Do Angiotensin Type 2 Receptors Modulate Haemodynamic Effects of Type 1 Receptors in Conscious Newborn Lambs?

Abstract

Introduction and hypothesis:

It was hypothesized that in the immediate newborn period, when the renin–angiotensin system (RAS) is activated, angiotensin type 2 receptors (AT2Rs) buffer the haemodynamic effects of angiotensin type 1 receptors (AT1Rs), as occurs in adult animals when the RAS is activated.

Materials and methods:

Arterial (systolic, diastolic, and mean) pressures (SAP, DAP, MAP), mean venous pressures (MVP) and renal blood flows (RBF) were measured in conscious, chronically instrumented lambs aged ~1 (8±2 days, N=8) and 6 weeks (41±4 days, N=11). In each animal, measurements were made before and after administration of the selective AT1R antagonist ZD 7155 (experiment one) and the selective AT2R antagonist PD123319 (experiment two) as well as both antagonists, ZD 7155 and PD 123319 (experiment three).

Results:

Haemodynamic responses to combined inhibition of both AT1Rs and AT2Rs were similar to inhibition of AT1Rs alone: there was a significant decrease in SAP, DAP, and MAP and a significant increase in RBF within minutes of concomitant administration of ZD 7155 and PD 123319 in both age groups. These responses were similar to responses to ZD 7155 alone, whereas PD 123319 alone did not alter any of the measured variables.

Conclusions:

AT2Rs do not counterbalance the pressor and renal vasoconstrictor effects elicited by activation of AT1Rs in the immediate newborn period. During this time, AT1Rs appear to predominate in eliciting the haemodynamic effects of angiotensin II (ANG II), whereas the role of the upregulated AT2Rs remains elusive.

Keywords

Neonate cardiovascular AT1R AT2R ANG II physiology haemodynamics

Introduction

The renin–angiotensin system (RAS) bioenzymic cascade is a key regulator of cardiovascular homeostasis, through its main effector peptide, angiotensin II (ANG II). By activating type 1 receptors (AT1Rs) on vascular smooth muscle cells, ANG II elicits vasoconstriction, leading to an increase in arterial pressure,^1–4 and also, through activation of AT1Rs at sympathetic nerve terminals, exerts facilitatory effects on neural transmission.^5,6 In contrast, type 2 receptors (AT2Rs) appear to modulate renal afferent arteriolar resistance as well as intra-renal production of bradykinin and nitric oxide⁷ and oppose the aforementioned vasoconstrictor effects of ANG II on AT1Rs when the RAS is activated in adult animals.^8,9

Interestingly, during the perinatal period, the RAS is also activated. For example, there are increased plasma levels of renin, angiotensin converting enzyme (ACE), and ANG II at birth and a decline as postnatal maturation proceeds.^10–17 The expression of AT1Rs and AT2Rs is also developmentally regulated, with AT2Rs predominating in the brain, adrenal gland and kidney of fetal and newborn rats, fowl, rabbits, sheep and humans.^18–31 In near-term fetal sheep and newborn lambs, AT2Rs are primarily expressed in systemic artery vascular smooth muscle cells.³² After 2 weeks of postnatal life, there is a transition from AT2Rs to AT1Rs that is complete by 3 months of age in sheep, when AT1Rs are the predominant receptor expressed in the systemic vasculature.^25,32 The physiological significance of this preponderance of AT2Rs in major blood vessels during the perinatal period is not known, as reviewed recently by us.³³

Previously, we explored the effects of endogenously produced ANG II on systemic and renal hemodynamics in conscious, developing lambs, as well as the individual receptor subtype through which ANG II exerts these effects.^34–36 Our results showed that acute bolus administration of ANG II to conscious lambs was associated with an age- and dose-dependent increase in mean arterial pressure and decrease in renal blood flow. These responses to ANG II were mediated exclusively by activation of AT1Rs in conscious newborn lambs under physiological conditions, whereas AT2Rs did not alter these effects. From these observations, we postulated that, in the newborn period when the RAS is normally activated, AT2Rs may buffer the haemodynamic effects of AT1Rs, as they do later in life.^37,38

