Vasoactive intestinal peptide evokes endothelium-dependent relaxation and cyclic AMP accumulation in rat aorta

Pepttdes, Vol 9, pp 853-858 ©PergamonPress plc, 1988 Pnntedm the U S.A

0196-9781/88$3 00 + 00

Vasoactive Intestinal Peptide Evokes Endothelium-Dependent Relaxation and Cyclic AMP Accumulation in Rat Aorta TAKEYOSHI SATA, I JOEL LINDEN/LE-WEN E T S U J I K U B O T A 3 A N D S A M I I. S A I D 4

LIU,

D e p a r t m e n t o f Medicine, University o f Illinois College o f Medicine and West Side VA Medical Center, Chicago, IL 60612 R e c e i v e d 27 N o v e m b e r 1987 SATA, T , J LINDEN, L.-W L1U, E. KUBOTA AND S 1 SAID Vasoacme mte~tmalpepttde evo~e~ endothehumdependent relaxatton and cychc AMP a¢cumulatton tn rat aorta. PEPTIDES 9(4) 853-858, 1988 --We have investigated V1P-mduced relaxation and cychc AMP accumulation m rat thoracic aorta strips, and the importance of endothehum to both actions The relaxation was greatly attenuated by removal ofendothehum, but was unaltered by cyclo-oxygenase or hpoxygenase mhlbltors Similarly, cychc AMP formatmn was nearly abohshed with loss of endothehum, but was largely unaffected by mhlbltors of arachidonate pathways, cytochrome P450 or guanylate cyclase VIP may stimulate the release of a diffusible factor from endothehum (an EDRI~), which activates adenylate cyclase and relaxes aortic smooth muscle EDRF

Vascular smooth muscle

Vasoddatlon

VIP

VASOACT1VE intestinal peptide (V1P), a widely distributed neuropepttde, relaxes vascular smooth muscle of the systemic and pulmonary circulations. The vasodllator effect of VIP Is independent of adrenergic, cholinergic, serotonergic, or hlstamlnergic receptors and, for the most part, ~s not mediated by cyclo-oxygenase metabolites [29]. One possible mechanism by which VIP might b n n g about vascular relaxatlon is through elevation of cychc AMP levels in vascular smooth muscle cells. A close correlation between VIPinduced relaxation and cyclic AMP accumulation has been observed in isolated rat aorta [31] and in cat cerebral artery [8]. It is not clear, however, whether VIP relaxes vascular smooth muscle cells by a direct action on these cells or by stimulating the release of another substance which then relaxes smooth muscle cells. Endothelium plays an obligatory role in the relaxation of vascular smooth muscle in response to acetylchohne (Ach) and other vasodilators [9,10]. These vasodilators stimulate endothelium to release an Endothelium-Derived Relaxing Factor (EDRF) which diffuses to and relaxes vascular smooth muscle through cyclic GMP accumulation [9--11]. The present experiments were designed (a) to examine

whether VIP relaxes rat aorta smooth muscle by releasing a diffusible factor from endothellum, and (b) if so, to characterize this factor. METHOD

Preparation of Rat Thoract~ Aorta Strips and Relaxation Experiment~ Rats (Sprague-Dawley, male, 230-350 g) were euthanized and their thoracic aortas carefully removed without stretchmg or lnjunng the mtIma. The aortas were immersed m ~ce-cold Krebs tOnger Buffer solution (KRB), containing in raM: NaC1 117.6, KC1 5.4, MgSO4 1.2, NaHCO.~ 25.0, NaHzPO4 1.0, glucose 11.1 and CaCI2 2.5. Each aorta was cleaned of any adherent fat or connective tissue, cut along its length with Wescott tenotomy scissors, and further cut into rmgs. Each preparation was attached to an isometric force transducer (Harvard, model 363) by fine metal needles, and tension was recorded on a Beckman Type R Dynograph. Each strip was placed in a 5-ml organ bath filled with KRB equilibrated with 95% O., and 5% CO2. The solution was maintained at 37°C and exchanged continuously at a rate of 2 ml/min by an infusion

