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Responses of human basilar arteries to vasoactive intestinal polypeptide
Life Sciences, Vol. 41, pp. 1155-1163 Printed in the U.S.A.
Pergamon Journals
RESPONSES OF HUMAN BASILAR ARTERIES TO VASOACTIVE INTESTINAL POLYPEPTIDE Richard P. White Departments of Pharmacology and Neurosurgery U n i v e r s i t y of Tennessee Medical Center Memphis, TN 38163 (Received in final form June 19, 1987)
Summary The responses to 9X10-7M vasoactive i n t e s t i n a l polypept i d e (VIP) by isolated human b a s i l a r a r t e r i e s of 30 i n d i v i d uals were studied to f u r t h e r elucidate the role the peptide might play in modifying cerebrovascular tone normally and in disease. In most experiments the a r t e r y was precontracted with prostaglandin F2~(PGF2~), e i t h e r with i or 2XlO-6M or with IO-5M PGF2~. The course of action to VIP was observed f o r 15 min following i t s application to the contracted vessel. Some a r t e r i e s f a i l e d to respond to VIP (13%), otherwise the a r t e r i e s relaxed 44% when the contraction was induced by IO-5M PGF2~ and 67.6% a f t e r the lower concentractions of PGF2~. There was no s i g n i f i c a n t decrement in the vasorelaxant e f f e c t of VIP throughout the period of observation. A second and t h i r d application of VIP to the precontracted a r t e r y produced s i g n i f i c a n t l y less of an e f f e c t than the f i r s t , but no consistent progressive pattern of tachyphylaxis was evident. In additional experiments, indomethacin (IO-5M) did not prevent the vasorelaxant e f f e c t of VIP, suggesting that prostanoid synthesis was not involved. Pretreatment of the a r t e r y with VIP did not prevent the contractions generated by 10, 30, 50 and 90 mM KCl while antithrombin I I I (I.2XlO-/M) did, i n d i c a t ing fundamental differences between these two vasorelaxants. In conclusion, VIP w i l l i n h i b i t contraction of isolated human cerebral a r t e r i e s for prolong periods and could be a s i g n i f i cant f a c t o r regulating cerebral blood flow in humans. Introduction Numerous reports indicate that vasoactive i n t e s t i n a l polypeptide (VIP) is present in nerves that supply cerebral a r t e r i e s and that the peptide may be important p h y s i o l o g i c a l l y as a v a s o d i l a t o r peptide ( 1 , 2 , 3 , 4 , 5 , 6 , 7 ) . Results obtained in laboratory animals have shown that VIP acts d i r e c t l y on vascular smooth muscle to induce relaxation (1,3) and that i n h i b i t o r s of cyclooxygenase block t h i s e f f e c t of VIP (5). Moreover, antiserum to VIP w i l l i n h i b i t the d i l a t o r e f f e c t of transmural nerve stimulation of f e l i n e cerebral a r t e r i e s (8). I t has also been proposed that the i n h i b i t o r y e f f e c t of VIP contributes to the delay in the onset of cerebral vasospasm (9) that, on median, occurs 7 days a f t e r subarachnoid hemorrhage (SAH) in patients ( i 0 ) .
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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I t has become evident in recent years that isolated human cerebral a r t e r i e s often respond d i f f e r e n t l y to vasoactive agents when compared with corresponding a r t e r i e s of laboratory animals. These agents include amines, polypeptides, and proteins. For instance, histamine, bradykinin, and thrombin relax human a r t e r i e s but c o n s t r i c t those of laboratory animals ( 1 1 , 1 2 , 1 3 ) . Angiotensin I I c o n s t r i c t s the human a r t e r y (14) but i t s main e f f e c t on the canine a r t e r y is d i l a t i o n (15). The c o n s t r i c t i o n e l i c i t e d by arachidonate in human cerebral a r t e r i e s is not blocked by indomethacin (16) but i t is blocked in the canine vessel (17). Moreover, whether agents l i k e VIP produce desensit i z a t i o n in human a r t e r i e s , as VlP does in canine vessels (18), is c r i t i c a l in assessing t h e i r roles in c l i n i c a l phenomenan. I t seemed important, therefore, to study the e f f e c t of VIP on the human vessel and to determine whether desens i t i z a t i o n or tachyphylaxis might l i m i t i t s effectiveness as a physiological d i l a t o r and as an agent that might delay the onset of cerebral vasospasm. The present study was performed to ascertain whether VIP might i n h i b i t contractions of human b a s i l a r a r t e r i e s , whether the a r t e r i e s manifests desensit i z a t i o n or tachyphylaxis to VlP, and to determine the e f f e c t of the cyclooxygenase i n h i b i t o r , indomethacin, on the responses induced by VIP. Methods Handling of the b a s i l a r a r t e r y . The b a s i l a r a r t e r i e s were obtained w i t h i n 24 hrs of death from 30 victims of accidents and other unnatural causes. The average age was 28.2±4.8 years. These vessels were found to be normal by gross inspection. The average O.D. was 3.48 mm. The vessels were placed in cold Krebs-Henseleit buffer at autopsy. A 4 mm segment ( r i n g ) was mounted onto two prongs, one fixed to the i0 ml tissue chamber and one movable, from which isometric contractions were recorded. The Krebs-Henseleit buffer used in the bath had the following compos i t i o n (mM):NaCI, 118.3; KCI, 4.7; MgS04, 1.2; KH2PO4, 1.2; CaCI2, 2.5; NaHCO3, 25 and glucose, I I . 0 . The a r t e r i a l segment was aerated with 95% 02 and 5% C02, and kept at 37°C. The bath pH was 7.35. The passive tension i n i t i a l l y placed on the a r t e r y was 2g. I f the vessel relaxed more than lg in response to the i n i t i a l stretch ( s t r e s s - r e l a x a t i o n ) , additional stretch was applied as necessary to establish a baseline tone of between I and 2g. One hr l a t e r the responses e l i c i t e d by i0, 30, 50 and 90 mM KCI were recorded. This was repeated every 20 min u n t i l the maximal contract i l e response was obtained ( i 0 ) . The response to K+ was used as a standard f o r comparing other c o n t r a c t i l e responses because i t is independent of receptors, and because of the great v a r i a t i o n in response among isolated a r t e r i e s (19). In t h i s regard, there was no r e l a t i o n s h i p between the time since death (within 24 hrs) and the magnitude of the response to K+. The response to repeated applications of K÷ by human b a s i l a r a r t e r i e s increases progressively u n t i l the fourth t r i a l , a f t e r which i t is stable (13). The vessels used in these experiments manifested t h i s behavior and the maximum contraction e l i c i t e d by K+ was 7.13±9.8 g. The basal tone of the a r t e r i e s studied rose on average 0.16 g/hr, which is c h a r a c t e r i s t i c of t h i s a r t e r y (13). This r i s e did not a f f e c t the responses to the agonists. Histological examination by hematoxylin and eosin stain demonstrated that the endothelium was present before and a f t e r the experimental period. In a d d i t i o n , at the end of the experimental period, thrombin (1 u n i t / m l ) was applied to a r t e r i e s precontracted with 10-5M PGF2~ to induce vasorelaxation as a t e s t f o r the presence of a functional endothelium (13).
