The experimental protocol was approved by the committee on Animal Experiments of the Academic Medical Center Amsterdam. Male New Zealand White rabbits aged 12 to 14 and 35 to 38 weeks (young and adult, respectively) were used.
The rabbits were anesthetized with Hypnorm® (fentanyl/fluanisone) 2.5 mg/kg i.m. Subsequently, heparin 875 IE/kg i.v. was injected and the rabbits were killed with Nembutal® (pentobarbital) 30 mg/kg i.v. The thoracic aorta was removed and placed in physiological salt solution (PSS) and gassed with a mixture of 95% O2 and 5% CO2 at room temperature.
Rabbit Thoracic Aorta Preparations
Segments of thoracic aorta with a length of ∼4 mm each were dissected and transferred into an organ bath set-up. The medium was composed as follows (mM): NaCl 118, Na2HPO4 1.2, NaHCO3 25, KCl 4.7, CaCl2 1.6, MgSO4 1.2, and glucose 11.0. Ascorbic acid (0.3) and Na2EDTA (0.03) were added to prevent oxidation of noradrenaline.
The present study involved two groups: (i) isolated thoracic aortic ring preparations obtained from young rabbits (12–14 weeks) and (ii) isolated thoracic aortic ring preparations obtained from adult rabbits (35–38 weeks). Three different experiments concerning each group were performed.
Experiment 1. Electrical Field Stimulation + Angiotensin II and Eprosartan
These experiments were performed in a noradrenaline spillover-model, thus allowing the selective measurement of sympathetic outflow using tritium labeled noradrenaline.
Radiolabeling of Noradrenergic Transmitter Stores
To label their noradrenergic transmitter stores, the aortic rings were incubated for 45 minutes in 2.0 mL of physiological salt solution containing 0.1 μmol/l l-[7,8 -3H]noradrenaline (specific activity 28.8 to 52.0 Ci/mmol) in a 5-mL glass-jacketed organ bath. The medium was continuously bubbled with carbogen and maintained at a temperature of 37°C.
After the incubation period the isolated aortic rings were washed with [3H]noradrenaline-free physiological salt solution (10- × 2 mL and 4- × 5 mL) to remove superficially bound, non-neuronal radioactivity before the experimental procedures were started, and mounted vertically between platinum wire electrodes (2 cm) placed along either side of the preparations in a 25-mL organ bath and subjected to a tension of 0.5 g. The organ bath contained 20.0 mL physiological salt solution. Desipramine (0.6 μmol/l) and corticosterone (40 μmol/l) were added to rule out uptake-1 and uptake-2 of [3H]noradrenaline, respectively. Yohimbine (1 μmol/l) was added to rule out any α2-adrenergic auto-inhibitory effects on [3H]noradrenaline release. The aortic rings were equilibrated for a total of 48 minutes. After an initial period of 18 minutes the preparations were subjected to a 2-minute period of EFS with a train of 3 milliseconds rectangular bipolar wave pulses of 150 mA, at a frequency of 2 Hz. (S1) (Danish Myo Technology Current Stimulator, model CS 200). This `priming' stimulation has proven to increase the reliability and stability of the subsequent basal and electrical field stimulation-induced [3H]noradrenaline spillover.
Stimulation of Intrinsic Sympathetic Nerves
After the equilibration period the aortic preparations were subjected to 2 periods of electrical field stimulation (see above). The first period of stimulation (S2) was applied directly after the equilibration period of 48 minutes and the radioactivity thus evoked was taken as control value. Subsequently, a second period (S3) was applied 24 minutes after S2.
Measurement of Tritium Outflow
Samples of 0.5 mL were repeatedly taken from the organ bath starting 36 minutes after washout. Since the organ bath medium was not changed and its total volume decreased stepwise by repeated drawing of samples, the actual outflow of radioactivity could be obtained by calculating the incremental accumulation in each sample corrected for the reduced volume.
The mean basal efflux of radioactivity/min preceding S2 and S3 was determined as the mean outflow/min of radioactivity in two 6-minute samples before each period of stimulation. The release/minute evoked by electrical field stimulation (S2 and S3, 2 minutes samples) was calculated by subtracting the corresponding mean basal efflux/minute from the apparent evoked efflux/minute. At the end of the experiment the residual radioactivity of the tissue was measured. By adding the total released radioactivity, the initial content of tritium-label was calculated. The effect of electrical field stimulation on the release could then be expressed as a fraction of the total tissue content present at the time at which the stimulation period was applied or `fractional release' of radioactivity (FR2 and FR3). Accordingly, the effects of pharmacological interventions are expressed as the ratio FR3/FR2.
