DOSE-DEPENDENT SYSTEMIC AND RENAL HAEMODYNAMIC EFFECTS OF ANGIOTENSIN II (ANG II) IN CONSCIOUS LAMBS: ROLE OF AT1 AND AT2 RECEPTORS by Mona Lynne Chappellaz Francine Gabriel Smith from Departments of Physiology & Biophysics/Medicine University of Calgary Calgary, Alberta, T2N 4N1 CANADA Running Title: ANG II and the developing newborn Address for correspondence: Francine G. Smith, Ph.D. Professor Department of Physiology & Biophysics Faculty of Medicine University of Calgary 3330 Hospital Drive, NW Calgary, Alberta, T2N 4N1 CANADA Telephone: (403)220-4090 Email: fsmith@ucalgary.ca Physiology in Press; published online on August 9, 2005 as 10.1113/expphysiol.2005.031195 Copyright 2005 by The Physiological Society ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 1 ABSTRACT The present experiments were designed to measure the effects of acute administration of ANG II on mean arterial pressure (MAP) and renal blood flow (RBF) in conscious, chronically instrumented lambs at two different stages of postnatal maturation, and to determine the receptors through which these effects of ANG II are elicited. Experiments consisted of haemodynamic measurements for 10 seconds before (Control) and for sixty sec after intravenous (I.V.) administration of one of 11 doses of ANG II (0 to 200 ng·kg-1). 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 one and six weeks, and a dose-dependent decrease in RBF to the EC100 of 50 ng·kg -1 in one week old lambs and 25 ng·kg-1 in lambs aged six weeks. Responses to EC50 ANG II were also measured in the presence of the specific ANG II AT1 receptor antagonist, ZD 7155, the specific AT2 receptor antagonist, PD 123319, and vehicle. 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. In addition, MAP decreased and RBF increased in both age groups of lambs after administration of ZD 7155, but not PD 123319, effects being similar in both age groups. These data provide new information that pressor and renal vasoconstrictor effects of ANG II during the first six weeks of postnatal life in lambs are elicited by activation of AT1 but not AT2 receptors. Key words: newborn, renal vascular tone, perinatal, ZD 7155, PD 123319 ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 2 INTRODUCTION The renin-angiotensin system plays an important role in regulating cardiovascular as well as fluid and electrolyte homeostasis, through its physiologically active peptide, angiotensin II (ANG II). This peptide’s effects are mediated by receptor coupling to intracellular messenger systems (Dudley et al., 1990; Speth & Kim, 1990) and activation of specific receptors which are pharmacologically distinct, the two most predominant receptor subtypes being AT1 and AT2. In adult animals, although one subtype predominates in specific tissues (e.g. AT1 receptors – VSM cells; AT2 receptors – uterus) several tissues including the kidney contain both AT1 and AT2 receptors ( Whitebread et al., 1989; Dudley et al., 1990; Speth & Kim, 1990). These two receptor subtypes differ in their affinities for ANG II, mechanisms of transmembrane signalling, tissue-specific expression, and physiological roles ( Dudley et al., 1990; Speth & Kim, 1990; Smith et al., 1992; Bottari et al., 1993; Ferguson & Washburn, 1998; Siragy, 2000; de Gasparo et al., 2000). Although it is well known that activation of AT1 receptors results in an increase in blood pressure, AT2 receptors also modulate pressor responses to ANG II, as first described by Scheuer & Perrone (1993). In fact, AT2 receptors appear to mediate a bradykinin - nitric oxide - cyclic GMP vasodilator cascade (Siragy & Carey, 2001). This suggests that AT2 receptors play an important role in blood pressure regulation by abrogating the vasoconstrictor responses to ANG II activation of AT1 receptors (Carey, Wang & Siragy, 2000; Siragy, 2000; Siragy & Carey, 2001; Li & Widdop, 2004). During the perinatal period, the renin-angiotensin system is activated, with increased plasma levels of renin and ANG II before birth and a further increase following the transition to newborn life; as postnatal maturation proceeds, renin and ANG II levels ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 3 decline (Trimper & Lumbers, 1972; Broughton Pipkin et al., 1974; Pelayo et al. 1981; Rosenfeld et al., 2003; Monument & Smith, 2003). In addition, the expression of AT1 and AT2 receptors is developmentally regulated, with AT2 receptors predominating in the brain, adrenal gland and kidney of fetal and newborn rats, fowl, rabbits, sheep and humans ( Millan et al., 1991; Gröne et al., 1992; Bensoussan et al., 1993; Ciuffo et al., 1993; Robillard et al., 1994; Robillard et al., 1995; Arens et al., 1998; Wintour et al., 1998; Kaiser et al., 1998; Wintour et al., 1999; Burrell et al., 2001; Bagby et al., 2002a; Bagby et al., 2002b; Kintscher & Unger, 2003). Except for umbilical VSM cells, the AT2 receptor is the primary receptor subtype expressed in systemic artery VSM cells of near- term fetal sheep and newborn lambs (Cox & Rosenfeld, 1999). After two weeks of postnatal life, there is a transition from AT2 to AT1 receptors that is complete by three months of age in sheep (Cox & Rosenfeld, 1999; Kaiser et al., 1998). This is similar for major blood vessels of the developing swine (Bagby et al., 2002b). To date, however, the physiological impact of (a) elevated ANG II levels, and (b) increased AT2 receptor expression early in life is poorly understood. The present experiments were designed to test the hypothesis that AT2 receptors buffer the vasoconstrictor response to AT1 receptor activation by ANG II, in an age- dependent manner, therefore making the vasculature of newborns less sensitive to ANG II. To test this hypothesis dose-dependent effects of acute administration of ANG II on systemic and renal haemodynamics were measured in lambs aged ~one week when circulating levels of renin (Thomsen & Smith, 2004; Monument & Smith, 2003) and ANG II ( Wilson et al., 1981b; Rosenfeld et al., 2003) as well as the expression of AT2 receptors in VSM cells ( Kaiser et al., 1998; Cox & Rosenfeld, 1999) are high, and ~six ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 4 weeks when renin ( Monument & Smith, 2003; Thomsen & Smith, 2004) and ANG II levels (Wilson et al., 1981b; Rosenfeld et al., 2003;) as well as AT2 receptors in VSM cells (Cox & Rosenfeld, 1999; Kaiser et al., 1998) are decreased. In additional experiments, to determine whether responses to ANG II are mediated by activation of AT1 or AT2 receptors, EC50 ANG II was also tested in the presence of the specific ANG II AT1 receptor antagonist, ZD 7155, the specific AT2 receptor antagonist, PD 123319, and vehicle. