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Differential patterns of relaxation by synthetic atrial peptide (APII) in the pulmonary artery, ascending and distal abdominal aorta
Regulatory Peptides, 28 (1990) 283-292 Elsevier
283
REGPEP 00909
Differential patterns of relaxation by synthetic atrial peptide (APII) in the pulmonary artery, ascending and distal abdominal aorta S. Aardal, M. Br~tveit a n d K . B . Helle Department of Physiology, University of Bergen. Bergen (Norway) (Received 16 October 1989; revised 29 January 1990; accepted 31 January 1990) Key words: Atriopeptin; VIP; el-Adrenoceptor; fl2-Adrenoceptor; Rat; Vascular smooth muscle
Summary The vasodilatory effects of the synthetic rat atriopeptin (APII) have been studied in vitro in agonist-contracted, endothelium-denuded segments of the rat pulmonary artery, the ascending, and the distal abdominal aorta. In the pulmonary artery the contractures to methoxamine were inhibited more potently by A P I I (pD 2 = 9.10 + 0.40, n = 6) than by the vasodilatory neuropeptide VIP (pD 2 = 7.37 + 0.66, n = 6). The intrinsic activity of A P I I was 0.46 + 0.16 (n = 6). In segments previously exposed to either VIP or the fl2-agonist salbutamol, A P I I was a near complete agonist (~ = 0.82 + 0.17, n = 7 and 0.84 + 0.14, n = 6, respectively) without significant changes in the potencies. A P I I was a complete agonist also for the inhibition of the e-agonist-contracted segments of the aorta, however, with potencies 10-fold lower than in the pulmonary artery. VIP was without functionally significant effects in the aorta. The tachykinins (CGRP, SP, Neurokinins A and B) were without effects in all segments tested. In the ascending aorta, A P I I induced a long-lasting tachyphylaxis to the e-agonists, nearly completely abolishing the subsequent responsiveness to NA and methoxamine for more than 4 h.
Introduction The atriopeptins comprise a newly discovered family of peptide hormones occurring in atrial secretory granules [ 1 ]. Atriopeptin is synthesized as prepro-atrionatriuretic Correspondence: S. Aardal, Department of Physiology, PKI, Arstadvei 19, 5009 Bergen, Norway 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
284 factor (prepro-ANF), stored as pro-ANFl_126 in these granules [2], and released into the circulation as a mature hormone, containing 28 amino acid residues. There is now ample evidence for atriopeptin (AP) being intimately involved in fluid, electrolyte and blood volume homeostatis [3-11 ]. Volume loading acts as one of the functional stimuli for AP release [ 12,13 ], enhancing plasma AP from 0.6 to 2.5 nM in the left carotid artery of the rat [ 14] and after induced paroxysmal tachycardia from 0.5 to 1.8 nM in the human coronary sinus [15]. Since most of the earlier studies were performed with pharmacologically high doses of the hormone, leading to distorted conclusions concerning the potency and biological role of the atriopeptins [16], it is essential to characterize in particular those responses which occur in the 0.1-10 nM range. Studies on isolated arteries from the rat and a number of other mammals show marked species differences with respect to regional selectivity for AP relaxation [ 17-20]. Vasodilation with AP is largely endothelium-independent and associated with cGMP, via receptor stimulation coupled to the membrane-bound guanyl cyclase [21-25 ]. The peptide is, on the other hand, poorly active against K + -contracted rabbit aorta while actively inhibiting intracellular release of Ca 2 + in agonist-contracted preparations [26,27]. The effector responses to elevated cAMP and c G M P are turned off by multiple molecular forms of phosphodiesterases [28,29], with discrete roles in modulating diverse metabolic processes. Interactions may thus be anticipated between AP and the agonists acting via cAMP in the various segments of the vascular tree. As the circulating levels of API and APIII are low, they are unlikely to be physiologically important as vasodilators [ 19]. APII is the circulating, active form of the hormone [30,31] and was therefore used in these experiments. We have now characterized responses to APII, VIP, and C G R P in denuded arterial segments closest to the atrial release, i.e., the pulmonary artery, the ascending and distal abdominal aorta of the rat. The/~2-agonist salbutamol (S al) has been used as reference for non-peptide vasodilatory responses to the contractants noradrenalin (NA) and the cq-selective agonist methoxamine. Part of these findings have been presented in a brief report [32].
