- Email: [email protected]
Relaxation of isolated bovine coronary arteries by vasoactive intestinal peptide
European Journal of Pharmacology, 181 (1990) 199-205
199
Elsevier EJP 51333
Relaxation of isolated bovine coronary arteries by vasoactive intestinal peptide Hiroo Itoh 1, Karl P. Lederis and Otto P. Rorstad Endocrine Research Group, Department of Medicine, Department of Pharmacology and Therapeutics, The University of Calgary, Calgary, Alberta, Canada T2N 4N1
Received 27 November 1989, revised MS received 5 March 1990, accepted 13 March 1990
The relaxant action of vasoactive intestinal peptide (VIP) was investigated using helical strips of four major branches of bovine coronary arteries. The concentration of VIP causing 50 percent of maximal relaxation ranged from 23 to 90 nM. Preincubation of arterial strips with VIP shifted the concentration-response curves for contractions elicited by potassium chloride or prostaglandin F2a to the right. The relaxant effect of VIP was retained following removal of the vascular endothelium or in the absence of extracellular calcium. The structurally homologous peptides porcine and human peptide histidine isoleucine (PHI) were less potent than was VIP. It is concluded that there are functional receptors for VIP in bovine coronary arteries. VIP (vasoactive intestinal peptide); PHI (peptide histidine isoleucine); Coronary artery; Vasorelaxation
1. Introduction The 28 residue neuropeptide, vasoactive intestinal peptide (VIP), was originally isolated on the basis of its systemic vasodilator action (Said and Mutt, 1970). A number of subsequent studies have indicated that VIP may be a physiologically relevant vasoactive neurotransmitter (Bevan and Brayden, 1987). Anatomical and physiological studies have suggested a role for VIP as an endogenous coronary vasodilator. Coronary arteries are accompanied by VIP-immunoreactive nerves (Della et al., 1983; Forssmann et al., 1988; Reinecke and Forssmann, 1984; Weihe et al., 1984). Systemic administration of VIP induces an increase in coronary blood flow in dogs (Blitz and Charbon,
1 Present address: Department of Medicine, Mie Prefecture Shima Hospital, Mie, Japan. Correspondence: O.P. Rorstad, Department of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
1983; Smitherman et al., 1982; Unverferth et al., 1985) and humans (Smitherman et al., 1989). In previous studies of isolated dog and porcine coronary arteries, comparatively weak relaxation to VIP has been observed (Unverferth et al., 1985) or concentration-response curves have not been generated, perhaps due to tachyphylaxis to VIP (Beny et al., 1986; Forssmann et al., 1988). Specific receptors for VIP have been demonstrated on bovine coronary arteries by radioligand binding techniques (Huang and Rorstad, 1987). In the present study, an attempt has been made to characterize the relaxant effect of VIP and the homologous porcine and h u m a n peptide histidine isoleucine (PHI) peptides on several major branches of isolated bovine coronary arteries. Considering that calcium ions m a y play a role in the action of VIP in other tissues (Bjoro et al., 1987; Bolton et al., 1981; Brostrom et al., 1983; Etgen and Browning, 1987; Prysor-Jones et al., 1987), we investigated the effect of removal of extracellular calcium on VIP-induced relaxation.
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
200 T h e d e p e n d e n c y of V I P - i n d u c e d r e l a x a t i o n o n the e n d o t h e l i u m was e x a m i n e d in the light of the different results r e p o r t e d in the literature a m o n g a variety of a n i m a l species a n d arterial sources ( S a t a et al., 1986; 1988).
T h e arterial strips were allowed to e q u i l i b r a t e for 90 m i n p r i o r to s t a r t i n g e x p e r i m e n t s . D u r i n g the e q u i l i b r a t i o n period, the b a t h s o l u t i o n was c h a n g e d every 20 min. T h e c h a n g e s in a r t e r i a l t e n s i o n were r e c o r d e d i s o m e t r i c a l l y with a f o r c e - d i s p l a c e m e n t t r a n s d u c e r ( S t a t h a m No. U C 2 ) a n d r e c o r d e d on a polygraph. Calcium-free Krebs-Henseleit solution was m a d e b y a d d i n g 1 m M E G T A i n s t e a d of 2.5 m M CaC12. T h e r e l a x a n t effect of V I P was s t u d i e d after c o n t r a c t i n g arterial strips w i t h p r o s t a g l a n d i n F2~ ( P G F 2 , ) , or KC1 as p r e v i o u s l y d e s c r i b e d (Suzuki et al., 1984). These agents p r o d u c e d an increase in tension which was stable d u r i n g the e x p e r i m e n t a l period. E a c h agent was a d d e d to the tissue b a t h in a c u m u l a t i v e m a n n e r to g e n e r a t e a c o n c e n t r a t i o n r e s p o n s e curve, unless i n d i c a t e d otherwise. C a r e was t a k e n to ensure t h a t the r e s p o n s e to a p a r t i c u lar agent h a d stabilized b e f o r e a d d i n g the subseq u e n t c o n c e n t r a t i o n . T h e r e l a x a n t effect of p e p t i d e s was expressed as a p e r c e n t a g e of the m a x i m a l r e l a x a n t r e s p o n s e of the s a m e arterial strip to 0.1 m M p a p a v e r i n e . This p r o c e d u r e perm i t t e d c o m p a r i s o n b e t w e e n the responses to different arterial strips which, w h e n expressed in
2. Materials and methods Bovine h e a r t s were o b t a i n e d f r o m a local a b a t toir a n d were k e p t in ice-cold K r e b s - H e n s e l e i t s o l u t i o n of the following c o m p o s i t i o n ( m M ) : NaC1 115; KC1 4.7; CaC12 2.5; MgC12 1.2; N a H C O 3 25; K H 2 P O 4 1.2; a n d glucose 10. T h e original o u t e r d i a m e t e r s of the arteries b e f o r e p r e p a r i n g helical strips were: left a n t e r i o r descending, 3-4 m m ; p o s t e r i o r d e s c e n d i n g , 3-4 m m ; circumflex, 5.5-7 m m ; a n d right c o r o n a r y artery, 4-5.5 mm. Bovine c o r o n a r y arterial segments were dissected a n d helical strips, 1 m m in w i d t h a n d 6 m m in length, were p r e p a r e d after r e m o v a l of fat a n d c o n n e c t i v e tissue. T h e strips were s u s p e n d e d vertically in a 3 ml o r g a n b a t h u n d e r a resting tension of 1.5 g. T h e b a t h m e d i u m was m a i n t a i n e d at 37 ° C a n d b u b b l e d with a m i x t u r e o f 95% 0 2 a n d 5% C O 2.
