasma [AII] rose during hypoxia (HV experiment; Fig. 2) and returned towards pre-hypoxic levels in the subsequent recovery period. In vehicle-infused CSD fetuses there was a variable rise in [AII] during hypoxia which did not reach significance (HV; Fig. 2), although there was no difference in the magnitude of the change in plasma [AII] between intact and CSD groups (55 ± 9 vs. 98 ± 29 pg ml-1, respectively). In intact and CSD fetuses infused with captopril, plasma [AII] did not rise during hypoxia (HC; Fig. 2). Thus, in CSD, although not intact, fetuses plasma [AII] was significantly lower during the 3 h protocol with captopril than with vehicle infusion (P < 0·05, ANOVA; Fig. 2).
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Figure 2. The effect of captopril on plasma angiotensin II during normoxia and hypoxia in intact and CSD fetuses
Values are means ± S.E.M. of intact and CSD fetal plasma AII responses to 1 h of hypoxia (shaded region) during vehicle ( ) and captopril ( ) infusion. n = 5, except where indicated by numbers in parentheses. * P < 0·025, significantly different from normoxia (45 min).
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There was a small but significant fall in [AII] at the onset of hypoxia with L-NAME infusion, in contrast to the rise in [AII] observed during hypoxia with vehicle infusion (Fig. 3). Over the course of the 3 h experiment, plasma [AII] was lower with the infusion of L-NAME than with the infusion of vehicle (P < 0·05, ANOVA; Fig. 3).
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Figure 3. The effect of L-NAME on plasma angiotensin II during normoxia and hypoxia in intact fetuses
Values are means ± S.E.M. of intact fetal plasma AII responses to 1 h of hypoxia (shaded region) during vehicle () and L-NAME () infusion. n = 5, except where indicated by numbers in parentheses. * P < 0·025, significantly different from normoxia (45 min).
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Cardiovascular measurements
Normoxia
During normoxia, there was no difference between cardiovascular measurements made with vehicle infusion and those made with captopril infusion (Figs 4, 5 and 6). However, there was a significant fall in FHR after the onset of captopril infusion in CSD fetuses (HC experiment; Fig. 4). Furthermore, captopril infusion during normoxia produced a fall in MAP in the NC experiment (Table 2), but not in the HC experiment (Fig. 4). In intact fetuses, CBF was elevated following the onset of both vehicle and captopril infusion during normoxia (HV and HC experiments; Fig. 6), but neither CBF nor CVR were altered by captopril infusion in the NC experiment (Table 2).
Table 2. Cardiovascular responses to captopril infusion during 3 h of normoxia in intact and CSD fetuses
| | n | Pre-infusion | Hour 1 | Hour 2 | Hour 3 |
| FHR (beats min-1) | Intact | 6 | 162 ± 11 | 161 ± 12 | 166 ± 14 | 156 ± 11 |
| CSD | 8 | 183 ± 6 | 176 ± 8 | 177 ± 5 | 180 ± 4 |
| MAP (mmHg) | Intact | 4 | 47 ± 1 | 42 ± 1* | 41 ± 2 | 40 ± 3 |
| CSD | 7 | 39 ± 2 | 37 ± 2 | 37 ± 2 | 33 ± 2 |
| FBF (ml min-1) | Intact | 6 | 48 ± 6 | 45 ± 7 | 45 ± 7 | 44 ± 8 |
| CSD | 7 | 55 ± 6 | 52 ± 6 | 53 ± 5 | 53 ± 6 |
| FVR (mmHg min ml-1) | Intact | 4 | 1·2 ± 0·3 | 1·3 ± 0·3 | 1·3 ± 0·3 | 1·4 ± 0·4 |
| CSD | 6 | 0·8 ± 0·1 | 0·8 ± 0·1 | 0·8 ± 0·1 | 0·6 ± 0·1 |
| CBF (mmHg) | Intact | 4 | 99 ± 14 | 97 ± 9 | 95 ± 10 | 93 ± 11 |
| CSD | 7 | 80 ± 23 | 83 ± 19 | 85 ± 18 | 82 ± 20 |
| FVR (mmHg min ml-1) | Intact | 3 | 0·5 ± 0·1 | 0·4 ± 0·1 | 0·5 ± 0·1 | 0·5 ± 0·4 |
| CSD | 6 | 0·6 ± 0·2 | 0·6 ± 0·2 | 0·6 ± 0·1 | 0·6 ± 0·2 |
Values are means ± S.E.M. of intact and CSD FHR, MAP, FBF, FVR, CBF and CVR with captopril infusion during 3 h normoxia (NC experiment. * P < 0·05, significantly different from pre-infusion by Student's paired t test.