Materials and methods
Tissue collection Female albino rats of the Wistar strain (230-280 g b.w.) were anaesthetized with 0.35 ml pentobarbital (50 mg/ml) i.p. Segments of the extrapulmonary branch of the pulmonary artery, the ascending aorta and the distal abdominal aorta were denuded by twisting an intraluminal catheter [33] and placed in the oxygenated Krebs-Henseleits medium. Recording of contractile tension in the isolated vascular preparations The 3-4 mm long segments were mounted as circular preparations on two 4 mm long stainless steel wires, each with a diameter of 0.2 mm, in an organ bath [34] containing 2.5 ml Krebs-Henseleits medium, oxygenated with 95~o oxygen and 5~o CO2 at 37 °C. The medium contained (in mM): NaC1 118, KC1 4.7, CaC12 2.5, MgSO 4 1.2, NaHCO3 25, KH2PO 4 1.2 and glucose 10.1. Tension was recorded isometrically by a
285 15mN
1:
'l"l Ir ..........................
i ....
t Methoxamine IOmin
Fig. 1. Rat pulmonary artery contracted by methoxamine (3.3.10 -6 M) in the presence of atropine (0.6.10-6 M) Grass Force-Displacement Transducer (type FTo3 ) connected to a Grass Polygraph (model 7D), and quantitated as previously described [35,36]. The preparations were passively stretched stepwise to a preload giving maximal contraction (15 m N ) with methoxamine (3.2- 10- 6 M) and equilibrated for 1 h before addition of drugs. Atropine ( 0 . 6 . 1 0 - 6 M) was added to all preparations 10 min before contraction with maximally effective concentrations of methoxamine ( 3 . 2 . 1 0 - 6 M ) or N A ( 2 . 6 . 1 0 - 6 M). The contractures stabilized within 8-15 min and were maintained for at least 1 h unless vasodilatory agents were added. Relaxation was quantified as the decline in contractile tension 3 - 4 min after addition of the agonist and expressed in percent of the maximal relaxation, i.e., back to the level of the resting tension (100 ~/o). Agonists were added in cumulative concentrations to determine potencies (pD2) and intrinsic activities (~). Potencies were expressed as the negative logarithm to the agonist concentrations giving half-maximal inhibitory effect [37]. The intrinsic activity was calculated as the ratio of the maximal effect of the agonist to that of the maximally obtainable relaxation. After each contraction and subsequent relaxation period, the segments were washed every 5 min until the tension had returned to the resting level and at least twice after this. Each segment was exposed to 4 or 5 cycles of agonist additions in order to detect tachyphylactic or potentiating effects of receptor activation. Contractile activity was maintained for longer than 5 h in the control experiments.
A 1.0-
Sal
PD2 APII
B
10
0.88
APII dP
APII__
0.66-
VIP
0.4.-
4-
0.2-
2-
0.0
0
Pulm.art
Dist.eorte
Pulm.a~
Dist.ao~a
Fig. 2. Comparison of intrinsic activities (ct) (left) and potencies (pD2) (right) of Atriopeptin II (APII), vasoactive intestinal peptide (VIP), and salbutamol (Sal) on isolated preparations of the rat pulmonary artery (n = 6) and distal abdominal aorta (n = 6).
286
Statistical analyses The results are expressed as means + S.D. Significance for differences between means were calculated by the Wilcoxon rank test for non-parametric, independent groups. Statistical significance was assumed when ** P < 0.01, * P < 0.05, and nonsignificance when P > 0.05. Drugs Atropine was obtained from Norges Apoteker Forening A/S, Norway; mebumal (pentobarbital) and noradrenalin from Svaneapoteket, Bergen, Norway; methoxamine (Vasoxine) from Wellcome Foundation Ltd. London, U.K. ; salbutamol (Ventolin) from Glaxo Operations Ltd. U.K.; APII-Rat, VIP (Human, porcine), C G R P I, C G R P II, Substance P (SP), Neurokinin A and B from Penninsula Laboratories Inc., U.S.A.
Results
Relaxation of methoxamine contractures in the pulmonary artery by APII, VIP and salbutamol As shown in Fig. 1, methoxamine elicited a rapid and stable contracture which was almost fully reversed by the selective fl2-receptor agonist salbutamol (Sal) (Fig. 2a). APII was a partial agonist when added as the first relaxant to the pulmonary artery, while a near complete relaxant in preparations previously exposed to cumulative doses of either Sal or VIP (Table I). VIP relaxed the pulmonary artery less efficiently than APII (~ --- 0.36-0.40), and was not altered by previous exposures to either Sal or APII (Table II). TABLE I Effect of APII on methoxamine contracted segments of the rat pulmonary artery, the ascending and distal abdominal aorta Tissues
APII (1 st drug)
APII (2nd drug) after Sal
Pulmonary artery Ascending aorta Distal abd. aorta
after VIP
pD 2
2
pD 2
a
pD 2
9.10 ± 0.40 (6) 7.57 + 0.17 (6) 7.78_+0.38 (6)
0.45 ± 0.16 (6) 0.90 ± 0.07 (6) 0.86_+0.12 (6)
8.44 ± 0.39 b (6)
0.84 + 0.14"* (6)
8.79 + 0.69 b (7)
.
.
7.89 ± 0.62 b (6)
.
0.77 ± 0.13 b (6)
0.82 + 0.17"* (7)
.