1Present address. Intensive Care Umt, Kyushu University Hospital, Fukuoka, Japan 2present address' Department of Physiology, Umverslty of Vlrgima School of Medicine, Charlottesvdle, VA 22908. 3present address Intensive Care Unit, Umverslty of Occupational & Environmental Health, Kltakyushu, Japan 4Requests for replants should be addressed to Saml I Said, Umverslty of Ilhnols, M/C 789, Chicago, IL 60612

853

854

SATA ET AL. I00

80 ¢-

._o

so

x ~) O:

40

20

I 8

I 7

-log [ VlP (M)]

I 6

FIG. 1 VIP-mduced relaxation (means±SEM) ol the rat aorta strips, w~th or without endothehum, precontracted with PGH,analog (PGH-A) (N=6) Cumulative molar concentration of VIP added is plotted against the degree of relaxation of aorta strips, expressed as percent reduction of high tension induced by PGHe-A All values of relaxation after loss of endothehum were significantly lower ~-~<0 01) than corresponding values with intact endothellum

pump (Cole-Parmer). An initial tension load of 1.0 g was applied. The strips were allowed to equilibrate for 40-60 rain The flow of the solution was stopped and contraction was elicited by the addition of 5 ng/ml of the synthetic prostaglandln He analog (PGH_,-A, 9, l l-dideoxy-llc~, 9c~-epoxymethano-prostaglandln F2c~, The Upjohn Co., Kalamazoo, M1), this concentration induced approximately 60% of the maximal contractile response to the drug. Subsequently, 6 cumulative additions of porcine VIP (Karolinska I n s t i t u t e , Stockholm, Sweden) were applied to the strips at 5-10 rain Intervals. Vascular relaxation was calculated as percent decrease of PGH.,-A-induced tone. When the endothehum was removed, it was done by gently rubbing the intimal surface with a metal spatula, with the vessel placed in a Petri dish filled with KRB at 37°C Removal of the endothelium was pharmacologically confirmed by loss of the relaxant response to Ach and to adenosine triphosphate (ATP), with preservation of the relaxant response to sodium nltroprusside (SNP) Indomethacin (Sigma Chemical Co., St Louis, MO) was dissolved in 0.1 N NaOH. Eicosatetraynoic acid (ETYA, gift of Hoffmann-La Roche, lnc , Nutley, NJ) and nordihydroguamretic acid (NDGA, Sigma) were dissolved in ethanol, the final concentration of ethanol did not exceed 0.02%. SKF-525A, a gift of Smith Kline and French (Philadelphia, PA), was dissolved in H.,O. These inhlbltors were added to organ baths after stable contractions were obtained. Tissues were then incubated for 10 mln before cumulative additions of VIP (Table 2).