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Experimental dru 9 solutions. Vasoactive intestinal polypeptide (VIP), prostaglandin F2~(PGF2~), indomethacin, and antithrombin I l l (AT I l l ) used in the experiments were obtained from Sigma Chem. Co. (St. Louis, MO). The indomethacin was solubilized with sodium carbonate (I part by wt. to 3 parts indomethacin). The VIP and PGF2~ were dissolved in buffer. Except for AT I l l , the volume of the drug solutions added to the 10 ml bath was 10 or 100 ~I. AT I I I was dissolved in I ml buffer taken from the bath and returned so that the volume of the bath was unaltered during the observation period.
A.
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FIG.1 Tracings showing differences in the e f f e c t of VIP in three d i f f e r e n t a r t e r i e s . AI i l l u s t r a t e s a control response to PGF2~ (W=wash) while A2 shows an abrupt response to VIP in the same vessel precontracted with PGF2~. Tracing B i l l u s t r a t e s a slower response to VIP while C shows an a r t e r y r e f r a c t o r y to VIP but not to sodium n i t r i t e . Basic experimental procedures. Each a r t e r y was then exposed several times to I0-6M and 10-5M of PGF2~ in an accumulative manner to see i f the response to the prostaglandin was reproducible and to determine whether the IO-6M concentration produced an e f f e c t judged to be s u f f i c i e n t for experimental purposes. I f not, 2XlO-6M PGF2e alone
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was applied in some experiments. The maximum response to 1 or 2X10-6M PGF2~ averaged 8.63±0.7 g while IO-~M PGF2~ produced a response of 12.85±1.1 g maximum. Any decay in the c e i l i n g e f f e c t was observed for 15 min. T y p i c a l l y the responses to IO-5M PGF2~ were more sustained than those obtained with the lower concentrations. Later, the capacity of VIP to i n h i b i t the contraction e l i c i t e d by PGF2~ was determined. In most experiments VIP was applied 2 to 6 min a f t e r the response to 1 or 2XlO-6M or to IO-5M PGF2~ had peaked. The maximum drop in c o n t r a c t i l e force obtained with VIP was noted and compared with the contraction present during the same i n t e r v a l of time in the absence of VIP (compare tracings AI and A2, Fig. 1, f o r an example). Preliminary experiments with IO-8M and 10-7M VIP indicated that some vessels would not respond to these concentrations. Therefore, the concentration of VIP used throughout these experiments was 9XIO-7M (3 ~g/ml) with the expectation that most a r t e r i e s would manifest a response. Moreover, previous reports indicate that t h i s concentration would produce the maximum e f f e c t on human cerebral a r t e r i e s precontracted with PGF2~ (7,20). Further d e t a i l s of the experimental design are presented in Results. Responses to the agonists were expressed as mean ± S.E.M. The level of significance of the response was assessed by the appropriate Student t - t e s t or by Pearson's Results Description of the VIP response. Figure 1 i l l u s t r a t e s the precipitous drop in c o n t r a c t i l e force (tracing A2) as well as the slower decline in contraction (tracing B) that was induced by 9XlO-7M VIP in d i f f e r e n t a r t e r i e s . The type of response e l i c i t e d by VIP was consistent f o r each a r t e r y studied. The vasorelaxant e f f e c t persisted throughout the observation period (Fig. I ) , although in 3 of 16 a r t e r i e s the response waned on average about 19% a f t e r i t had peaked. About 13% of the a r t e r i e s (4 of 30) f a i l e d to respond to VIP (Fig. 1, tracing C). The f a i l u r e was not due to i n a c t i v e VIP since the same solution of VIP e l i c i t e d responses in suseptible vessels nor due to an impaired endothelium since i unit/ml thrombin relaxed the a r t e r i e s on average 27% (n=4). Magnitude of the vasorelaxant e f f e c t of VIP. Figure 2 shows that the r e l a x a t i o n produced by 9X10-7M VIP was greater in the vessels precontracted with I or 2X10-6M PGF2~ than those contracted beforehand with IO-5M PGF2~, being 67.6±9.5% and 44.1±6.7%, r e s p e c t i v e l y (P
a r t e r i e s precontracted with 1 or 2X10-6M PGF2~(n=5) or 10-5M PGF2~ exposed to 9XIO-7M f o r 15 min. After washout, and an i n t e r v a l of 20 the procedure was repeated. In t h i s manner the a r t e r i e s were VIP three times.
Figure 3 shows that the f i r s t response to VIP was c l e a r l y the greatest of the three responses to VIP (P
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much less than the second. Hence, there was no c o r r e l a t i o n between the peak responses obtained with the second and t h i r d t r i a l of VlP (Pearson's r : 0 . 0 4 2 ) . Moreover, the peak response of the second and t h i r d response did not p e r s i s t as long as t h a t seen with the f i r s t in 4 of 8 a r t e r i e s , but again there was no consistency in t h i s v a r i a t i o n . Nevertheless, a l l of the responses obtained during the f i r s t t r i a l were greater than those seen with e i t h e r the second or the t h i r d a p p l i c a t i o n s of VIP.