-
All segments were precontracted with 3.2- 10 6 M methoxamine in the presence of 0.6.10 6 M atropine. Values are means + S.D. of (n) preparations. a The methoxamine contraeture was 88 _+5~o (n = 6) inhibited in segments previously relaxed by APII. h p > 0.05. ** P < 0.01.
287
TABLE II Potencies (pD2) and intrinsic activities (~) for VIP in methoxamine contracted segments of the pulmonary artery and distal abdominal aorta of the rat Tissues
VIP (lst drug)
VIP (2nd drug) after Sal
Pulmonary artery Distal abd. aorta
after APII
pD 2
~
pD 2
a
pD 2
7.37 ± 0.66 (6) 4.32 ± 3.30
0.36 ± 0.16 (6) 0.08 ± 0.09
7.12 ± 0.77 a (8)
0,40 ± 0.19 a (8)
7.74 ± 0.66 a (6)
(6)
(6)
All segments were precontracted with 3.2.10- 6 M methoxamine in the presence of 0.6.10 Values are means + S.D. of (n) preparations. a p > 0.05.
0.40 ± 0.15 a (6)
6 M atropine.
C G R P I, C G R P II, SP, Neurokinin A and Neurokinin B were without relaxing effect in peptide concentrations below 10- 7 M.
Differential effects of APII, VIP and Sal in the pulmonary artery, the ascending and the distal aorta Since there were no clear differences between the thoracic and distal abdominal segments of the aorta, only the data for the distal abdominal aorta are presented. Fig. 2a compares the intrinsic activities of A P I I relative to VIP and Sal as first drugs. Sal was a near complete relaxant only in the pulmonary artery. The order of intrinsic activities in this segment was: Sal > > A P I I > VIP, while in the distal abdominal aorta the order was: APII > Sal > > VIP. The order of potencies (pD2) for the peptides were the same in the pulmonary artery and in the distal abdominal aorta (Fig. 2b). However, the potencies and intrinsic activities were too low to indicate a functional role for VIP in the distal aorta. The potency for Sal was also lower in this segment and C G R P I and II, SP and neurokinin A and B were without effects in all segments of the aorta, revealing A P I I as the main relaxing peptide for this vessel. A P H inhibition of ot-adrenoceptor contractures in the ascending aorta The contractile responses to methoxamine and noradrenalin (NA) in the ascending aorta are shown in Figs. 3 a - f and 4 a - d , respectively. Both agonists contracted this segment forcefully and repeatedly prior to the A P I I exposure (Figs. 3 a - c and 4a-c). Maximal development of tension in each contracture took more than 12 min. A P I I was not only an efficient relaxant (Table I), but also induced spontaneous oscillations with a frequency of 1-3 min during the relaxation (Figs. 3c and 4c). On the other hand, following the A P I I relaxation the repeatedly washed segments were no longer responsive to methoxamine or NA, being inhibited by 87.9 + 6 . 8 ~ (n = 6) and by 88.5 + 5.3~o
288
a 15 m N
] y---~-
]( J
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iii
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lii']]
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Met
AP
d 15mN
f
e
I
,~l~imllmllll~
-q'mmlmr..... ,
,'
I
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' ...........
;
-.-~-~'- ::"'lJl~Li
,..................
m
:,
]
mm
,,
iT
!
!
t
t
t
t
Met
Met
VIP
Met
]
Fig. 3. Rat ascending aorta contracted repeatedly by 3.2.10-6 M methoxamine (Met) before (a-c) and after (d-f) relaxation by a cumulative series of atriopeptin (AP) from 0.17 riM-1.7 #M. In e VIP was added in cumulative series from 0.12 nM-1.2 #M.
15 m N
a
b
i, -
,! .............
i~
i I.......
t
t
NA
NA
C 15 m N
iN
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d
~i .............. i '''E.... J"l..... i"'("~'"i"(""'"':~mm'~-t NA
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NA
Fig. 4. Rat ascending aorta contracted repeatedly by 2.6" I 0 - 6 M noradrenaline (NA) before (a-c) and after (d) relaxation by a cumulative series of atriopeptin (AP) from 0.17 nM-1.7 #M.
289
£//
15raN ~
"
~
/
b
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I
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!'I i .......
I ..................
Met c
[[ii'r
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I
Sal
15mN
t
~
,
,~l~...,irr
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!r'"l'111II'r~/111111
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t
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Fig. 5. Rat ascending aorta contracted repeatedly by 3.2.10-6 M methoxamine (Met) in the presence of atropine (0.6.10 -6 M). Each segment was relaxed first by a maximal dose of(a) salbutamol (8.4.10 - 6 M)
followed by (b) atriopeptin II (APII) or first by (c) vasoactive intestinal peptide 1.2.10 6 M (VIP) followed by APII (d).