of rat aorta In response to VIP. The preparations were essentially as described by Rubanyl, Vanhoutte and others [26--28]. Briefly, ring segments of rat aorta were opened and the endothelium removed as described above. One cut edge of the circumferential strip was fixed by two fine metal needles to the bottom of a vertically positioned plastic plate in 5-ml organ baths. The upper end of the strip was attached to an isometric force transducer. An initial load of 1.0 g was apphed. After a 40-60 min equilibration period, contraction was induced by PGH2-A (5 ng/ml) The removal of the endothehum was verified by loss of relaxation in response to 10 ~'M Ach. The passive tension of the circumferential strip (recipient) was then released and a longitudinal tissue with intact endothelium (donor) was placed between the recipient and the plate so that the lnttma of the recipient was closely apposed to that of the donor An initial load of 1.0 g was again applied, and the layered preparations were allowed to equlhbrate for 40-60 rain before PGH,-A was added to increase the vascular tone Because the donor tissue was not attached to a force transducer, only the tone change of the recipient aorta strip was recorded. After the tissues reached a stable contraction, 4 cumulative additions of VIP were made. The preparations were then washed for 40-60 min by continuous exchange of KRB at 2 ml/min. Contraction was then again ehclted by PGHz-A. After stabilization of the raised tone, methylene blue (5x 10-'~ M) was added, and the relaxation protocol was repeated. This concentration completely abolished the relaxant effect of 10-~ Ach In control experiments, aorta strips without endothelium were used as donors. ('y~ lt~ Ntt(leottde Measurements Ring segments of thoracic aorta with or without endothehum were prepared as described above. Each segment was incubated, with or without VIP, in 5-ml of KRB equilibrated with 95% O., and 5% CO.2 at 37°C. After 60 sec of incubation, the aorta segments were freeze-clamped between brass plates precooled in hquld nitrogen. When necessary, lnhlbltors of arachldonate metabohsm, SKF-525 A or methylene blue were added for 10 mln before and during incubation of tissues with VIP. The frozen tissues were homogenized in 1 ml of 0.1 N HCI with a tissue disruptor (Polytron, 20 sec at setting 8), and stored at -20°C. After centrifugatlon at 20,000xg for 30 min, the HCI extracts were concentrated by lyophilizatlon, resuspended in 0.5 ml of 50 mM sodium acetate, pH 4.75, and cyclic nucleotldes measured by Gammaflo automated radiotmmunoassay [4]. Pellets were dissolved in 2 ml of 2 N NaOH and their protein content assayed by fluorescence after the addition of fluorescamine (Sigma) as described by Linden et al [22]. Statism al Analy~ts All values were expressed as means+_SEM and were analyzed either by Student's t-test (Table 1, Figs. 1, 3 and 4) or by Analysis of Variance, followed by Dunnett's or Tukey's test (Table 2) [13] Differences were judged to be significant at the p < 0 05 level RESULTS

Layered Preparations These expenments were designed to demonstrate the release of a diffusible relaxing factor(s) from the endothelium

Dependence on Endothehum oJ VIP-lnduced Relaxation oJ Rat Aorta Cumulative additions of VIP caused dose-dependent de-

VIP EFFECT ON THE RAT AORTA

855

A) Endothelium Intact

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B) Endothelium Removed

lOmln -41 -S 4xlOM IOM PGH2-A Ach ATP

-e -7 1.2xlOId 6xlOM VlP 8NP

FIG. 2. RelaxaUon by VIP and other vasodflators of rat aorta' In the presence of intact endothelmm (panel A), aomc segments preconstrlcted wath PGH-A relaxed wath Ach (4× 10-~' M), ATP (10 M), VIP (7 2× 10-~ M), and SNP (5× 10_7 M) After removal of endothelmm, Ach (4× 10-6 M), ATP (10 ~ M) and VIP (1 2× 10 6 M) faded to reduce relaxatmn, but relaxant response to SNP was unaffected Vemcal line shows tension scale and horizontal lane, tame scale Similar observatmns were obtained m 6 experiments.

TABLE 1 VIP-STIMULATION OF CYCLIC AMP FORMATION IN RAT AORTA, WITH AND WITHOUT ENDOTHELIUM (MEAN _+ SEM, N=8)

100

Cychc AMP (pMol/mg protein)

80 Addmon

With Endothehum

Without Endothehum

1.94 _+ 0 19 4505_+ 540

1 67 - 0 18 3 38 +- 0 6 8

Z

_o

None V 1 P ( 3 x l 0 5M)

60

x < ._1 LU EE 4 0 o~

I 8

1 7

I 6

VIP, - l o g (M)

FIG. 3. Effects of mdomethacm (final concentration 5x 10 6 M) and ETYA (final concentration 2 5x 10-~ M) on VIP-induced relaxation of rat aorta stnps with Intact endothelium (means_+SEM) Plot as m Fig. 1. Closed circles are values for V1P alone (N = 8), squares are for VIP m the presence of ETYA (N=6), and triangles are for VIP + mdomethacln (N=6) No slgmficant difference between the 3 sets of data.