I
I
l
10
20
i
~
i
I
i
I
10
100
1°°I A. KCI 7s
o°
2s
0 t 5
I
L.-
40
0.1
CONCENTRATION(mM)
I
1.0
CONCENTRATION (,uM)
Fig. 1. Effect of VIP on the concentration-response curves for coronary artery contraction by KC1 (A) and PGF2~ (B). Helical strips of bovine anterior descending coronary arteries were preincubated with differing concentrations of VIP for 10 min prior to cumulative addition of increasing concentrations of KCI (A) or PGF2~ (B). Symbols refer to control (e); 10 nM VIP (©); 30 nM VIP (A); and 100 nM VIP (r,). Contractions were expressed as percentages of the maximal contractions induced by 40 mM KC1 (3.92_+ 0.16 g, mean+ S.E.M.) or 30/~M PGF2~ (4.00 :t: 0.34 g). The arterial preparations used for control and experimental groups were similar in terms of maximal contraction. Each data point represents the mean of five to eight experiments and the vertical lines indicate the S.E.M.
201 TABLE 1 Relaxation of isolated coronary arterial strips by papaverine and VIP. The relaxation by VIP was expressed as the - log IC50, defined as the concentration of VIP which caused 50% of the maximal relaxation achieved by 0.1 m M papaverine. There were no significant differences a m o n g the relaxations of the different arteries to papaverine or the IC50 of relaxation in response to VIP (P > 0.05, one way analysis of variance). In a given artery, there were no significant differences in the relaxant responses to papaverine between arteries which had been precontracted by KCI or PGF2~. Likewise, there were no significant differences in the IC50 values for VIP relaxation between arteries precontracted by KC1 or PGF2~ (P > 0.05, Students' t-test), n, n u m b e r of experiments. Artery
Left anterior Posterior descending Circumflex Right coronary
Relaxation by papaverine g (mean 5: S.E.M.) (n)
ICs0 of VIP relaxation - l o g M (mean + S.E.M.) (n)
KCl-contracted arteries
PGF2=-contracted arteries
KCl-contracted arteries
PGF2~-contracted arteries
2.91 + 0.33 3.01 + 0.44 2.97 _+0.58 2.34+0.52
3.13 + 0.32 2.96 + 0.45 2.54 4- 0.41 3.004-0.54
7.03 _+0.20 7.38 + 0.16 7.19 _ 0.19 7.044-0.28
7.41 4- 0.14 7.64 _+0.80 7.30 4- 0.10 7.50+0.03
(5) (5) (5) (6)
(8) (7) (5) (5)
terms of absolute tension measurements, displayed greater variability between experiments. Endothelial cells were removed by gently rubbing the strips for 1 min, with intimal surface down, over a sheet of filter paper wetted with Krebs-Henseleit solution (Furchgott and Zawadzld, 1980). Statisti-
(5) (5) (5) (6)
(8) (7) (5) (5)
cal analysis was by one way analysis of variance of Student's t-test as appropriate. VIP was purchased from Bachem Inc. (lot R7124). pPHI (porcine peptide histidine isoleucine), hPHI (human peptide histidine isoleucine) and secretin were obtained from Peninsula
,
~ !
.
,
.
,
o-4
.
°
A.
B.
Z
_o
50
X W IT-
100
0
8
7 CONCENTRATION
6 OF
0 PEPTIDES
8
7
6
(-Iog[Ml)
Fig. 2. Relaxation of isolated helical strips of bovine left anterior descending coronary artery by VIP (O), pPHI (A), hPHI (Ix) and secretin (o). Arterial tension had been raised by pretreatment with 16 m M KC1 (A) or 3 jaM PGF2= (B). The relaxant response to 0.1 m M papaverine of KCl-contracted arteries was 2.71 + 0.28 g (mean + S.E.M., n = 12) and of PGF2=-contracted arteries was 2.84 + 0.24 g (n = 16). Relaxations by VIP and the homologous peptides were normalized as percentages of the relaxation responses to 0.1 m M papaverine. The n u m b e r of experiments were: (a) KCI: VIP 5, pPHI 5, hPHI 2, and (b) PGF2~: VIP 8, pPHI 6, hPHI 2. Secretin at the only high concentration tested had no relaxant action. Each data point is the m e a n and the vertical lines indicate the S.E.M.
202
Laboratories. Papaverine, acetylcholine and PGF2~ were from Sigma Chemical Co. Nifedipine was from the Bayer Co.
tO min r ~ a ~ /
.IFEDIPINE 10-6M 10"8 • ~
,
VW 3x10 "8 v
10"7 M v
PAPAVERINE
10"4M •
o
Ca2÷.!REE 3;~0~6R[qlPGF2~
3. Results
The isolated bovine coronary artery preparations were highly responsive to both the contracting agents, KCI and PGF2~ (fig. 1). Based on the concentration-response curves, 16 mM KC1 and 3 /tM PGF2, were selected as appropriate for precontraction of the arterial strips. VIP and papaverine relaxed the precontracted coronary artery strips (table 1, fig. 2). There was no tachyphylaxis to VIP. The concentration-response curves for contraction of the left anterior descending coronary artery by KC1 and PGF2,~were shifted to the right by preincubation of the arterial strip with 10 to 100 nM VIP (fig. 1). Four branches of the bovine coronary artery, namely the left anterior descending, the posterior descending, the circumflex, and the right coronary branches, all relaxed equally to papaverine or VIP (table 1). Use of either 16 mM KC1 or 3 /~M PGF2, was comparable in terms of establishing an augmented tension from which the relaxant effect of VIP could be demonstrated (table 1). Among the four coronary artery branches studied, the ICs0 values of relaxation by VIP ranged from 23 to 90 nM (table 1). VIP relaxed helical strips of the left anterior descending artery with equal ICs0 values in the presence or absence of a functional endothelium (table 2). In contrast, relaxation by acetylcholine was eliminated by removal of the TABLE 2 Effect of removal of endothelium on VIP-induced relaxation of bovine anterior descending coronary arteries. The basal tensions of the arterial strips induced by 3 /~M PGF2a were 3.13 4-0.32 g (mean 4- S.E.M.) with intact endothelium and 2.70 +_0.35 g with the endothelium removed. Acetylcholine (1 /~M) decreased the tension induced by 3 /~M PGF2,~ by 4 8 _ 8 % (mean ± S.E.M.) in the presence of intact endothelium. Endothelium
n
Relaxation by 1 ~ M acetylcholine
ICso of VIP relaxation - l o g M (mean + S.E.M.)