(n = 6) respectively. This attenuation (Figs. 3 d - f and 4d) lasted at least 4 h and appeared only in the ascending aorta. There were no detectable effects of VIP (Fig. 3e) or Sal on the frequency or amplitudes of these contractures. Neither Sal nor VIP (Fig. 5a and c) given as the first relaxant at maximally efficient concentrations had any inhibitory effect on the following methoxamine (Fig. 5b and d) or NA contractures.
Discussion
The pulmonary artery is the first vascular target for the right atrial release of atriopeptins. We have now shown that the al-adrenoceptor mediated contractures in the rat pulmonary artery, not included in previous reports [20,38 ], are efficiently relaxed by the synthetic rat APII. The potency value in the pulmonary artery was (pD 2 = 9.10 + 0.40) relevant for the 0.6-2.5 nM range of plasma AP in the carotic artery in volume loaded rats [ 14]. Analogously, the ascending aorta is the first possible vascular target for the APII released from the left atrium. Previous reports refer to APII relaxation in unspecified thoracic or abdominal segments [ 17,38-40]. We have shown that the APII potencies for relaxation of :~-adrenoceptor contractures were 10-fold higher in the pulmonary artery than in the three different segments of the aorta. Our experiments have unmasked tissue-selective and qualitatively different interactions between APII and adrenoceptor activated responses in the pulmonary artery and the ascending aorta. Taking into account that the pulmonary APII vasodilation was substantially enhanced by previous exposure to VIP and the fl2-adrenoceptor agonist,
290 the observed high pD 2 value for A P I I indicates a functional role for A P I I in regulating the tension of the pulmonary artery. In the ascending aorta A P I I inhibited the subsequent ~-adrenoceptor elicited contractures at normal extracellular Ca 2 +. This is analogous to the A P I I inhibitions of the N A contracture in the rabbit thoracic aorta seen at low extracellular Ca 2 + concentration [27], although longer lasting than so far reported for the rabbit aorta [26]. The enhanced intrinsic activity of A P I I in the pulmonary artery segments previously exposed to the c A M P enhancing vasoinhibitors [41-43], VIP and the/~2-agonist suggests a synergism between c G M P - and cAMP-activated responses in this particular segment. This may possibly reflect significant differences between the pulmonary artery and the ascending aorta with respect to agonist-coupled release of Ca 2 + from the sarcoplasmic stores [44]. In vivo experiments have shown long-lasting reductions in blood-pressure by atriopeptin apart from the primary vasodilation [45]. Exogenous A P has also been shown to antagonize ~-adrenergic pressor responses in the pithed rat model [46,47]. Interaction with the sympathetic neuro effector system at several levels may thus represent a complementary mechanism for the cardiovascular effects of A P I I , as reflections of long lasting attenuation of N A contractures presently reported for the rat proximal aorta. In conclusion, our present findings are consistent with A P I I being a relaxant of the :¢-adrenoceptor contractures in the rat pulmonary artery at functionally relevant, nanomolar concentrations. In the ascending aorta, A P I I is a relaxant at 10-fold higher concentrations. The most important role for A P I I in this segment appears to be the persistent inhibition of further methoxamine and N A contractures. The significant differences in A P I I responses in the various segments of the rat aorta may have bearing on the complex embryological origin of this vessel [48].
Acknowledgements This work has been supported by grants from the National Council on Cardiovascular Diseases.