The present experiments were, therefore, carried out to investigate the potential role of AT2Rs in modulating the responses to AT1R activation in conscious lambs, by exploring the haemodynamic effects of combined inhibition of both ATRs. To this end, haemodynamic effects of an AT1R antagonist alone, an AT2R antagonist alone, and antagonists to both AT1R and AT2R combined were measured in conscious lambs aged ~1 week – when circulating levels of ANG II are increased and AT2Rs predominate in the conduit arteries – and ~6 weeks – when circulating ANG II levels as well as AT2R expression are decreased.

Methods

Experiments were carried out in two separate age groups of conscious chronically instrumented lambs aged ~1 and ~6 weeks at the time of experiments (Table 1). Animals were obtained from a local source (Woolfit Acres, Olds, Alberta, Canada) and housed with their mothers in individual pens in the vivarium of the Health Sciences Centre of the University of Calgary, where they were provided with standard food and water, except during surgery, training, and experiments. All surgical and experimental procedures described herein were carried out in accordance with the Guide to the Care and Use of Experimental Animals provided by the Canadian Council on Animal Care and with the approval of the Animal Care Committee of the University of Calgary.

Table 1.

Demographics and baseline variables in conscious lambs.

Group	1 week	6 weeks
N	8	11
Sex	3♀/5♂	7♀/4♂
Age (days)	8 ± 2	41 ± 4^†
Weight (kg)	7.6 ± 1.7	15.4 ±2.9^†
Kidney weight (g)	59.6 ± 11.5	80.2 ± 9.3^†
MAP (mmHg)	72 ± 6	77 ± 5^†
SAP (mmHg)	100 ± 5	106 ± 6^†
DAP (mmHg)	50 ± 6	53 ± 4
MVP (mmHg)	2 ± 2	6 ± 2^†
HR (beats·min⁻¹)	200 ± 25	130 ± 26^†
RBF (mL·min⁻¹·g⁻¹)	1.8 ± 0.4	3.4 ±1.4^†
RVR (mmHg.ml⁻¹.g⁻¹.min⁻¹)	38.8 ± 7.2	18.3 ± 7.1^†

Values are mean ± SD. DAP: diastolic arterial pressure; HR: heart rate; MAP: mean arterial pressure; MVP: mean venous pressure; RBF: renal blood flow; RVR: renal vascular resistance; SAP: systolic arterial pressure.

†

p<0.05 compared with 1 week.

Surgical procedures: Surgery was carried out using aseptic techniques as previously described.^39,40 Briefly, anaesthesia was induced with a mask and isoflurane (~5%) in oxygen, the trachea was intubated, and then anaesthesia was maintained by ventilating the lungs with isoflurane (1.5–2.5% in a mixture of nitrous oxide and oxygen (3:2)). Under sterile conditions, catheters (Tygon® Microbore Tubing 0.04″ ID–0.07″ OD, arterial catheter and 0.05″ ID–0.09″ OD, venous catheter) were inserted into femoral vessels bilaterally and advanced to the aorta and inferior vena cava, respectively, for later pressure measurements, as well as I.V. injections of drugs and solutions. Catheters were tunnelled subcutaneously to exit the lamb on the right and left flank, respectively. The right kidney was then approached retroperitoneally, and a pre-calibrated ultrasonic flow transducer (size 3S–6S, Transonic Systems Inc., Ithaca, New York, USA) was placed around the renal artery for later measurement of renal blood flow (RBF) as previously described.⁴¹ After closure of incisions, catheters and the flow transducer cable were contained in pouches on a lamb body jacket (Lomir Inc., Montréal, QC, Canada) for safe storage between experiments.

