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Reactivity of isolated bovine cerebral arteries to biogenic amines
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REACTIVITY O F I S O L A T E D B O V I N E CEREBRAL ARTERIES TO B I O G E N I C A M I N E S J. B. CHENG and S. SHIBATA University of Hawaii, School of Medicine, Department of Pharmacology, Honolulu, Hawaii 96822 U.S.A. (Received 19 September 1977) Abstract 1. Serotonin and histamine produced dose-dependent contraction in bovine anterior, middle, posterior cerebral and basilar arteries. Their sensitivity to serotonin was greater than that to histamine. Basilar arteries were the least sensitive to serotonin. 2. The maximal contractile response of serotonin was the same as that of histamine in middle, posterior cerebral and basilar but not in anterior cerebral arteries. 3. Only high concentrations of norepinephrine, methoxamine and isoproterenol caused a barely detectable contractile response in cerebral arteries. However, in the middle cerebral arteries, isoproterenol and norepinephrine (6 out of 10 strips) sometimes elicited a detectable relaxation which was blocked by propranolol. 4. No response to acetylcholine, carbachol, nicotine or transmural stimulation was seen. Tyramine (10 4 M) only induced a small tonic contraction. 5. These data suggest that adrenergic and cholinergic receptors may not play a major role in regulating the muscular tone of bovine cerebral arteries. In addition, the regional differences of the cerebrovascular reactivity to the above mentioned biogenic amines were observed.
INTRODUCTION
Despite extensive study, there is still c o n s i d e r a b l e disa g r e e m e n t c o n c e r n i n g the vascular responsiveness to vasoactive substances in cerebral b l o o d vessels. A l t h o u g h it is well k n o w n that cerebral b l o o d vessels receive rich adrenergic and cholinergic innervation, s o m e d o u b t r e m a i n s c o n c e r n i n g the eliciting of m e c h a n i c a l r e s p o n s e in cerebral arteries by stimulation o f adrenergic and cholinergic receptors (Raper et al., 1972~ T o d a & Fujita, 1973: Mitchell et al., 1975). T h e r e are at least t w o possible reasons for the conflicting results o b t a i n e d by various investigators. O n e m i g h t be attributed to species differences. In an earlier study, it was found that n o r e p i n e p h r i n e was a very p o t e n t s p a s m o g e n in h u m a n cerebral arteries (Shibata et al., 1977), whereas a relatively lower p o t e n c y was observed in dog (Toda & Fujita, 1973), cat (Nielsen & O w m a n , 1971) and goat (Urquilla et al., 1975). A n o t h e r reason for the differing p h e n o m e n a is a c h a n g e in vascular responsiveness b e t w e e n different regions of cerebral arteries. As this regional difference might p r o v i d e s o m e i n f o r m a t i o n in the regulation o f c e r e b r o v a s c u l a r tone, the vascular reactivity to bio\ genic amines was m e a s u r e d in the bovine anterior, middle, p o s t e r i o r cerebral and basilar arteries.
MATERIALS
AND
METHODS
Bovine brains (Hereford-Bos taurus, weighing 300--450 kg) were obtained from a local slaughterhouse and were removed immediately after sacrifice of the animal. The anterior, middle, posterior cerebral and basilar arteries were dissected from the brain. Before arrival in the laboratory, arteries were kept in cold Krebs-Ringer solution for about 25 rain. 189
The cerebral arteries were prepared in helical strips under the dissecting microscope and mounted vertically in a tissue bath containing 20 ml of Krebs-Ringer solution of the following composition (mM): NaC1, 120.3; KC1, 4.8; CaCI2, 1.2; MgSO4.7H20, 1.3; NaHCO 3, 24.2; and glucose, 5.5 bubbled with a gas mixture of 95'% O2, 5~g CO2 and maintained at 3 7 C _+ 1 C, pH = 7.4. A resting tension of 1 g was initially applied to the strips and maintained throughout the experiment. The preparations were allowed to equilibrate for 90 min in the bathing medium before the experiment commenced. Isometric tension changes were recorded through a force-displacement transducer (Grass FT.03) which was connected to a Grass polygraph. The full, cumulative dose-dependent curves were obtained by a stepwise increase in concentration of the agonist. A second dose was added, as soon as a steady response was obtained from the preceding dose. After a full dose-response for an angonist was obtained, the preparation was then allowed to rest for 30 min with changes of the bath fluid every 10 min before introducing a second different agonist. To determine the relaxation effect, some strips were first contracted with 5-HT (10 -4 M) to provide the tonic contraction upon which the test agents were studied. Crossdesensitization between the different agonists were not observed. Unless specified, the maximum contraction evoked by each agonist served as the 100'~o control to determine the median effective contraction (EDso). Numerical values are expressed as the mean _+ S.E.M. and significant differences are indicated by P-value < 0.05 in the Student's t-test. The following agents were used: 5-hydroxytryptamine creatine sulfate, l-noradrenaline bitartrate, methoxamine hydrochloride,/-nicotine (base), histamine dihydrochloride, isoproterenol hydrochloride, tyramine hydrochloride, acetylcholine chloride, carbachol, papaverine hydrochloride, phentolamine hydrochloride, propranolol hydrochloride, diphenhydramine hydrochloride and burimamide. The concentration of agents was always expressed as the final concentration in the tissue bath.