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292 32 Aardal, S., Brgttveit, M. and Helle, K.B., Atriopeptin modulations of adrenoceptor responses in the pulmonary artery and ascending aorta in the rat, Abstract Int. Congr. Physiol. Sci. 1989. 33 Furchgott, R.F. and Zawadizki, J. V., The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature, 228 (1980) 373-376. 34 Hogestatt, E.D., Anderson, K.E. and Edvinson, L., Mechanical properties of rat cerebral arteries as studied by a sensitive device for recording mechanical activity in isolated small blood vessels, Acta Physiol. Scand.. 117 (1983) 49-61. 35 BrS.tveit, M., Rydningen, H.T. and Helle, K.B., Comparison of atriopeptins II and llI, VIP and fl2-adrenoceptor-evoked relaxations in the two layers of smooth muscle in the rat portal vein, Acta Physiol. Sand., 130 (1987) 593-599. 36 Rydningen, H.T., Omland, T., Opstad, P.K. and Helle, K.B., Comparison of VIP and fl2-adrenoceptor induced relaxations in the circular and longitudinal smooth muscle layers of the rat portal vein, Acta Physiol. Scand., 130 (1987) 601-606. 37 Van Rossum, J.M., Cumulative dose response curves II. Technique for the making of dose response curves for isolated organs and the evaluation of drug parameters, Arch. Int. Pharmacodyn., 108 (1963) 299-330. 38 Winquist, R.J., Pharmacological effects of atrial natriuretic peptide, Endocrinol. Metabol. Clin. North Am. 16 (1987) 163-182. 39 Kleinert, H. D., Maack, T., Atlas, S. A., Januszewicz, A., Sealey, J. E. and Laragh, J. H., Atrial natriuretic factor inhibits angiotensin, norepinephrine and potassium-induced vascular contractility, Hypertension, 6 (1984) I143-I147. 40 Rapoport, R. M., Waldman, S. A., Schwartz, K., Winquist, R. and Mural, F., Effects of atrial natriuretic factor, sodium nitroprusside and acetylcholine on cyclic GMP levels and relaxation in rat aorta, Eur. J. Pharmacol., 115 (1985) 219-229. 41 Kubota, M., Moseley, J.M., Butera, L., Dusting, G.J., MacDonald, P. S. and Martin, T.J., Calcitonin gene-related peptide stimulates cyclic AMP formation in rat aortic smooth muscle cells, Biochem. Biophys. Res. Commun. 132 (1985) 88-94. 42 Wilson, S.P., Vasoactive intestinal peptide elevates cyclic AMP levels and potentiates secretion in bovine adrenal chromaffin cells, Neuropeptides, 11 (1988) 17-21. 43 Amenta, D., Iacopino, L. and Amenta, F., Vasoactive intestinal polypeptide-sensitive cyclic adenosine monophosphate generating system in the rat portal vein, Arch. Int. Pharmacodyn., 291 (1988) 88-95. 44 Devine, C.E., Somlyo, A.V. and Somlyo, A.P., Sarcoplasmic reticulum and exitation-concentration coupling in mammalian smooth muscle, J. Cell. Biol., 52 (1972) 690-718. 45 Lappe, R.W., Smits, J.F.M., Todt, J.A., Debets, J.J.M. and Wendt, R.L., Failure of atriopeptin II to cause arterial vasodilation in the conscious rat, Circ. Res., 56 (1985) 606-612. 46 Gellai, M. and Ruffolo, J., Renal effects of selective alpha-I and alpha-2 adrenoceptor agonists in conscious normotensive rats, J. Pharm. Exp. Ther., 240 (1986) 723-728. 47 Zukowska-Grojec, Z., Haass, M., Kopin, l.J. and Zamir, N., Interactions of atrial natriuretic peptide with the sympathetic and endocrine systems in the pithed rat, J. Pharmacol. Exp. Ther., 239 (1986) 480-487. 48 Barry, A., The aortic arch derivatives in the human adult, Anatomical Record, 111 (1951) 221-38.
283
REGPEP 00909
Differential patterns of relaxation by synthetic atrial peptide (APII) in the pulmonary artery, ascending and distal abdominal aorta S. Aardal, M. Br~tveit a n d K . B . Helle Department of Physiology, University of Bergen. Bergen (Norway) (Received 16 October 1989; revised 29 January 1990; accepted 31 January 1990) Key words: Atriopeptin; VIP; el-Adrenoceptor; fl2-Adrenoceptor; Rat; Vascular smooth muscle
Summary The vasodilatory effects of the synthetic rat atriopeptin (APII) have been studied in vitro in agonist-contracted, endothelium-denuded segments of the rat pulmonary artery, the ascending, and the distal abdominal aorta. In the pulmonary artery the contractures to methoxamine were inhibited more potently by A P I I (pD 2 = 9.10 + 0.40, n = 6) than by the vasodilatory neuropeptide VIP (pD 2 = 7.37 + 0.66, n = 6). The intrinsic activity of A P I I was 0.46 + 0.16 (n = 6). In segments previously exposed to either VIP or the fl2-agonist salbutamol, A P I I was a near complete agonist (~ = 0.82 + 0.17, n = 7 and 0.84 + 0.14, n = 6, respectively) without significant changes in the potencies. A P I I was a complete agonist also for the inhibition of the e-agonist-contracted segments of the aorta, however, with potencies 10-fold lower than in the pulmonary artery. VIP was without functionally significant effects in the aorta. The tachykinins (CGRP, SP, Neurokinins A and B) were without effects in all segments tested. In the ascending aorta, A P I I induced a long-lasting tachyphylaxis to the e-agonists, nearly completely abolishing the subsequent responsiveness to NA and methoxamine for more than 4 h.