Detection of Tritium in the Samples and Tissue
After the experiment the tissues were kept overnight in 2 mL of 0.5 M quaternary ammonium hydroxide solved in toluene (Soluene, Packard). Radioactivity was measured by liquid scintillation counting (Tri Carb 2900TR, Packard) in 20-mL aliquots (with either samples or tissue) after addition of 5 mL of the scintillation mixture (Ultima Gold, Packard). Corrections for counting efficiency were made by external automatic standardization.
To investigate the influence of angiotensin II (10 pM–0.1 μM) on EFS-evoked noradrenaline release angiotensin II was added in one particular concentration to the medium 150 seconds before S3. To indicate the effect of angiotensin II the ratio FR3/FR2 was used.
In another series of preparations, the influence of multiple concentrations of eprosartan (0.1 nM–0.1 μM) on the interaction between angiotensin II (10 nM) and stimulation-induced sympathetic outflow was investigated to address the AT1-receptor role concerning facilitation in relation to maturation. Either vehicle or eprosartan was added in one particular concentration to the medium, 20 minutes before S3. Angiotensin II (10 nM) was added 150 seconds before S3. To characterize the sympatho-inhibitory effects of eprosartan the ratio FR3/FR2 was used.
Experiment 2. Concentration-Response Curves of Angiotensin II and Eprosartan
The aortic rings were mounted between 2 triangular stainless steel hooks and placed into an organ bath setup with isometric tension recording. The medium, consisting of 5-mL PSS, was continuously bubbled with carbogen and maintained at a temperature of 37°C. Nω-nitro-L-arginine (L-NNA, 0.1 mM) was added to exclude the influence of endothelium-derived nitric oxide.
Isometric tension was measured by means of isometric force transducers (A.D. instruments, Castle Hill, Australia), connected to a MacLab/8 computer system. The aortic rings of both groups were equilibrated in PSS for 30 minutes at a resting tension of 20 mN, which was maintained throughout the experiment. The equilibration period was followed by a priming procedure that consisted of a single application of KPSS for 3 minutes (PSS containing 60 mM KCL; equimolar substitution for NaCl), a single concentration of angiotensin II (0.1 μM), followed by 2 subsequent KPSS-induced depolarizations. Each stimulus was applied 15 minutes after the preparations had been washed out repeatedly and had returned to a resting tension of 20 mN.
Concentration-response curves to angiotensin II (1 nM–1 μM) were obtained by single additions with half-log increments 35 minutes subsequent to the priming procedure. To investigate the influence of eprosartan on angiotensin II-elicited contractions, a CRC was constructed for angiotensin II in the presence of one particular concentration of eprosartan to be tested, or the vehicle. The antagonist was added to the medium 20 minutes before the administration of angiotensin II was started.
Non-linear regression was carried out to calculate the maximal effect (Emax) and the concentration of angiotensin II that caused half-maximal effects (EC50). The effects of the different concentrations of eprosartan were expressed as percentage of the maximum contractile force elicited by angiotensin II alone (% Emax).
Experiment 3. Concentration-Response Curves of Noradrenaline and Angiotensin II
In addition, concentration-response curves of noradrenaline (1 nM–0.1 mM), in the presence of angiotensin II (1 nM, sub-pressor concentration, added 2 minutes before the CRC was started) or the vehicle, were obtained by single additions with half-log increments 35 minutes subsequent to the priming procedure. The results were calculated and expressed as reported for eprosartan.
Drugs and Chemicals
Desipramine HCl, yohimbine HCl, and Nω-nitro-L-arginine (Sigma, USA) were dissolved in distilled water. Corticosterone (Bufa, The Netherlands) was dissolved in dimethylsulfoxide (DMSO). Stock solutions of desipramine (0.6 mM), yohimbine (1 mM), and corticosterone (40 mM) were further diluted with physiological salt solution.
Angiotensin II (Bachem, Bubendorf, Switzerland, synthetic human sequence) was dissolved in distilled water. Stock solutions of angiotensin II (0.1 mM) were stored in 50-μL aliquots at −20°C. Tritiated levo-[7,8 -3H]noradrenaline (Amersham Pharmacia Biotech, Little Chalfont, England) had a specific radioactivity of 28.8 to 52.0 Ci/mmol and a radioactive concentration of 1.0 mCi/ml. Soluene and Ultima Gold solutions were obtained from Packard (Groningen, The Netherlands). Eprosartan (Solvay, Hannover, Germany) was dissolved in NaOH 1M. Using HCl 1M, the pH of the solution was adjusted to 7.5. (-)-Noradrenaline bitartrate (Sigma, USA) was dissolved in distilled water containing L(+) ascorbic acid 1 mg/ml.
BIBS 222 represents 2-n-butyl-1-[4-(6-carboxy-2,5-dichlorobenzoylamino)-benzyl]-6-N-(methylamino-carbonyl)-n-pentylamino-benzimidazole.