190
,I. B. (?HFY(; and S. SnlBATA Table 1, The internal diameter and walt thickness values in various portions of bovinc cerebral arteries Wall thickness (ram)
Blood vessels Anterior cerebral arteries Middle cerebral arteries Posterior cerebral arteries Basilar arteries
0.40 0.67 0.41 0.57
Internal diameter (ram)
+ 0.03 (8) _+ 0.06" (8) + 0.03 (8) + 0.05 (8)
0.99 + 0.08 (8) 1.39 _+ 0.03" (8) 1.02 + 0.07 (8~ 1.15 + 0.00 (8)
" Value significantly different from that of anterior and posterior and posterior cerebral arteries (P < 0.05).
R ES U LTS
Internal diameter and wall thickness The internal diameter and wall thickness of bovine cerebral arteries (anterior, middle, posterior cerebral and basilar arteries) were measured by the method of Bevan et al. (1974). Using an ocular micrometer, the internal diameter was derived from the longitudinal length, wet weight of the tissue and tissue gravity (1.05). To loosen the muscular tone, bovine cerebral arteries were incubated by Krebs Ringer medium containing papaverine (10 4 MI for 1 hr, and then cut longitudinally. The order of internal diameter magnitude of the blood vessels was: middle cerebral arteries > basilar arteries > posterior cerebral arteries + anterior cerebral arteries. The order of vascular wall thickness was: middle cerebral arteries + basilar arteries > posterior cerebral arteries + anterior cerebral arteries (Table I).
E~'ect ~] serotonin (5-HT), Norepinephrine, isoproterenol, methoxamine and histamine Figure 1 illustrates representative traces of contractile response to 5-HT, methoxamine, norepinephrine and isoproterenol on middle cerebral arteries. In the bovine middle cerebral arteries (N = 13 16 for each experiment), 5-HT and histamine (10 a 10 5 M) produced a dose-dependent contraction, whereas norepinephrine, methoxamine and isoproterenol (10 8 I0 "* M) caused only a negligible or a barely detectable effect. Similar results were observed in basilar, anterior and posterior cerebral arteries (N = 13 16 for each experiment). In some cases (6 out of 10 strips), isoproterenol and norepinephrine (10 -~ 1 0 - 5 M ) caused a small relaxation in the bovine middle cerebral arteries (Fig. 2), but not in
5-HT
•
8
~
7
ME
•
8
• 7
the anterior, posterior cerebral and basilar arteries. Pretreatment with propranolol (10 ~' M1 completely blocked these relaxations, Table 2 shows the median effect concentration (EDso) and the m a x i m u m contraction (maximum active tension) of 5-HT and histamine on the different portions of bovine cerebral arteries. In the bovine cerebral arteries, the potency of 5-HT (based on the EDso) was greater than that of histamine; based on the m a x i m u m contractile response, the contractile efficacy of 5-HT was not different from that of histamine except in the anterior cerebral arteries. In the anterior cerebral arteries, the efficacy of histamine was twice as great as that of 5-HT. Among the cerebral arteries used, the basilar arteries showed less sensitivity to 5-HT than the others. O n the other hand, all bovine cerebral arteries showed the same sensitivity to histamine. After treatment of diphenhydramine (10 " M I ( H ~ - blocker) for 10min, the EDso (2.1 _+ 1.0 x 10 a M ) for histamine in the cerebral arteries (N = 12) was increased to 9.0_+2.0 x 1 0 SM. O n the other hand, pretreatment with burimamide 110 -~' M ) ( H 2 - - blocker) for 10min failed to alter the sensitivity to histamine; there was no significant alteration in the EDso values (4.0 + 0.8 x 10 ~ M) for histamine after treatment in comparison with control value (3.5_+ 0.3 x 10 v M). Neither diphenhydramine nor b u r i m a m i d e treatment significantly altered the m a x i m u m contractile response of cerebral arteries to histamine.