Lambs were allowed to recover from the effects of surgery and anaesthesia in a small animal critical care unit (Shor-line, Kansas City, KS, USA) for ~30 to 60 min, after which time they were returned to the vivarium, where they were housed with their mothers until the time of the experiment at least 4 days later. Antibiotics (Excenel® (Ceftriofur) 2.2 mg/kg (Pfizer, Kirkland, QC, Canada)) were administered intramuscularly at 24 h intervals beginning the day before surgery for a total period of 4 days. During the recovery after surgery, animals were trained daily for ~1h to allow them to accommodate to the supportive sling in which they were housed during experiments. This training period ensured that animals were resting quietly and accustomed to their surroundings during experiments in the laboratory environment.

Experimental details: On the day of the experiment, the animals were placed in the supportive sling in the laboratory environment for at least 60 min. During this time, an intravenous (I.V.) infusion of 5% dextrose in 0.9% sodium chloride at a rate of 4.17 ml·kg⁻¹·h⁻¹ through the right femoral venous catheter was initiated and continued for the duration of the experiment to assist in maintaining fluid and electrolyte balance. The left femoral catheters were connected to pressure transducers (Model P23XL, Statham, West Warwick, RI, USA) to continuously measure arterial and venous pressures. The flow transducer cable was connected to a flowmeter (T101, Transonics Systems) to continuously measure RBF in real-time using ultrasound technology. These haemodynamic variables were recorded onto a polygraph (Model 7, Grass Instruments Inc., West Warwick, RI, USA) and simultaneously digitized at 200 Hz using the data acquisition and analysis software package, PolyVIEW™ (AstroMed Inc., USA).

At the end of each experiment, lambs were returned to the vivarium, where they were housed with the ewe until the next experimental day. After all experiments, animals were administered a lethal dose of sodium pentobarbitone. Following postmortem inspection for verifying catheters and flow transducer placement, the zero offset of the flow transducer was recorded for RBF measurement correction. The kidneys were removed, examined grossly and weighed to normalize renal haemodynamic measurements.

Experimental protocol: Experiments were carried out in each animal in random order and at minimum intervals of 48 h in order to ensure adequate time for drug clearance between experiments.³⁴ Each experiment consisted of continuous haemodynamic measurements for 30 min before (Control) and 60 min after I.V. administration of antagonists selective to the AT1R, ZD 7155, and the AT2R, PD 123319. Two additional experiments consisting of continuous haemodynamic measurements for 30 min before (Control) and 60 min after I.V. administration of either ZD 7155 or PD 123319 alone were also performed in each animal. The three experiments were carried out in random order and at minimum intervals of 48 h. ZD 7155 was administered as an I.V. infusion of 70 µg·kg⁻¹·h⁻¹ following an I.V. bolus of 100 µg·kg⁻¹; PD 123319 was administered as a bolus of 100 µg·kg⁻¹, followed by an infusion of 70 µg·kg⁻¹·h⁻¹,³⁶ using a microprocessor-controlled syringe pump (Mi 60-1B, World Precision Instruments Inc., Sarasota, FL, USA) at a rate of 4.17 ml·kg⁻¹·h⁻¹. Dose-response experiments for ZD 7155 as well as PD 123319 were previously conducted over the range of doses 0 to 1600 µg/kg for ZD 7155 and 0 to 400 µg/kg for PD 123319. In these previous experiments, measured cardiovascular variables were examined for 120 min after inhibition of ATRs. Before and at 5, 30, 60, 90 and 120 min after administration of either ZD 7155 or PD 123319, ANG II was administered (EC₅₀ selected from previous ANG II dose: mean arterial pressure (MAP) response study), to test the magnitude and duration of any effects of ZD 7155 and PD 123319 on the pressor response to ANG II. For each ANG II test, measurements were made for 10 sec before and 30 sec after I.V. injection of ANG II infused over 10 sec using the same microprocessor controlled syringe pump. The doses used in these experiments were therefore carefully selected from these previous dose–response experiments, allowing us to be certain that ATRs were inhibited by the administered compounds.

