The constrictor effects caused by angiotensin II (Ang II) in various arterial vascular beds have been studied in great detail. In all vascular tissues so far investigated, the Ang II-induced constrictor effect is mediated by angiotensin receptors of the AT1-subtype (1-3). The constrictor effect induced by Ang II in the rat portal vein also is mediated by AT1-receptor stimulation (4). However, limited attention has been paid to the constrictor effects of Ang II in capacitance vessels, although the contraction of such vessels under the influence of Ang II can be imagined to occur under clinical circumstances.
High plasma Ang II levels may occur under certain clinical conditions, and it may be expected that higher concentrations of the Ang II degradation products angiotensin III (Ang III) and angiotensin IV (Ang IV) will accompany such conditions as well. For instance, antihypertensive treatment with the AT1-receptor antagonist losartan is known to be associated with high Ang II levels in plasma (5). Elevated levels of Ang II have also been observed in patients with severe congestive heart failure (6), during and after cardiopulmonary bypass surgery, and in postoperative hypertension (7,8). Furthermore, it has been suggested that circulating Ang II may unfavorably influence the patency of venous bypass grafts (9).
In a series of previous investigations, we compared the vascular effects of Ang II with those of its degradation products Ang III and Ang IV, but these studies were limited to preparations from isolated arteries, isolated cardiac tissues, and to pithed rats (10,11). For this reason, we thought it of interest to investigate the effects of Ang II, Ang III, and Ang IV in the venous vascular bed.
In this comparative study, we investigated the effects of Ang II, Ang III, and Ang IV in human isolated saphenous vein (SV) preparations obtained during aortocoronary artery bypass surgery. The receptor subtypes involved in the responses to angiotensin peptides were analyzed by means of the selective angiotensin receptor antagonists losartan (12) and PD123177 (13), respectively.
Angiotension (I-VII), a heptapeptide formed from either angiotensin I (Ang I) or Ang II in vivo (14), which causes biphasic blood pressure responses in pithed rats and renal hypertensive dogs (15,16), also was included in this study.
Several studies have indicated that endogenous degradative proteases may interfere with the effects of angiotensin peptides (17,18). In our study, the effects of angiotensin peptides in human SV were therefore compared in the absence and presence of amastatin, an inhibitor of aminopeptidase-A and -M.
Ang II-induced prostacyclin release has been found to occur in endothelium-denuded human SV (9). We therefore also studied the influence of the cyclooxygenase inhibitor indomethacin on the responses to angiotensin peptides in isolated endothelium-denuded human SV preparations.
Human isolated SV preparations
Human isolated SVs were obtained from 20 patients, 14 men and six women (age range, 50-81 years; mean ± SEM, 66 ± 2 years) who were subjected to aortocoronary artery bypass surgery. All patients had chronic stable angina and were taking oral antianginal medication. The medication consisted of β-adrenoceptor blockers, long-acting nitrates (mostly isosorbide dinitrate), and calcium antagonists, in various combinations. Patients with unstable angina or those treated with angiotensin-converting enzyme (ACE)-inhibitors were excluded. The venous preparation to be studied was separated from the surgically excised SV. Immediately when the samples became available, they were placed in a Krebs solution of the following composition (mM): NaCl, 119.0; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; NaHCO3, 25; KH2PO4, 1.2; and glucose, 5.5; gassed with 95% O2 + 5% CO2 (at room temperature). After adhesive fat and connective tissue had been removed, the veins were preserved maximally for 5 days in the University of Wisconsin (UW) solution at 4°C. The composition of the UW solution has been described elsewhere in detail (19).