Acetyh:holine and carbachol In this experiment, 5 strips of bovine anterior, middle, posterior cerebral and basilar arteries were used. None of the preparations showed any detectable contractile or relaxing response to acetylcholine or earba-
NE
•
6
•
5
6•
ISO
•
5
•
5
4•
IQ
rain
Fig. l. The representative contractile response of bovine middle cerebral arteries to 5-HT. methoxamine iMEI, norepinephrine (NE) and isoproterenol IISOI. Concentration is in log M.
Reactivity of isolated bovine cerebral arteries NE
Ig
7
ISO
6
5
7
• 6
• 5
rain
Fig. 2. The relaxing effect of norepinephrine (NE) and isoprotcrenol (1SO) on the bovine middle cerebral arteries (6 out of 10 strips). Concentration is in log M.
chol (10 9-10-5 M). Even in the presence of active tension (5-HT, 10 -~ M), both agents failed to elicit any relaxation of the bovine cerebral arteries (N = 4). Electrical t r a n s m u r a l stimulation
The effect of electrical stimulation (5 60V, 10--100Hz, 0.3 10msec in duration) was tested in 12 bovine arterial preparations. The transmural stimulation failed to evoke a relaxation or contraction in all arteries tested. N i c o t i n e and t y r a m i n e
In each of the 5 cerebral arteries, nicotine (10 ~ 10 4 M) failed to elicit demonstrable contractile or relaxing effect in all preparations without or with active tension (5-HT, 10 -4 M). The administration oftyramine in 10 -4 M but not in 10 5 M elicited a small tonic contractile response in the anterior, middle, posterior cerebral and basilar arteries. The maximum contractile response for tyramine was 0.25 + 0.01 g (N = 9) in the arterial strips. After treatment with pheutolamine (10- 6 M), however, tyramine failed to elicit a detectable response in the cerebral arteries.
DISCUSSION
The present experiment demonstrated that bovine cerebral arteries showed extremely low reactivity to alpha adrenergic agonists even in the comparison of low sensitivity to alpha adrenergic agonists in the cerebral arteries of dog (Toda & Fujita, 1973), cat (Nielsen & Owman, 1971) and goat (Urquilla et al., 1975). Unlike experiment animals, human cerebral arteries showed a much more potent response to norepinephrine (Shibata et al., 1977). It is interesting that among the bovine cerebral arteries, only a few middle cerebral arteries showed detectable relaxing response to isoproterenol and norepinephrine, which was effectively antagonized by beta adrenergic blocking agents. Thus, the relaxing effect is attributed to the stimulation of the beta receptors in the vascular smooth muscle of the bovine cerebral arteries. The different responses of the middle cerebral arteries could just as well be explained by non-reactive preparations rather than by different distribution of the beta receptors. However, isoproterenol barely caused a detectable response or had no effect on cerebroarterial strips from human (Shibata et a l , 1977) dog (Toda, 1974) and goat (Urquilla et • al., 1975). The antagonistic effect of diphenhydramine but not of burimamide on the histamine-induced contraction
191
of bovine cerebral arteries suggests that the receptors for histamine in cerebral arteries is of the H~-type. A similar class of histamine receptors in the goat cerebral arteries was also observed (Urquilla et al., 1975). Bovine cerebral arteries showed regional differences in the vascular reactivity to different vasoactive agents tested. However, it seems unlikely that this regional difference is reflected in the size of the arteries since vascular reactivity, in particular of the bovine cerebral arteries, is not directly correlated to the internal diameter of wall thickness of blood vessels. Recently, the regional differences support the idea that a reactivity of one cerebral artery cannot be generalized to other cerebral arteries of the same animal. Wei et aL (1975) reported that pial arteries larger than 1001tin in diameter contracted in response to topical application of norepinephrine, but, smaller pial arteries were unresponsive to norepinephrine. It was recently found that contractile response of serotonin was greater in distal than in middle portions of the human basilar arteries: however for norepinephrine, the contractions were