c r e a s e s o f P G H 2 - A - l n d u c e d t o n e of rat aorta. In strips w i t h i n t a c t e n d o t h e h u m , V I P elicited a m a x i m a l r e l a x a t i o n of 81-+ 5% o f the p l a t e a u t e n s i o n (0.82_+ 0.28 g). In strips w i t h o u t e n d o t h e h u m , h o w e v e r , t h e m a x i m a l r e l a x a t i o n w a s only 9_+3% (Fig. 1). R e m o v a l o f e n d o t h e l i u m did n o t significantly affect the PGH2-A-induced tone, a n d the strips r e t a i n e d their r e l a x a n t r e s p o n s e to s o d i u m n i t r o p r u s s i d e b u t n o l o n g e r rel a x e d w i t h A c h o r A T P (Fig 2). T h e viability a n d s m o o t h muscle responsiveness of endothelium denuded preparations w e r e f u r t h e r c o n f i r m e d b y r e s t o r a t i o n o f t h e i r r e s p o n s e s in l a y e r i n g e x p e r i m e n t s (see b e l o w )

lzffe~ts o f lnhtbitors o f Arachidonate Metabolism on Relaxation I n d o m e t h a c i n (5× 10-6 M) h a d n o effect o n p l a t e a u tension e v o k e d b y PGH2-A. E T Y A (2.5× 10-5 M) d e c r e a s e d P G H 2 - A - l n d u c e d t o n e b y 10-20%, w h e n t h e drug w a s a d d e d

S A T A ET AL.

856

TABLE 2 I N F L U E N C E OF VARIOUS INHIBITORS ON CYCLIC AMP A C C U M U L A T I O N I N D U C E D BY VIP IN RAT AORTA W I T H INTACT E N D O T H E L I U M (MEAN +_ SEM)

Cychc AMP (pmol/mg protein) Additions

N

-VIP

N

+VIP (3× 10 7 M)*

None Indomethacln (5× 10 ~' M) (Cyclo-oxygenase inhibitor) E T Y A ( 2 5 × I 0 ~M) (Llpoxygenase inhibitor) NDGA (5×10 ~ M) (Llpoxygenase inhibitor) SKF-525A (5×10 ~ M) (Cytochrome P450 inhibitor) Methylene Blue (5 × 10 ~'M) (Guanylate cyclase inhibitor)

15 6

1 39 _+ 0 16 1 68 _+ 0 26

15 6

24 16 _+ 2 26 25.88 _+ 5 44

8

171_+012

8

2045_+212

8

I 64 _+ 0 23

8

l0 05 _+ 0 63

8

1 68 _+ 0 68

8

22 83 + 2 69

6

166+_047

6

2780_+459

ETYA eicosatetrayno~c acid, NDGA nordlhydroguamreUc acid *VIP increased cAMP levels slgmficantly F(5,45)=4 2, p<0.01, with or without mhlbltors (ANOVA and Dunnett's test), with NDGA the increase was less pronounced than m other preparations (p<0 05, Tukey's test)

50

-

I

layered

40

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© -

30

X

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o~

20

layered + methylene blue

10

0

wRhout endothetlum

-

i

i

I

I

8.3

7

7.3

6

VIP, - l o g (M)

FIG. 4 Dependence of VIP-mduced relaxanon of rat aorta strips on intact endothellum (means_+SEM) Open circles are values for vessels with denuded endothehum (N =4). closed circles are for layered preparations in which intact endothehum was restored (N=5), and trmngles are for methylene blue addmon to layered preparations (N =5) Endothehum-deficlent aorta relaxed poorly at all concentrauons of VIP, relative to corresponding values with intact (layered) endothehurn (p<0 01). Methylene blue had no slgmficant influence

