Br. J. Pharmacol. (1990), 101, 200-204
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Macmillan Press Ltd, 1990 I
Contractile and relaxant responses of the canine isolated spinal artery to vasoactive substances K. Shirai, Y. Kawai & 'T. Ohhashi 1st Department of Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan
1 Effects of vasoactive substances were investigated in the canine isolated spinal branch of the intercostal artery (SBICA).
2 Addition of angiotensin II (All), vasopressin, noradrenaline (NA), adrenaline, 5-hydroxytryptamine (5HT), and dopamine each produced concentration-dependent contraction in the SBICA, whereas prostaglandin F2., histamine, and tyramine caused only slight contraction. The decreasing order of the potency of contractile agents was All > vasopressin = NA > 5-HT > adrenaline > dopamine. 3 Although the pD2 value for phenylephrine (5.31 0.36) was smaller than that for NA (6.48 + 0.13), there was no significant difference in EmaX value between these two agonists in the SBICA. On the other hand, xylazine produced only a slight contraction, the pD2 value being 3.59 + 0.08. Phentolamine (10-810-6 M) and prazosin (10-810-6 M) competitively inhibited the NA-induced contraction, while yohimbine (10-8-10-6M) did not. 4 Acetylcholine (ACh), sodium nitroprusside (SNP), ATP, ADP, and adenosine caused concentrationdependent relaxations in SBICA following contraction with NA. On the other hand, isoprenaline up to 1i-4M did not produce any relaxation. The decreasing order of potency of the relaxant agents was ACh > SNP > ATP = ADP = adenosine. 5 The ACh-induced relaxation was competitively inhibited by atropine and was abolished by mechanical removal of the endothelium. Aspirin (5 x 10- 5M) did not affect the relaxant response to ACh, while oxyhaemoglobin (10- 5 M) and methylene blue (10- 5 M) produced significant attenuation. 6 These results suggest that NA produces contraction of the isolated canine SBICA which is mainly mediated via a1-adrenoceptors and that ACh causes a relaxation of the SBICA due to release of endothelium-derived relaxing factor (EDRF) from the endothelial cells.
Introduction
Methods
The neural and humoral regulation of regional blood flow in the central nervous system is of both physiological and clinical interest. Many studies, both in vivo and in vitro, have been carried out to evaluate the mode of regulation of cerebral circulation (see Edvinsson & MacKenzie, 1977; Heistad & Kontos, 1983 for review). On the other hand, studies on the regulation of spinal cord blood flow have been limited. Blood flow in the spinal cord has been measured by means of in vivo techniques including hydrogen clearance (Korbine et al., 1974), 133Xe clearance (Smith et al., 1969; Griffiths, 1973), the microsphere method (Marcus et al., 1977), and [14C]-iodoantipyrine autoradiography (Sandler & Tator, 1976). Furthermore, new methods such as Laser-Doppler flowmetry (Sakamoto et al., 1988) and the ['4C]-butanol 'indicatorfractionation' technique (Lindsberg et al., 1989) have been validated for quantitating spinal blood flow in vivo. These studies have suggested that neural, chemical, metabolic, and autoregulatory factors may play important roles in the regulation of spinal cord blood flow. In contrast to the in vivo studies, there are few in vitro studies that have examined directly the effects of vasoactive substances on isolated blood vessels which supply blood to the spinal cord. The spinal cord receives its blood supply from arteries which are branches of either the vertebral artery, intercostal artery, lumbar artery, or iliolumbar artery. The present study was undertaken to compare the effects of various vasoactive substances on the isolated spinal branch of the intercostal artery (SBICA) of the dog, and to investigate pharmacologically the mechanisms of smooth muscle contraction and relaxation induced by noradrenaline and acetylcholine, respectively.