greater in the middle segment (Shibata et al., 1977). This phenomena has also been established in the peripheral vascular bed, which shows a remarkable degree of variation in different parts of the circulation (Bevan & Su, 1973). Perhaps, the different functional pharmacological receptors in the cerebrovasculature may account for the different responses. In spite of regional differences in vascular reactivity, most of the bovine cerebral arteries showed relatively high sensitivity to 5-HT as compared to other test agents. These data are consistent with previously observed results found in the cat (Nielsen & Owman, 1971), dog (Toda & Fujita, 1973) goat (Urquilla et al., 1975) and human (Shibata et al., 1977). A lack of responses of electrical transmural stimulation in the bovine cerebral arteries might be explained by the sparse adrenergic innervation associated with inadequate release of norepinephrine or insufficient density of alpha receptors to cause a response in bovine cerebral arteries. In addition, they showed a lack of response to both acetylcholine and carbachol; thus, suggesting that there is a negligible role of the cholinergic mechanism in the regulation of vascular tone in the cerebral arteries. it is known that nicotine depolarized neuronal membrane of sympathetic nerve endings, consequently releasing a chemical transmitter (Su & Bevan, 1970). In addition, nicotine caused contraction of the peripheral vascular smooth muscle by this neurogenic mechanism (Shibata, 1969). It is reported that nicotine caused either contraction in human cerebral arteries (Shibata et al., 1977) or relaxation in dog cerebral arteries (Toda, 1975). The contractions or relaxations blocked by phentolamine and bretylium suggest an involvement of 2 different neurogenic mechanisms in the cerebral arteries of dog and human. In the present experiment, however, bovine cerebral arteries failed to respond to nicotine indicating the nicotine-induced response is species-dependent. Acknowledgements This work was research grant from the Hawaii Heart greatly appreciate the cooperation of the Ltd. from where bovine cerebral vessels
supported by a Association. We Hawaii Meat Co, were obtained.
2.6 ± 1.2 × 10 7u (13) 2.9 ± 1.2 x 10 ~' (141
0.61' ± 0.1 1.25 ¢ _+ 0.1
2.0 ± 1.2 x 10 ='' (15) 2.1 ± 1.0 x 10 t, (15)
1.65 ± 0.2 1.60 ± 11.2
M i d d l e cerebral artery EDso Max. tension (M) tg)
~' Each value represents the m e a n ± S.E.M. of 13 16 experiments. ~' Value significantly lower t h a n h i s t a m i n e (P < 0.05). Value significantly different. d Value significantly different from that of anterior, middle, and p o s t e r i o r cerebral ateries.
ltistamine
5-HT
Anterior cerebral artery EDso M a x tension (M) (g) 3.4 + 1.5 x 10 v~" (16) 2.8 ± 0.8 × 10 ~' {16)
1.05 ±0.14 1.10 ±0.6
P o s t e r i o r cerebral artery EDso Max. tension (M) {g)
Table 2. E D 5 ~alucs and m a x i m u m c o n t r a c t i o n of 5-HT and h i s t a m i n e in bo,,ine cerebral arteries"
9.5 _+ 2.2 × 10 7 .... (15) 3.8 + 1.7 x 10 t, (15)
1.80 + 0.3 1.86 +_ 0.3
Basilar artery EDs Max. tension IM) (g)
Z
=
Reactivity of isolated bovine cerebral arteries REFERENCES
BEVAN J. A. & Su C. (59731 Sympathetic mechanisms in blood vessels: Nerve and muscle relationships. A. Rer. Pharmac. 13, 269.-285. BEVAN J. A. HOSMER D. W., LJUNG B., PEt;RAM B. L. & Su C. (1974) Innervation pattern and neurogenic response of rabbit veins. Blood Vessels I I , 172 182. MITCHELL G., SCRIVEN D. R. L. & ROSENDORE~ C. (5975} Adrenoeeptors in intracerebral resistance vessels. Br. J. Pharmac. 54, 51 55. NIELSEN K. C. & OWMAN C. (1971) Contractile response and amine receptor mechanisms in isolated middle cerebral artery of the cat. Brain Res. 27, 33 42. RAPER A. J., KONTOS H. A., WEI E. P. & PATTERSON J. L. (1972) Unresponsiveness of pial precapillary vessels to catecholamines and sympathetic nerve stimulation. Circulation Res. 31, 257 266. SHIBATA S. 11969) Effect of prolonged cold storage on the contractile response of strips of rabbit aorta to various agents. Circulation Res. 24, 170 187. SHmATA S., CHENG J. B. & MURAKAMI W. (1977) Reactivity of isolated h u m a n cerebral arteries to biogenic amines. Blood Vessels 14, 356 365.