Present Absent
8 6
Present Absent
7.41+0,14 7.434-0,21
z
g
100
L-(C o"
L
,
8
,
7 -log IM]
Fig. 3. Relaxation by VIP of isolated helical strips of bovine left anterior descending coronary arteries preincubated for 10 rain in a calcium-free Krebs-Henseleit solution containing 1 m M E G T A then contracted with 3 jaM PGF2,. The relaxation induced by 0.1 m M papaverine was 0.56+0.06 g (mean_+ S.E.M., n = 6). The tracing of a representative experiment shows that 1/LM nifedipine failed to cause relaxation.
endothelium, pPHI and hPHI also relaxed the left anterior descending coronary artery, but were less potent than VIP (fig. 2). Secretin had no effect on coronary artery tension. Use of a calcium-free solution, which prevented a vascular response to 1 /xM nifedipine, did not inhibit relaxation by VIP (ICs0 of 50 nM) (fig. 3).
4. Discussion
VIP immunoreactive nerve fibers have been identified in the adventitial-medial layers of coronary arteries of the dog, rat, guinea pig and tupaia (Della et al., 1983; Forssmann et al., 1988; Reinecke and Forssmann, 1984; Weihe et al., 1984). Bruin et al. (1986) were able to measure VIP-like immunoreactivity by radioimmunoassay in the main branches of dog coronary arteries. In contrast, Taguchi et al. (1986) identified few VIP immunoreactive fibers in the left anterior descending coronary artery of the dog. U d d m a n et al. (1981) found that VIP immunoreactive nerves were
203 absent in cat coronary arteries. Forsgren (1989) identified VIP-immmunoreactive nerve fibers associated with bovine sinoatrial and atrioventricular nodes and in the walls of cardiac arteries. Previous studies have demonstrated that VIP has significant effects on cardiac function, including coronary dilation and stimulation of heart rate and contraction. Intravenous administration of VIP or direct infusion of the peptide into coronary arteries induces coronary dilation in the dog (Smitherman et al., 1982; Brum et al., 1986) and human (Smitherman et al., 1989). Several investigators have directly measured an increase in dog coronary artery blood flow in response to intravenous administration of VIP (Blitz and Charbon, 1983; Smitherman et al., 1982; Unverferth et al., 1985; Anderson et al., 1988). Although systemically administered VIP reduces blood pressure and increases flood flow to a number of organs, it appears to have a preferential effect in enhancing flow to the heart over other organs (Smitherman et al., 1982; Unverferth et al., 1985). Using the guinea pig isolated heart, Bachelard et al. (1986) observed a reduction of the myocardial perfusion pressure by VIP, which was interpreted to be due to dilation of the coronary vasculature. A small number of studies have examined the effect of VIP on isolated coronary arteries. Unverferth et al. (1985) observed that VIP relaxed dog anterior descending coronary arteries which had been precontracted with KC1. Beny et al. (1986) and Forssmann et al. (1988) showed that VIP relaxed tings or helical strips of porcine coronary arteries precontracted by 10 /~M acetylcholine. In comparison with the present study, bovine coronary arteries were more responsive to VIP than were dog coronary arteries (Unverferth et al., 1985). Furthermore, bovine coronary arteries did not exhibit the marked tachyphylaxis to VIP that has been observed with porcine coronary arteries (Beny et al., 1986; Huang et al., 1989). This characteristic permitted generation of concentration-response curves utilizing cumulatively increasing concentrations of VIP in the present study. The demonstration that VIP relaxed the main branches of bovine coronary arteries is pertinent in light of the presence of VIP receptors and
VIP-responsive adenylate cyclase in arteries from this species (Huang and Rorstad, 1984; 1987). The VIP receptor has also been localized to the medial layer of bovine arteries by autoradiography (Poulin et al., 1986). Studies of the endothelial dependence of VIP vasorelaxation in arteries other than the coronary have generated differing results depending on the animal species and the anatomical site (Sata et al., 1986; 1988). Beny et al. (1986) and Forssmann et al. (1988) showed that VIP relaxation of porcine coronary arteries was independent of the endothelium. In the bovine coronary arteries used in the present study, VIP relaxation was also retained in the absence of a functional endothelium. The vasodilator effect of VIP was not dependent on the presence of extracellular calcium. Therefore, it appears unlikely that VIP would act significantly on plasma membrane calcium channels to account for its relaxant action on coronary arteries. The homologous PHI peptides also have vasodilator actions (reviewed in Huang et al., 1989). In the majority of studies, the PHI peptides have shown lower potency than VIP, including observations on segments of bovine cerebral arteries (Suzuki et al., 1984), bovine pulmonary arteries (Greenberg et al., 1987), porcine basilar artery (Suzuki et al., 1984), cat middle cerebral artery (Edvinsson and McCulloch, 1985), and human celiac and pulmonary arteries (Thom et al., 1987). The present study demonstrating concentrationresponse curves for PHI peptides of lower potency compared to that of VIP agrees with the lower potency of the PHI peptides for binding to the bovine vascular receptor (Huang and Rorstad, 1987) and for stimulating bovine vascular adenylate cyclase (Huang and Rorstad, 1984). Currently available evidence suggests that the PHI peptides exert their vascular effects by interacting with lower affinity at the level of a VIP-preferring receptor (Huang et al., 1989), In conclusion, the present findings together with the previous study of radioligand binding (Huang and Rorstad, 1987), support the view that bovine coronary arteries possess functional receptors for VIP. The presence of perivascular VIP immunoreactive nerves of coronary arteries raises the possibility that the VIP system may play a role in
204
the regulation of coronary blood flow. The particular contribution of VIP among other vasoactive factors to the physiology of coronary circulation remains to be determined.
Acknowledgements We thank Ms. Lisa Potapoff for preparing the manuscript. This study was supported by grants from the Alberta Heart and Stroke Foundation and the Medical Research Council of Canada (MRC). H.I. was a fellow of the Alberta Heritage Foundation for Medical Research (AHFMR). K.L. is a career investigator of the MRC. O.P.R. is a medical scholar of the AHFMR.