Mongrel dogs of either sex, weighing 6-13 kg, were anaesthetized with sodium pentobarbitone (25mgkg-1, i.v.) and killed by bleeding. Spinal branches of the 10th, 11th, and 12th intercostal arteries (0.6-0.8mm outer diameter) were quickly dissected out. After the arteries were cleaned of fat and connective tissue, they were cut into cylindrical segments (2mm long) under a dissecting microscope. Two tungsten wires were run through the lumen of the arterial segment. The preparations were suspended in 10 ml organ baths which were perfused with Krebs solution at a constant rate of 4 ml min -. The composition of the solution (mM) was as follows: NaCl 120.0, KCl 5.9, NaHCO3 25.0, NaH2PO4 1.2; CaCI2 2.5, MgCl2 1.2 and glucose 5.5. The solution was maintained at 36.0 + 0.50C and aerated with a gas mixture of 95% 02 and 5% CO2 to give a pH of 7.3-7.4. The upper end of the preparation was connected to a force-displacement transducer (Shinko Tsushin UL-10), and the lower end was fixed to the bottom of the bath. The resting tension was adjusted to 1.0g, which was optimal for obtaining maximum contractile response to 80 mm KCl solution.
' Author for
correspondence.
Experimental protocol All arterial segments were allowed to equilibrate for 90min. After the equilibration period, cumulative concentrationresponse curves for vasoconstrictor agents except angiotensin II (All) and vasopressin were obtained by stepwise increase in concentration of the drug by a factor of 10, as soon as a steady response to the previous administration had been achieved. Concentration-response curves for All and vasopressin were obtained by the addition of single concentration of the peptides every 30-120min, long enough to avoid tachyphylaxis. At the beginning and end of each experiment, the
RESPONSES OF ISOLATED SPINAL ARTERY
contractile response to 80 mm KCl solution was obtained. The extent of contraction induced by an agonist was expressed as a percentage of the KCl-induced contraction in each preparation. When a-adrenoceptor antagonists were tested, the drugs had been dissolved in oxygenated Krebs solution and perfused at least 30min before the concentration-response curves for a-adrenoceptor agonists were determined. Controls were always run with no antagonist to observe timedependent changes in the sensitivity of the preparations to the agonists. When responses to vasorelaxant agents were observed, the preparations were contracted by 5 x 10-6-10-5M noradrenaline (NA), which resulted in 40-60% of the 80mM KClinduced maximum contraction in each preparation. After the contraction reached a plateau, cumulative concentrationresponse curves for the relaxants were obtained in the same way as for constrictor agents. The extent of relaxation was expressed as a percentage of maximum relaxation induced by 10- M sodium nitroprusside. In some experiments, the effects of atropine, aspirin, methylene blue, and oxyhaemoglobin on the relaxant responses to acetylcholine (ACh) were investigated. These inhibitors had been perfused at least 30min before ACh was added. Oxyhaemoglobin was prepared by the technique described by Martin et al. (1985). The responses to ACh were also examined in preparations from which the endothelial cells had been removed by mechanically rubbing the intimal surface with cotton sticks. In four rubbed canine SBICA preparations, the absence of endothelial cells was confirmed histologically by the silver staining procedure (Poole et al., 1958).
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10
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-log [agonists] (M)
Figure 1 The contractile responses of canine isolated spinal branches of intercostal arteries to angiotensin II (*), vasopressin (K), noradrenaline (A), 5-hydroxytryptamine (0), adrenaline (A), dopamine (V), prostaglandin F2. (-), histamine (El), and tyramine (0). On the ordinate scale, contractions induced by 80mM KCl solution were taken as 100% in each experiment. All points represent the mean of 5 observations, and the vertical lines indicate the standard error of the mean.