193
Su C. & BEVAN J. A. (1970) Blockade of the nicotineinduced norepinephrine released by cocaine, phenoxybenzamine and desipramine. J. Pharmac. exp. Ther. 175, 533 540. TODA N. (1974) The action of vasodilating drugs on isolated basilar, coronary and mesenteric arteries of the dog. J. Pharmac. exp. Ther. 191, 139 546. TODA N. (1975) Nicotine-induced relaxation in isolated canine cerebral arteries. J. Pharmac. exp. Ther. 193, 37(~384. TODA N. & FUJITA Y. (1973) Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephrine and transmural electrical stimulation. Circulation Res. 33~ 98 104. URQbqLLA P. R., MARCO E. J. & LLUCn S. (19751 Pharmacological receptors of the cerebral arteries of the goat. Blood Vessels 12, 53 67. WEI E. P., RAPER A. J., KONTOS H. A. & PATTERSON J. L., JR. (19751 Determinants of response of pial arteries to norepinephrine and sympathetic nerve stimulation. Stroke 6, 654-658.
0306-362 ~, 7 8 0 6 0 1 - 0 1 8 9 0 2 . 0 0
0
REACTIVITY O F I S O L A T E D B O V I N E CEREBRAL ARTERIES TO B I O G E N I C A M I N E S J. B. CHENG and S. SHIBATA University of Hawaii, School of Medicine, Department of Pharmacology, Honolulu, Hawaii 96822 U.S.A. (Received 19 September 1977) Abstract 1. Serotonin and histamine produced dose-dependent contraction in bovine anterior, middle, posterior cerebral and basilar arteries. Their sensitivity to serotonin was greater than that to histamine. Basilar arteries were the least sensitive to serotonin. 2. The maximal contractile response of serotonin was the same as that of histamine in middle, posterior cerebral and basilar but not in anterior cerebral arteries. 3. Only high concentrations of norepinephrine, methoxamine and isoproterenol caused a barely detectable contractile response in cerebral arteries. However, in the middle cerebral arteries, isoproterenol and norepinephrine (6 out of 10 strips) sometimes elicited a detectable relaxation which was blocked by propranolol. 4. No response to acetylcholine, carbachol, nicotine or transmural stimulation was seen. Tyramine (10 4 M) only induced a small tonic contraction. 5. These data suggest that adrenergic and cholinergic receptors may not play a major role in regulating the muscular tone of bovine cerebral arteries. In addition, the regional differences of the cerebrovascular reactivity to the above mentioned biogenic amines were observed.
INTRODUCTION
Despite extensive study, there is still c o n s i d e r a b l e disa g r e e m e n t c o n c e r n i n g the vascular responsiveness to vasoactive substances in cerebral b l o o d vessels. A l t h o u g h it is well k n o w n that cerebral b l o o d vessels receive rich adrenergic and cholinergic innervation, s o m e d o u b t r e m a i n s c o n c e r n i n g the eliciting of m e c h a n i c a l r e s p o n s e in cerebral arteries by stimulation o f adrenergic and cholinergic receptors (Raper et al., 1972~ T o d a & Fujita, 1973: Mitchell et al., 1975). T h e r e are at least t w o possible reasons for the conflicting results o b t a i n e d by various investigators. O n e m i g h t be attributed to species differences. In an earlier study, it was found that n o r e p i n e p h r i n e was a very p o t e n t s p a s m o g e n in h u m a n cerebral arteries (Shibata et al., 1977), whereas a relatively lower p o t e n c y was observed in dog (Toda & Fujita, 1973), cat (Nielsen & O w m a n , 1971) and goat (Urquilla et al., 1975). A n o t h e r reason for the differing p h e n o m e n a is a c h a n g e in vascular responsiveness b e t w e e n different regions of cerebral arteries. As this regional difference might p r o v i d e s o m e i n f o r m a t i o n in the regulation o f c e r e b r o v a s c u l a r tone, the vascular reactivity to bio\ genic amines was m e a s u r e d in the bovine anterior, middle, p o s t e r i o r cerebral and basilar arteries.