All data are expressed as means ± SEM. Student t test (two-tailed, unpaired) was used to evaluate statistical significance of differences between means of control and treatment groups. An ANOVA followed by Bonferroni Multiple Comparison test was used for multiple comparisons with a control group. Differences of P less than 0.05 were considered statistically significant.
Experiment 1. Electrical Field Stimulation + Angiotensin II and Eprosartan
In control experiments, in both groups (young versus adult), we observed no change of the basal efflux of tritium-label between the different periods of stimulation. Stimulation of the aortic rings resulted in a marked increase in radioactivity spillover by approximately a factor 6 in rings obtained from rabbits of both groups (P < 0.05 compared with basal outflow, P > 0.05 between young and adult). Additionally, no difference was observed in the absolute tritium-labeled noradrenaline release of both groups (data not shown).
Furthermore, for control experiments in both groups the fractional release of EFS-evoked tritium labeled noradrenaline remained constant throughout the experiment (young; FR3/FR2 0.96 ± 0.04, n = 8 and adult; FR3/FR2 0.99 ± 0.04, n = 8).
Tetrodotoxin (1 μM) nearly abolished the EFS-evoked tritium-labeled noradrenaline release (data not shown), which advocates the notion that the evoked radioactivity is released from nerve endings.
In both groups, angiotensin II added to the organ bath in one particular concentration 150 seconds before S3, did not alter the efflux at rest. However, it caused a concentration-dependent increase of the EFS-evoked sympathetic outflow (young rabbits; FR3/FR2 2.00 ± 0.11 produced by 1 nM angiotensin II (n = 10) and adult rabbits; FR2/FR1 2.03 ± 0.11 produced by 10 nM angiotensin II (n = 7)). Interestingly, we observed approximately a log increment difference in potency, although the degree of facilitation was the same in both groups (Fig. 1A).
Addition of eprosartan to the medium 20 minutes before S3 neither influenced the basal efflux of tritium-label nor the noradrenaline spillover evoked by electrical field stimulation (data not shown). However, eprosartan concentration-dependently attenuated the subsequent angiotensin II-enhanced (10 nM) sympathetic outflow (Fig. 2A and 2B). Although we used the same concentration of angiotensin II, the inhibitory potency of eprosartan differed between the groups of the study (young; pIC50 7.91 ± 0.12 and adult; pIC50 8.81 ± 0.31, P < 0.05).
Experiment 2. Concentration-Response Curves of Angiotensin II and Eprosartan
Angiotensin II (1 nM–0.3 μM) caused a concentration-dependent increase in contractile force of rabbit thoracic aortic rings in both groups (young rabbits; Emax 20.62 ± 2.24 mN, pD2 8.16 ± 0.04, n = 10 and adult rabbits; Emax 21.64 ± 3.86 mN, pD2 7.63 ± 0.02, n = 7). Interestingly, we observed approximately a half log increment difference in potency, although the maximal absolute contraction was similar in both groups (Fig. 1B).
In aortic rings obtained from young rabbits, eprosartan (0.1 nM–0.1 μM) influenced the angiotensin II contractions in a competitive manner (pA2 8.90 ± 0.11, n = 24) (Fig. 3A). Eprosartan (0.1 nM–0.1 μM) inhibited angiotensin II-induced contractions in preparations from adult rabbits displaying a mixed profile of antagonism (Fig. 3B).
Experiment 3. Concentration-Response Curves of Noradrenaline and Angiotensin II
Noradrenaline caused the same concentration-dependent increase in contractile force of rabbit thoracic aortic rings in both groups (young rabbits; EC50 −6.95 ± 0.05 log M, Emax 129.8 ± 2.0% of contractions by KPPS (Emax 36.85 ± 1.07 mN) n = 6, and adult rabbits; EC50 −6.93 ± 0.05 log M, Emax 130.7 ± 2.5% of contractions by KPPS (Emax 31.08 ± 5.49 mN), n = 6). Vasoconstrictor responses to noradrenaline remained unchanged by the presence of angiotensin II 1 nM in both groups. A P value more than 0.05 compared with control for both Emax- and EC50- values (Fig. 4A and 4B).
The present study clearly demonstrates that, in the rabbit isolated thoracic aorta, the responses to angiotensin II at both the prejunctional and the postjunctional site are maturation-dependent. At both sites we observed a rightward shift of the concentration-response curve to angiotensin II without a depression of the maximal response. This phenomenon may possibly be explained by a decreased AT1-receptor expression, although other changes could also contribute.