Hypoxia
In intact vehicle-infused fetuses, there was a rapid initial fall in FHR and FBF, and a rise in FVR at the onset of hypoxia (HV experiment; Figs 4, 5 and 6). As hypoxia proceeded, FHR returned towards pre-hypoxic levels, while the FBF and FVR changes were sustained for the duration of hypoxia. In addition, there was a slower rise in MAP during hypoxia (Fig. 4). There was a tendancy for CBF to rise during hypoxia, but this did not reach significance.
In captopril-infused intact fetuses the FHR response to hypoxia was no different from that during vehicle infusion. Furthermore, MAP and FVR, and FBF levels during hypoxia with captopril infusion were similar to those achieved during vehicle infusion, although the rise in FVR during hypoxia was quite variable and did not reach significance.
In vehicle-infused CSD fetuses the magnitude of the initial fall in FHR was significantly less than in the corresponding intact fetuses (P < 0·05). In contrast, the rise in MAP during hypoxia in these fetuses was similar to that of vehicle-infused intact fetuses. In addition, FBF fell and FVR rose during hypoxia. However, the rise in FVR tended to be smaller than in the vehicle-infused intact group, indeed the rapid initial rise in FVR observed in intact fetuses (60 vs. 65 min, P < 0·05) did not reach significance in the CSD group.
The infusion of captopril (HC experiment) did not alter the FHR response of the CSD group to hypoxia. However, the rise in MAP seen in vehicle-infused CSD during hypoxia was absent following the infusion of captopril (Fig. 4). There was an initial fall in FBF (60 vs. 65 min, P < 0·05) at the onset of hypoxia in CSD fetuses infused with vehicle and captopril. As hypoxia continued, this fall was maintained in the vehicle-infusion experiment (HV), but with captopril infusion there was no difference in FBF or FVR between normoxia and hypoxia. During hypoxia, CBF tended to rise in all groups, but this only reached significance in captopril-infused CSD group and was accompanied by a fall in CVR (Fig. 6).
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Figure 5. The effect of captopril on femoral blood flow and vascular resistance during normoxia and hypoxia in intact and CSD fetuses
Values are means ± S.E.M. Intact and CSD FBF (n = 6 and n = 9, respectively) and FVR (n = 5 and n = 8, respectively) responses to 1 h of hypoxia (shaded region) during vehicle (HV experiment;  ) and captopril (HC experiment;  ) infusion. The dotted line denotes the onset of infusion at -5 min. The horizontal bars show the time period over which data was reduced to a summary measure. ² P < 0·05 and ²² P < 0·01, significantly different from normoxia in vehicle group; ³³ P < 0·01, significantly different from normoxia in captopril group.
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Figure 6. The effect of captopril on carotid blood flow and vascular resistance during normoxia and hypoxia in intact and CSD fetuses
Values are means ± S.E.M. of intact and CSD fetal CBF (n = 4 and n = 8, respectively) and CVR (n = 3 and n = 6, respectively) responses to 1 h of hypoxia (shaded region) during vehicle (HV experiment; ) and captopril (HC experiment; ) infusion. The dashed line denotes the onset of infusion at -5 min. The horizontal bars show the time period over which data were reduced to a summary measure. ² P < 0·05, significantly different from normoxia in vehicle group; ³ P < 0·05 and ³³ P < 0·01, significantly different from normoxia in captopril group.
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DISCUSSION |
In this study we confirm that a rise in fetal plasma [AII] occurs during hypoxia, but show that it is not likely to be mediated by carotid chemoreflex mechanisms. This rise in [AII] is inhibited by the infusion of captopril, an ACE inhibitor, and by the infusion of L-NAME, a NO synthesis inhibitor. However, AII does not appear to contribute to the cardiovascular changes of the intact fetus during hypoxia, since peripheral vascular resistance and MAP rise, and FHR changes, to a similar level with and without the infusion of captopril or L-NAME (see Green et al. 1996). It is not until carotid chemoreflex mechanisms have been removed that a role for AII in the regulation of peripheral vascular tone becomes apparent.