Human Basilar Arteries p,. II
150 B Control [ ] VIP 9 x 10~M
X (:
:Z 100 o~ t~ t~ cO 0m o
50
cO
o
0 P G F 2 ~ 10 "s M
1 o r 2 x 1 0 "e M
FIG. 2 l l l u s t r a t i o n of the magnitude of the r e l a x a n t e f f e c t of VlP in a r t e r i e s precontracted with d i f f e r e n t concentrations of PGF2~. A s t e r i s k s = s i g n i f i c a n t d i f f e r e n c e from control (P
on contractions 9enerated by K+.
In these experiments VIP (9XlO-7M) was added to the bath 8 min p r i o r to the cumulative a d d i t i o n of i 0 , 30, 50 and 90 mM of K+ in order to ascertain whether VIP might influence any portion of the concentration-response curve of K+(n=5). Figure 4 shows t h a t VIP had no e f f e c t on the K+ responses and f u r t h e r shows t h a t AT I I I (I.2X10-7M) markedly i n h i b i t e d K+.
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Repeat Trials
0.
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1st
i-
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VIP 9 x 10 .7 M)
2nd
3rd
.o_ m 4"*
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t
._:
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o (n o
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FIG. 3 Summary of the effect of VIP applied 3 times to arteries precontracted with PGF2~. Asterisk=significant difference between f i r s t and subsequent responses (P
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~
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9 x 107M Control
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FIG. 4 Figure shows that pretreatment of arteries with VIP failed to prevent the contractions produced by different concentrations of KCI while pretreatment with antithrombin I l l did.
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In ~other experiments, contractions produced in 4 a r t e r i e s with 90 mM K+ not s i g n i f i c a n t l y i n h i b i t e d (0.0-4.3%) by the addition of 9XlO-7M VIP to the bath. In contrast, AT I I I s i g n i f i c a n t l y i n h i b i t s a r t e r i e s precontracted w~,th 90 mM K+(IO). The f a i l u r e of VIP to i n h i b i t K+ was not due to i n d i v i d u a l differences among the a r t e r i e s since in these same vessels VIP i n h i b i t e d the contractions generated by PGF2~. Influence of indomethacin on VIP induced r e l a x a t i o n . In these experiments the a r t e r y was exposed to 10-5M indomethacin f o r 15 min p r i o r to the addition of e i t h e r I or 2XlO-bM PGF2~ (n=2) or IO-5M PGF2~ (n=4) to precontract the vessel. Then 9XIO-7M VlP was added to the bath 2-5 min a f t e r the contraction had peaked. To minimize the role tachyphylaxis might play in the response to VIP, in three a r t e r i e s the e f f e c t of VlP was observed in the absence of indomethacin and then again in the presence of the cyclooxygenase i n h i b i t o r . In three other a r t e r i e s the procedure was reversed: f i r s t the response to VIP was obtained in the presence of indomethacin and again l a t e r in i t s absence. The results showed that the percent drop in c o n t r a c t i l e force due to VIP was the same in the presence (58.9±9.1%) or absence (53.3±12.8%) of indomethacin. Discussion Previous studies have shown that VlP produces vasorelaxation or vasodilat a t i o n in cerebral a r t e r i e s of the r a b b i t (2), cat ( 1 , 3 , 5 ) , dog (6), baboon (4), pig, cow (7), and human (7,20). The l a t t e r were branches of the middle cerebral a r t e r y obtained from patients during surgery f o r tumors or i n t r a c t a b l e epilepsy. The v a r i a t i o n in the ED50 (5.3 f o l d ) f o r VIP, and in the maximum i n h i b i t i o n produced (1.6 f o l d ) , obtained in these pial a r t e r i e s is most l i k e l y due to the differences in the concentrations of prostaglandin F2~ (from 0.3 to 2.5X10-6M) used by the i n v e s t i g a t o r s to precontract the vessel rather than a difference in the preparation ( s p i r a l s t r i p s versus segments) (7,20). In t h i s regard, i t is evident in Fig. 2 that VIP is more e f f e c t i v e when the concentration of prostaglandin F2~ used produced submaximal contractions. In any case, previous reports agree that the maximum i n h i b i t i o n produced by VIP in human pial a r t e r i e s is obtained at 3XlO-6M (7,20). Questions remained, however, as to whether the i n h i b i t i o n persisted or was t r a n s i e n t and whether indomethacin a f f e c t s the response. The present study demonstrates that the larger b a s i l a r a r t e r y of the human also relaxes in response to VlP. The r e s u l t s f u r t h e r show that t h i s e f f e c t p e r s i s t s , so that the phenomenon of d e s e n s i t i z a t i o n need not l i m i t the e f f e c tiveness of VIP, at l e a s t f o r 15 min. Tachyphylaxis to VIP was apparent in some a r t e r i e s but was so v a r i a b l e t h a t , on average, the second and t h i r d applications of VlP produced a s i g n i f i c a n t v a s o d i l a t a t i o n , though less than the first. Tachyphylaxis may have been c l e a r l y demonstrable i f VlP had been applied f o r longer periods. In any case, i t was not as evident in the human b a s i l a r a r t e r y as i t is in the corresponding vessel of the canine (18). Moreover, in contrast to porcine and bovine cerebral a r t e r i e s (7), VlP did not i n h i b i t the contractions generated by K+ in the human b a s i l a r a r t e r y . Why some a r t e r i e s f a i l e d to respond to VIP is unknown, but i n d i v i d u a l v a r i a t i o n in responses to some agonists is c h a r a c t e r i s t i c of human cerebral a r t e r i e s , even "fresh" ones (21). Perhaps lower concentrations of VlP would have increased while higher concentrations would have decreased the f a i l u r e rate of 13%. In any case, the r e l a t i v e l y small branches of the human middle cerebral a r t e r y appear to respond r e l i a b l y to VIP (7,20). I t is possible that