In rabbit isolated thoracic aortic rings, angiotensin II concentration-dependently enhanced the EFS-evoked noradrenaline release in both groups, as was reported previously by us and others (Fig. 1A). 13–15 Additionally, the process of maturation appeared not to influence the relative or the absolute angiotensin II-augmented responses. Interestingly, the facilitation concentration-effect curve was shifted rightward by 1 unity log M increment. Several factors that determine the response to angiotensin II (potency) must be taken into account; namely the affinity of the ligand (angiotensin II) for the AT1-receptor, the desensitization of AT1-receptor, several signaling, and the angiotensin AT1-receptor density, respectively.
Although little is known concerning maturation in relation to angiotensin II-affinity for the AT1-receptor several, studies indicate that the binding affinity is not influenced by age. 16,17 Therefore, it appears unlikely that the rightward shift can be explained by an age-induced decrease in affinity of angiotensin II for the AT1-receptor. Nonetheless, this possibility can not be disregarded entirely. Potency differences without depression of the maximal response can be associated with the process of desensitization. Desensitization of the AT1-receptor has been described repeatedly 18–20 and involves G-protein–uncoupling. Additionally, a decreased density or expression of AT1-receptors may explain the observed rightward shift, although this must be substantiated by means of binding studies. The existence of AT1-spare receptors was demonstrated to occur repeatedly. 21,22 By means of the Furchgott analysis 23 a receptor reserve of 9% was demonstrated for the AT1-receptor in the rabbit thoracic aorta using sarile, a slowly dissociating peptide angiotensin-receptor antagonist. 22 Additionally, maturation-dependent changes of angiotensin receptor expression have been reported recently. 24
No maturation-dependent difference was observed concerning the maximal postjunctional responses to angiotensin II, which is at accordance with several previous studies, in human and rodent tissue. 6,25 We did observe a rightward shift of the angiotensin II-mediated vasoconstriction curve with maturation (Fig. 1B). As for the prejunctional AT1-receptor a desensitization or decreased density of the postjunctional AT1-receptor may explain this phenomenon, although this must be substantiated by means of binding studies.
Interestingly, the maturation-dependent rightward shift to angiotensin II differed between the responses mediated by the neuronal (log M difference) and vascular (0.5 log M difference) AT1-receptor, respectively (Fig. 1A and 1B). Different AT1-receptor subtypes have been suggested to exist at the sympathetic nerve terminal and the vascular smooth muscle, respectively. 15,26,27 AT1-receptor subtype differences may explain our observations, although this remains to be fully elucidated.
Eprosartan concentration-dependently attenuated the angiotensin II-enhanced sympathetic nerve traffic in both groups (Fig. 2A and 2B), thus confirming the concept that angiotensin II-facilitated sympathetic outflow is mediated by the AT1-receptor independent of age. The inhibitory potency of eprosartan at angiotensin II (10 nM) differed significantly (young; pIC50 7.91 ± 0.12 and adult; pIC50 8.81 ± 0.31, P < 0.05). However, this is exactly what one would predict from the difference in angiotensin concentration-response curves and therefore does not imply anything concerning sensitivity of the angiotensin receptor in these tissues to the antagonist.
At the level of the vasculature eprosartan concentration-dependently inhibited the constrictor response to angiotensin II (Fig. 3A and 3B). In aortic rings of young rabbits eprosartan behaved as a competitive antagonist (pA2 8.90 ± 0.11). Potent competitive antagonism (nM range) of eprosartan has been observed in the rabbit thoracic aorta 28 and in renal arterioles. 29 Conversely, eprosartan displayed a mixed profile of antagonism in aortic rings of adult animals, combining a rightward shift of the concentration-effect curve and a suppression of Emax. This phenomenon is complex, precluding any clear interpretation about maturation-related changes in receptor function, expression, or coupling. The difference in binding with maturation may be a function of the chemical moiety of eprosartan rather than a class effect of the AT1-receptor antagonist. However, it was demonstrated in the rat portal vein that differences in the type of antagonism displayed by the specific non-peptide AT1-receptor antagonist BIBS 222 might be explained by reduction or abolishment of the AT1-receptor expression or reserve. 22
Concerning both age groups, no differences were observed between the vasoconstrictor responses to exogenous noradrenaline, as reported previously in the rat preparation. 30 Additionally, we could not confirm a facilitatory role of angiotensin II (1 nM) on postsynaptic α-adrenoceptor mediated responses, as reported by others. 31,32 In the rat mesenteric artery and the rabbit ear artery, as in the present study, no effect of angiotensin II on noradrenaline responses was observed. 33,34
In conclusion, it appears that the responses to angiotensin II at both the prejunctional and the postjunctional site are maturation-dependent. In the adult group, at both sites studied, we observed a rightward shift of the concentration-response curve to angiotensin II without a depression of the maximal response compared with the young group. This phenomenon could be explained by decreased expression of AT1-receptors, or post-receptor changes in signaling pathways. This additionally may offer a possible explanation for the different types of antagonism induced by eprosartan in young compared with adult rabbits.
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