Drugs: Both ZD 7155 (5,7-Diethyl-3,4-dihydro-1-[[2’-(1H-tetrazol-5-yl)[1,1’-biphenyl]-4-yl]methyl]-1,6-napthyridin-2(1H)-one hydrochloride) and PD 123319 (1-[[4-(Dimethylamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate) were manufactured by Tocris Cooksons Ltd (Bristol, UK). On the morning of each experiment, ZD 7155 and PD 123319 were weighed and immediately dissolved in a sterile solution of 0.9% sodium chloride.

Data analyses: Haemodynamic measurements of systolic (SAP), diastolic (DAP) and mean arterial pressure (MAP), mean venous pressure (MVP) and RBF were analysed over consecutive 1 min intervals using the analyses component of PolyVIEW™. Heart rate (HR) was calculated from the systolic peak of the arterial pressure waveform using PolyVIEW™. RBF measured in the two age groups was normalized per gram of kidney weight. Renal vascular resistance (RVR) was calculated as (MAP−MVP)/RBF. All haemodynamic data were averaged over consecutive 10 min intervals before and after treatment and presented as such.

There was no significant sex difference in systemic and renal haemodynamic responses to the ATR antagonists that were used to test our research hypothesis. These findings confirm previous studies conducted in our laboratory that show no gender related differences in cardiovascular or renal function in young lambs. Because there were no effects of sex on any of the measured variables, data from both male and female lambs were combined.

Sample size (N=8) was determined by applying a power calculation and based upon our previous experimental findings, with a projected difference of ~20% between means, using a power of 0.8 and α=0.05. In the older age group, the sample size was adjusted to N=11, due to the technical limitations that influenced the success rate for direct RBF measurements (i.e. transducer malfunctions).

The normal distribution of the data was evaluated with the Kolmogorov–Smirnov test. Non-paired Student’s t-tests were used to compare group differences for demographic and baseline variables. Effects on the measured and calculated variables were evaluated using statistical software (Sigmastat, Version 3.5, Systat Software Inc., San Jose, California) and applying three-way analysis of variance, factors being time, age and treatment. Where the F value was significant, Holm–Sidak multiple comparison procedures were applied to determine where the significant differences occurred, using a 90% confidence interval. Data presented in the text, tables and figures are expressed as mean±SD.

Results

Baseline HR and RVR were higher in 1-week-old as compared with 6-week-old lambs (Table 1) whereas MVP, MAP, and RBF were lower.

For MAP, there was an overall effect of age (F=133.8, p<0.001), and treatment (F=20.84, p<0.001), which was reflected in an altered baseline but no interaction between age and treatment (p=0.26). MAP decreased within 10 min of administration of ZD 7155 and remained decreased for the entire 60 min period of infusion. This response was the same with concomitant administration of both ZD 7155 and PD 123319, whereas MAP remained constant after PD 123319 alone in both age groups. The decrease in MAP after ZD 7155 and after ZD 7155 + PD 123319 resulted from a predominant decrease in SAP (Figure 1 left), as well as a decrease in DAP (Figure 1 right) in both age groups. When data were also tested as change from control to remove any effects of the altered baseline, there was also no interaction, whereas the treatment effect remained.

Figure 1.

Effects of ATR antagonists on systolic arterial pressure (SAP) and diastolic arterial pressure (DAP).