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branches of the c i r c l e of W i l l i s may respond more c o n s i s t e n t l y to VIP since the nerves to some of these branches in humans contain abo,t twice as much immunor e a c t i v e VIP than the b a s i l a r ~ r t e r y (20). The r e s u l t obtained with indomethacin suggests t h a t the e f f e c t of VIP on human cerebral a r t e r i e s is not dependent on the production of prostanoids. In c o n t r a s t , v a s o d i l a t a t i o n of VIP in cat cerebral a r t e r i e s is i n h i b i t e d by indomethacin, but not i t s e f f e c t on peripheral flow (5). This also appears to be the case with bradykinin (22). Thus, cat cerebral a r t e r i e s may synthesize prostanoids as a means of responding to c e r t a i n v a s o d i l a t o r s while peripheral a r t e r i e s may not. In any case, indomethacin does not i n h i b i t the e f f e c t of VIP on a l l a r t e r i e s tested and the present f i n d i n g suggests t h a t i n h i b i t o r s of cyclooxygenase would not d i s r u p t a v a s o d i l a t o r response to VIP in humans cerebral a r t e r i e s normally or i f VIP was a c o n t r i b u t o r to vascular headache. The mechanism of action of VIP on human cerebral a r t e r i e s must d i f f e r from t h a t of AT I I I because only the l a t t e r blocked the usual c o n t r a c t i l e response to K+. The i n h i b i t o r y e f f e c t of AT I I I is not dependent on the presence of the endothelium (13), as is the case with many v a s o d i l a t o r s (22). AT I I I dots not s t i m u l a t e the sodium e l e c t r o g e n i c pump to e f f e c t v a s o r e l a x a t i o n nor produce tachyphylaxis (10). In 3XlO-8M concentration, representing 0.7% of the plasma concentration (4.5xlO-6M), AT I I I completely reverses the r i s e in i n t r a c e l l u l a r calcium e l i c i t e d by 50mM K+ in cultured vascular smooth muscle from r a t aortae (A. Hassid and R.P. White, unpublished data). This fundamental e f f e c t of AT I I I may account f o r i t s i n h i b i t i o n of a wide v a r i e t y of c o n t r a c t i l e agents l i k e UTP, s e r o t o n i n , and the bloody CSF of p a t i e n t s with SAH (10,13,23). I t is unknown whether VIP is e f f e c t i v e when blood is present in the CSF f o l l o w i n g SAH as proposed by others ( 9 ) , but the abundance of AT I I I in blood, and i t s marked i n h i b i t o r y q u a l i t i e s , makes i t a leading candidate as a f a c t o r which would prevent, or delay the onset o f , cerebral vasospasm of p a t i e n t s (10,13). There is evidence t h a t the cerebral a r t e r i e s which are most responsive to VIP are those t h a t are supplied with VIP immunoreactive nerves (1,8) and w i l l d i l a t e upon transmural nerve s t i m u l a t i o n by a mechanism independent of a f u n c t i o n a l endothelium or the release of a c e t y l c h o l i n e ( 1 , 3 ) . I t has been f u r t h e r argued t h a t the primary e f f e c t of a c e t y l c h o l i n e released by nerves supplying cerebral vessels is v a s o c o n s t r i c t i o n so that any v a s o d i l a t a t i o n produced by nerve s t i m u l a t i o n would r e s u l t from the release of v a s o d i l a t o r substances l i k e VIP (24). The primary e f f e c t of a c e t y l c h o l i n e on i s o l a t e d human cerebral a r t e r i e s is unclear, with vasorelaxation (25), mixed e f f e c t s ( i i ) , and v a s o c o n s t r i c t i o n (19) being reported. In c o n t r a s t , the only e f f e c t of VIP on the human vessel, observed by independent i n v e s t i g a t o r s , has been v a s o r e l a x a t i o n . This f i n d i n g , together with the above mentioned histochemical data, support the hypothesis t h a t VIPergic nerves supply human cerebral a r t e r i e s and t h a t VIP could serve as a v a s o d i l a t o r of those a r t e r i e s in health and disease. Acknowledgements This work was supported in part by grant NS 21405 of the National I n s t i t u t e s of Health, USPHS. The author thanks Dr. F. Curtis Dohan, Mr. Craig Lahren, Mrs. Marion Johnson and Mrs. Gwendolyn Stornes f o r t h e i r e s s e n t i a l assistance. References 1. 2.
S.P. DUCKLES and S . I . SAID, Eur. J. Pharmacol. 78 371-374 (1982). D.D. HEISTAD, M.L. MARCUS, S . I . SAID and P.M. GROSS, Am. J. Physiol. 239 H13-H80 (1980).
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3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16.
17. 18. 19. 20. 21.
22. 23.
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T.J.-F. LEE, A. SAITO and I. BEREZIN, Science 224 898-901 (1984). J. McCULLOCH and L. EDVINSSON, Am. J. Physiol. 238 H449-H456 (1980). E.P. WEI, H.A. KONTOSand S.I. SAID, Am. J. Physiol. 239 H765-H768 (1980). D.A. WILSON, J.T. O'NEILL, S.I. SAID and R.J. TRAYSTMAN, Circ. Res. 48 138-148 (1981). Y. SUZUKI, D. McMASTER, K. LEDERIS and O.P. RORSTAD, Brain Res. 322 9-16 (1984). J.E. BRAYDENand J.A. BEVAN, Stroke 16 136 (1985). J.A. BEVAN, S.G. CUTLER and H.H. SCHMIDEK, Trends Pharmacol. Sci. 7 345347 1986). R.P. WHITE, Stroke 17 207-213 (1986). J.E. HARDEBO, J. HAN~O and C. OWMAN, Gen. Pharmac. 14 133-134 (1983). N. TODA, Am. J. Physiol. 232 H267-H274 (1977). -R.P. WHITE, J. Cereb. Bloo~Flow Metabol. 7 68-73 (1987). K.S. PAUL, E.T. WHALLEY, C. FORSTER, R. LYE and J. DUTTON, J. Neurosurg. 57 334-340 (1982). N~ TODA and M. MIYAZAKI, Am. J. Physiol. 240 H247-H254 (1981). L. BRANDT, B. LJUNGGREN, K.-E. ANDERSSON, B. HINDFELT and T. USKI, J. Neurosurg. 55 877-883 (1981). R.P. WHITE,~. SHIRASAWA and L. CAGEN, Life Sci. 34 923-931 (1984). N. TODA, Am. J. Physiol. 243 HI45-H153 (1982). -G.S. ALLEN, C.J. GROSS, L.A. FRENCH and S.N. CHOU, J. Neurosurg. 44 594600 (1976). L. EDVINSSON and R. EKMAN, Peptides 5 329-331 (1984). L. BRANDT, B. LJUNGGREN, K.-E. ANDERSSONand B. HINDFELT, J. Neurosurg. 55 431-437, (1981). R.F. FURCHGOTT,Ann. Rev. Pharmacol. Toxicol. 24 175-197 (1984). R.P. WHITE, R.M. MACLEODand M.S. MUHLBAUER, Surg. Neurol. 27 237-242 (1987).