Change,~ m ('y~ he Nu~ leotlde LeveLs m Rat Aorta I n c u b a U o n o f e n d o t h e h u m - m t a c t ring s e g m e n t s of rat a o r t a with 0 . 3 / x M V I P for 60 sec at 37°C r e s u l t e d m a 23-fold i n c r e a s e m c A M P o v e r basal levels (Table 1). S t i m u l a t i o n of c A M P p r o d u c t i o n b y V I P was c o n c e n t r a t i o n - d e p e n d e n t . At t h r e e c o n c e n t r a t i o n s of V1P, 3 × 1 0 ~ M, 3× 10 7 M a n d 3× 10_5 M, the m e a n levels o f c A M P w e r e , r e s p e c t i v e l y , 7 45 ( N = 2 ) , 24.16+_2.26 ( N = I 5 ) , a n d 45.05+_5 40 ( N = 8 ) p m o l / m g protem R e m o v a l of e n d o t h e h u m c a u s e d only a small, nonsignific a n t d e c r e a s e of b a s a l levels o f c A M P A l t h o u g h V I P elicited a two-fold i n c r e a s e In c A M P levels t p < 0 . 0 5 ) in t i s s u e s without e n d o t h e l i u m , this i n c r e a s e was m a r k e d l y a n d slgmfic a n d y less t h a n m t i s s u e s with e n d o t h e h u m ( p < 0 . 0 0 1 ) T h u s , V I P - i n d u c e d c A M P a c c u m u l a t i o n , h k e the r e l a x a t i o n , was largely d e p e n d e n t on e n d o t h e l i u m . Cyclic G M P levels were not significantly altered by VIP, w h e t h e r e n d o t h e l i u m was p re sent (from 1.04_+ 0 11 to 1.11 _+0.15 pmol/mg protein, N = 15) or a b s e n t (from 0.69-+0.05 to 0.72+_0.12, N = 6 ) . B e c a u s e e n d o t h e h u m - d e p e n d e n t r e l a x a t i o n of v a s c u l a r s m o o t h m u s c l e m r e s p o n s e to a c e t y l c h o l i n e a n d s o m e o t h e r v a s o d d a t o r s has b e e n s h o w n to b e s u s c e p t i b l e to inh~bitors o f h p o x y g e n a s e , c y t o c h r o m e P450 o r g u a n y l a t e cyclase [ 10], we e x a m i n e d t h e effects o f s e v e r a l s e l e c t e d m h i b l t o r s on c A M P a c c u m u l a t i o n e v o k e d b y V I P m ring s e g m e n t s with e n d o t h e h u m (Table 2). E x c e p t for N D G A (one of t w o h p o x y g e n a s e m h l b i t o r s used), w h i c h r e d u c e d c A M P acc u m u l a t i o n i n d u c e d b y VIP, n o n e o f the i n h l b i t o r s h a d a n y significant influence.

Layered Preparattons a f t e r tensxon h a d r e a c h e d a p l a t e a u . D i l u e n t a l o n e (0.025% e t h a n o l ) did n o t affect the c o n t r a c t i o n . B o t h m d o m e t h a c i n a n d E T Y A , a d d e d for 10 m i n b e f o r e a n d d u r i n g t h e c u m u l a tive a d d i t i o n s o f VIP, h a d n o effect o n the V I P doser e s p o n s e r e l a t i o n s h i p (Fig. 3).

C o n t r a c t i o n s of rat a o r t a strips r e d u c e d b y PGH2-A m l a y e r e d p r e p a r a t i o n s were similar (0.76+_0.12 g, N = 5 ) to t h o s e o f strxps t h a t w e r e i n c u b a t e d a l o n e (0.82+_0.28 g, N=I4) E n d o t h e l i u m - d e n u d e d c i r c u m f e r e n t i a l strips t h a t w e r e layered w i t h tissues w i t h o u t e n d o t h e h u m relaxed by