For RBF, there was an overall effect of age (F=241.4; p<0.001) and of treatment (F=2.3; p=0.067) but no interaction between age and treatment (p=0.20). As illustrated in Figure 2(left), RBF increased within 10 min of administration of ZD 7155 alone in both age groups, whereas there were no effects after PD 123319 alone. The RBF response to ZD 7155 was not altered by combined treatment with PD 123319. When data were also tested as change from control to remove any effects of the altered baseline, there was also no interaction, whereas the treatment effect remained. The RVR response to ZD 7155 mirrored that of RBF: there was an overall effect of age (F=229.1, p<0.001) and treatment (F=12.2, p<0.001) but no interaction between age and treatment (p=0.65). As illustrated in Figure 2(right), RVR decreased soon after administration of ZD 7155 alone, whereas there were no effects after PD 123319 alone in either age group.

Figure 2.

Effects of ATR antagonists on renal blood flow (RBF) and renal vascular resistance (RVR).

There were no significant effects of any ATR antagonists alone or combined on MVP in either age group. There were also no significant effects on HR of ZD 7155 alone, PD 123319 alone, or combined treatment with ZD 7155 + PD 123319, at 1 or 6 weeks of age in conscious lambs.

Discussion

The present study was designed to investigate the role of AT2Rs in buffering the haemodynamic effects of AT1Rs in the newborn period. To this end, parameters of systemic and renal haemodynamics were measured in conscious lambs aged ~1 and ~6 weeks, before and after combined inhibition of both AT1Rs and AT2Rs. Novel findings of this study are that in both age groups: (a) the decrease in arterial pressure which occurs following inhibition of AT1Rs was not altered by concomitant inhibition of AT2Rs; (b) the decrease in RVR and increase in RBF which occur after inhibition of AT1Rs were also not altered by concomitant inhibition of AT2Rs; (c) there were no effects of combined treatment with ZD 7155 and PD 123319 on HR or MVP. Together, these data show that, under physiological conditions, AT2Rs, which are highly expressed in systemic blood vessels early in life, do not appear to modulate the haemodynamic effects of AT1Rs postnatally. Therefore, we conclude that there is no interaction between AT1Rs and AT2Rs in modulating systemic and renal haemodynamics in the newborn period under physiological conditions.

Velaphi et al. (2002)⁴² showed in conscious young sheep that systemic ANG II infusion elicits a dose-dependent increase in arterial pressure, but, when administered locally into the hind limb, it does not elicit a vasoconstrictor response until 2 weeks after birth, which supports earlier findings of a preponderance of AT2Rs in major arteries in newborn lambs. Chappellaz and Smith (2005) also measured the pressor and renal vasoconstrictor responses to exogenous ANG II in conscious lambs.³⁴ Administration of ANG II was associated with a dose-dependent increase in MAP to the EC₁₀₀ of 100 ng·kg⁻¹ in lambs aged both 1 and 6 weeks, and a dose-dependent decrease in RBF to the EC₁₀₀ of 50 ng·kg⁻¹ in 1-week-old lambs and 25 ng·kg⁻¹ in lambs aged 6 weeks. Administration of ZD 7155, but not PD 123319 or vehicle, abolished the MAP and RBF responses to ANG II in both age groups of lambs.³⁵ These data provided information that pressor and renal vasoconstrictor effects of ANG II during the first 6 weeks of postnatal life in lambs are elicited by activation of AT1Rs but not AT2Rs. More recently, Wehlage and Smith (2012) confirmed the important role of AT1Rs in influencing cardiovascular homeostasis as well as the arterial baroreflex control of heart rate early in life.³⁶

Under certain conditions in adult animals and humans, ANG II counterbalances its own vasoconstrictor effects of AT1R activation through vasorelaxation mediated by activation of AT2Rs. These effects of AT2Rs are, however, revealed only when (a) the RAS is activated (i.e. by a low salt diet or haemorrhage), (b) arterial pressure is elevated, and (c) AT1Rs are pharmacologically inhibited.^9,37,38,43 Experiments carried out in adult rats in the renovascular hypertension model as well as under conditions of salt depletion have provided evidence that AT2Rs are involved in blood pressure regulation through crosstalk with AT1Rs, mediating a vasodilation that counterbalances the vasoconstrictor effects of AT1R through activation of the bradykinin–nitric oxide–cGMP pathway.^44,45 Under these experimental conditions, arterial pressure was considerably elevated above resting levels. Therefore, the responses we observed in conscious newborn lambs in the present study could result from the fact that resting arterial pressure was not elevated, although the aforementioned conditions (a) and (c) were met. It is, therefore, possible that the known crosstalk between AT1Rs and AT2Rs is only elicited in conditions of a prevailing increase in arterial pressure. This premise warrants further investigation.