Because the functional endothelium is likely to have been partially damaged by the surgical manipulation (9), the endothelium was removed completely at the beginning of every experiment by gentle rubbing of the intimal surface with a wooden rod, in Krebs solution. Each piece of SV was cut into 4-mm-length rings. The rings were fixed between two L-shaped stainless steel hooks and suspended in a 10-ml organ bath with Krebs solution at 37°C (pH = 7.4). Each preparation was connected to a force transducer (UFI Co., CA, U.S.A.) by a silk thread, and the isometric force was recorded with a MacLab/8-computer system (A.D. Instruments, London, U.K.). A resting tension of 3.5 g was maintained throughout.
After an equilibration period of 90 min, the vascular rings were exposed to a depolarizing potassium solution twice with an interval of 20 min to test the viability and to prime the preparations. The depolarizing potassium solution had the same composition as the Krebs buffer used, except for the NaCl, which had been completely replaced by an equimolar amount of KCl. Once the contraction had reached a plateau, the tissues were washed with Krebs solution 4 times. The contraction induced by the second potassium challenge was taken as the maximal (100%) response of the vascular ring, and the responses to angiotensin peptides were expressed as a percentage of this contraction.
Forty minutes after the second potassium-induced contraction, cumulative concentration-response curves (CRCs) were constructed for Ang II (0.1-100 nM), Ang III (1 nM-3 μM), Ang IV (0.3 μM-0.1 mM), and angiotensin (I-VII) (1 μM-1 mM). In some experiments, before the effects of angiotensin peptides were tested, the rings were incubated for 30 min with losartan (10 and 100 nM), PD123177 (1 μM), amastatin (10 μM), or indomethacin (10 μM), respectively. To avoid tachyphylaxis, only a single CRC for any of the angiotensin peptides was obtained in each ring preparation. Appropriate controls were run at the same time in different rings obtained from the same SV.
In separate experiments, after cumulative CRCs for Ang II, Ang III, or Ang IV had been obtained, the rings were rinsed 4 times and reequilibrated for 1 h. The second series of cumulative CRCs for Ang II, Ang III, or Ang IV was then constructed to study the phenomenon of tachyphylaxis. Angiotensin peptide-induced contractions were expressed as a percentage of the maximal responses obtained in the first series of experiments.
At the end of each experiment, after the drugs had been washed out with Krebs solution 4 times, the vessel was precontracted with noradrenaline (3 μM). Subsequently methacholine (1 μM) was added, and the vasodilation thus induced was used to test the presence or absence of functional endothelium. A marked reduction of the noradrenaline-induced tone was taken as evidence that a significant amount of functional endothelium was present. Conversely, the absence of a relaxant response was judged to be indicative for the disappearance of functional endothelium.
Ang II, Ang III, Ang IV, and angiotensin (I-VII) were obtained from Saxon (Hannover, Germany); losartan and PD123177 (1-[(4-amino-3-methylphenyl) methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1 H-imidazole[4,5-C] pyridine-6-carboxylic acid) from Thomae GmbH (Biberach/Riss, Germany); and amastatin from ICN Biomedicals B. V. (Zoetermeer, the Netherlands). Indomethacin and noradrenaline were from Sigma (St. Louis, MO, U.S.A.) and methacholine bromide from Janssen Chimica (Beerse, Belgium). All drugs were dissolved in saline except for indomethacin and PD123177, which were taken up in a small volume of 0.1 M Na2CO3 and 1 M NaOH, respectively, and subsequently diluted with saline.
Analysis of experimental data
The data are presented as mean ± SEM for n observations. The CRCs for the agonists were analyzed by means of a computer program (GraphPad; Institute for Scientific Information, San Diego, CA, U.S.A.), and the pD2 value [-log effective concentration that produces 50% of the maximal effect (EC50)], as well as the maximal effect (Emax) were thus obtained. The pA2 values were calculated by using Shild regressions: log(r − 1) = log[B] - logKB. The concentration ratio (r) represents the EC50 value of the agonist in the presence of the antagonist divided by the EC50 value of the agonist in the absence of the antagonist. [B] is the concentration of the antagonist. The statistical significance of differences was analyzed by means of one-way analysis of variance (ANOVA) or Student's t test, and values of p < 0.05 were considered significant.