References Anderson, F.L., A.C. Kralios, R. Herchberger and M.R. Bristow, 1988, Effect of vasoactive intestinal peptide on myocardial contractility and coronary blood flow in the dog: comparison with isoproterenol and forskolin. J. Cardiovasc. Pharmacol. 12, 365. Bachelard, H., S. St. Pierre and F. Rioux, 1986, The coronary vasodilator effect of neurotensin in the guinea pig isolated heart, Peptides 7, 431. Beny, J.-L., P.C. Brunet and H. Huggel, 1986, Effect of mechanical stimulation, substance P and vasoactive intestinal polypeptide on the electrical and mechanical activities of circular smooth muscle from pig coronary arteries contracted with acetylcholine: role of the endothelium, Pharmacology 33, 61. Bevan, J.A. and J.E. Brayden, 1987, Nonadrenergic neural vasodilator mechanisms, Circ. Res. 60, 309. Bjoro, T., B.C. Ostberg, O. Sand, J. Gordeladze, J.-G. Iversen, P.A. Torjesen, K.M. Gautvik and E. Haug, 1987, Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4C1) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca 2+, Mol. Cell Endocrinol. 49, 119. Blitz, W. and G.A. Charbon, 1983, Regional vascular influences of vasoactive intestinal peptide, Stand. J. Gastroenterol. 18, 755. Bolton, T.B., R.J. Lang and B. Ottesen, 1981, Mechanism of action of vasoactive intestinal polypeptide on myometrial smooth muscle of rabbit and guinea pig, J. Physiol. 318, 41. Brostrom, M.A., C.O. Brostrom, L.A. Brotman and S. Green, 1983, Regulation of Ca2+-dependent cyclic AMP accumulation and Ca 2+ metabolism in intact pituitary tumor cells by modulators of prolactin production, Mol. Pharmacol. 23, 399. Brum, J.M., A.A. Bove, Q. Sufan, W. Reilly and V.L.W. Go, 1986, Action and localization of vasoactive intestinal
peptide in the coronary circulation: evidence for nonadrenergic, noncholinergic coronary regulation, J. Am. Coll. Cardiol. 7, 406. Della, N.G., R.E. Papka, J.B. Furness and M. Costa, 1983, Vasoactive intestinal peptide-like immunoreactivity in nerves associated with the cardiovascular system of guinea-pigs, Neuroscience 9, 605. Edvinsson, L. and L. McCulloch, 1985, Distribution and vasomotor effects of peptide HI (PHI) in feline cerebral blood vessels in vitro and in situ, Reg. Pept. 10, 345. Etgen, A.M. and E.T. Browning, 1987, Calcium dependence of vasoactive intestinal peptide-stimulated cyclic AMP accumulation in rat hippocampal slices, J. Neurochem. 49, 303. Forsgren, S., 1989, Vasoactive intestinal polypeptide-like immunoreactivity in the bovine heart: high degree of coexistence with neuropeptide Y-like immunoreactivity, Cell Tissue Res. 256, 125. Forssmann, W.G., J. Triepel, C. Daffner, C. Heym, P. Cuevas, M.I.M. Noble and N. Yanaihara, 1988, Vasoactive intestinal peptide in the heart, Ann. N.Y. Acad. Sci. 527, 405. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial ceils in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Greenberg, B., K. Rhoden and P.J. Barnes, 1987, Relaxant effects of vasoactive intestinal peptide and peptide histidine isoleucine in human and bovine pulmonary arteries, Blood Vessels 24, 45. Huang, M. and O.P. Rorstad, 1984, Cerebral vascular adenylate cyclase: evidence for coupling to receptors for vasoactive intestinal peptide and parathyroid hormone, J. Neurochem. 43, 849. Huang, M., H. Itoh, K. Lederis and O.P. Rorstad, 1989, Evidence that the vascular actions of PHI are mediated by a VIP-preferring receptor, Peptides 10, 993. Huang, M. and O.P. Rorstad, 1987, VIP receptors in mesenteric and coronary arteries: a radioligand binding study, Peptides 8, 477. Poulin, P., Y. Suzuki, K. Lederis and O.P. Rorstad, 1986, Autoradiographic localization of binding sites for vasoactive intestinal peptide (VIP) in bovine cerebral arteries, Brain Res. 381, 382. Prysor-Jones, R.A., J.J. Silverlight and J.S. Jenkins, 1987, Vasoactive intestinal peptide increases intracellular free calcium in rat and human pituitary tumour cells in vitro, J. Endocrinol. 114, 119. Reinecke, M. and W.G. Forssmann, 1984, Regulatory peptides (SP, NT, VIP, PHI, ENK) of autonomic nerves in the guinea pig heart, Clin. Exper. Hypert. A6, 1867. Said, S.I. and V. Mutt, 1970, Potent peripheral and splanchnic vasodilator peptide from normal gut, Nature 225, 863. Sata, T., H.P. Misra, E. Kubota and S.I. Said, 1986, Vasoactive intestinal polypeptide relaxes pulmonary artery by an endothelium-independent mechanism, Peptides 7 (Suppl. 1), 225. Sata, T., J. Linden, L.-W. Liu, E. Kubota and S.I. Said, 1988, Vasoactive intestinal peptide evokes endothelium-dependent relaxation and cyclic AMP accumulation in rat aorta, Peptides 9, 853.
205 Smitherman, T.C., J.J. Popma, S.I. Said, G.J. Krejs and G.J. Dehmer, 1989, Coronary hemodynamic effects of intravenous vasoactive intestinal peptide in humans, Am. J. Physiol. 257, H1254. Smitherman, T.C., H. Saiko, A.M. Geumei, T. Yoshida, M. Oyamada and S.I. Said, 1982, Coronary vasodilator action of VIP, in: Vasoactive Intestinal Peptide, ed. S.I. Said (Raven Press, New York), p. 169. Suzuki, Y., D. McMaster, K. Lederis and O.P. Rorstad, 1984, Characterization of the relaxant effects of vasoactive intestinal peptide (VIP) and PHI on isolated brain arteries, Brain Res. 322, 9. Taguchi, T., Y. Ishii, F. Matsubara and K. Tanaka, 1986, Intimal thickening and the distribution of vasomotor nerves in the mechanically injured dog coronary artery, Exp. Mol. Pathol. 44, 138.
Thom, S., A. Hughes, G. Martin and P.S. Sever, 1987, Endothelium-dependent relaxation in isolated human arteries and veins, Clin. Sci. 73, 547. Uddman, R., J. Alumets, L. Edvinsson, R. Hakanson and F. Sundler, 1981, VIP nerve fibres around peripheral blood vessels, Acta. Physiol. Scand. 112, 65. Unverferth, D., T.M. O'Dorisio, W.W. Muir, J. White, M.M. Miller, R.L. Hamlin and R.D. Magorien, 1985, Effect of vasoactive intestinal polypeptide on the canine cardiovascular system, J. Lab. Clin. Med. 106, 542. Weihe, E., M. Reinecke and W.G. Forssmann, 1984, Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart. Interrelation with neurotensin- and substance P-like immunoreactive nerves, Cell Tiss. Res. 236, 527.