Plasma angiotensin II during normoxia and hypoxia
We observed that plasma [AII] rose during hypoxia in intact fetuses to levels in accordance with previous studies (Broughton Pipkin et al. 1974). We did not measure plasma renin activity (PRA) in the present study, but there is evidence that moderate hypoxia does not significantly increase fetal sheep PRA (Wood, Kane & Raff, 1990). In the sheep fetus, increased PRA during haemorrhage is unaffected by bilateral vago-sympathectomy (Wood, Chen & Bell, 1989), although peripheral arterial chemoreceptors are implicated in the AII response to hypoxia with hypercapnia (Wood et al. 1990). In the present study a variable rise in [AII] was observed during hypoxia in vehicle-infused CSD fetuses, although no difference in [AII] was observed between vehicle-infused intact and CSD groups during normoxia and hypoxia. These findings suggest that carotid chemo- and baroreceptors do not mediate the rise in [AII] during hypoxia. Thus AII is not likely to be a suitable candidate for the, as yet unidentified, reflexly released vascoconstrictor substance thought to complement the -adrenergic efferent limb of the reflex arc at the onset of hypoxia (Giussani et al. 1993).
During normoxia there was no apparent change in plasma [AII] after the onset of captopril infusion. One possible explanation is low basal fetal RAS activity since, while plasma renin is higher in the near-term fetus than in the mother, fetal plasma [AII] is relatively low (Broughton Pipkin et al. 1974; Raimbach & Thomas, 1990). Moreover, levels of pulmonary ACE (Raimbach & Thomas, 1990) are low in the fetus compared with the adult. Alternatively, it does not seem likley that our results are due to inadequate ACE inhibition since the rise in plasma [AII] during hypoxia and the pressor response to an I.V. AI bolus were absent following captopril infusion. However, we cannot rule out the possibility that measurement of circulating levels of AII may not accurately reflect local vascular [AII]. The pressor response to AI observed before captopril infusion was greater in the HC experiment than in the subsequent NC experiment. This may be due to slow clearance of captopril from the fetal circulation (Broughton Pipkin, Symonds & Turner, 1982) especially since the kidneys, a major site of captopril clearance, are functionally immature in the fetus compared with the adult (Lumbers, 1983).
A number of studies have investigated the interaction between NO and AII synthesis. Previously we reported a large rise in MAP after the onset of L-NAME infusion during normoxia in the same fetuses (Green et al. 1996). It is possible that this rise in MAP contributed to the variable, but non-significant, decline in [AII] after the onset of L-NAME infusion in the present study. During hypoxia, the rise in [AII] seen during vehicle infusion was absent when L-NAME was infused. Possible explanations are that (i) the rise in [AII] during hypoxia was dependent on NO synthesis, either at the level of renin release or the conversion of AI to AII, or (ii) L-NAME administration initiated other physiological processes which in turn decreased AII synthesis. It does not seem likely that MAP differences can account for our findings since we reported previously that MAP rose to a similar level during hypoxia with vehicle and L-NAME infusion (Green et al. 1996).
Angiotensin II and chemoreflexes during normoxia
Previous fetal studies show that AII receptor blockade (Mattioli, Chien, Vassenon, Crist & Lynn, 1979) does not alter MAP, while ACE inhibition produces a fall in MAP during normoxia (Broughton Pipkin et al. 1982; Robillard, Weismann, Gomez, Ayers, Lawton & Van Orden, 1983). In the present study we did not find a consistent effect of ACE inhibition on basal cardiovascular parameters since, in intact fetuses, the onset of captopril infusion during normoxia caused a fall in MAP in the NC experiment, but not in the HC experiment, whereas in CSD fetuses, we observed a fall in FHR following captopril during normoxia in the HC experiment, but not in the subsequent NC experiment. A role for AII in maintaining vascular tone in fetal peripheral (Iwamoto & Rudolph, 1979) and renal (Robillard et al. 1983) circulations has been suggested. However, in the present study the onset of captopril infusion during normoxia did not alter FBF, and the fall in CBF with captopril infusion was also observed after vehicle infusion. Indeed, the variable effect of captopril on MAP and FHR, with no change in blood flow distribution, in the present study is consistent with the lack of effect of captopril infusion on plasma [AII] during normoxia. Moreover, we observed no difference in cardiovascular measurements between intact and CSD groups during normoxia, in agreement with our previous findings (Giussani et al. 1993). Thus, the results of the present study, in which fetuses were subjected to CSD alone or combined with ACE inhibition, suggest that neither chemoreflex mechanisms nor the RAS are pivotal in maintaining basal fetal cardiovascular control. This agrees with studies on adults which suggest that just one of the -adrenergic, AVP and RAS systems needs to remain intact in order for basal blood pressure to be maintained (Paller & Linas, 1984).