24. 25.
T.J.-F. LEE, In: J.E. Hardebo, pp T. TSUKAHARA, H. HANDA, Stroke 17
Neural Regulation of Brain Circulation, Eds. C. Owman and 285-296, Elsevier, Amsterdam (1986). USUI, T. TANIGUCHI, S. SHIMOHAMA, M. FUJIWARA and H. 300-304 (1986).
Pergamon Journals
RESPONSES OF HUMAN BASILAR ARTERIES TO VASOACTIVE INTESTINAL POLYPEPTIDE Richard P. White Departments of Pharmacology and Neurosurgery U n i v e r s i t y of Tennessee Medical Center Memphis, TN 38163 (Received in final form June 19, 1987)
Summary The responses to 9X10-7M vasoactive i n t e s t i n a l polypept i d e (VIP) by isolated human b a s i l a r a r t e r i e s of 30 i n d i v i d uals were studied to f u r t h e r elucidate the role the peptide might play in modifying cerebrovascular tone normally and in disease. In most experiments the a r t e r y was precontracted with prostaglandin F2~(PGF2~), e i t h e r with i or 2XlO-6M or with IO-5M PGF2~. The course of action to VIP was observed f o r 15 min following i t s application to the contracted vessel. Some a r t e r i e s f a i l e d to respond to VIP (13%), otherwise the a r t e r i e s relaxed 44% when the contraction was induced by IO-5M PGF2~ and 67.6% a f t e r the lower concentractions of PGF2~. There was no s i g n i f i c a n t decrement in the vasorelaxant e f f e c t of VIP throughout the period of observation. A second and t h i r d application of VIP to the precontracted a r t e r y produced s i g n i f i c a n t l y less of an e f f e c t than the f i r s t , but no consistent progressive pattern of tachyphylaxis was evident. In additional experiments, indomethacin (IO-5M) did not prevent the vasorelaxant e f f e c t of VIP, suggesting that prostanoid synthesis was not involved. Pretreatment of the a r t e r y with VIP did not prevent the contractions generated by 10, 30, 50 and 90 mM KCl while antithrombin I I I (I.2XlO-/M) did, i n d i c a t ing fundamental differences between these two vasorelaxants. In conclusion, VIP w i l l i n h i b i t contraction of isolated human cerebral a r t e r i e s for prolong periods and could be a s i g n i f i cant f a c t o r regulating cerebral blood flow in humans. Introduction Numerous reports indicate that vasoactive i n t e s t i n a l polypeptide (VIP) is present in nerves that supply cerebral a r t e r i e s and that the peptide may be important p h y s i o l o g i c a l l y as a v a s o d i l a t o r peptide ( 1 , 2 , 3 , 4 , 5 , 6 , 7 ) . Results obtained in laboratory animals have shown that VIP acts d i r e c t l y on vascular smooth muscle to induce relaxation (1,3) and that i n h i b i t o r s of cyclooxygenase block t h i s e f f e c t of VIP (5). Moreover, antiserum to VIP w i l l i n h i b i t the d i l a t o r e f f e c t of transmural nerve stimulation of f e l i n e cerebral a r t e r i e s (8). I t has also been proposed that the i n h i b i t o r y e f f e c t of VIP contributes to the delay in the onset of cerebral vasospasm (9) that, on median, occurs 7 days a f t e r subarachnoid hemorrhage (SAH) in patients ( i 0 ) .
0024-3205/87 $3.00 + .00 Copyright (c) 1987 Pergamon Journals Ltd.