Isolated SV preparations, set up as described in Methods, maintained a stable vascular tone for several hours. Functional endothelium was absent in the endothelium-denuded human SV preparations, as confirmed by the lack of vasodilator effect of 1 μM methacholine after precontraction with 3 μM noradrenaline. The comparable maximal responses to the depolarizing potassium solution and 3 μM noradrenaline challenge were obtained after preservation of the veins in UW solution for ≤5 days.
In endothelium-denuded SV preparations, the cumulative addition of Ang II (0.1-100 nM), Ang III (1 nM-3 μM), and Ang IV (0.3 μM-0.1 mM) caused concentration-dependent contractions with comparable maximal responses (Fig. 1 and Table 1). Ang III was 16 times less active than Ang II, whereas Ang IV was ∼2,700-fold less potent than Ang II. The slope of the CRC of Ang III was significantly less steep (p < 0.05) than that of the Ang II curves (Fig. 1 and Table 1). Similar slopes were observed for the CRCs of Ang II and Ang IV, respectively. Angiotensin (I-VII) was inactive in this preparation, causing neither contraction nor relaxation.
The AT1-subtype selective antagonist losartan (10 and 100 nM) concentration-dependently shifted the Ang II, Ang III, and Ang IV curves to the right, without changing the maximal responses (Fig. 2). The pA2 values of losartan for Ang II, Ang III, and Ang IV curves amounted to 8.79 ± 0.12, 8.48 ± 0.13, and 8.85 ± 0.05, respectively. However, the AT2-subtype selective antagonist PD123177 did not influence the CRCs for angiotensin peptides (Fig. 2).
Preincubation with the aminopeptidase-A and -M inhibitor amastatin (10 μM) did not significantly change the CRCs for Ang II. However, 10 μM amastatin significantly shifted the Ang III and Ang IV CRCs to the left and increased the slopes of the angiotensin curves (Fig. 3 and Table 1). Accordingly, in the presence of amastatin, the potency of Ang III and Ang IV was increased by ∼16 and 12 times, respectively. Incubation with the cyclooxygenase inhibitor indomethacin (10 μM) did not influence the CRCs for any of the angiotensin peptides studied (Table 1).
In separate experiments, angiotensin peptides appeared to cause concentration-dependent increases in contractile force 60 min after the first CRCs had been constructed (Fig. 4). The Emax, pD2 and slope values of the second Ang III and Ang IV curves were not significantly different from those of the first CRCs (Fig. 4B and C). However, the second cumulative Ang II curves were shifted to the right, the spasmogenic threshold concentration for Ang II was increased, and the maximal responses were decreased by ∼50% (Fig. 4A).
Human SV preparations as obtained from routine aortocoronary artery bypass surgery interventions and set up as described in our study proved suitable for pharmacologic in vitro studies. The preparations remained functional and stable for several hours, and the pharmacodynamic measurements were as reproducible and precise as in animal vessels in vitro.
The endothelium of the SV used in our study was removed because the endothelium is likely to have been partially or totally damaged by the surgical manipulation (9). In the preliminary experiments, we found no differences in the maximal responses to Ang II between the endothelium-intact (unremoved) rings and the endothelium-denuded vascular rings obtained from the same patient (data not shown).
The UW solution used for the preservation of the vessels has been demonstrated to be very suitable for isolated canine arteries and human subcutaneous arteries (20-22). In our study, we also observed that the contractile responses to depolarizing potassium and noradrenaline remained fully intact after preservation of the human SV preparations in UW solution for 5 days.