VIP E F F E C T ON T H E RAT AORTA

857

7_+ 2% in response to VIP (10-6). Ach (10-6 M) failed to relax the recipient strips under these conditions. When recipient tissues were layered with endothelium-intact donor tissues, however, VIP relaxed recipient tissues in a dose-dependent fashion (Fig. 4). in this preparation, Ach (10 -6 M) also induced relaxation (46.6-+7.5%, N=4). The addition of methylene blue (5× 10-5 M), a guanylate cyclase inhibitor, to tissues previously contracted by PGH2-A further increased basal tone (by 3 7+- 15%, N=3), but methylene blue did not affect the VIP dose-response relationship in layered preparations (Fig. 4). DISCUSSION Our results indicate that VIP-lnduced relaxation of rat thoracic aorta is largely dependent on the presence of intact endothelium. The persistence of the relaxant response to nitroprusside after removal of endothelium confirms the responsivenes of vascular smooth muscle to vasodilators other than VIP. This responsiveness is also demonstrated by the restoration of VIP-lnduced relaxation in layered preparations. Endothelium-dependent vascular relaxation by VIP has been reported by other investigators working with rat aorta [6], rabbit mesenteric artery [19], human celiac artery [32], and cultured vascular smooth muscle cells [12]. Schoeffter and Stoclet [31], on the other hand, described endotheliumindependent relaxation of isolated rat aorta in response to VIP The reason for this discrepancy is not clear, but the VIP-induced relaxation obtained by the latter investigators, even in the presence of endothelium, was strikingly small (16% of serotonin-induced tone). On the other hand, endothelium-independent relaxations due to VIP have uniformly been described in cat cerebral arteries [7], dog coronary artery [5], and pulmonary arteries of rat, rabbit, guinea pig [30], and humans [15]. VIP has been shown to stimulate the formation of cyclic A M P in blood vessels [8,18], including rat aorta [31], as in a variety of other tissues [1]. Our data demonstrate that both the cyclic AMP accumulation and the relaxation of isolated rat aorta evoked by VIP are greatly reduced by removing endothelium. The close association between the cyclic AMP levels and relaxation suggests a causal relationship. The vascular relaxant effect of several other vasodllators that depend on endothehum is similarly associated with the accumulation of cyclic A M P in smooth muscle. Thus, relaxations of rat aorta by adenosine and of rat femoral artery by isoproterenol are greatly attenuated, but not abolished, by removing endothehum [20]. Rabanyl and Vanhoutte [27] suggested that the endothelium of the canine coronary artery produces a signal in response to the betaadrenoceptor agonists isoproterenol and norepinephrine and to adenosine, which facihtates the direct relaxant effects of these compounds on vascular smooth muscle cells. Both beta-adrenoceptor agonists and adenosine stimulate adenylate cyclase activity in vascular smooth muscle cells [2,17].

The neuropeptide calcltonin gene-related peptide (CGRP) has also been shown to be a potent activator of adenylate cyclase in cultured rat smooth muscle cells [12], and to require the presence of endothelium in relaxing rat aorta in vitro [3]. On the other hand, strong evidence suggests that the vasodilator action of several other endothelium-dependent vasodilators, including acetylcholine, histamine and bradykinin, as well as the endothelium-independent sodium nitropruss~de and atrial natriuretic peptlde, may be mediated by cyclic GMP [16,23]. Thus, both cyclic nucleotides are considered likely mediators of the same biologic effect, vascular relaxation, produced by different vasodilators. The fadure of cyclic A M P stimulation by VIP after the removal of endothelium may mean (a) that VIP stimulates cyclic A M P production mainly in endothelium, or (b) that endothelium is required for the generation of cyclic AMP in smooth muscle cells. It is unlikely that the predominant elevation of aortic cyclic AMP levels in response to VIP occurs in endothehum, in view of the relatively small mass of endothelial cells compared to that of smooth muscle cells. Cyclic A M P elevation in tissues without endothelium may be due to a direct action of VIP on smooth muscle cells. Functional receptors for VIP on cultured smooth muscle cells from rat aorta [17], and cychc A M P stimulation by VIP in these cells, have been demonstrated [17,24]. Since both relaxation and cyclic AMP accumulation in rat aorta in response to VIP were markedly attenuated, though not abolished, in endothelium-denuded aorta, both effects were probably mediated in large measure by an endothelium-dependent mechanism, but in part also by a direct action on vascular smooth muscle. The findings in layered preparations provide direct evidence that VIP induces the release of a dlfftlsible relaxing factor (or factors) from the endothehum. The chemical nature of this factor remains to be determined, but it is clearly not the originally described EDRF, which recent evidence suggests may be nitric oxide [25]: (1) Neither VIP-induced relaxation nor cyclic AMP accumulation was inhibited by methylene blue (Table 2), which inactivates E D R F [10,23], (2) VIP stimulated cyclic A M P accumulation but did not alter cyclic GMP levels; and (3) lipoxygenase lnhlbitors either did not inhibit VIP-induced relaxation of rat aorta, or reduced It only moderately. The possiblity is not excluded that the cyclic AMP generated by VIP diffuses to and relaxes smooth muscle, although cyclic AMP does not penetrate the plasma membrane. The recent demonstration that CGRP-lnduced, endothelium-dependent relaxation of aortic rings is not accompanied by cyclic GMP accumulation [14] supports the likelihood of the existence of additional types of EDRF. In conclusion, our results suggest that VIP stimulates the release of a factor from endothehum that reaches smooth muscle where it induces cyclic AMP accumulation and relaxation. The factor is not a metabolite of cyclooxygenase, lipoxygenase, or cytochrome P450 pathways.