Recent experiments in adult rodents and humans have also revealed that the haemodynamic roles for AT2Rs may be species specific: in cultured cells and isolated vessels, as well as in vivo experiments in mice and rats, AT2Rs have been implicated in counteracting the effects of AT1Rs.³⁸ In pregnant C57BL/6J mice, the contractile response of isolated umbilical arteries to ANG II is increased in the presence of the AT2R inhibitor, PD 123319,⁴⁶ thus supporting a vasodilatory role for AT2Rs. In contrast, recent studies in isolated umbilical arteries removed from pregnant and non-pregnant women showed that ANG II induced vascular contractions were not altered in the presence of AT2R inhibition.⁴⁷ This may implicate species differences in the role of AT2Rs in modulating haemodynamic effects of AT1Rs.

The protective AT2R-mediated renal vasodilation^48,49 appears to occur by the renal production of bradykinin, nitric oxide and cGMP as well as prostaglandins.⁵⁰ Our hypothesis regarding a potential regulatory role for AT2Rs in the newborn period was based upon such an assumption, that there would also be a developmentally regulated crosstalk between the AT1Rs and AT2Rs in mediating renal haemodynamic effects of ANG II. This is supported by a developmentally regulated intra-renal expression and distribution of ATRs in various species.^27,51 The temporal and spatial profile of ATR expression in the ovine kidney microvasculature during postnatal development has not yet been characterized. It is conceivable, however, that ATR expression, signalling pathways and/or modulatory effects of other vasoactive peptides may also be developmentally regulated.

The results of the present study are intriguing, since AT2Rs appear to predominate in the systemic vasculature of developing lambs.^25,32 AT2Rs are the primary subtype expressed in the systemic vasculature near term and during the first 2 weeks of postnatal life in sheep, after which there is a near complete transition from AT2Rs to AT1Rs by 3 months postnatally. Based upon these observations, we would expect there to be a preponderance of AT2R activity in the systemic vasculature in the 1-week-old lambs in the present study, whereas in older lambs we would expect the transition to AT1Rs to be in place. The physiological impact of increased ANG II levels during the perinatal period, as well as the preponderance of AT2Rs in the vasculature early in life, is not yet known.

AT2Rs may function as a backup mechanism in regulating high blood pressure in the newborn period, whereas AT1Rs predominate in regulating both cardiovascular homeostasis and the arterial baroreflex.^34–36 AT2Rs may also be predominantly involved in growth processes at cellular and subcellular level at this age, while a haemodynamic component of their function may develop later in life. While this assumption is interesting, more studies are required to elucidate the perhaps more complex physiological roles for AT2Rs in the newborn period. The current findings may impact therapeutic choices in human infants with high prevailing blood pressure, as well as contribute a new perspective to explaining the developmental origins of chronic adult disease.

Footnotes

Acknowledgements

The authors gratefully acknowledge the expertise of Dr Wei Qi for surgically instrumenting the animals, and the technical assistance provided by Miss Lucy Yu.

Conflict of interest

The author declares that there is no conflict of interest.

Funding

This work was supported by an Operating Grant provided by the Canadian Institutes for Health Research. At the time of these studies, Dr Angela E. Vinturache was a doctoral candidate supported by graduate scholarships provided by the CIHR Training Program in Genetics, Child Development, and Health as well as the University of Calgary.

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