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I t has become evident in recent years that isolated human cerebral a r t e r i e s often respond d i f f e r e n t l y to vasoactive agents when compared with corresponding a r t e r i e s of laboratory animals. These agents include amines, polypeptides, and proteins. For instance, histamine, bradykinin, and thrombin relax human a r t e r i e s but c o n s t r i c t those of laboratory animals ( 1 1 , 1 2 , 1 3 ) . Angiotensin I I c o n s t r i c t s the human a r t e r y (14) but i t s main e f f e c t on the canine a r t e r y is d i l a t i o n (15). The c o n s t r i c t i o n e l i c i t e d by arachidonate in human cerebral a r t e r i e s is not blocked by indomethacin (16) but i t is blocked in the canine vessel (17). Moreover, whether agents l i k e VIP produce desensit i z a t i o n in human a r t e r i e s , as VlP does in canine vessels (18), is c r i t i c a l in assessing t h e i r roles in c l i n i c a l phenomenan. I t seemed important, therefore, to study the e f f e c t of VIP on the human vessel and to determine whether desens i t i z a t i o n or tachyphylaxis might l i m i t i t s effectiveness as a physiological d i l a t o r and as an agent that might delay the onset of cerebral vasospasm. The present study was performed to ascertain whether VIP might i n h i b i t contractions of human b a s i l a r a r t e r i e s , whether the a r t e r i e s manifests desensit i z a t i o n or tachyphylaxis to VlP, and to determine the e f f e c t of the cyclooxygenase i n h i b i t o r , indomethacin, on the responses induced by VIP. Methods Handling of the b a s i l a r a r t e r y . The b a s i l a r a r t e r i e s were obtained w i t h i n 24 hrs of death from 30 victims of accidents and other unnatural causes. The average age was 28.2±4.8 years. These vessels were found to be normal by gross inspection. The average O.D. was 3.48 mm. The vessels were placed in cold Krebs-Henseleit buffer at autopsy. A 4 mm segment ( r i n g ) was mounted onto two prongs, one fixed to the i0 ml tissue chamber and one movable, from which isometric contractions were recorded. The Krebs-Henseleit buffer used in the bath had the following compos i t i o n (mM):NaCI, 118.3; KCI, 4.7; MgS04, 1.2; KH2PO4, 1.2; CaCI2, 2.5; NaHCO3, 25 and glucose, I I . 0 . The a r t e r i a l segment was aerated with 95% 02 and 5% C02, and kept at 37°C. The bath pH was 7.35. The passive tension i n i t i a l l y placed on the a r t e r y was 2g. I f the vessel relaxed more than lg in response to the i n i t i a l stretch ( s t r e s s - r e l a x a t i o n ) , additional stretch was applied as necessary to establish a baseline tone of between I and 2g. One hr l a t e r the responses e l i c i t e d by i0, 30, 50 and 90 mM KCI were recorded. This was repeated every 20 min u n t i l the maximal contract i l e response was obtained ( i 0 ) . The response to K+ was used as a standard f o r comparing other c o n t r a c t i l e responses because i t is independent of receptors, and because of the great v a r i a t i o n in response among isolated a r t e r i e s (19). In t h i s regard, there was no r e l a t i o n s h i p between the time since death (within 24 hrs) and the magnitude of the response to K+. The response to repeated applications of K÷ by human b a s i l a r a r t e r i e s increases progressively u n t i l the fourth t r i a l , a f t e r which i t is stable (13). The vessels used in these experiments manifested t h i s behavior and the maximum contraction e l i c i t e d by K+ was 7.13±9.8 g. The basal tone of the a r t e r i e s studied rose on average 0.16 g/hr, which is c h a r a c t e r i s t i c of t h i s a r t e r y (13). This r i s e did not a f f e c t the responses to the agonists. Histological examination by hematoxylin and eosin stain demonstrated that the endothelium was present before and a f t e r the experimental period. In a d d i t i o n , at the end of the experimental period, thrombin (1 u n i t / m l ) was applied to a r t e r i e s precontracted with 10-5M PGF2~ to induce vasorelaxation as a t e s t f o r the presence of a functional endothelium (13).
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Experimental dru 9 solutions. Vasoactive intestinal polypeptide (VIP), prostaglandin F2~(PGF2~), indomethacin, and antithrombin I l l (AT I l l ) used in the experiments were obtained from Sigma Chem. Co. (St. Louis, MO). The indomethacin was solubilized with sodium carbonate (I part by wt. to 3 parts indomethacin). The VIP and PGF2~ were dissolved in buffer. Except for AT I l l , the volume of the drug solutions added to the 10 ml bath was 10 or 100 ~I. AT I I I was dissolved in I ml buffer taken from the bath and returned so that the volume of the bath was unaltered during the observation period.
A.
Human Baallar Arteries W
PGF=~
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1;"M IO"M L
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FIG.1 Tracings showing differences in the e f f e c t of VIP in three d i f f e r e n t a r t e r i e s . AI i l l u s t r a t e s a control response to PGF2~ (W=wash) while A2 shows an abrupt response to VIP in the same vessel precontracted with PGF2~. Tracing B i l l u s t r a t e s a slower response to VIP while C shows an a r t e r y r e f r a c t o r y to VIP but not to sodium n i t r i t e . Basic experimental procedures. Each a r t e r y was then exposed several times to I0-6M and 10-5M of PGF2~ in an accumulative manner to see i f the response to the prostaglandin was reproducible and to determine whether the IO-6M concentration produced an e f f e c t judged to be s u f f i c i e n t for experimental purposes. I f not, 2XlO-6M PGF2e alone
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was applied in some experiments. The maximum response to 1 or 2X10-6M PGF2~ averaged 8.63±0.7 g while IO-~M PGF2~ produced a response of 12.85±1.1 g maximum. Any decay in the c e i l i n g e f f e c t was observed for 15 min. T y p i c a l l y the responses to IO-5M PGF2~ were more sustained than those obtained with the lower concentrations. Later, the capacity of VIP to i n h i b i t the contraction e l i c i t e d by PGF2~ was determined. In most experiments VIP was applied 2 to 6 min a f t e r the response to 1 or 2XlO-6M or to IO-5M PGF2~ had peaked. The maximum drop in c o n t r a c t i l e force obtained with VIP was noted and compared with the contraction present during the same i n t e r v a l of time in the absence of VIP (compare tracings AI and A2, Fig. 1, f o r an example). Preliminary experiments with IO-8M and 10-7M VIP indicated that some vessels would not respond to these concentrations. Therefore, the concentration of VIP used throughout these experiments was 9XIO-7M (3 ~g/ml) with the expectation that most a r t e r i e s would manifest a response. Moreover, previous reports indicate that t h i s concentration would produce the maximum e f f e c t on human cerebral a r t e r i e s precontracted with PGF2~ (7,20). Further d e t a i l s of the experimental design are presented in Results. Responses to the agonists were expressed as mean ± S.E.M. The level of significance of the response was assessed by the appropriate Student t - t e s t or by Pearson's Results Description of the VIP response. Figure 1 i l l u s t r a t e s the precipitous drop in c o n t r a c t i l e force (tracing A2) as well as the slower decline in contraction (tracing B) that was induced by 9XlO-7M VIP in d i f f e r e n t a r t e r i e s . The type of response e l i c i t e d by VIP was consistent f o r each a r t e r y studied. The vasorelaxant e f f e c t persisted throughout the observation period (Fig. I ) , although in 3 of 16 a r t e r i e s the response waned on average about 19% a f t e r i t had peaked. About 13% of the a r t e r i e s (4 of 30) f a i l e d to respond to VIP (Fig. 1, tracing C). The f a i l u r e was not due to i n a c t i v e VIP since the same solution of VIP e l i c i t e d responses in suseptible vessels nor due to an impaired endothelium since i unit/ml thrombin relaxed the a r t e r i e s on average 27% (n=4). Magnitude of the vasorelaxant e f f e c t of VIP. Figure 2 shows that the r e l a x a t i o n produced by 9X10-7M VIP was greater in the vessels precontracted with I or 2X10-6M PGF2~ than those contracted beforehand with IO-5M PGF2~, being 67.6±9.5% and 44.1±6.7%, r e s p e c t i v e l y (P
a r t e r i e s precontracted with 1 or 2X10-6M PGF2~(n=5) or 10-5M PGF2~ exposed to 9XIO-7M f o r 15 min. After washout, and an i n t e r v a l of 20 the procedure was repeated. In t h i s manner the a r t e r i e s were VIP three times.