199
Elsevier EJP 51333
Relaxation of isolated bovine coronary arteries by vasoactive intestinal peptide Hiroo Itoh 1, Karl P. Lederis and Otto P. Rorstad Endocrine Research Group, Department of Medicine, Department of Pharmacology and Therapeutics, The University of Calgary, Calgary, Alberta, Canada T2N 4N1
Received 27 November 1989, revised MS received 5 March 1990, accepted 13 March 1990
The relaxant action of vasoactive intestinal peptide (VIP) was investigated using helical strips of four major branches of bovine coronary arteries. The concentration of VIP causing 50 percent of maximal relaxation ranged from 23 to 90 nM. Preincubation of arterial strips with VIP shifted the concentration-response curves for contractions elicited by potassium chloride or prostaglandin F2a to the right. The relaxant effect of VIP was retained following removal of the vascular endothelium or in the absence of extracellular calcium. The structurally homologous peptides porcine and human peptide histidine isoleucine (PHI) were less potent than was VIP. It is concluded that there are functional receptors for VIP in bovine coronary arteries. VIP (vasoactive intestinal peptide); PHI (peptide histidine isoleucine); Coronary artery; Vasorelaxation
1. Introduction The 28 residue neuropeptide, vasoactive intestinal peptide (VIP), was originally isolated on the basis of its systemic vasodilator action (Said and Mutt, 1970). A number of subsequent studies have indicated that VIP may be a physiologically relevant vasoactive neurotransmitter (Bevan and Brayden, 1987). Anatomical and physiological studies have suggested a role for VIP as an endogenous coronary vasodilator. Coronary arteries are accompanied by VIP-immunoreactive nerves (Della et al., 1983; Forssmann et al., 1988; Reinecke and Forssmann, 1984; Weihe et al., 1984). Systemic administration of VIP induces an increase in coronary blood flow in dogs (Blitz and Charbon,
1 Present address: Department of Medicine, Mie Prefecture Shima Hospital, Mie, Japan. Correspondence: O.P. Rorstad, Department of Medicine, 3330 Hospital Drive N.W., Calgary, Alberta, Canada T2N 4N1.
1983; Smitherman et al., 1982; Unverferth et al., 1985) and humans (Smitherman et al., 1989). In previous studies of isolated dog and porcine coronary arteries, comparatively weak relaxation to VIP has been observed (Unverferth et al., 1985) or concentration-response curves have not been generated, perhaps due to tachyphylaxis to VIP (Beny et al., 1986; Forssmann et al., 1988). Specific receptors for VIP have been demonstrated on bovine coronary arteries by radioligand binding techniques (Huang and Rorstad, 1987). In the present study, an attempt has been made to characterize the relaxant effect of VIP and the homologous porcine and h u m a n peptide histidine isoleucine (PHI) peptides on several major branches of isolated bovine coronary arteries. Considering that calcium ions m a y play a role in the action of VIP in other tissues (Bjoro et al., 1987; Bolton et al., 1981; Brostrom et al., 1983; Etgen and Browning, 1987; Prysor-Jones et al., 1987), we investigated the effect of removal of extracellular calcium on VIP-induced relaxation.
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
200 T h e d e p e n d e n c y of V I P - i n d u c e d r e l a x a t i o n o n the e n d o t h e l i u m was e x a m i n e d in the light of the different results r e p o r t e d in the literature a m o n g a variety of a n i m a l species a n d arterial sources ( S a t a et al., 1986; 1988).
T h e arterial strips were allowed to e q u i l i b r a t e for 90 m i n p r i o r to s t a r t i n g e x p e r i m e n t s . D u r i n g the e q u i l i b r a t i o n period, the b a t h s o l u t i o n was c h a n g e d every 20 min. T h e c h a n g e s in a r t e r i a l t e n s i o n were r e c o r d e d i s o m e t r i c a l l y with a f o r c e - d i s p l a c e m e n t t r a n s d u c e r ( S t a t h a m No. U C 2 ) a n d r e c o r d e d on a polygraph. Calcium-free Krebs-Henseleit solution was m a d e b y a d d i n g 1 m M E G T A i n s t e a d of 2.5 m M CaC12. T h e r e l a x a n t effect of V I P was s t u d i e d after c o n t r a c t i n g arterial strips w i t h p r o s t a g l a n d i n F2~ ( P G F 2 , ) , or KC1 as p r e v i o u s l y d e s c r i b e d (Suzuki et al., 1984). These agents p r o d u c e d an increase in tension which was stable d u r i n g the e x p e r i m e n t a l period. E a c h agent was a d d e d to the tissue b a t h in a c u m u l a t i v e m a n n e r to g e n e r a t e a c o n c e n t r a t i o n r e s p o n s e curve, unless i n d i c a t e d otherwise. C a r e was t a k e n to ensure t h a t the r e s p o n s e to a p a r t i c u lar agent h a d stabilized b e f o r e a d d i n g the subseq u e n t c o n c e n t r a t i o n . T h e r e l a x a n t effect of p e p t i d e s was expressed as a p e r c e n t a g e of the m a x i m a l r e l a x a n t r e s p o n s e of the s a m e arterial strip to 0.1 m M p a p a v e r i n e . This p r o c e d u r e perm i t t e d c o m p a r i s o n b e t w e e n the responses to different arterial strips which, w h e n expressed in
2. Materials and methods Bovine h e a r t s were o b t a i n e d f r o m a local a b a t toir a n d were k e p t in ice-cold K r e b s - H e n s e l e i t s o l u t i o n of the following c o m p o s i t i o n ( m M ) : NaC1 115; KC1 4.7; CaC12 2.5; MgC12 1.2; N a H C O 3 25; K H 2 P O 4 1.2; a n d glucose 10. T h e original o u t e r d i a m e t e r s of the arteries b e f o r e p r e p a r i n g helical strips were: left a n t e r i o r descending, 3-4 m m ; p o s t e r i o r d e s c e n d i n g , 3-4 m m ; circumflex, 5.5-7 m m ; a n d right c o r o n a r y artery, 4-5.5 mm. Bovine c o r o n a r y arterial segments were dissected a n d helical strips, 1 m m in w i d t h a n d 6 m m in length, were p r e p a r e d after r e m o v a l of fat a n d c o n n e c t i v e tissue. T h e strips were s u s p e n d e d vertically in a 3 ml o r g a n b a t h u n d e r a resting tension of 1.5 g. T h e b a t h m e d i u m was m a i n t a i n e d at 37 ° C a n d b u b b l e d with a m i x t u r e o f 95% 0 2 a n d 5% C O 2.