Introduction The atriopeptins comprise a newly discovered family of peptide hormones occurring in atrial secretory granules [ 1 ]. Atriopeptin is synthesized as prepro-atrionatriuretic Correspondence: S. Aardal, Department of Physiology, PKI, Arstadvei 19, 5009 Bergen, Norway 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
284 factor (prepro-ANF), stored as pro-ANFl_126 in these granules [2], and released into the circulation as a mature hormone, containing 28 amino acid residues. There is now ample evidence for atriopeptin (AP) being intimately involved in fluid, electrolyte and blood volume homeostatis [3-11 ]. Volume loading acts as one of the functional stimuli for AP release [ 12,13 ], enhancing plasma AP from 0.6 to 2.5 nM in the left carotid artery of the rat [ 14] and after induced paroxysmal tachycardia from 0.5 to 1.8 nM in the human coronary sinus [15]. Since most of the earlier studies were performed with pharmacologically high doses of the hormone, leading to distorted conclusions concerning the potency and biological role of the atriopeptins [16], it is essential to characterize in particular those responses which occur in the 0.1-10 nM range. Studies on isolated arteries from the rat and a number of other mammals show marked species differences with respect to regional selectivity for AP relaxation [ 17-20]. Vasodilation with AP is largely endothelium-independent and associated with cGMP, via receptor stimulation coupled to the membrane-bound guanyl cyclase [21-25 ]. The peptide is, on the other hand, poorly active against K + -contracted rabbit aorta while actively inhibiting intracellular release of Ca 2 + in agonist-contracted preparations [26,27]. The effector responses to elevated cAMP and c G M P are turned off by multiple molecular forms of phosphodiesterases [28,29], with discrete roles in modulating diverse metabolic processes. Interactions may thus be anticipated between AP and the agonists acting via cAMP in the various segments of the vascular tree. As the circulating levels of API and APIII are low, they are unlikely to be physiologically important as vasodilators [ 19]. APII is the circulating, active form of the hormone [30,31] and was therefore used in these experiments. We have now characterized responses to APII, VIP, and C G R P in denuded arterial segments closest to the atrial release, i.e., the pulmonary artery, the ascending and distal abdominal aorta of the rat. The/~2-agonist salbutamol (S al) has been used as reference for non-peptide vasodilatory responses to the contractants noradrenalin (NA) and the cq-selective agonist methoxamine. Part of these findings have been presented in a brief report [32].
Materials and methods
Tissue collection Female albino rats of the Wistar strain (230-280 g b.w.) were anaesthetized with 0.35 ml pentobarbital (50 mg/ml) i.p. Segments of the extrapulmonary branch of the pulmonary artery, the ascending aorta and the distal abdominal aorta were denuded by twisting an intraluminal catheter [33] and placed in the oxygenated Krebs-Henseleits medium. Recording of contractile tension in the isolated vascular preparations The 3-4 mm long segments were mounted as circular preparations on two 4 mm long stainless steel wires, each with a diameter of 0.2 mm, in an organ bath [34] containing 2.5 ml Krebs-Henseleits medium, oxygenated with 95~o oxygen and 5~o CO2 at 37 °C. The medium contained (in mM): NaC1 118, KC1 4.7, CaC12 2.5, MgSO 4 1.2, NaHCO3 25, KH2PO 4 1.2 and glucose 10.1. Tension was recorded isometrically by a
285 15mN
1:
'l"l Ir ..........................
i ....
t Methoxamine IOmin
Fig. 1. Rat pulmonary artery contracted by methoxamine (3.3.10 -6 M) in the presence of atropine (0.6.10-6 M) Grass Force-Displacement Transducer (type FTo3 ) connected to a Grass Polygraph (model 7D), and quantitated as previously described [35,36]. The preparations were passively stretched stepwise to a preload giving maximal contraction (15 m N ) with methoxamine (3.2- 10- 6 M) and equilibrated for 1 h before addition of drugs. Atropine ( 0 . 6 . 1 0 - 6 M) was added to all preparations 10 min before contraction with maximally effective concentrations of methoxamine ( 3 . 2 . 1 0 - 6 M ) or N A ( 2 . 6 . 1 0 - 6 M). The contractures stabilized within 8-15 min and were maintained for at least 1 h unless vasodilatory agents were added. Relaxation was quantified as the decline in contractile tension 3 - 4 min after addition of the agonist and expressed in percent of the maximal relaxation, i.e., back to the level of the resting tension (100 ~/o). Agonists were added in cumulative concentrations to determine potencies (pD2) and intrinsic activities (~). Potencies were expressed as the negative logarithm to the agonist concentrations giving half-maximal inhibitory effect [37]. The intrinsic activity was calculated as the ratio of the maximal effect of the agonist to that of the maximally obtainable relaxation. After each contraction and subsequent relaxation period, the segments were washed every 5 min until the tension had returned to the resting level and at least twice after this. Each segment was exposed to 4 or 5 cycles of agonist additions in order to detect tachyphylactic or potentiating effects of receptor activation. Contractile activity was maintained for longer than 5 h in the control experiments.
A 1.0-
Sal
PD2 APII
B
10
0.88
APII dP
APII__
0.66-
VIP
0.4.-
4-
0.2-
2-
0.0
0
Pulm.art
Dist.eorte
Pulm.a~
Dist.ao~a
Fig. 2. Comparison of intrinsic activities (ct) (left) and potencies (pD2) (right) of Atriopeptin II (APII), vasoactive intestinal peptide (VIP), and salbutamol (Sal) on isolated preparations of the rat pulmonary artery (n = 6) and distal abdominal aorta (n = 6).