In endothelium-denuded SV preparations, Ang III and Ang IV each elicited concentration-dependent contractions, with a similar maximal effect as Ang II, indicating comparable efficacy of these three peptides in this preparation. Ang III was ∼16 times less potent than Ang II, whereas a much lower potency for Ang IV was found. The slope of the Ang III CRCs proved more shallow than that of the Ang II curves, whereas the slopes of the CRCs for Ang II and Ang IV were the same. This discrepancy may be explained by the rapid enzymatic degradation of Ang III (17). This presumption is supported by the observations in our study that amastatin, an inhibitor of aminopeptidase-A and -M, significantly increased the slope of Ang III CRCs and the potency of Ang III. The lack of effect of amastatin on Ang II responses may be explained by its much stronger inhibitory effects on aminopeptidase-M activity than on aminopeptidase-A activity (23). Our data are consistent with those of Robertson et al. (17) and Ahmad and Ward (23), who found that the contractile responses to Ang III, but not Ang II, were potentiated by the presence of amastatin. Although the proteases responsible for the degradation of Ang IV so far remain unknown in detail, they probably also belong to the aminopeptidase category, as indicated by the amastatin-induced increase in constrictor potency of Ang IV.
Angiotensin receptors are subdivided into at least two different subtypes: AT1- and AT2-receptor subpopulations (24). Most cardiovascular and neural effects of Ang II are shown to be mediated by AT1-receptors, whereas the role of AT2-receptors so far remains unclear. Our functional experiments also strongly suggest that the contractile effects of Ang II, Ang III, and Ang IV in human SV are mediated by AT1-receptors but not AT2-receptors, because the AT1-receptor-selective antagonist losartan competitively antagonized the contractile responses to Ang II, Ang III, and Ang IV, whereas the AT2-receptor-selective antagonist PD123177 did not influence the responses to these three peptides. Similar results have been obtained in rat aorta (10).
Unlike that in canine SV, basal prostacyclin release of human SV is known to be very small and uninfluenced by functional endothelium (9,25). Ang II can induce prostacyclin release only in endothelium-denuded human SV, and this effect proved more pronounced in the veins that had been subjected to surgical manipulation (9). In our study, neither the basal tension of the veins nor angiotensin peptides-induced contractions were changed by the cyclooxygenase inhibitor indomethacin, suggesting that prostacyclin caused no vasodilator activity in this preparation. The lack of effect of indomethacin on Ang II-induced contractions has also been reported in rabbit renal arteries and mesenteric arteries (26,27).
The well-known Ang II-induced tachyphylaxis has been reported to occur also in human isolated placental arteries and detrusor muscles (28-30), and cyclooxygenase products were shown to be not involved in the Ang II tachyphylaxis (30). In this study, we observed tachyphylaxis induced by Ang II but by neither Ang III nor Ang IV in endothelium-denuded human SV preparations. Similar findings were obtained in endothelium-denuded rat aorta (10). In agreement with many other investigations (31,32), we propose to explain the Ang II tachyphylaxis as the result of a downregulation of Ang II receptors, induced by the interaction with a regulatory "tachyphylactic" site, which is probably not present in the conformation of Ang III and Ang IV.
Our findings demonstrate that in endothelium-denuded human SV, Ang II and its degradation products Ang III and Ang IV cause significant vasoconstrictor effects with similar efficacy. Endogenous aminopeptidase activity is likely to counteract the effects of the angiotensin peptides. The contractile responses to angiotensin peptides are mediated by angiotensin AT1-receptors but not AT2-receptors. Cyclooxygenase products are not involved in the contractile effects of the angiotensin peptides in human SV. In the absence of the endothelium, Ang II showed substantial tachyphylaxis, whereas Ang III and Ang IV did not.
The potent constrictor effect of Ang II in isolated veins substantiates the contribution of this peptide to vascular tone in capacitance vessels (33), especially when circulating Ang II concentrations are increased, as discussed in the Introduction. Conversely, ACE inhibitors and AT1-receptor antagonists are known to cause dilation of the venous vascular bed in conditions of severe congestive heart failure (6), as reflected by the reduction of cardiac preload induced by both categories of drugs.
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