MATERIALS
AND
METHODS
Bovine brains (Hereford-Bos taurus, weighing 300--450 kg) were obtained from a local slaughterhouse and were removed immediately after sacrifice of the animal. The anterior, middle, posterior cerebral and basilar arteries were dissected from the brain. Before arrival in the laboratory, arteries were kept in cold Krebs-Ringer solution for about 25 rain. 189
The cerebral arteries were prepared in helical strips under the dissecting microscope and mounted vertically in a tissue bath containing 20 ml of Krebs-Ringer solution of the following composition (mM): NaC1, 120.3; KC1, 4.8; CaCI2, 1.2; MgSO4.7H20, 1.3; NaHCO 3, 24.2; and glucose, 5.5 bubbled with a gas mixture of 95'% O2, 5~g CO2 and maintained at 3 7 C _+ 1 C, pH = 7.4. A resting tension of 1 g was initially applied to the strips and maintained throughout the experiment. The preparations were allowed to equilibrate for 90 min in the bathing medium before the experiment commenced. Isometric tension changes were recorded through a force-displacement transducer (Grass FT.03) which was connected to a Grass polygraph. The full, cumulative dose-dependent curves were obtained by a stepwise increase in concentration of the agonist. A second dose was added, as soon as a steady response was obtained from the preceding dose. After a full dose-response for an angonist was obtained, the preparation was then allowed to rest for 30 min with changes of the bath fluid every 10 min before introducing a second different agonist. To determine the relaxation effect, some strips were first contracted with 5-HT (10 -4 M) to provide the tonic contraction upon which the test agents were studied. Crossdesensitization between the different agonists were not observed. Unless specified, the maximum contraction evoked by each agonist served as the 100'~o control to determine the median effective contraction (EDso). Numerical values are expressed as the mean _+ S.E.M. and significant differences are indicated by P-value < 0.05 in the Student's t-test. The following agents were used: 5-hydroxytryptamine creatine sulfate, l-noradrenaline bitartrate, methoxamine hydrochloride,/-nicotine (base), histamine dihydrochloride, isoproterenol hydrochloride, tyramine hydrochloride, acetylcholine chloride, carbachol, papaverine hydrochloride, phentolamine hydrochloride, propranolol hydrochloride, diphenhydramine hydrochloride and burimamide. The concentration of agents was always expressed as the final concentration in the tissue bath.
190
,I. B. (?HFY(; and S. SnlBATA Table 1, The internal diameter and walt thickness values in various portions of bovinc cerebral arteries Wall thickness (ram)
Blood vessels Anterior cerebral arteries Middle cerebral arteries Posterior cerebral arteries Basilar arteries
0.40 0.67 0.41 0.57
Internal diameter (ram)
+ 0.03 (8) _+ 0.06" (8) + 0.03 (8) + 0.05 (8)
0.99 + 0.08 (8) 1.39 _+ 0.03" (8) 1.02 + 0.07 (8~ 1.15 + 0.00 (8)
" Value significantly different from that of anterior and posterior and posterior cerebral arteries (P < 0.05).
R ES U LTS
Internal diameter and wall thickness The internal diameter and wall thickness of bovine cerebral arteries (anterior, middle, posterior cerebral and basilar arteries) were measured by the method of Bevan et al. (1974). Using an ocular micrometer, the internal diameter was derived from the longitudinal length, wet weight of the tissue and tissue gravity (1.05). To loosen the muscular tone, bovine cerebral arteries were incubated by Krebs Ringer medium containing papaverine (10 4 MI for 1 hr, and then cut longitudinally. The order of internal diameter magnitude of the blood vessels was: middle cerebral arteries > basilar arteries > posterior cerebral arteries + anterior cerebral arteries. The order of vascular wall thickness was: middle cerebral arteries + basilar arteries > posterior cerebral arteries + anterior cerebral arteries (Table I).