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 5 METHODS Experiments were carried out at least four days after surgery in conscious, chronically instrumented lambs aged ~one (n=14) and ~six weeks (n=28) of postnatal life. Details of animals studied are provided in Table 1. Lambs were obtained from a local source (Woolfitt Acres, Olds, Alberta) and housed with their mothers in individual pens in the vivarium of the Health Sciences Center, except during training, surgery and experiments. All surgical and experimental procedures described herein were carried out according to the “Guidelines for the Care and Use of Laboratory Animals” provided by the Canadian Council on Animal Care, and were approved by the Animal Care Committee of the University of Calgary. Surgical procedures: Surgery was performed on lambs using sterile techniques, as previously described (Smith & Abraham, 1995). Briefly, anaesthesia was induced with a mask and halothane (3-4%) in oxygen, the trachea was intubated and anaesthesia maintained with halothane (1-1.5%) in a mixture of nitrous oxide and oxygen (3:1). The femoral veins were cannulated (PE 160, Intramedic) and the catheters advanced to the inferior vena cava for intravenous (I.V.) infusions during experiments and for measurements of mean venous pressure. The left femoral artery was also cannulated (PE 160, Intramedic) and the catheter advanced to the abdominal aorta for measurement of arterial pressure during experiments. Catheters were tunnelled subcutaneously to exit the lamb on the right and left flanks. Through a left flank incision to expose the left kidney, a precalibrated ultrasonic flow transducer (3 - 6 mm, Transonics Systems Inc.) was placed around the left renal artery for measurement of renal blood flow (RBF) during experiments, as previously described (de Wildt & Smith, 1997). All incisions were ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 6 sutured, and the catheters and flow transducer cable were secured in pouches on a body jacket (Lomir Inc.), for safe storage. Lambs were allowed to recover from the effects of surgery and anaesthesia in a critical care unit for small animals (Shor-line, Schroer Manufacturing Co.) with adjustable oxygen supply and, when standing, were returned to their mothers in the vivarium. Antibiotics (Synergistin, sulbactam benzathine 3.3 mg·kg-1/ampicillin trihydrate 6.6 mg·kg-1) were administered intramuscularly on the day before surgery and during the recovery period at 24 h intervals thereafter, for an additional 48 h. During the recovery period, lambs were trained for at least 60-min daily to rest quietly in a supportive sling in the laboratory environment. Experimental details: On the day of an experiment the lamb was removed from the vivarium and placed in the same supportive sling in the laboratory environment for 60- min. During this equilibration period, an I.V. infusion of 5% dextrose and 0.9% sodium chloride was started and continued throughout the experiment at 4.17 mL·h-1·kg-1, to assist in maintaining fluid balance during experiments. Catheters were connected to pressure transducers (Statham, P23XL) for pressure measurements; the flow transducer was connected to a flowmeter (T101, Transonics Systems Inc.) for measurement of RBF. Pressures and RBF were recorded onto a polygraph (Grass Instruments, Model 7) and simultaneously digitized at 200 Hz using the data acquisition and analysis software package, PolyVIEW® (AstroMed Inc.). After experiments were completed, lambs were administered a lethal dose of sodium pentobarbitone. Placement of catheters and the flow transducer was confirmed by post mortem inspection, and the zero offset of the flow transducer was determined; both kidneys were removed and immediately weighed. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 7 Detailed protocols: There were two protocols carried out as detailed below: Protocol I Experiments consisted of haemodynamic measurements for ten seconds before (Control) and for 60 sec after administration of one of 11 doses of ANG II (0, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100, and 200 ng·kg-1). Each dose was administered twice, in random order, with five to seven min between each dose, based upon the known T1/2 of ANG II of ~20 sec measured in sheep (Rosenfeld et al., 1995), and to allow the haemodynamic variables to return to control levels between each dose. For each experiment, all doses were administered in the same volume (0.03 – 0.04 mL·kg-1) and infused I.V. over ten seconds using a microprocessor controlled syringe pump (Model 100, Stoelting, KD Scientific). This protocol was used to select the EC50 ANG II dose: MAP response for protocol II. Protocol II Responses to the EC50 dose of ANG II selected from protocol I were measured. This was followed by I.V. administration of 400 µg·kg-1 of the specific ANG II AT1 receptor antagonist, ZD 7155, 400 µg·kg-1 of the specific ANG II AT2 receptor antagonist, PD 123319, or vehicle. (Doses of ZD 7155 and PD 123319 were selected from preliminary dose response studies). ZD 7155 or PD 123319 were administered only once and separate animals were used for each experiment. For each dose of ZD 7155 or PD 123319, as well as for vehicle, the same volume was administered (0.48 to 1.33 mL·kg-1) over two minutes using a microprocessor controlled syringe pump (Model 100, Stoelting, KD Scientific). After 30 min, the MAP and RBF responses to EC50 ANG II were again measured. MAP and RBF were continuously digitized at 200 Hz during the experimental period. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 8 Drugs: ANG II (Sigma-Aldrich Canada Ltd.) was dissolved in a sterile solution of 0.9% sodium chloride to make a stock solution of 1 mg·mL-1. Doses of ANG II were constituted by serial dilution, and stored at -20°C in 1 mL aliquots. 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 trifluoroacete) 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, as required. The same volume of 0.9% sodium chloride was used as vehicle. Selection of drugs: A potent, highly selective, and reversible antagonist for the AT1 receptor, ZD 7155 does not generate an active metabolite such as is the case for losartan (EXP 3174 has non-competitive antagonistic effects on the AT1 receptor) (Timmermans et al., 1993). ZD 7155 is ~ten-fold more potent than losartan in antagonizing ANG II- induced pressor effects in conscious rats (Junggren et al., 1996), and it readily displaces [125I]-ANG II from its binding sites on guinea pig adrenal gland membranes in a concentration-dependent manner (Wong et al., 1992).] PD 123319 is an highly selective, reversible antagonist for the AT2 receptor and therefore a suitable drug choice for haemodynamic measurements in conscious animals (Siragy & Carey, 2001). Data analyses: Systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) were determined for each experiment using the analyses component of PolyVIEW®. RBF measured in the two age groups was normalized per gram (g) of kidney weight. Renal vascular resistance (RVR) was calculated as: (MAP – ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 9 MVP)/RBF, where MAP and MVP refer to mean arterial and venous pressures respectively. Statistical tests were carried out using Sigmastat Version 3.0 (Jandel Scientific) and a confidence interval of 95%; normality of distribution was confirmed prior to testing. All data in the text, tables and figures are expressed as mean±one standard deviation. Protocol I Data were averaged over the ten second control period to one control measurement. Using the analyses component of PolyVIEW®, the maximum response to ANG II, as well as the time (in seconds) to maximum after ANG II, was determined for each trial at each dose; the two trials at each dose were averaged and data pooled. Dose- response relationships were constructed for ANG II dose: MAP response and ANG II dose: RBF response in both age groups. The EC100 was selected for both variables for both age groups as well as the time to peak response; the EC50 was calculated as the dose that elicited a half-maximal change in MAP or RBF. Statistical differences were evaluated using two-way ANOVA, factors being age, and dose. Where the F value was significant, Holm-Sidak multiple comparison procedures (Ludbrook, 1991) were applied to determine where the significant differences occurred. Protocol II MAP and RBF responses to ANG II measured before and after ZD 7155, PD 123319, or vehicle, were evaluated using ANOVA with factors, age (one week, six weeks), treatment (vehicle, ZD 7155, PD 123319), and time (Control, 30 min). Where the F value was significant, Holm-Sidak multiple comparison procedures were applied to determine where significant differences occurred (Ludbrook, 1991). Comparisons between baseline variables were made using non-paired t-tests. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 10 RESULTS Protocol I Table 1 shows demographics of conscious lambs studied in protocol I. MAP was lower at one week (71±7 mmHg) compared to six weeks (77±8 mmHg) (p<0.001); RBF was also lower at one week (2.8±0.8 mL·min-1·g-1) compared to six weeks (3.25±1.1 mL·min- 1 ·g-1 (p=0.024). There was an effect of dose (F=122.8, p<0.001) and age (F=47.4, p<0.001) on the MAP response to ANG II as well as an interaction between dose and age (F=4.2, p<0.001). 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 one and six weeks (Figure 1). The increase in MAP was significantly greater than 0 for doses of ≥12.5 ng·kg-1 at one week and ≥3.125 ng·kg-1 at six weeks. Details of MAP responses at EC100 are provided in Table 2. Changes in MAP resulted from age- and dose-dependent increases in both SAP and DAP, the profiles of which mirrored the MAP responses to ANG II in both age groups. There was also an effect of dose (F=38.5, p<0.001) and age (F=30.5, p<0.001) as well as an interaction between dose and age (F=3.1, p<0.001) on the RBF response to ANG II (Figure 2). Administration of ANG II was associated with a dose-dependent decrease in RBF to the EC100 of 50 ng·kg -1 in one week old lambs and 25 ng·kg-1 in lambs aged six weeks (Figure 2). The decrease in RBF was significantly greater than 0 for doses of ≥12.5 ng·kg-1 at one week and ≥3.125 ng·kg-1 at six weeks. Details of RBF responses at EC100 are provided in Table 2. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 11 The time to peak of the MAP response to ANG II was increased when compared to the time to peak of the RBF response (F=32.9, p<0.001) (Table 2), and was greater at six than one week of age (F=81.4, p<0.001). Protocol II Administration of ZD 7155 but not PD 123319 or vehicle abolished by 100% the MAP and RBF responses to ANG II in both age groups of lambs measured at 30 min compared to Control (Figures 3 - 4). Table 3 provides variables measured during Control and 30 min after administration of vehicle, ZD 7155, and PD 123319. In both age groups of lambs, MAP decreased after administration of ZD 7155, but not PD 123319, effects being similar in both age groups. There was also an increase in RBF in both age groups of lambs after administration of ZD 7155 and no significant effects of PD 123319 on RBF. There were also no significant effects of ZD 7155 and PD 123319 on MVP, and no significant effects of vehicle on any of the measured variables (Table 3). ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 12 DISCUSSION It was the purpose of the present experiments to investigate the effects of acute administration of ANG II on MAP and RBF in conscious, chronically instrumented lambs at two different stages of postnatal maturation, and to determine the receptors through which these effects of ANG II are elicited. Novel findings of our study are as follows: (a) acute bolus administration of ANG II to conscious lambs was associated with both an age- and dose-dependent increase in MAP and an age- and dose-dependent decrease in RBF; (b) these responses to ANG II were mediated by activation of AT1 receptors; (c) administration of ZD 7155 was associated with a decrease in MAP and an increase in RBF at both one- and six weeks of age, suggesting that AT1 receptors modulate haemodynamics early in life. Although our findings do indicate that the vasculature of the newborns is less sensitive to ANG II, these effects do not appear to result from activation of AT2 receptors. In fact, the ANG II pressor and RBF responses were not enhanced by PD 123319. Therefore, we reject our hypothesis that AT2 receptors appear to buffer the vasoconstrictor response to AT1 receptor activation by ANG II, in an age- dependent manner, thereby making the vasculature of newborns less sensitive to ANG II. During fetal life, the renin-angiotensin system is activated, levels of renin and ANG II being high near-term (Trimper & Lumbers, 1972; Broughton Pipkin et al., 1974; Pelayo et al., 1981; Rosenfeld et al., 2003). The transition from fetal to newborn life is associated with a further activation of the renin-angiotensin system (Lumbers & Reid, 1977; Davidson, 1987); after birth, renin and ANG II levels decline ( Broughton Pipkin et al., 1974; Pelayo et al., 1981; Rosenfeld et al., 2003; Monument & Smith, 2003) Binder & Anderson (1992) demonstrated a negative correlation between plasma renin activity ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 13 (PRA) and arterial pressure measured in fetal sheep near term, in newborn lambs studied in the 24 h after delivery by caesarean section and during the first week of postnatal life. In developing sheep, PRA decreases from 15.7±8.4 ng AI·mL-1·h-1 at ~one week to 2.7±1.6 ng AI·mL-1·h-1 at ~six weeks and to adult levels of 1.6±1.5 ng AI·mL-1·h-1 at ~12 weeks (Monument & Smith, 2003) during which time, arterial pressure increases slightly yet renal vascular resistance decreases dramatically. Except for umbilical VSM cells, the AT2 receptor is the primary receptor subtype expressed in systemic artery VSM cells of near-term fetal sheep and newborn lambs (Cox & Rosenfeld, 1999). After two weeks of postnatal life, there is a transition from AT2 to AT1 receptors that is complete by three months of age in sheep ( Kaiser et al., 1998; Cox & Rosenfeld, 1999). This is similar for major blood vessels of the developing swine (Bagby et al., 2002b). AT2 receptor expression is also present at the onset of kidney development and decreases during gestation in developing sheep (Gimonet et al., 1998; Butkus et al., 1997) as in other species (Shanmugam et al., 1994; Kakuchi et al., 1995). In sheep, however, where nephrogenesis is complete before birth, AT2 receptor mRNA expression is almost undetectable after 120 days of gestation, whereas in mouse and rat, AT2 mRNA persists until nephrogenesis is complete in the weeks after birth. AT1 mRNA expression is also present during fetal life in sheep (Robillard et al., 1994; Butkus et al., 1997; Gimonet et al., 1998) as in other species (Tufro-McReddie et al., 1993; Shanmugam et al., 1994; Kakuchi et al., 1995) yet persists after nephrogenesis is complete. Furthermore, it is localized in differentiated structures (Gimonet et al., 1998) suggesting more of a functional role in contributing to mesangial and/or smooth muscle cell formation. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 14 In previous studies, blood pressure responses to exogenous administration of ANG II have been measured in fetal and newborn animals (Iwamoto & Rudolph, 1981; Siegel, 1981; Wilson et al., 1981a; Wilson et al., 1981b; Velaphi et al., 2002; Shi, Yao, Stewart et al., 2004;): For example, in α-chloralose anaesthetised lambs, infusion of ANG II at a rate of 0.1 µg·kg-1·min-1 over several minutes increased blood pressure by ~40% (Goetzman & Bennett, 1991). Davidson (1987) infused 0.1 to 2.3 µg·min-1 of ANG II for two minutes to conscious lambs in the hours after birth and observed an increase in blood pressure which correlated with plasma ANG II. Siegel (1981) measured blood pressure responses to two doses of ANG II - 0.025 and 0.25 µg·kg-1·min-1 - infused over 60 min to lambs as well as ewes studied 48 h after surgery. Ten-fold the concentration of ANG II was required in lambs to raise blood pressure to the same extent as ewes providing evidence that the pressor response to ANG II is reduced early in life. This is similar to our present observations that MAP responses to ANG II were greater at six- than one-week of postnatal age in conscious lambs. Velaphi et al., (2002) also measured blood pressure responses to four doses of ANG II ranging from 0.04 to 0.4 µg·kg-1·min-1, each infused I.V. over seven minutes at three postnatal ages in conscious lambs. A dose-dependent increase in arterial pressure that was similar across the three age groups was reported, confirming results from a previous study by Wilson et al., (1981b) in which blood