Drugs The following drugs were used: acetylcholine chloride (Daiichi Seiyaku), atropine sulphate (Tanabe), ATP disodium (Wako), isoprenaline hydrochloride (Nikken Kagaku), methylene blue (Merck), phentolamine mesylate (Ciba-Geigy), prazosin hydrochloride (Pfizer), prostaglandin F2. (Ono Yakuhin), sodium nitroprusside (Merck), and xylazine hydrochloride (Bayer). (-)-Adrenaline bitartrate, angiotensin II, aspirin, dopamine hydrochloride, haemoglobin, histamine dihydrochloride, 5hydroxytryptamine creatinine sulphate, (-Y)noradrenaline bitartrate, tyramine hydrochloride, vasopressin, and yohimbine hydrochloride were obtained from Sigma Chemical Co. Adenosine, ADP disodium, and phenylephrine hydrochloride were purchased from Kowa Co. The concentrations of drugs are expressed in log molar terms.
Figure 2 demonstrates concentration-response curves of aadrenoceptor agonists in five preparations. Phenylephrine (a1-agonist) produced a concentration-dependent contraction. Although the pD2 value for phenylephrine was smaller (P < 0.05) than that for NA, there was no significant difference in the Emx values of these two agonists (Table 1). On the other hand, xylazine (Ox2-agonist) produced only a small contraction (Figure 2). The pD2 and Em.x values for xylazine were significantly smaller (P < 0.01) than those for NA (Table 1). Phentolamine (10-8-10-6M) and prazosin (10-8-10-6M) inhibited the responses to NA in a concentration-dependent manner (Figure 3a,b). On the other hand, yohimbine in the concentration range 10 8M-106 M did not affect the contrac50 r
Statistics Experimental values shown in the text, figures, and tables are mean + standard error of the mean (s.e.mean). The negative logarithm of ED50, the concentration of agonists causing one half of the maximum contraction, is expressed as the pD2 value. Statistical analyses were performed by use of Student's t test or one-way analysis of variance (ANOVA) followed by Dunnett's test. The difference in means were considered significant when P < 0.05.
c
0 IL)
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Results Contractile responses of isolated spinal arteries Administration of angiotensin II (AII), vasopressin, noradrenaline (NA), 5-hydroxytryptamine (5-HT), adrenaline, and dopamine produced concentration-dependent contractions in spinal branches of intercostal arteries (SBICA) of the dog (Figure 1). The order of potency of the contractile agents was AII > vasopressin = NA > 5-HT > adrenaline > dopamine. Prostaglandin F2, (PGF2G), histamine, and tyramine caused only very small contractions in these preparations (Figure 1).
0 9
8
7
6
5
4
3
-log [agonists] (M) Figure 2 The contractile responses of canine isolated spinal branches of intercostal arteries to noradrenaline (A), phenylephrine (0), and xylazine (0). The ordinate scale is the same as in Figure 1. All points represent the mean of 5 observations, and the vertical lines indicate s.e.mean.
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Table 1 pD2 and Em.. values for various a-adrenoceptor agonists in the canine spinal branch of the intercostal artery
-log [Agonists] (M) 9
10.
E mnax a
8 a
.
7
6
5
4
0i
Agonist
n
pD2
(%)
Noradrenaline Phenylephrine Xylazine
5 5 5
6.48 + 0.13 5.31 + 0.36* 3.59 + 0.08**
43.4 + 5.6 45.7 + 6.9Ns 3.7 + 1.6**
a Maximum contraction produced by a-agonists as a percentage of KCI response. * Significantly different from the value for noradrenaline, P < 0.05, ** P SNP > ATP = ADP = adenosine. Addition of isoprenaline ranging from 10 -8M to I0- M did not produce any relaxation in these preparations (Figure 4). Atropine (10--1i0-6M) caused a parallel shift to the right of the concentration-response curve to ACh. Schild plot
analysis showed that the slope was 1.11 + 0.12, which did not differ from unity and the pA2 value was 10.6 + 0.54 (n = 6). As shown in Figure 5b, addition of ACh (10 1- -10-3 M) did not cause relaxation in preparations without endothelium, but SNP 10- 5M did. Pretreatment with 5 x 10-5M aspirin did not affect ACh-induced relaxation (Figure Sc). On the other hand, the effect of ACh was significantly attenuated by pretreatment with 10- 5M methylene blue (Figure Sd). Pretreatment with oxyhaemoglobin 10- M increased the threshold concentration and decreased the maximum relaxation produced by ACh. These results are summarized in Figure 6.