E~'ect ~] serotonin (5-HT), Norepinephrine, isoproterenol, methoxamine and histamine Figure 1 illustrates representative traces of contractile response to 5-HT, methoxamine, norepinephrine and isoproterenol on middle cerebral arteries. In the bovine middle cerebral arteries (N = 13 16 for each experiment), 5-HT and histamine (10 a 10 5 M) produced a dose-dependent contraction, whereas norepinephrine, methoxamine and isoproterenol (10 8 I0 "* M) caused only a negligible or a barely detectable effect. Similar results were observed in basilar, anterior and posterior cerebral arteries (N = 13 16 for each experiment). In some cases (6 out of 10 strips), isoproterenol and norepinephrine (10 -~ 1 0 - 5 M ) caused a small relaxation in the bovine middle cerebral arteries (Fig. 2), but not in
5-HT
•
8
~
7
ME
•
8
• 7
the anterior, posterior cerebral and basilar arteries. Pretreatment with propranolol (10 ~' M1 completely blocked these relaxations, Table 2 shows the median effect concentration (EDso) and the m a x i m u m contraction (maximum active tension) of 5-HT and histamine on the different portions of bovine cerebral arteries. In the bovine cerebral arteries, the potency of 5-HT (based on the EDso) was greater than that of histamine; based on the m a x i m u m contractile response, the contractile efficacy of 5-HT was not different from that of histamine except in the anterior cerebral arteries. In the anterior cerebral arteries, the efficacy of histamine was twice as great as that of 5-HT. Among the cerebral arteries used, the basilar arteries showed less sensitivity to 5-HT than the others. O n the other hand, all bovine cerebral arteries showed the same sensitivity to histamine. After treatment of diphenhydramine (10 " M I ( H ~ - blocker) for 10min, the EDso (2.1 _+ 1.0 x 10 a M ) for histamine in the cerebral arteries (N = 12) was increased to 9.0_+2.0 x 1 0 SM. O n the other hand, pretreatment with burimamide 110 -~' M ) ( H 2 - - blocker) for 10min failed to alter the sensitivity to histamine; there was no significant alteration in the EDso values (4.0 + 0.8 x 10 ~ M) for histamine after treatment in comparison with control value (3.5_+ 0.3 x 10 v M). Neither diphenhydramine nor b u r i m a m i d e treatment significantly altered the m a x i m u m contractile response of cerebral arteries to histamine.
Acetyh:holine and carbachol In this experiment, 5 strips of bovine anterior, middle, posterior cerebral and basilar arteries were used. None of the preparations showed any detectable contractile or relaxing response to acetylcholine or earba-
NE
•
6
•
5
6•
ISO
•
5
•
5
4•
IQ
rain
Fig. l. The representative contractile response of bovine middle cerebral arteries to 5-HT. methoxamine iMEI, norepinephrine (NE) and isoproterenol IISOI. Concentration is in log M.
Reactivity of isolated bovine cerebral arteries NE
Ig
7
ISO
6
5
7
• 6
• 5
rain
Fig. 2. The relaxing effect of norepinephrine (NE) and isoprotcrenol (1SO) on the bovine middle cerebral arteries (6 out of 10 strips). Concentration is in log M.
chol (10 9-10-5 M). Even in the presence of active tension (5-HT, 10 -~ M), both agents failed to elicit any relaxation of the bovine cerebral arteries (N = 4). Electrical t r a n s m u r a l stimulation
The effect of electrical stimulation (5 60V, 10--100Hz, 0.3 10msec in duration) was tested in 12 bovine arterial preparations. The transmural stimulation failed to evoke a relaxation or contraction in all arteries tested. N i c o t i n e and t y r a m i n e
In each of the 5 cerebral arteries, nicotine (10 ~ 10 4 M) failed to elicit demonstrable contractile or relaxing effect in all preparations without or with active tension (5-HT, 10 -4 M). The administration oftyramine in 10 -4 M but not in 10 5 M elicited a small tonic contractile response in the anterior, middle, posterior cerebral and basilar arteries. The maximum contractile response for tyramine was 0.25 + 0.01 g (N = 9) in the arterial strips. After treatment with pheutolamine (10- 6 M), however, tyramine failed to elicit a detectable response in the cerebral arteries.