I
I
l
10
20
i
~
i
I
i
I
10
100
1°°I A. KCI 7s
o°
2s
0 t 5
I
L.-
40
0.1
CONCENTRATION(mM)
I
1.0
CONCENTRATION (,uM)
Fig. 1. Effect of VIP on the concentration-response curves for coronary artery contraction by KC1 (A) and PGF2~ (B). Helical strips of bovine anterior descending coronary arteries were preincubated with differing concentrations of VIP for 10 min prior to cumulative addition of increasing concentrations of KCI (A) or PGF2~ (B). Symbols refer to control (e); 10 nM VIP (©); 30 nM VIP (A); and 100 nM VIP (r,). Contractions were expressed as percentages of the maximal contractions induced by 40 mM KC1 (3.92_+ 0.16 g, mean+ S.E.M.) or 30/~M PGF2~ (4.00 :t: 0.34 g). The arterial preparations used for control and experimental groups were similar in terms of maximal contraction. Each data point represents the mean of five to eight experiments and the vertical lines indicate the S.E.M.
201 TABLE 1 Relaxation of isolated coronary arterial strips by papaverine and VIP. The relaxation by VIP was expressed as the - log IC50, defined as the concentration of VIP which caused 50% of the maximal relaxation achieved by 0.1 m M papaverine. There were no significant differences a m o n g the relaxations of the different arteries to papaverine or the IC50 of relaxation in response to VIP (P > 0.05, one way analysis of variance). In a given artery, there were no significant differences in the relaxant responses to papaverine between arteries which had been precontracted by KCI or PGF2~. Likewise, there were no significant differences in the IC50 values for VIP relaxation between arteries precontracted by KC1 or PGF2~ (P > 0.05, Students' t-test), n, n u m b e r of experiments. Artery
Left anterior Posterior descending Circumflex Right coronary
Relaxation by papaverine g (mean 5: S.E.M.) (n)
ICs0 of VIP relaxation - l o g M (mean + S.E.M.) (n)
KCl-contracted arteries
PGF2=-contracted arteries
KCl-contracted arteries
PGF2~-contracted arteries
2.91 + 0.33 3.01 + 0.44 2.97 _+0.58 2.34+0.52
3.13 + 0.32 2.96 + 0.45 2.54 4- 0.41 3.004-0.54
7.03 _+0.20 7.38 + 0.16 7.19 _ 0.19 7.044-0.28
7.41 4- 0.14 7.64 _+0.80 7.30 4- 0.10 7.50+0.03
(5) (5) (5) (6)
(8) (7) (5) (5)
terms of absolute tension measurements, displayed greater variability between experiments. Endothelial cells were removed by gently rubbing the strips for 1 min, with intimal surface down, over a sheet of filter paper wetted with Krebs-Henseleit solution (Furchgott and Zawadzld, 1980). Statisti-
(5) (5) (5) (6)
(8) (7) (5) (5)
cal analysis was by one way analysis of variance of Student's t-test as appropriate. VIP was purchased from Bachem Inc. (lot R7124). pPHI (porcine peptide histidine isoleucine), hPHI (human peptide histidine isoleucine) and secretin were obtained from Peninsula
,
~ !
.
,
.
,
o-4
.
°
A.
B.
Z
_o
50
X W IT-
100
0
8
7 CONCENTRATION
6 OF
0 PEPTIDES
8
7
6
(-Iog[Ml)
Fig. 2. Relaxation of isolated helical strips of bovine left anterior descending coronary artery by VIP (O), pPHI (A), hPHI (Ix) and secretin (o). Arterial tension had been raised by pretreatment with 16 m M KC1 (A) or 3 jaM PGF2= (B). The relaxant response to 0.1 m M papaverine of KCl-contracted arteries was 2.71 + 0.28 g (mean + S.E.M., n = 12) and of PGF2=-contracted arteries was 2.84 + 0.24 g (n = 16). Relaxations by VIP and the homologous peptides were normalized as percentages of the relaxation responses to 0.1 m M papaverine. The n u m b e r of experiments were: (a) KCI: VIP 5, pPHI 5, hPHI 2, and (b) PGF2~: VIP 8, pPHI 6, hPHI 2. Secretin at the only high concentration tested had no relaxant action. Each data point is the m e a n and the vertical lines indicate the S.E.M.
202
Laboratories. Papaverine, acetylcholine and PGF2~ were from Sigma Chemical Co. Nifedipine was from the Bayer Co.
tO min r ~ a ~ /
.IFEDIPINE 10-6M 10"8 • ~
,
VW 3x10 "8 v
10"7 M v
PAPAVERINE
10"4M •
o
Ca2÷.!REE 3;~0~6R[qlPGF2~
3. Results
The isolated bovine coronary artery preparations were highly responsive to both the contracting agents, KCI and PGF2~ (fig. 1). Based on the concentration-response curves, 16 mM KC1 and 3 /tM PGF2, were selected as appropriate for precontraction of the arterial strips. VIP and papaverine relaxed the precontracted coronary artery strips (table 1, fig. 2). There was no tachyphylaxis to VIP. The concentration-response curves for contraction of the left anterior descending coronary artery by KC1 and PGF2,~were shifted to the right by preincubation of the arterial strip with 10 to 100 nM VIP (fig. 1). Four branches of the bovine coronary artery, namely the left anterior descending, the posterior descending, the circumflex, and the right coronary branches, all relaxed equally to papaverine or VIP (table 1). Use of either 16 mM KC1 or 3 /~M PGF2, was comparable in terms of establishing an augmented tension from which the relaxant effect of VIP could be demonstrated (table 1). Among the four coronary artery branches studied, the ICs0 values of relaxation by VIP ranged from 23 to 90 nM (table 1). VIP relaxed helical strips of the left anterior descending artery with equal ICs0 values in the presence or absence of a functional endothelium (table 2). In contrast, relaxation by acetylcholine was eliminated by removal of the TABLE 2 Effect of removal of endothelium on VIP-induced relaxation of bovine anterior descending coronary arteries. The basal tensions of the arterial strips induced by 3 /~M PGF2a were 3.13 4-0.32 g (mean 4- S.E.M.) with intact endothelium and 2.70 +_0.35 g with the endothelium removed. Acetylcholine (1 /~M) decreased the tension induced by 3 /~M PGF2,~ by 4 8 _ 8 % (mean ± S.E.M.) in the presence of intact endothelium. Endothelium
n
Relaxation by 1 ~ M acetylcholine
ICso of VIP relaxation - l o g M (mean + S.E.M.)