286
Statistical analyses The results are expressed as means + S.D. Significance for differences between means were calculated by the Wilcoxon rank test for non-parametric, independent groups. Statistical significance was assumed when ** P < 0.01, * P < 0.05, and nonsignificance when P > 0.05. Drugs Atropine was obtained from Norges Apoteker Forening A/S, Norway; mebumal (pentobarbital) and noradrenalin from Svaneapoteket, Bergen, Norway; methoxamine (Vasoxine) from Wellcome Foundation Ltd. London, U.K. ; salbutamol (Ventolin) from Glaxo Operations Ltd. U.K.; APII-Rat, VIP (Human, porcine), C G R P I, C G R P II, Substance P (SP), Neurokinin A and B from Penninsula Laboratories Inc., U.S.A.
Results
Relaxation of methoxamine contractures in the pulmonary artery by APII, VIP and salbutamol As shown in Fig. 1, methoxamine elicited a rapid and stable contracture which was almost fully reversed by the selective fl2-receptor agonist salbutamol (Sal) (Fig. 2a). APII was a partial agonist when added as the first relaxant to the pulmonary artery, while a near complete relaxant in preparations previously exposed to cumulative doses of either Sal or VIP (Table I). VIP relaxed the pulmonary artery less efficiently than APII (~ --- 0.36-0.40), and was not altered by previous exposures to either Sal or APII (Table II). TABLE I Effect of APII on methoxamine contracted segments of the rat pulmonary artery, the ascending and distal abdominal aorta Tissues
APII (1 st drug)
APII (2nd drug) after Sal
Pulmonary artery Ascending aorta Distal abd. aorta
after VIP
pD 2
2
pD 2
a
pD 2
9.10 ± 0.40 (6) 7.57 + 0.17 (6) 7.78_+0.38 (6)
0.45 ± 0.16 (6) 0.90 ± 0.07 (6) 0.86_+0.12 (6)
8.44 ± 0.39 b (6)
0.84 + 0.14"* (6)
8.79 + 0.69 b (7)
.
.
7.89 ± 0.62 b (6)
.
0.77 ± 0.13 b (6)
0.82 + 0.17"* (7)
.
-
All segments were precontracted with 3.2- 10 6 M methoxamine in the presence of 0.6.10 6 M atropine. Values are means + S.D. of (n) preparations. a The methoxamine contraeture was 88 _+5~o (n = 6) inhibited in segments previously relaxed by APII. h p > 0.05. ** P < 0.01.
287
TABLE II Potencies (pD2) and intrinsic activities (~) for VIP in methoxamine contracted segments of the pulmonary artery and distal abdominal aorta of the rat Tissues
VIP (lst drug)
VIP (2nd drug) after Sal
Pulmonary artery Distal abd. aorta
after APII
pD 2
~
pD 2
a
pD 2
7.37 ± 0.66 (6) 4.32 ± 3.30
0.36 ± 0.16 (6) 0.08 ± 0.09
7.12 ± 0.77 a (8)
0,40 ± 0.19 a (8)
7.74 ± 0.66 a (6)
(6)
(6)
All segments were precontracted with 3.2.10- 6 M methoxamine in the presence of 0.6.10 Values are means + S.D. of (n) preparations. a p > 0.05.
0.40 ± 0.15 a (6)
6 M atropine.
C G R P I, C G R P II, SP, Neurokinin A and Neurokinin B were without relaxing effect in peptide concentrations below 10- 7 M.
Differential effects of APII, VIP and Sal in the pulmonary artery, the ascending and the distal aorta Since there were no clear differences between the thoracic and distal abdominal segments of the aorta, only the data for the distal abdominal aorta are presented. Fig. 2a compares the intrinsic activities of A P I I relative to VIP and Sal as first drugs. Sal was a near complete relaxant only in the pulmonary artery. The order of intrinsic activities in this segment was: Sal > > A P I I > VIP, while in the distal abdominal aorta the order was: APII > Sal > > VIP. The order of potencies (pD2) for the peptides were the same in the pulmonary artery and in the distal abdominal aorta (Fig. 2b). However, the potencies and intrinsic activities were too low to indicate a functional role for VIP in the distal aorta. The potency for Sal was also lower in this segment and C G R P I and II, SP and neurokinin A and B were without effects in all segments of the aorta, revealing A P I I as the main relaxing peptide for this vessel. A P H inhibition of ot-adrenoceptor contractures in the ascending aorta The contractile responses to methoxamine and noradrenalin (NA) in the ascending aorta are shown in Figs. 3 a - f and 4 a - d , respectively. Both agonists contracted this segment forcefully and repeatedly prior to the A P I I exposure (Figs. 3 a - c and 4a-c). Maximal development of tension in each contracture took more than 12 min. A P I I was not only an efficient relaxant (Table I), but also induced spontaneous oscillations with a frequency of 1-3 min during the relaxation (Figs. 3c and 4c). On the other hand, following the A P I I relaxation the repeatedly washed segments were no longer responsive to methoxamine or NA, being inhibited by 87.9 + 6 . 8 ~ (n = 6) and by 88.5 + 5.3~o
288
a 15 m N
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]( J
t Met b
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iii
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Met
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e
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,'
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;
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,..................
m
:,
]
mm
,,
iT
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!
t
t
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t
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Met
VIP
Met
]
Fig. 3. Rat ascending aorta contracted repeatedly by 3.2.10-6 M methoxamine (Met) before (a-c) and after (d-f) relaxation by a cumulative series of atriopeptin (AP) from 0.17 riM-1.7 #M. In e VIP was added in cumulative series from 0.12 nM-1.2 #M.