Angiotensin II and chemoreflexes during hypoxia
In the present study we observed the well-characterized rapid fall in FHR at the onset of hypoxia which is known to be primarily a carotid arterial chemoreflex response (Bartelds et al. 1993; Giussani et al. 1993); indeed the magnitude of the fall was reduced in the CSD group during both vehicle and captopril infusion. The rise in FHR towards pre-hypoxic levels as hypoxia continued is attributed to a rise in circulating levels of catecholamines (Jones & Robinson, 1975). Carotid sinus denervation also removes baroreceptor afferents, but it seems unlikely that they contribute to the rapid initial fall in FHR since the rise in MAP seen during hypoxia was too slow. Indeed, previous studies on the fetus have shown that if the rise in MAP and central venous pressure during hypoxia is blocked by infusion of nitroprusside the initial fall in FHR during hypoxia is not altered (Raff & Wood, 1992). The initial rapid rise in FVR during hypoxia was absent in the CSD group infused with vehicle infusion, in agreement with Giussani et al. (1993), but not with captopril infusion. As hypoxia continued FVR rose and FBF fell to similar levels in intact and CSD groups.
The rise in plasma [AII] is not altered by CSD and therefore AII does not appear to be a likely mediator of rapid carotid reflex responses to hypoxia. However, the rise in plasma [AII] during hypoxia may contribute to the slower cardiovascular responses that occur during hypoxia. Studies on adult dogs implicate AII in the maintained rise in MAP seen during hypoxia with acidosis (Rose, Vance, Dacus, Brashers, Peach & Carey, 1991), and blockade of AII receptors using saralasin blunts the fetal hypertensive response to hypoxia (Mattioli et al. 1979). However, we have shown that MAP and peripheral blood flow changes during hypoxia were similar with vehicle and captopril infusion. Furthermore, in the present study, L-NAME infusion in intact fetuses blocked the rise in plasma [AII] during hypoxia. But, as we have reported previously, the rise in MAP and femoral vasoconstriction during hypoxia in these fetuses is unaltered by the infusion of L-NAME (Green et al. 1996). Thus, these results when combined do not support a role for AII in the cardiovascular responses to hypoxia in intact fetuses.
Rather, our data show that only after the afferent carotid reflex limb is removed does a role for AII during hypoxia becomes apparent, since in the CSD group, but not in the intact group, captopril infusion blunted the hypertensive response, and attenuated the fall in FBF and rise in FVR during hypoxia. We cannot address the relative contribution of carotid chemo- and baroreceptors to these results. However, we have shown that MAP rose to a similar extent in vehicle-infused intact and CSD fetuses during hypoxia. Similarly, late gestation ovine fetal cardiovascular control during haemorrhage is not critically dependent on one mechanism, but autonomic and hormonal mechanisms, namely the autonomic nervous system and the RAS, may act in concert (Scroop, Stankewytsch-Janusch & Marker, 1992).
In summary, we confirm that plasma [AII] rises during hypoxia, but show that this is not mediated by carotid reflex mechanisms. We have also shown that angiotensin converting enzyme inhibition does not alter plasma [AII], nor does AII appear to contribute to cardiovascular control, during normoxia. On the other hand, our results suggest that carotid reflex mechanisms exert a major influence on cardiovascular control during hypoxia and that it is once these are removed that a role for AII in regulating MAP and peripheral blood flow becomes apparent.
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Acknowledgements
This work was supported by The Wellcome Trust. L. R. G was a Medical Research Council Scholar. We thank Clare Crowe for her technical assistance. We acknowledge the statistical advice of Professor S. Senn, Department of Statistical Science and Department of Epidemiology and Public Health, University College London, in the interpretation of these data.
Corresponding author
M. A. Hanson: Department of Obstetrics and Gynaecology, University College London, 86-96 Chenies Mews, London WC1E 6HX, UK.
Email: m.hanson@ucl.ac.uk
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Introduction