Figure 3 shows that the f i r s t response to VIP was c l e a r l y the greatest of the three responses to VIP (P
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Human Basilar Arteries and VIP
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much less than the second. Hence, there was no c o r r e l a t i o n between the peak responses obtained with the second and t h i r d t r i a l of VlP (Pearson's r : 0 . 0 4 2 ) . Moreover, the peak response of the second and t h i r d response did not p e r s i s t as long as t h a t seen with the f i r s t in 4 of 8 a r t e r i e s , but again there was no consistency in t h i s v a r i a t i o n . Nevertheless, a l l of the responses obtained during the f i r s t t r i a l were greater than those seen with e i t h e r the second or the t h i r d a p p l i c a t i o n s of VIP.
Human Basilar Arteries p,. II
150 B Control [ ] VIP 9 x 10~M
X (:
:Z 100 o~ t~ t~ cO 0m o
50
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FIG. 2 l l l u s t r a t i o n of the magnitude of the r e l a x a n t e f f e c t of VlP in a r t e r i e s precontracted with d i f f e r e n t concentrations of PGF2~. A s t e r i s k s = s i g n i f i c a n t d i f f e r e n c e from control (P
on contractions 9enerated by K+.
In these experiments VIP (9XlO-7M) was added to the bath 8 min p r i o r to the cumulative a d d i t i o n of i 0 , 30, 50 and 90 mM of K+ in order to ascertain whether VIP might influence any portion of the concentration-response curve of K+(n=5). Figure 4 shows t h a t VIP had no e f f e c t on the K+ responses and f u r t h e r shows t h a t AT I I I (I.2X10-7M) markedly i n h i b i t e d K+.
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Human Basilar Arteries and VIP
Repeat Trials
0.
with
1st
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Vol. 41, No. 9, 1987
VIP 9 x 10 .7 M)
2nd
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._:
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FIG. 3 Summary of the effect of VIP applied 3 times to arteries precontracted with PGF2~. Asterisk=significant difference between f i r s t and subsequent responses (P
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.-e
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FIG. 4 Figure shows that pretreatment of arteries with VIP failed to prevent the contractions produced by different concentrations of KCI while pretreatment with antithrombin I l l did.
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Human Basilar Arteries and VIP
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In ~other experiments, contractions produced in 4 a r t e r i e s with 90 mM K+ not s i g n i f i c a n t l y i n h i b i t e d (0.0-4.3%) by the addition of 9XlO-7M VIP to the bath. In contrast, AT I I I s i g n i f i c a n t l y i n h i b i t s a r t e r i e s precontracted w~,th 90 mM K+(IO). The f a i l u r e of VIP to i n h i b i t K+ was not due to i n d i v i d u a l differences among the a r t e r i e s since in these same vessels VIP i n h i b i t e d the contractions generated by PGF2~. Influence of indomethacin on VIP induced r e l a x a t i o n . In these experiments the a r t e r y was exposed to 10-5M indomethacin f o r 15 min p r i o r to the addition of e i t h e r I or 2XlO-bM PGF2~ (n=2) or IO-5M PGF2~ (n=4) to precontract the vessel. Then 9XIO-7M VlP was added to the bath 2-5 min a f t e r the contraction had peaked. To minimize the role tachyphylaxis might play in the response to VIP, in three a r t e r i e s the e f f e c t of VlP was observed in the absence of indomethacin and then again in the presence of the cyclooxygenase i n h i b i t o r . In three other a r t e r i e s the procedure was reversed: f i r s t the response to VIP was obtained in the presence of indomethacin and again l a t e r in i t s absence. The results showed that the percent drop in c o n t r a c t i l e force due to VIP was the same in the presence (58.9±9.1%) or absence (53.3±12.8%) of indomethacin. Discussion Previous studies have shown that VlP produces vasorelaxation or vasodilat a t i o n in cerebral a r t e r i e s of the r a b b i t (2), cat ( 1 , 3 , 5 ) , dog (6), baboon (4), pig, cow (7), and human (7,20). The l a t t e r were branches of the middle cerebral a r t e r y obtained from patients during surgery f o r tumors or i n t r a c t a b l e epilepsy. The v a r i a t i o n in the ED50 (5.3 f o l d ) f o r VIP, and in the maximum i n h i b i t i o n produced (1.6 f o l d ) , obtained in these pial a r t e r i e s is most l i k e l y due to the differences in the concentrations of prostaglandin F2~ (from 0.3 to 2.5X10-6M) used by the i n v e s t i g a t o r s to precontract the vessel rather than a difference in the preparation ( s p i r a l s t r i p s versus segments) (7,20). In t h i s regard, i t is evident in Fig. 2 that VIP is more e f f e c t i v e when the concentration of prostaglandin F2~ used produced submaximal contractions. In any case, previous reports agree that the maximum i n h i b i t i o n produced by VIP in human pial a r t e r i e s is obtained at 3XlO-6M (7,20). Questions remained, however, as to whether the i n h i b i t i o n persisted or was t r a n s i e n t and whether indomethacin a f f e c t s the response. The present study demonstrates that the larger b a s i l a r a r t e r y of the human also relaxes in response to VlP. The r e s u l t s f u r t h e r show that t h i s e f f e c t p e r s i s t s , so that the phenomenon of d e s e n s i t i z a t i o n need not l i