DISCUSSION
The present experiment demonstrated that bovine cerebral arteries showed extremely low reactivity to alpha adrenergic agonists even in the comparison of low sensitivity to alpha adrenergic agonists in the cerebral arteries of dog (Toda & Fujita, 1973), cat (Nielsen & Owman, 1971) and goat (Urquilla et al., 1975). Unlike experiment animals, human cerebral arteries showed a much more potent response to norepinephrine (Shibata et al., 1977). It is interesting that among the bovine cerebral arteries, only a few middle cerebral arteries showed detectable relaxing response to isoproterenol and norepinephrine, which was effectively antagonized by beta adrenergic blocking agents. Thus, the relaxing effect is attributed to the stimulation of the beta receptors in the vascular smooth muscle of the bovine cerebral arteries. The different responses of the middle cerebral arteries could just as well be explained by non-reactive preparations rather than by different distribution of the beta receptors. However, isoproterenol barely caused a detectable response or had no effect on cerebroarterial strips from human (Shibata et a l , 1977) dog (Toda, 1974) and goat (Urquilla et • al., 1975). The antagonistic effect of diphenhydramine but not of burimamide on the histamine-induced contraction
191
of bovine cerebral arteries suggests that the receptors for histamine in cerebral arteries is of the H~-type. A similar class of histamine receptors in the goat cerebral arteries was also observed (Urquilla et al., 1975). Bovine cerebral arteries showed regional differences in the vascular reactivity to different vasoactive agents tested. However, it seems unlikely that this regional difference is reflected in the size of the arteries since vascular reactivity, in particular of the bovine cerebral arteries, is not directly correlated to the internal diameter of wall thickness of blood vessels. Recently, the regional differences support the idea that a reactivity of one cerebral artery cannot be generalized to other cerebral arteries of the same animal. Wei et aL (1975) reported that pial arteries larger than 1001tin in diameter contracted in response to topical application of norepinephrine, but, smaller pial arteries were unresponsive to norepinephrine. It was recently found that contractile response of serotonin was greater in distal than in middle portions of the human basilar arteries: however for norepinephrine, the contractions were greater in the middle segment (Shibata et al., 1977). This phenomena has also been established in the peripheral vascular bed, which shows a remarkable degree of variation in different parts of the circulation (Bevan & Su, 1973). Perhaps, the different functional pharmacological receptors in the cerebrovasculature may account for the different responses. In spite of regional differences in vascular reactivity, most of the bovine cerebral arteries showed relatively high sensitivity to 5-HT as compared to other test agents. These data are consistent with previously observed results found in the cat (Nielsen & Owman, 1971), dog (Toda & Fujita, 1973) goat (Urquilla et al., 1975) and human (Shibata et al., 1977). A lack of responses of electrical transmural stimulation in the bovine cerebral arteries might be explained by the sparse adrenergic innervation associated with inadequate release of norepinephrine or insufficient density of alpha receptors to cause a response in bovine cerebral arteries. In addition, they showed a lack of response to both acetylcholine and carbachol; thus, suggesting that there is a negligible role of the cholinergic mechanism in the regulation of vascular tone in the cerebral arteries. it is known that nicotine depolarized neuronal membrane of sympathetic nerve endings, consequently releasing a chemical transmitter (Su & Bevan, 1970). In addition, nicotine caused contraction of the peripheral vascular smooth muscle by this neurogenic mechanism (Shibata, 1969). It is reported that nicotine caused either contraction in human cerebral arteries (Shibata et al., 1977) or relaxation in dog cerebral arteries (Toda, 1975). The contractions or relaxations blocked by phentolamine and bretylium suggest an involvement of 2 different neurogenic mechanisms in the cerebral arteries of dog and human. In the present experiment, however, bovine cerebral arteries failed to respond to nicotine indicating the nicotine-induced response is species-dependent. Acknowledgements This work was research grant from the Hawaii Heart greatly appreciate the cooperation of the Ltd. from where bovine cerebral vessels
supported by a Association. We Hawaii Meat Co, were obtained.
2.6 ± 1.2 × 10 7u (13) 2.9 ± 1.2 x 10 ~' (141
0.61' ± 0.1 1.25 ¢ _+ 0.1
2.0 ± 1.2 x 10 ='' (15) 2.1 ± 1.0 x 10 t, (15)
1.65 ± 0.2 1.60 ± 11.2
M i d d l e cerebral artery EDso Max. tension (M) tg)
~' Each value represents the m e a n ± S.E.M. of 13 16 experiments. ~' Value significantly lower t h a n h i s t a m i n e (P < 0.05). Value significantly different. d Value significantly different from that of anterior, middle, and p o s t e r i o r cerebral ateries.
ltistamine
5-HT
Anterior cerebral artery EDso M a x tension (M) (g) 3.4 + 1.5 x 10 v~" (16) 2.8 ± 0.8 × 10 ~' {16)
1.05 ±0.14 1.10 ±0.6
P o s t e r i o r cerebral artery EDso Max. tension (M) {g)
Table 2. E D 5 ~alucs and m a x i m u m c o n t r a c t i o n of 5-HT and h i s t a m i n e in bo,,ine cerebral arteries"
9.5 _+ 2.2 × 10 7 .... (15) 3.8 + 1.7 x 10 t, (15)
1.80 + 0.3 1.86 +_ 0.3
Basilar artery EDs Max. tension IM) (g)
Z
=
Reactivity of isolated bovine cerebral arteries REFERENCES
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