Present Absent
8 6
Present Absent
7.41+0,14 7.434-0,21
z
g
100
L-(C o"
L
,
8
,
7 -log IM]
Fig. 3. Relaxation by VIP of isolated helical strips of bovine left anterior descending coronary arteries preincubated for 10 rain in a calcium-free Krebs-Henseleit solution containing 1 m M E G T A then contracted with 3 jaM PGF2,. The relaxation induced by 0.1 m M papaverine was 0.56+0.06 g (mean_+ S.E.M., n = 6). The tracing of a representative experiment shows that 1/LM nifedipine failed to cause relaxation.
endothelium, pPHI and hPHI also relaxed the left anterior descending coronary artery, but were less potent than VIP (fig. 2). Secretin had no effect on coronary artery tension. Use of a calcium-free solution, which prevented a vascular response to 1 /xM nifedipine, did not inhibit relaxation by VIP (ICs0 of 50 nM) (fig. 3).
4. Discussion
VIP immunoreactive nerve fibers have been identified in the adventitial-medial layers of coronary arteries of the dog, rat, guinea pig and tupaia (Della et al., 1983; Forssmann et al., 1988; Reinecke and Forssmann, 1984; Weihe et al., 1984). Bruin et al. (1986) were able to measure VIP-like immunoreactivity by radioimmunoassay in the main branches of dog coronary arteries. In contrast, Taguchi et al. (1986) identified few VIP immunoreactive fibers in the left anterior descending coronary artery of the dog. U d d m a n et al. (1981) found that VIP immunoreactive nerves were
203 absent in cat coronary arteries. Forsgren (1989) identified VIP-immmunoreactive nerve fibers associated with bovine sinoatrial and atrioventricular nodes and in the walls of cardiac arteries. Previous studies have demonstrated that VIP has significant effects on cardiac function, including coronary dilation and stimulation of heart rate and contraction. Intravenous administration of VIP or direct infusion of the peptide into coronary arteries induces coronary dilation in the dog (Smitherman et al., 1982; Brum et al., 1986) and human (Smitherman et al., 1989). Several investigators have directly measured an increase in dog coronary artery blood flow in response to intravenous administration of VIP (Blitz and Charbon, 1983; Smitherman et al., 1982; Unverferth et al., 1985; Anderson et al., 1988). Although systemically administered VIP reduces blood pressure and increases flood flow to a number of organs, it appears to have a preferential effect in enhancing flow to the heart over other organs (Smitherman et al., 1982; Unverferth et al., 1985). Using the guinea pig isolated heart, Bachelard et al. (1986) observed a reduction of the myocardial perfusion pressure by VIP, which was interpreted to be due to dilation of the coronary vasculature. A small number of studies have examined the effect of VIP on isolated coronary arteries. Unverferth et al. (1985) observed that VIP relaxed dog anterior descending coronary arteries which had been precontracted with KC1. Beny et al. (1986) and Forssmann et al. (1988) showed that VIP relaxed tings or helical strips of porcine coronary arteries precontracted by 10 /~M acetylcholine. In comparison with the present study, bovine coronary arteries were more responsive to VIP than were dog coronary arteries (Unverferth et al., 1985). Furthermore, bovine coronary arteries did not exhibit the marked tachyphylaxis to VIP that has been observed with porcine coronary arteries (Beny et al., 1986; Huang et al., 1989). This characteristic permitted generation of concentration-response curves utilizing cumulatively increasing concentrations of VIP in the present study. The demonstration that VIP relaxed the main branches of bovine coronary arteries is pertinent in light of the presence of VIP receptors and
VIP-responsive adenylate cyclase in arteries from this species (Huang and Rorstad, 1984; 1987). The VIP receptor has also been localized to the medial layer of bovine arteries by autoradiography (Poulin et al., 1986). Studies of the endothelial dependence of VIP vasorelaxation in arteries other than the coronary have generated differing results depending on the animal species and the anatomical site (Sata et al., 1986; 1988). Beny et al. (1986) and Forssmann et al. (1988) showed that VIP relaxation of porcine coronary arteries was independent of the endothelium. In the bovine coronary arteries used in the present study, VIP relaxation was also retained in the absence of a functional endothelium. The vasodilator effect of VIP was not dependent on the presence of extracellular calcium. Therefore, it appears unlikely that VIP would act significantly on plasma membrane calcium channels to account for its relaxant action on coronary arteries. The homologous PHI peptides also have vasodilator actions (reviewed in Huang et al., 1989). In the majority of studies, the PHI peptides have shown lower potency than VIP, including observations on segments of bovine cerebral arteries (Suzuki et al., 1984), bovine pulmonary arteries (Greenberg et al., 1987), porcine basilar artery (Suzuki et al., 1984), cat middle cerebral artery (Edvinsson and McCulloch, 1985), and human celiac and pulmonary arteries (Thom et al., 1987). The present study demonstrating concentrationresponse curves for PHI peptides of lower potency compared to that of VIP agrees with the lower potency of the PHI peptides for binding to the bovine vascular receptor (Huang and Rorstad, 1987) and for stimulating bovine vascular adenylate cyclase (Huang and Rorstad, 1984). Currently available evidence suggests that the PHI peptides exert their vascular effects by interacting with lower affinity at the level of a VIP-preferring receptor (Huang et al., 1989), In conclusion, the present findings together with the previous study of radioligand binding (Huang and Rorstad, 1987), support the view that bovine coronary arteries possess functional receptors for VIP. The presence of perivascular VIP immunoreactive nerves of coronary arteries raises the possibility that the VIP system may play a role in
204
the regulation of coronary blood flow. The particular contribution of VIP among other vasoactive factors to the physiology of coronary circulation remains to be determined.
Acknowledgements We thank Ms. Lisa Potapoff for preparing the manuscript. This study was supported by grants from the Alberta Heart and Stroke Foundation and the Medical Research Council of Canada (MRC). H.I. was a fellow of the Alberta Heritage Foundation for Medical Research (AHFMR). K.L. is a career investigator of the MRC. O.P.R. is a medical scholar of the AHFMR.