Table 2 Slope and pA2 values of a-adrenoceptor antagonists against noradrenaline in canine spinal branch of intercostal artery n
Slope
pA2
Phentolamine Prazosin Yohimbine
5 5 5
1.07 + 0.12s 1.07 + 0.14S No effect
8.79 + 0.39 10.20 + 0.47 No effect
L
Figure 4 The relaxant responses to acetylcholine (El), sodium nitroprusside (U), ATP (A), ADP (0), adenosine (A), and isoprenaline (*) in canine isolated spinal branches of intercostal arteries contracted by 5 x 10--0-10`M noradrenaline. On the ordinate scale, relaxations induced by 10- M sodium nitroprusside were taken as 100% in each experiment. All points represent the mean of 5 observations, and the vertical lines indicate s.e.mean.
Relaxant responses of isolated spinal arteries
Antagonist
50 .
a)
Discussion Contractile responses of isolated spinal arteries
NS The value does not significantly differ from unity. The values are mean + s.e.mean, and n is the number of
The present results revealed that vasoactive substances such as AII, vasopressin, NA, 5-HT, adrenaline and dopamine
observations.
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100
100
100
50
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3
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Figure 3 The effects of various a-adrenoceptor antagonists on contractile respones to noradrenaline in canine isolated spinal branches of intercostal arteries; (a) control (A), 10- I M (A), 10- 7 M (V), 10-6 M (V), phentolamine (n = 7); (b) control (A), 10-8M (A), I0- I M (V), 10- I m (V), prazosin (n = 6); (c) control (A), 10- 8 M (A), 10- 7 M (V), 10-6 M (V), yohimbine (n = 6). The ordinate scale is the same as in Figure 1. The vertical lines indicate the standard error of the mean.
RESPONSES OF ISOLATED SPINAL ARTERY a
-Iog[SNPI(M) 5
b 5mm I
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0.2g
AA ~~~8
5 min
7AAA
A
6 514 3 - log[AChl(M)
c
d
0.2
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AAAA
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log[AChl(M)
Figure 5 Typical responses of isolated canine spinal branches of intercostal arteries to acetylcholine; (a) control; (b) responses to acetylcholine (ACh) and sodium nitroprusside (SNP) in the preparation from which endothelial cells were removed; (c) pretreatment with 5 x I0- M aspirin; (d) pretreatment with 10- 5 M methylene blue. All concentrations in this figure are given as negative logs.
caused a concentration-dependent contraction in the canine isolated spinal branch of the intercostal artery (SBICA). Among these substances, All was the most potent vasoconstrictor (Figure 1). In general, All is regarded as a potent vasoconstrictor agent in isolated small arterial preperations. However, there is a regional difference in the response of isolated arteries to All, and it produces only a slight contraction in canine cerebral arteries (Toda & Miyazaki, 1978). In canine isolated cerebral arteries, All also causes a relaxation in preparations contracted by PGF2. (Toda & Miyazaki, 1981). This relaxation seems to be mediated mainly by the release of prostacyclin from the endothelial cells. In contrast to the cerebral arteries, pretreatment with 5 x 10-5M aspirin produced no effect on the All-induced contraction in the SBICA. Thus, there may be a difference in AII-induced release of endogenous vasodilator prostaglandins between cerebral and spinal arteries. PGF2. produces a marked contraction in isolated cerebral arteries of various species (White & Hagen, 1982). However, PGF2. up to 10-0M elicited only a small contraction of the -log[ACh](M) 11a
10 -
9
8 EI 7-II 6 a
5
4
3
0
,o1
X 50
Y y y y
100
Figure 6 The effects of aspirin 5 x lo- M (A, n = 7), methylene blue 10-5M (V, n = 12), oxyhaemoglobin 10-5M (0, n = 7) and removal of endothelium (O, n = 6) on relaxant responses to acetylcholine in isolated canine spinal branches of intercostal arteries. The control concentration-response curve to acetylcholine is represented by (-, n = 5). The vertical lines indicate s.e.mean.