ACKNOWLEDGEMENTS Supported in part by the Medical Research Service of the Veterans Admimstration and by NIH Research Grants HL-30450, HL31039, and HL-35656. J.L Is an Estabhshed Investigator of the American Heart Association We thank Manlyn Satkiewlcz and Bridget Reilly for help with preparation of the manuscript.

858

SATA ETAL

REFERENCES 1 Amwanoff, B and G Rossehn VIP receptors amd control of cychc AMP production In' Vasoa~ttve Intesttnal Pepttde~ New York Raven Press, 1982, pp 307-322 2. Anand-Srivastava, M B. and D. J Franks. Stimulation of adenylate cyclase by adenosine and other agomsts m mesentnc artery smooth muscle cells m culture. Life Set 37: 857-867, 1985. 3 Bram, S . D , T . J Wflhams, J R. Tlppms, H . R Morns a n d I Maclntyre Calcltonln-gene-related peptide ~s a potent vasoddator. Nature 313: 54-56, 1985 4 Brooker, G , W L Terasak~ and M G Price Gammaflow A completely automated radlolmmunoassay system St wn~ e 194: 270-276, 1976. 5 Brum, J M , V . M W Go, P M V a n h o u t t e a n d A A Bove Evidence for VIP-erglc control of coronary vasoregulatlon RegM Pept 3: 37, 1985 6 Daws, J M and K I Wdhams Endothehum-dependent relaxant effects of vasoachve intestinal polypeptlde and arachldomc acid m rat aorhc strtps Prostaglandms 27: 195-202, 1984 7 Duckies, S P and S I Said Vasoactlve intestinal peptlde as a neurotransmltter m the cerebral circulation Em J Pharmat ol 78: 371-374, 1982 8 Edwnsson, L , B B l~redholm, E Hamel, l Jansen and C Verrecchla Penvascular peptldes relax cerebral arteries concomltant w~th stimulation of cychc adenosine monophosphate accumulation or release of an endothehum-denved relaxing factor m the cat Neuros(t Lett 58: 213-217, 1985 9 Furchgott, R F Role of endothehum m responses of vascular smooth muscle Cw( Reds 53: 557-573, 1983 l0 Furchgott, R F. The role of endothehum m the responses of vascular smooth muscle to drugs Annu Rev Pharmat ~)l To.r~t ol 24: 175-197, 1984 11 Furchgott, R F and J. V Zawadzk~ The obhgatory role of endothehal cells m the relaxation of arterial smooth muscle by acetylchohne Nature 288: 373-376, 1980 12 Ganz, P , A W Sandrock, S. C Landis, J Leopold, M A G~mbrone, Jr and R W Alexander. Vasoactive intestinal pept~de. vasodilatlon and cychc AMP generation Am J Physu)l 250: H755-H760, 1986 13. Godfrey, K Statistics in practice, comparing the means of several groups N E n g l J Med 313: 1450-1456, 1985 14 Grace, G C , G J Dusting, B E Kemp and T J Martm Endothehum and the vasodllator action of rat calotonln generelated pept~de (CGRP) Br J Pharma~ ol 91: 729-733, 1987 15 Greenberg, B , K Rhoden and P Barnes Endothellum relaxant factor ~s present m human pulmonary artery Ctr~ulat~on 72: 111-265, 1985 16 Grlffith, T M , D H Edward, M J Lew~s and A H Henderson Evidence that cychc guanosme monophosphate (cGMP) medmtes endothehum-dependent relaxatmn Eur I Pharma~ ol 112: 195-204, 1985 17 Hirata, Y , M Tom~ta, S Takata and T Fujlta Functional receptors for vasoactive intestinal pept~de m cultural vascular smooth muscle cells from rat aorta Bto¢hem Btophv.s Rea Commun 132: 107%1087, 1985