m i t the e f f e c tiveness of VIP, at l e a s t f o r 15 min. Tachyphylaxis to VIP was apparent in some a r t e r i e s but was so v a r i a b l e t h a t , on average, the second and t h i r d applications of VlP produced a s i g n i f i c a n t v a s o d i l a t a t i o n , though less than the first. Tachyphylaxis may have been c l e a r l y demonstrable i f VlP had been applied f o r longer periods. In any case, i t was not as evident in the human b a s i l a r a r t e r y as i t is in the corresponding vessel of the canine (18). Moreover, in contrast to porcine and bovine cerebral a r t e r i e s (7), VlP did not i n h i b i t the contractions generated by K+ in the human b a s i l a r a r t e r y . Why some a r t e r i e s f a i l e d to respond to VIP is unknown, but i n d i v i d u a l v a r i a t i o n in responses to some agonists is c h a r a c t e r i s t i c of human cerebral a r t e r i e s , even "fresh" ones (21). Perhaps lower concentrations of VlP would have increased while higher concentrations would have decreased the f a i l u r e rate of 13%. In any case, the r e l a t i v e l y small branches of the human middle cerebral a r t e r y appear to respond r e l i a b l y to VIP (7,20). I t is possible that
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branches of the c i r c l e of W i l l i s may respond more c o n s i s t e n t l y to VIP since the nerves to some of these branches in humans contain abo,t twice as much immunor e a c t i v e VIP than the b a s i l a r ~ r t e r y (20). The r e s u l t obtained with indomethacin suggests t h a t the e f f e c t of VIP on human cerebral a r t e r i e s is not dependent on the production of prostanoids. In c o n t r a s t , v a s o d i l a t a t i o n of VIP in cat cerebral a r t e r i e s is i n h i b i t e d by indomethacin, but not i t s e f f e c t on peripheral flow (5). This also appears to be the case with bradykinin (22). Thus, cat cerebral a r t e r i e s may synthesize prostanoids as a means of responding to c e r t a i n v a s o d i l a t o r s while peripheral a r t e r i e s may not. In any case, indomethacin does not i n h i b i t the e f f e c t of VIP on a l l a r t e r i e s tested and the present f i n d i n g suggests t h a t i n h i b i t o r s of cyclooxygenase would not d i s r u p t a v a s o d i l a t o r response to VIP in humans cerebral a r t e r i e s normally or i f VIP was a c o n t r i b u t o r to vascular headache. The mechanism of action of VIP on human cerebral a r t e r i e s must d i f f e r from t h a t of AT I I I because only the l a t t e r blocked the usual c o n t r a c t i l e response to K+. The i n h i b i t o r y e f f e c t of AT I I I is not dependent on the presence of the endothelium (13), as is the case with many v a s o d i l a t o r s (22). AT I I I dots not s t i m u l a t e the sodium e l e c t r o g e n i c pump to e f f e c t v a s o r e l a x a t i o n nor produce tachyphylaxis (10). In 3XlO-8M concentration, representing 0.7% of the plasma concentration (4.5xlO-6M), AT I I I completely reverses the r i s e in i n t r a c e l l u l a r calcium e l i c i t e d by 50mM K+ in cultured vascular smooth muscle from r a t aortae (A. Hassid and R.P. White, unpublished data). This fundamental e f f e c t of AT I I I may account f o r i t s i n h i b i t i o n of a wide v a r i e t y of c o n t r a c t i l e agents l i k e UTP, s e r o t o n i n , and the bloody CSF of p a t i e n t s with SAH (10,13,23). I t is unknown whether VIP is e f f e c t i v e when blood is present in the CSF f o l l o w i n g SAH as proposed by others ( 9 ) , but the abundance of AT I I I in blood, and i t s marked i n h i b i t o r y q u a l i t i e s , makes i t a leading candidate as a f a c t o r which would prevent, or delay the onset o f , cerebral vasospasm of p a t i e n t s (10,13). There is evidence t h a t the cerebral a r t e r i e s which are most responsive to VIP are those t h a t are supplied with VIP immunoreactive nerves (1,8) and w i l l d i l a t e upon transmural nerve s t i m u l a t i o n by a mechanism independent of a f u n c t i o n a l endothelium or the release of a c e t y l c h o l i n e ( 1 , 3 ) . I t has been f u r t h e r argued t h a t the primary e f f e c t of a c e t y l c h o l i n e released by nerves supplying cerebral vessels is v a s o c o n s t r i c t i o n so that any v a s o d i l a t a t i o n produced by nerve s t i m u l a t i o n would r e s u l t from the release of v a s o d i l a t o r substances l i k e VIP (24). The primary e f f e c t of a c e t y l c h o l i n e on i s o l a t e d human cerebral a r t e r i e s is unclear, with vasorelaxation (25), mixed e f f e c t s ( i i ) , and v a s o c o n s t r i c t i o n (19) being reported. In c o n t r a s t , the only e f f e c t of VIP on the human vessel, observed by independent i n v e s t i g a t o r s , has been v a s o r e l a x a t i o n . This f i n d i n g , together with the above mentioned histochemical data, support the hypothesis t h a t VIPergic nerves supply human cerebral a r t e r i e s and t h a t VIP could serve as a v a s o d i l a t o r of those a r t e r i e s in health and disease. Acknowledgements This work was supported in part by grant NS 21405 of the National I n s t i t u t e s of Health, USPHS. The author thanks Dr. F. Curtis Dohan, Mr. Craig Lahren, Mrs. Marion Johnson and Mrs. Gwendolyn Stornes f o r t h e i r e s s e n t i a l assistance. References 1. 2.
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