SBICA in the present experiments. Thus, the response of the SBICA to PGF2. seems to be markedly different from that of cerebral arteries. Electron microscopic (Iwayama et al., 1970; Nielsen et al., 1971; Sato & Suzuki, 1975) and histochemical studies (Nielsen & Owman, 1967; Hernandez-Perez & Stone, 1974; Kawai & Ohhashi, 1986) demonstrated a dense adrenergic innervation in cerebral arteries of various animals. Howevef, electrical stimulation of the sympathetic nerves produces only slight or no contraction of cerebral arteries (Raper et al., 1972; Toda & Fujita, 1973). Furthermore, contraction of cerebral arteries, including the internal carotid artery, induced by exogenous NA is considerably smaller than that produced by other agonists such as 5-HT and PGF2. (Bohr et al., 1961; Toda & Fujita, 1973; Kawai et al., 1984). Thus, the physiological significance of sympathetic nerves in the regulation of cerebral circulation is not clear. In the present study, the addition of tyramine produced no contraction in SBICA (Figure 1). This does not imply, however, that sympathetic nerves do not play an important role in the regulation of the spinal circulation. Morphological studies demonstrated the existence of sympathetic innervation of the smaller arteries supplying the spinal cord (Ohgushi, 1968; Marcus et al., 1977; McNicolas et al., 1980). The pharmacological characteristics of the a-adrenoceptors in SBICA were investigated with selective agonists and antagonists. The results showed that NA (al- and a2-agonist) and phenylephrine (a1-agonist) caused almost the same maximum contraction, although the pD2 value for phenylephrine was smaller than that for NA (Table 1). On the other hand, xylazine (a2-agonist) produced only a small contraction (Figure 2, Table 1). These results suggest that NA and phenylephrine contract the SBICA mainly through activation of a1-adrenoceptors on the smooth muscle cells. This view is also supported by the present results that phentolamine (at- and a2-antagonist) and prazosin (a1-antagonist) competitively inhibited the NA-induced contraction but yohimbine (a2-antagonist) did not (Figure 3). Previous work has suggested that the a-adrenoceptor subtype in smooth muscle cells of canine cerebral arteries was a2 in the middle cerebral and basilar arteries (Sakakibara et al., 1982; Skarby et al., 1983; Toda, 1983), and both al and a2 in the internal carotid arteries (Kawai et al., 1988). On the other hand, NA-induced contractions are mediated by a1-adrenoceptors in canine extracranial arteries of various tissues (Sakakibara et al., 1982; Toda, 1983). Our findings suggest that the a-adrenoceptor subtype in the SBICA is similar to that in the extracranial arteries rather than in the cerebral arteries of dogs.