18 Huang, M and O P Rorstad Effects of vasoactlve intestinal polypeptlde, monoammes, prostaglandms, and 2-chloroadenosme on adenylate cyclase m rat cerebral mlcrovessels J Neurot hem 40: 71%726, 1983 19 Itoh, T., T Sasagurl, Y Makita, Y Kanmura and H KurJyama Mechamsms of vasodflat~on reduced by vasoactJve intestinal polypeptlde m rabbit mesentenc artery Am J Phvmol 249: H231-H240, 1985 20. Komshl, M. and C Su Role ofendothehum m ddator responses of spontaneously hypertenswe rat arteries H_vpertenston 5: 881-886, 1983 21 Kubota, M , J. M Moseley, L Butera, G J Dusting, P S. MacDonald and T. J Martin Calcltomn gene-related peptlde s~mulates cychc AMP formation m rat aorhc smooth muscle cells Btoc hem B.~phys Re.~ Commun 132: 88-94, 1985 22, Linden, J . C E Hollen and A Patel. The mechamsm by which adenosine and chohnerglc agent reduce contractlhty m rat myocardlum Ctr~ Re,s 56: 728-735, 1985 23 Murad, F Cychc guanosme monophosphate as a medmtor of vasoddatmn J (Ira Invest 78:. 1-5, 1986 24 Nab~ka, T , Y Nara, Y Yamon, W Lovenberg and J. Endo Ang~otensm I1 and phorbol ester enhance lsoproterenol- and vasoachve intestinal pephde (VIP)-lnduced cychc AMP accumulation m vascular smooth muscle ceils. B~o~hem Btophy~ Re~ Commun 131: 30-36, 1985 25 Palmer, R M. J , A G. Fernge and S Moncada. Nitric oxide release accounts for the biological actlwty of endothehumderived relaxing factor Nature 327: 524-526, 1987 26 Rubanyl, G M and P M Vanhoutte Ouabaln inhibits endothehum-dependent relaxahons to arach~domc acid m canine coronary arteries I Pharma~ol E~p Ther 235: 81-86, 1985 27 Rubanyl, G and P M Vanhoutte Endothehum-removal decreases relaxations of canine coronary arteries caused by /3-adrenerglc agomsts and adenosine J Cardtova~ Pharma~ol 7: 13%144, 1985 28 Rubanyl, G M , R R Lorentzand P M Vanhoutte. Bmassay of endothehum-denved relaxing factor(s), lnactlvatmn by catecholamlnes Am J Phy~ud 249: H95-H101, 1985 29 Said, S I. Vasoddator action of VIP Introductmn and general conslderahons In. Va~oac ttve Intestinal Pepttde, edited by S i Said. New York Raven Press, 1982, pp 16%176. 30. Sara, T , H P. Mlsra, E Kubota and S I. Said Vasoactlve intestinal polypeptlde relaxes pulmonary artery by an endothehum-lndependent mechamsm Pepttdes 7: Suppl 1, 225-227, 1986 31 Schoeffter, P and J Stoclet Effect of vasoactlve intestinal polypephde (VIP) on cychc AMP level and relaxation in rat isolated aorta Eur .I Pharma~ ol 109: 275-279, 1985 32 Thorn, S , A. Hughes, G Martin and P S Sever. Endothehum-dependent relaxahon m isolated human arteries and veins Chn Set 73: 547-552, 1987