pressure increased following 30 min ANG II infusions, also in a dose-dependent manner, effects being similar in lambs aged one to seven weeks. Responses to such longer-term ANG II infusions may, however, be complicated by factors other than the acute, direct effects of ANG II. For example, an increased circulating level of ANG II is associated with stimulation of other vasoactive factors as ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 15 prostaglandins, nitric oxide, or kinins, inhibition of renin release, activation of the sympathetic nervous system, direct cardiac effects, and alterations in CNS control of the baroreceptor reflex as well as secondary effects of ANG II centrally on other systems such as arginine vasopressin (Breuhaus & Chimoskey, 1990; Lüscher, 1990; Butler et al., 1994; Brooks, 1997; Dendorfer et al., 1998; de Gasparo, 2002). Effects of endogenously produced ANG II have also been evaluated in developing animals. For example, in anaesthetised newborn pigs, angiotensin converting enzyme (ACE) inhibition following administration of enalaprilat, is associated with a reduction in arterial pressure as well as RBF (Nilsson & Friberg, 2000). In previous studies in conscious, chronically instrumented three week old lambs, (de Wildt & Smith, 1997; Smith et al., 1997) we showed that ACE inhibition by administration of captopril was associated with a decrease in DAP and MAP, and an increase in heart rate, as well as a decrease in RBF. More recently, we administered captopril to conscious lambs aged one- and six-weeks of postnatal life to assess the cardiovascular effects of endogenously produced ANG II during postnatal development (Monument & Smith, 2003). A decrease in MAP and an increase in heart rate occurred after ACE inhibition; these effects were greater in lambs aged one week compared to lambs aged six weeks. The fact the pressor and renal vascular responses to ANG II were not significantly altered by administration of PD 123319 in the present study is not surprising since AT2-dependent vasodilatation after exposure to exogenous ANG II involves bradykinin B2 receptor activation (Siragy, 2000). Widdop et al., (1992) measured blood pressure as well as flow to renal, mesenteric and hindquarters using Doppler shift in conscious Long Evans rats and assessed the effects of ANG II before and after administration of the AT1 receptor ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 16 antagonist, EXP 3174, and the AT2 receptor antagonist, PD 123319. EXP 3174 but not PD 123319 inhibited the pressor and haemodynamic responses to ANG II. This is in keeping with our present observations in conscious developing lambs. Scheuer & Perrone (1993) first described a role for AT2 receptors in modulating the blood pressure responses to ANG II. In that study, ANG II injected into anesthetized rats at doses of 100 to 1000 ng·kg-1 elicited a dose-dependent depressor response ~1 - 4 min after the initial pressor response. Blockade of the pressor responses to ANG II by the selective AT1 receptor antagonist, losartan, unmasked a more consistent depressor response which was abolished by combined AT1 and AT2 receptor blockade. In the present study in conscious lambs, the depressor response observed by Scheuer and Perrone (1993) in the minutes following high doses of ANG II was not seen even at the highest doses tested. This may have been due either to differences in species (sheep vs rat), experimental conditions (anesthetized vs conscious), and/or dose (0-200 vs 100-1000 ng·kg-1). Inhibition of AT1 receptors would also increase circulating renin and ANG II levels which might in turn activate AT2 receptors. Combined AT1 and AT2 receptor antagonism may reveal such an additive effect. In conclusion, the present experiments provide new information that acute ANG II administration to conscious lambs results in a dose-dependent increase in MAP and decrease in RBF. Effects of ANG II on both variables appear greater at six than one week of age, postnatally, in conscious lambs. Furthermore, both pressor and renal vasoconstrictor effects of ANG II during the first six weeks of postnatal life in lambs are elicited by activation of AT1 but not AT2 receptors. In addition, effects of endogenously produced ANG II on blood pressure and renal vascular tone appear to be modulated ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 17 exclusively by activation of AT1 receptors soon after birth. A physiological role for AT2 receptors in the immediate newborn period remains to be determined. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 18 ACKNOWLEDGEMENTS This work was supported by an Operating Grant provided by the Canadian Institutes for Health Research and a Grant-in-Aid of Research provided by the Heart and Stroke Foundation of Canada. During the tenure of these experiments Mona Chappellaz was supported by graduate scholarships provided by Alexa W. Church, William Davies, Province of Alberta and Department of Medical Science. Francine G. Smith was a Heritage Medical Senior Scholar supported by the Alberta Heritage Foundation for Medical Research. Portion of this work was presented in poster format to the annual meetings for Experimental Biology 2004 and 2005, and published in the proceedings (Chappellaz, M.L., Smith, F.G. Dose-dependent effects of ANG II on systemic and renal hemodynamics in conscious lambs. FASEB J. 18(4): A287, 2004; Chappellaz, M.L., Smith, F.G. Age- dependent effects of angiotensin II (ANG II) on systemic and renal haemodynamics in conscious lambs: Role of AT1 and AT2 receptors. FASEB J. 19: A153, 2005). The authors gratefully acknowledge the excellent assistance provided by Dr. Wei Qi and Ms. Karen Connolly. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 19 REFERENCES Arens, Y., Chapados, R.A., Coc, B.E., Kamm, K.E., & Rosenfeld, C.R. (1998). Differential development of umbilical and systemic arteries: II. contractile proteins. Am J Physiol 274, R1815-R1823. Bagby, S.P., Lebard, L.S., Luo, Z., Ogden, B.E., Corless, C.L., McPherson, E.D., & Speth, R.C. (2002a). ANG II AT1 and AT2 receptors in developing kidney of normal microswine. Am J Physiol Renal Physiol 283, F755-F764. Bagby, S.P., Lebard, L.S., Luo, Z., Speth, R.C., Ogden, B.E., & Corless, C.L. (2002b). Angiotensin II type 1 and 2 receptors in conduit arteries of normal developing swine. Arterioscler Thromb Vasc Biol 22, 1113-1121. Bensoussan, M., Lelongt, B., DeLauche, M., Ronco, P.M. Ontogeny of the type 1 (AT1) and type 2 (AT2) angiotensin II (AII) receptors in rat kidney organotypic cultures: Effects of specific AT1 and AT2 antagonists. J Am Soc Nephrol 4, 461. 1993. Binder , N.D., & Anderson, D.F. (1992). Plasma renin activity responses to graded decreases in renal perfusion pressure in fetal and newborn lambs. Am J Physiol 262, R524-R529. Bottari, S.P., De Gasparo, M., Steckelings, M., Levens, N.R. (1993). Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications. Front Neuroendocrinol 14(2), 123-171. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 20 Breuhaus B.A. & Chimoskey, J.E. (1990). Hemodynamic and behavioural effects of angiotensin II in conscious sheep. Am J Physiol Regul Integr Comp Physiol 258, R1230-R1237. Brooks, V.L. (1997). Interactions between angiotensin II and the sympathetic nervous system in the long-term control of arterial pressure. Clin Exp Pharmacol Physiol 24(1), 83-90. Broughton Pipkin, F., Kirkpatrick, S.M.L., Lumbers, E.R., & Mott, J.C. (1974). Renin and angiotensin-like levels in foetal, new-born and adult sheep. J Physiol 241, 575-588. Burrell, J.H., Hegarty, B.D., McMullen, J.R., & Lumbers, E.R. (2001). Effects of gestation on ovine fetal and maternal angiotensin receptor subtypes in the heart and major blood vessels. Exp Physiol 86, 71-82. Butkus, A., Albiston, A., Alcorn, D., Giles, M., McCausland, J., Moritz, K., Zhuo, J., & Wintour, E.M. (1997). Ontogeny of angiotensin II receptors, types 1 and 2, in ovine mesonephros and metanephros. Kidney Int 51, 628-636. Butler, D.G., Butt, D.A., Puskas, D., & Oudit, G.Y. (1994). Angiotensin II-mediated catecholamine release during the pressor response in rats. J Endocrinol 142, 19-28. Carey, R.M., Wang, Z.-Q., & Siragy, H.M. (2000). Role of the angiotensin type 2 receptor in the regulation of blood pressure and renal function. Hypertension 35, 155-173. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 21 Ciuffo, G.M., Viswanathan, M., Seltzer, A.M., Tsutsumi, K., & Saavedra, J.M. (1993). Glomerular angiotensin II receptors subtypes during development of rat kidney. Am J Physiol 265, F264-F271. Cox, B.E. & Rosenfeld, C.R. (1999). Ontogeny of vascular angiotensin II receptor subtype expression in ovine development. Pediatr Res 45, 414- 424. Davidson, D. (1987). Circulating vasoactive substances and hemodynamic adjustments at birth in lambs. J Appl Physiol 63, 676-684. de Gasparo, M. (2002). Angiotensin II and nitric oxide interaction. Heart Fail Rev 7, 347-358. de Gasparo, M., Catt, K.J., Inagami, T., Wright, J.W., & Unger, T. (2000). International Union of Pharmacology. XXIII. The angiotensin II receptors. Pharmacol Rev 52, 415-472. de Wildt, S.N. & Smith, F.G. (1997). Effects of the angiotensin converting enzyme (ACE) inhibitor, captopril, on the cardiovascular, endocrine and renal responses to furosemide in conscious lambs. Can J Physiol Pharmacol 75, 263-270. Dendorfer, A., Raasch, W., Tempel, K., & Dominiak, P. (1998). Interactions between the renin-angiotensin system (RAS) and the sympathetic system. Basic Res Cardiol 93, 24-29. Dudley, D.T., Panek, R.L., Major, T.C., Lu, G.H., Bruns, R.F., Klinkefus, B.A., Hodges, J.C., & Weishaar, R.E. (1990). Subclasses of angiotensin II ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 22 binding sites and their functional significance. Mol Pharmacol 38, 370- 377. Ferguson, A.V. & Washburn, D.L. (1998). Angiotensin II: a peptidergic neurotransmitter in central autonomic pathways. Prog Neurobiol 54, 169- 192. Gimonet, V., Bussieres, L., Medjebeur, A.A., Gasser, B., Lelongt, B., & Laborde, K. (1998). Nephrogenesis and angiotensin II receptor subtypes gene expression in the fetal lamb. Am J Physiol 274, F1062-F1069. Goetzman, B.W. & Bennett, S.H. (1991). Hemodynamic responses to angiotensin II in the newborn lamb. Dev Pharmacol Ther 17, 63-69. Gröne, H.J., Simon, M., & Fuschs, E. (1992). Autoradiographic characterization of angiotensin receptor subtypes in fetal and adult human kidney. Am J Physiol 262, F326-F331. Iwamoto, H.S. & Rudoph, A.M. (1981). Effects of angiotensin II on the blood flow and its distribution in fetal lambs. Circ Res 48, 183-189. Junggren, I.L., Zhao, X., Sun, X., & Hedner, T. (1996). Comparative cardiovascular effects of the angiotensin II type 1 receptor antagonists ZD 7155 and losartan in the rat. J Pharm Pharmacol 48, 829-833. Kaiser, J.R., Cox, B.E., Roy, T.A., & Rosenfeld, C.R. (1998). Differential development of umbilical and systemic arteries. I. ANG II receptor subtype expression. Am J Physiol Regul Integr Comp Physiol 274, R797- R807. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 23 Kakucki, J., Ichiki, T., Kiyama, S., Hogan, B.L., Fogo, A., Inagami, T., & Ichikawa, I. (1995). Developmental expression of renal angiotensin II receptor genes in the mouse. Kidney Int 47, 140-147. Kintscher, U. & Unger, T. (2003). Angiotensin II receptor expression: from maturation to pathogenesis. Am J Physiol Regul Integr Comp Physiol 285, R26-R27. Li, X.C. & Widdop, R.E. (2004). AT2 receptor-mediated vasodilation is unmasked by AT1 receptor blockade in conscious SHR. Brit J Pharmacol 142, 821-830. Ludbrook, J. (1991). Editorial review on making multiple comparisons in clinical and experimental pharmacology and physiology. Clin Exp Pharmacol Physiol 18, 392. Lumbers, E.R. & Reid, G.C. (1977). Effects of vaginal delivery and caesarean section on plasma renin activity and angiotensin II levels in human umbilical cord blood. Biol Neonate 31, 127-134. Lüscher, T.F. (1990). Imbalance of endothelium-derived relaxing and contracting factors. Am J Hypertens 3, 317-330. Millan, M.A., Jacobowitz, D.M., Aguilera, G., & Catt, K.J. (1991). Differential distribution of AT1 and AT2 angiotensin II receptor subtypes in the rat brain during development. Proc Natl Acad Sci, USA 88, 11440- 11444. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 24 Monument, M.J. & Smith, F.G. (2003). Age-dependent effects of captopril on the arterial baroreflex control of heart rate in conscious lambs. Exp Physiol 88, 761-768. Nilsson, A.B.M. & Friberg, F. (2000). Acute renal responses to angiotensin-converting enzyme inhibition in the neonatal pig. Pediatr Nephrol 14, 1071-1076. Pelayo, J.C., Eisner, G.M., & Jose, P.A. (1981). The ontogeny of the renin-angiotensin system. Clin Perinatol 8, 347-359. Robillard, J.E., Page, W.V., Mathews, M.S., Schutte, B.C., Nuyt, A.-M., & Segar, J.L. (1995). Differential gene expression and regulation of renal angiotensin II receptor subtypes (AT1 and AT2) during fetal life in sheep. Pediatr Res 38, 896-904. Robillard, J.E., Schutte B.C., Page, W.V., Fedderson, J.A., Porter, C.C., & Segar, J.L. (1994). Ontogenic changes and regulation of renal angiotensin II type 1 receptor gene expression during fetal and newborn life. Pediatr Res 36, 755-762. Rosenfeld, C.R., Gresores, A., Roy, T.A., & Magness, R.R. (1995). Comparison of ANG II in fetal and pregnant sheep: metabolic clearance and vascular sensitivity. Am J Physiol Endocrinol Metab 268, E237-E247. Rosenfeld, C.R., Velaphi, S., Despain, K., & Roy, T. (2003) Renin- angiotensin system (RAS) in conscious sheep during the first month after birth. FASEB J. 16, D174. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 25 Scheuer, D.A. & Perrone, M.H. (1993). Angiotensin type 2 receptors mediate depressor phase of biphasic pressure response to angiotensin. Am J Physiol 264, R917-R923. Shanmugam, S., Corvol, P., & Gasc, J.M. (1994). Ontogeny of the two angiotensin II type 1 receptor subtypes in rats. Am J Physiol 267, E828- E836. Shi, L., Yao, J., Stewart, L., & Xu, Z. (2004). Brain c-fos expression and pressor responses after I.V. or I.C.V. angiotensin in the near-term fetus. Neuroscience 126, 979-987. Siegel, S.R. (1981). Decreased vascular and increased adrenal and renal sensitivity to angiotensin II in the newborn lamb. Circ Res 48, 34-38. Siragy, H.M. (2000). AT1 and AT2 receptors in the kidney: Role in disease and treatment. Am J Kidney Dis 36, S4-S9. Siragy, H.M. & Carey, R.M. (2001). Angiotensin type 2 receptors: potential importance in the regulation of blood pressure. Curr Opin Nephrol Hypertens 10, 99-103. Smith, F.G. & Abraham, J. (1995). Renal and renin responses to furosemide in conscious lambs during postnatal maturation. Can J Physiol Pharmacol 73, 107-112. Smith, F.G., Chan, S., & de Wildt, S.N. (1997). Effects of renal denervation on cardiovascular and renal responses to ACE inhibition in conscious lambs. J Appl Physiol 83, 414-419. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 26 Smith, R.D., Chiu, A.T., Wong, P.C., Herblin, W.F., & Timmermans, P.B.M.W.M. (1992). Pharmacology of nonpeptide angiotensin II receptor antagonists. Ann Rev Pharmacol Toxicol 32, 135-165. Speth, R.C. & Kim, K.H. (1990). Discrimination of two angiotensin II receptor subtypes with a selective agonist analogue of angiotensin II, p- aminophenylalanine angiotensin II. Biochem Biophys Res Comm 169, 997- 1006. Thomsen, J.J. & Smith, F.G. (2004). Age-dependent cardiovascular, renal, and endocrine responses to furosemide in conscious lambs. Clin Exp Pharmacol Physiol 31, 70-75. Timmermans, P.B.M.W.M., Wong, P.C., Chiu, A.T., Herblin, W.F., Benfield, P., Carini, D.J., Lee, R.J., Wexler, R.R., Saye, J.A.M., & Smith, R.D. (1993). Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev 45, 205-251. Trimper, C.E. & Lumbers, E.R. (1972). The renin angiotensin system in foetal lambs. Pflügers Arch 336, 1-10. Tufro-McReddie, A., Harrison, J.K., Everett, A.D., & Gomez, R.A. (1993). Ontogeny of type 1 angiotensin II receptor gene expression in the rat. J Clin Invest 91, 530-537. Velaphi, S.C., Roy, T., Despain, K., & Rosenfeld, C.R. (2002). Differential responses to systemic and local angiotensin II infusions in conscious postnatal sheep. Pediatr Res 52, 333-341. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 27 Whitebread, S., Mele, M., Kamber, B., & de Gasparo, M. (1989). Preliminary biochemical characterization of two angiotensin II receptor subtypes. Biochem Biophys Res Comm 163, 284-291. Widdop, R.E., Gardiner, S.M., Kemp, P.A., & Bennett, T. (1992). Inhibition of the haemodynamic effects of angiotensin II in conscious rats by AT2-receptor antagonists given after the AT1-receptor antagonist, EXP 3174. Brit J Pharmacol 107, 873-880. Wilson, T.A., Kaiser, D.L., Wright, E.M., Ortt, E.M., Freedlender, A.E., Peach, M.J., & Carey, R.M. (1981a). Importance of plasma angiotensin concentrations in a comparative study of responses to angiotensin in the maturing newborn lamb. Hypertension 3, II18-II24. Wilson, T.A., Kaiser, D.L., Wright, E.M., Peach, M.J., & Carey, R.M. (1981b). Ontogeny of blood pressure and the renin-angiotensin- aldosterone system. Sequential studies in the newborn lamb. Circ Res 49, 416-423. WintouR, E.M., Alcorn, D., Albiston, A., Boon, W.C., Butkus, A., Earnest, L., Moritz, K., & Shandley, L. (1998). The renin-angiotensin system and the development of the kidney and adrenal in sheep. Clin Exp Pharmacol Physiol 25, S97-S100. Wintour E.M., Moritz, K., Butkus, A., Baird, R., Albiston, A., & Tenis, N. (1999). Ontogeny and regulation of the AT1 and AT2 receptors in the ovine fetal adrenal gland. Mol Cell Endocrinol 157, 161-170. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 28 Wong, P.C., Chiu, A.T., Duncia, J.V., Herblin, W.F., Smith, R.D., & Timmermans, P.B.M.W.M. (1992). Angiotensin II receptor antagonists and receptor subtypes. Trends Endocrinol Metabol 3, 211-217. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 29 FIGURE LEGENDS Figure 1 Dose-dependent effects of ANG II on mean arterial pressure (MAP) Maximum change in mean arterial pressure (MAP) from control after administration of ANG II to conscious lambs aged ~one week (open bars) and ~six weeks (solid bars). Values are mean±SD; n=4 – 10 for each dose. ‡EC100; *p<0.05 compared to 0; †p<0.05 compared to one week. Figure 2 Dose-dependent effects of ANG II on renal blood flow (RBF) Maximum change in renal blood flow (RBF) after administration of ANG II to conscious lambs aged ~one week (open bars) and ~six-weeks (solid bars). Values are mean±SD; n=4 – 11 for each dose. ‡EC100; *p<0.05 compared to 0; †p<0.05 compared to one week. Figure 3 Effects of AT1 and AT2 receptor antagonists on mean arterial pressure (MAP) responses to ANG II Mean arterial pressure (MAP) responses to EC50 ANG II measured before and after administration of ZD 7155 and PD 123319 to conscious lambs aged ~one week (open bars) and ~six-weeks (solid bars). *p<0.05 compared to Control. Values are mean±SD; n=3 – 4. Figure 4 Effects of AT1 and AT2 receptor antagonists on renal blood flow response to ANG II Renal blood flow (RBF) responses to EC50 ANG II measured before and after administration of ZD 7155 and PD 123319 to conscious lambs aged ~one week (open bars) and ~six-weeks (solid bars). *p<0.05 compared to Control. Values are mean±SD; n=3 – 4. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( 30 Table 1 Demographics of conscious lambs studied in protocols I and II Protocol I Protocol I Protocol II Protocol II One week Six weeks One week Six weeks N 5 16 9 12 Gender 2♀/3♂ 8♀/8♂ 4♀/5♂ 6♀/5♂ Body weight (kg) 7.3±0.8 14.5±1.6† 7.5±1.1 14.3±1.5† Age (days) 7±1 39±2† 8±1 42±3† Total kidney weight (g) 52.0±8.3 71.5±9.5† 60.2±15.8 69.6±9.2† Values are mean±SD. ) by guest on December 9, 2009ep.physoc.orgDownloaded from Exp Physiol ( 31 Table 2 Responses to ANG II in conscious lambs studied in protocol I One week Six weeks Time to maximum MAP at EC100 (sec) 15.4±1.9 19.3±2.7† Change in MAP at EC100 (mmHg) 25±3 36±12† MAP EC50 (ng·kg -1) 25 17 Time to maximum RBF at EC100 (sec) 8.1±2.4 10.2±1.7† Change in RBF at EC100 (mL·min -1·g-1) 2.6±1.0 3.8±1.2† RBF EC50 (ng·kg -1) 10 4 MAP, mean arterial pressure; RBF, renal blood flow; EC, effective concentration. Values are mean±SD. †p<0.05 compared to one week. N= 4 – 11 for each group. ) by guest on December 9, 2009ep.physoc.orgDownloaded from Exp Physiol ( 32 Table 3 Effects of vehicle, ZD 7155, and PD 123319 in conscious lambs One week Six weeks Time Vehicle ZD 7155 PD 123319 Vehicle ZD 7155 PD 123319 MAP (mmHg) Control 75±3 73±3 79±4 77±4 76±6 70±5 30 min 75±4 62±6† 80±3 75±3 68±7† 70±7 MVP (mmHg) Control 3±1 3±1 6±2 5±2 5±2 4±2 30 min 3±2 3±2 6±2 5±2 4±2 4±1 HR (beats·min-1) Control 175±9 190±41 186±16 108±23 114±22 97±11 30 min 164±25 183±31 162±9 112±23 113±20 102±13 RBF (mL·min-1·g-1) Control 2.8±0.7 2.5±0.2 2.7±1.2 3.2±0.7 3.6±1.7 4.1±1.2 30 min 2.7±0.7 3.0±0.4† 2.6±0.8 3.2±0.7 4.2±1.8† 3.8±1.1 RVR (mmHg/mL·g·min-1) Control 27.7±10.2 28.1±2.1 29.6±14.2 24.1±5.4 23.3±8.2 17.0±3.4 30 min 28.8±10.1 20.1±2.9 31.6±11.3 23.6±5.0 17.7±5.7 18.2±4.0 MAP, mean arterial pressure; MVP, mean venous pressure; RBF, renal blood flow; RVR, renal vascular resistance. †p<0.05 compared to Control. Values are mean±SD. N=3 – 4 for each group. ) by guest on December 9, 2009ep.physoc.orgDownloaded from Exp Physiol ( Figure 1 Dose-dependent effects of ANG II on mean arterial pressure (MAP) Dose ANG II (ng.kg-1) MAP ( ∆ , mmHg) 0 10 20 30 40 50 Six weeks One week ∗ 25 0 200 100 50 12.5 6.25 3.125 ∗ 1.563 † † † † ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ∗ ‡ ‡ Maximum change in mean arterial pressure (MAP) from control after administration of ANG II to conscious lambs aged ~one week (open bars) and ~six weeks (solid bars). Values are mean±SD; n=4 - 10 for each dose. ‡EC100; *p<0.05 compared to 0; †p<0.05 compared to one week. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( Figure 2 Dose-dependent effects of ANG II on renal blood flow (RBF) Dose ANG II (ng.kg-1) RBF ( ∆, mL . min -1 . g -1 ) 0.0 1.0 2.0 3.0 4.0 5.0 One week Six weeks 0 3.125 6.25 50 12.5 25 1.56 0.78 0.39 ‡ ‡ ∗ ∗ ∗ ∗ ∗ ∗ † † † 100 †∗ ∗ ∗ ∗ ∗ Maximum change in renal blood flow (RBF) after administration of ANG II to conscious lambs aged ~one week (open bars) and ~six-weeks (solid bars). Values are mean±SD; n=4 - 11 for each dose. ‡EC100; *p<0.05 compared to 0; †p<0.05 compared to one week. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( Figure 3 Effects of AT1 and AT2 receptor antagonists on mean arterial pressure (MAP) responses to ANG II Control 30 min Control 30 min MAP ( ∆, mmHg) 0 10 20 PD 123319 ZD 7155 ∗ ∗ Mean arterial pressure (MAP) responses to ANG II measured before and after administration of ZD 7155 and PD 123319 to conscious lambs aged ~one week (open bars) and ~six-weeks (solid bars). *p<0.05 compared to Control. Values are mean±SD; n=3 – 4. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol ( Figure 4 Effects of AT1 and AT2 receptor antagonists on renal blood flow response to ANG II Control 30 min Control 30 min RBF ( ∆ , mL.min -1 .g -1 ) -4.0 -3.0 -2.0 -1.0 0.0 PD 123319 ZD 7155 ∗ ∗ Renal blood flow (RBF) responses to ANG II measured before and after administration of ZD 7155 and PD 123319 to conscious lambs aged ~one week (open bars) and ~six- weeks (solid bars). *p<0.05 compared to Control. Values are mean±SD; n=3 – 4. ) by guest on December 9, 2009 ep.physoc.org Downloaded from Exp Physiol (