References Anderson, F.L., A.C. Kralios, R. Herchberger and M.R. Bristow, 1988, Effect of vasoactive intestinal peptide on myocardial contractility and coronary blood flow in the dog: comparison with isoproterenol and forskolin. J. Cardiovasc. Pharmacol. 12, 365. Bachelard, H., S. St. Pierre and F. Rioux, 1986, The coronary vasodilator effect of neurotensin in the guinea pig isolated heart, Peptides 7, 431. Beny, J.-L., P.C. Brunet and H. Huggel, 1986, Effect of mechanical stimulation, substance P and vasoactive intestinal polypeptide on the electrical and mechanical activities of circular smooth muscle from pig coronary arteries contracted with acetylcholine: role of the endothelium, Pharmacology 33, 61. Bevan, J.A. and J.E. Brayden, 1987, Nonadrenergic neural vasodilator mechanisms, Circ. Res. 60, 309. Bjoro, T., B.C. Ostberg, O. Sand, J. Gordeladze, J.-G. Iversen, P.A. Torjesen, K.M. Gautvik and E. Haug, 1987, Vasoactive intestinal peptide and peptide with N-terminal histidine and C-terminal isoleucine increase prolactin secretion in cultured rat pituitary cells (GH4C1) via a cAMP-dependent mechanism which involves transient elevation of intracellular Ca 2+, Mol. Cell Endocrinol. 49, 119. Blitz, W. and G.A. Charbon, 1983, Regional vascular influences of vasoactive intestinal peptide, Stand. J. Gastroenterol. 18, 755. Bolton, T.B., R.J. Lang and B. Ottesen, 1981, Mechanism of action of vasoactive intestinal polypeptide on myometrial smooth muscle of rabbit and guinea pig, J. Physiol. 318, 41. Brostrom, M.A., C.O. Brostrom, L.A. Brotman and S. Green, 1983, Regulation of Ca2+-dependent cyclic AMP accumulation and Ca 2+ metabolism in intact pituitary tumor cells by modulators of prolactin production, Mol. Pharmacol. 23, 399. Brum, J.M., A.A. Bove, Q. Sufan, W. Reilly and V.L.W. Go, 1986, Action and localization of vasoactive intestinal
peptide in the coronary circulation: evidence for nonadrenergic, noncholinergic coronary regulation, J. Am. Coll. Cardiol. 7, 406. Della, N.G., R.E. Papka, J.B. Furness and M. Costa, 1983, Vasoactive intestinal peptide-like immunoreactivity in nerves associated with the cardiovascular system of guinea-pigs, Neuroscience 9, 605. Edvinsson, L. and L. McCulloch, 1985, Distribution and vasomotor effects of peptide HI (PHI) in feline cerebral blood vessels in vitro and in situ, Reg. Pept. 10, 345. Etgen, A.M. and E.T. Browning, 1987, Calcium dependence of vasoactive intestinal peptide-stimulated cyclic AMP accumulation in rat hippocampal slices, J. Neurochem. 49, 303. Forsgren, S., 1989, Vasoactive intestinal polypeptide-like immunoreactivity in the bovine heart: high degree of coexistence with neuropeptide Y-like immunoreactivity, Cell Tissue Res. 256, 125. Forssmann, W.G., J. Triepel, C. Daffner, C. Heym, P. Cuevas, M.I.M. Noble and N. Yanaihara, 1988, Vasoactive intestinal peptide in the heart, Ann. N.Y. Acad. Sci. 527, 405. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial ceils in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Greenberg, B., K. Rhoden and P.J. Barnes, 1987, Relaxant effects of vasoactive intestinal peptide and peptide histidine isoleucine in human and bovine pulmonary arteries, Blood Vessels 24, 45. Huang, M. and O.P. Rorstad, 1984, Cerebral vascular adenylate cyclase: evidence for coupling to receptors for vasoactive intestinal peptide and parathyroid hormone, J. Neurochem. 43, 849. Huang, M., H. Itoh, K. Lederis and O.P. Rorstad, 1989, Evidence that the vascular actions of PHI are mediated by a VIP-preferring receptor, Peptides 10, 993. Huang, M. and O.P. Rorstad, 1987, VIP receptors in mesenteric and coronary arteries: a radioligand binding study, Peptides 8, 477. Poulin, P., Y. Suzuki, K. Lederis and O.P. Rorstad, 1986, Autoradiographic localization of binding sites for vasoactive intestinal peptide (VIP) in bovine cerebral arteries, Brain Res. 381, 382. Prysor-Jones, R.A., J.J. Silverlight and J.S. Jenkins, 1987, Vasoactive intestinal peptide increases intracellular free calcium in rat and human pituitary tumour cells in vitro, J. Endocrinol. 114, 119. Reinecke, M. and W.G. Forssmann, 1984, Regulatory peptides (SP, NT, VIP, PHI, ENK) of autonomic nerves in the guinea pig heart, Clin. Exper. Hypert. A6, 1867. Said, S.I. and V. Mutt, 1970, Potent peripheral and splanchnic vasodilator peptide from normal gut, Nature 225, 863. Sata, T., H.P. Misra, E. Kubota and S.I. Said, 1986, Vasoactive intestinal polypeptide relaxes pulmonary artery by an endothelium-independent mechanism, Peptides 7 (Suppl. 1), 225. Sata, T., J. Linden, L.-W. Liu, E. Kubota and S.I. Said, 1988, Vasoactive intestinal peptide evokes endothelium-dependent relaxation and cyclic AMP accumulation in rat aorta, Peptides 9, 853.
205 Smitherman, T.C., J.J. Popma, S.I. Said, G.J. Krejs and G.J. Dehmer, 1989, Coronary hemodynamic effects of intravenous vasoactive intestinal peptide in humans, Am. J. Physiol. 257, H1254. Smitherman, T.C., H. Saiko, A.M. Geumei, T. Yoshida, M. Oyamada and S.I. Said, 1982, Coronary vasodilator action of VIP, in: Vasoactive Intestinal Peptide, ed. S.I. Said (Raven Press, New York), p. 169. Suzuki, Y., D. McMaster, K. Lederis and O.P. Rorstad, 1984, Characterization of the relaxant effects of vasoactive intestinal peptide (VIP) and PHI on isolated brain arteries, Brain Res. 322, 9. Taguchi, T., Y. Ishii, F. Matsubara and K. Tanaka, 1986, Intimal thickening and the distribution of vasomotor nerves in the mechanically injured dog coronary artery, Exp. Mol. Pathol. 44, 138.
Thom, S., A. Hughes, G. Martin and P.S. Sever, 1987, Endothelium-dependent relaxation in isolated human arteries and veins, Clin. Sci. 73, 547. Uddman, R., J. Alumets, L. Edvinsson, R. Hakanson and F. Sundler, 1981, VIP nerve fibres around peripheral blood vessels, Acta. Physiol. Scand. 112, 65. Unverferth, D., T.M. O'Dorisio, W.W. Muir, J. White, M.M. Miller, R.L. Hamlin and R.D. Magorien, 1985, Effect of vasoactive intestinal polypeptide on the canine cardiovascular system, J. Lab. Clin. Med. 106, 542. Weihe, E., M. Reinecke and W.G. Forssmann, 1984, Distribution of vasoactive intestinal polypeptide-like immunoreactivity in the mammalian heart. Interrelation with neurotensin- and substance P-like immunoreactive nerves, Cell Tiss. Res. 236, 527.