15 m N
a
b
i, -
,! .............
i~
i I.......
t
t
NA
NA
C 15 m N
iN
i
......
d
~i .............. i '''E.... J"l..... i"'("~'"i"(""'"':~mm'~-t NA
AP
NA
Fig. 4. Rat ascending aorta contracted repeatedly by 2.6" I 0 - 6 M noradrenaline (NA) before (a-c) and after (d) relaxation by a cumulative series of atriopeptin (AP) from 0.17 nM-1.7 #M.
289
£//
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"
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,
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Fig. 5. Rat ascending aorta contracted repeatedly by 3.2.10-6 M methoxamine (Met) in the presence of atropine (0.6.10 -6 M). Each segment was relaxed first by a maximal dose of(a) salbutamol (8.4.10 - 6 M)
followed by (b) atriopeptin II (APII) or first by (c) vasoactive intestinal peptide 1.2.10 6 M (VIP) followed by APII (d).
(n = 6) respectively. This attenuation (Figs. 3 d - f and 4d) lasted at least 4 h and appeared only in the ascending aorta. There were no detectable effects of VIP (Fig. 3e) or Sal on the frequency or amplitudes of these contractures. Neither Sal nor VIP (Fig. 5a and c) given as the first relaxant at maximally efficient concentrations had any inhibitory effect on the following methoxamine (Fig. 5b and d) or NA contractures.
Discussion
The pulmonary artery is the first vascular target for the right atrial release of atriopeptins. We have now shown that the al-adrenoceptor mediated contractures in the rat pulmonary artery, not included in previous reports [20,38 ], are efficiently relaxed by the synthetic rat APII. The potency value in the pulmonary artery was (pD 2 = 9.10 + 0.40) relevant for the 0.6-2.5 nM range of plasma AP in the carotic artery in volume loaded rats [ 14]. Analogously, the ascending aorta is the first possible vascular target for the APII released from the left atrium. Previous reports refer to APII relaxation in unspecified thoracic or abdominal segments [ 17,38-40]. We have shown that the APII potencies for relaxation of :~-adrenoceptor contractures were 10-fold higher in the pulmonary artery than in the three different segments of the aorta. Our experiments have unmasked tissue-selective and qualitatively different interactions between APII and adrenoceptor activated responses in the pulmonary artery and the ascending aorta. Taking into account that the pulmonary APII vasodilation was substantially enhanced by previous exposure to VIP and the fl2-adrenoceptor agonist,
290 the observed high pD 2 value for A P I I indicates a functional role for A P I I in regulating the tension of the pulmonary artery. In the ascending aorta A P I I inhibited the subsequent ~-adrenoceptor elicited contractures at normal extracellular Ca 2 +. This is analogous to the A P I I inhibitions of the N A contracture in the rabbit thoracic aorta seen at low extracellular Ca 2 + concentration [27], although longer lasting than so far reported for the rabbit aorta [26]. The enhanced intrinsic activity of A P I I in the pulmonary artery segments previously exposed to the c A M P enhancing vasoinhibitors [41-43], VIP and the/~2-agonist suggests a synergism between c G M P - and cAMP-activated responses in this particular segment. This may possibly reflect significant differences between the pulmonary artery and the ascending aorta with respect to agonist-coupled release of Ca 2 + from the sarcoplasmic stores [44]. In vivo experiments have shown long-lasting reductions in blood-pressure by atriopeptin apart from the primary vasodilation [45]. Exogenous A P has also been shown to antagonize ~-adrenergic pressor responses in the pithed rat model [46,47]. Interaction with the sympathetic neuro effector system at several levels may thus represent a complementary mechanism for the cardiovascular effects of A P I I , as reflections of long lasting attenuation of N A contractures presently reported for the rat proximal aorta. In conclusion, our present findings are consistent with A P I I being a relaxant of the :¢-adrenoceptor contractures in the rat pulmonary artery at functionally relevant, nanomolar concentrations. In the ascending aorta, A P I I is a relaxant at 10-fold higher concentrations. The most important role for A P I I in this segment appears to be the persistent inhibition of further methoxamine and N A contractures. The significant differences in A P I I responses in the various segments of the rat aorta may have bearing on the complex embryological origin of this vessel [48].
Acknowledgements This work has been supported by grants from the National Council on Cardiovascular Diseases.
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