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Relaxant responses of isolated spinal arteries The addition of ACh, SNP, ATP, ADP, and adenosine produced concentration-dependent relaxations in SBICA which had been contracted by NA. On the other hand, isoprenaline up to IO- M did not cause any relaxation in the preparations. Stimulation of fi-adrenoceptors produces cerebral vasodilatation in vivo (Edvinsson & MacKenzie, 1977). In addition, fiadrenoceptor agonists induce a relaxation of isolated cerebral vessels when the preparations have intrinsic tone. These findings suggest that fi-adrenoceptors exist in cerebral arteries, contrasting with the present results in the SBICA. Among the vasodilators used, ACh was the most potent on the SBICA. This relaxation seems to be mediated via muscarinic receptors, since it was competitively inhibited by atropine. Relaxation induced by ACh, but not that induced by SNP, was abolished by mechanical removal of the endothelium. It is well known that endothelium-derived relaxing factor (EDRF) (Furchgott & Zawadzki, 1980) and prostacyclin (Moncada et al., 1977) are released from vascular endothelial cells and cause vascular smooth muscle cells to relax. In the present experiments, treatment with aspirin, an inhibi-
tor of cyclo-oxygenase, did not affect the ACh-induced relaxation, suggesting that prostacyclin did not play an important role in the ACh-induced relaxation. On the other hand, oxyhaemoglobin, an inhibitor of EDRF, and methylene blue, an inhibitor of guanylate cyclase, markedly suppressed the AChinduced relaxation in the spinal arteries. All these findings strongly suggest that the ACh-induced relaxation in the SBICA is mainly mediated through release of EDRF. A previous report suggests that ACh-induced endotheliumdependent relaxation is relatively small in canine cerebral arteries (Tsukahara et al., 1986). There may be a regional difference in endothelial function or sensitivity of vascular smooth muscles to EDRF between spinal and cerebral arteries. The present experimental results suggest that the pharmacological characteristics of SBICA are quite different from those of the isolated canine cerebral arteries reported in previous studies. In the canine SBICA, the addition of NA produces considerable contraction which is mainly mediated via a1-adrenoceptors. On the other hand, ACh causes a marked relaxation of the SBICA due to the release of EDRF from the endothelial cells.
References ARUNLAKSHANA, 0. & SCHILD, H.O. (1959) Some quantitative uses of drug antagonists. Br. J. Pharmacol. Chemother., 14, 48-59. BOHR, D.F., GOULET, P.L. & TAQUINI, A.C. Jr. (1961) Direct tension recording from smooth muscle of resistance vessels from various organs. Angiology, 12,478485. EDVINSSON? L. & MAcKENZIE, E.T. (1977) Amine mechanisms in the cerebral circulation. Pharmacol. Rev., 28, 275-348. FURCHGOTT, R.F. & ZAWADZKI, J.V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature, 288, 373-376. GRIFFITHS, I.R. (1973) Spinal cord blood flow in dogs. 1. The "Normal flow". J. Neurol. Neurosurg. Psychiatr., 36, 34-41. HEISTAD, D.D. & KONTOS, H.A. (1983) Cerebral circulation. In Handbook of Physiology, Sect. 2 Vol. 3 ed. Shephard, J. & Abboud, F.M. pp. 137-182, Bethesda: Am. Physiol. Soc. HERNANDEZ-PtREZ, M.J. & STONE, H.L. (1974) Sympathetic innervation of the circle of Willis in the macaque monkey. Brain Res., 80, 507-511. IWAYAMA, T., FURNESS, J.B. & BURNSTOCK, G. (1970) Dual adrenergic and cholinergic innervation of the cerebral arteries of the rat. Circ. Res., 26, 635-646. KAWAI, Y., KOBAYASHI, S. & OHHASHI, T. (1988) Existence of two types of postjunctional a-adrenoceptors in the isolated canine internal carotid artery. Can. J. Physiol. Pharmacol., 66, 655-659. KAWAI, Y. & OHHASHI, T. (1986). Histochemical studies of the adrenergic innervation of canine cerebral arteries. J. Autonom. Nerv. Syst., 15, 103-108. KAWAI, Y., OHHASHI, T. & AZUMA, T. (1984) Redistribution of flow between canine isolated internal and external carotid arteries by vasoactive substances. Jpn. J. Physiol., 34, 457-468. KORBINE, A.I., DOYLE, T.F. & MARTINS, A.N. (1974) Spinal cord blood flow in the rhesus monkey by the hydrogen clearance method. Surg. Neurol., 2, 197-200. LINDSBERG, P.J., O'NEILL, J.T., PAAKKARI, I.A., HALLENBECK, J.M. & FEUERSTEIN, G. (1989) Validation of laser-doppler flowmetry in measurement of spinal cord blood flow. Am. J. Physiol., 257, H674-H680. MARCUS, M.L, HEISTAD, D.D., EHRHARDT, J.C. & ABBOUD, F.M. (1977). Regulation of total and regional spinal cord blood flow. Circ. Res., 41, 128-134. MARTIN, W., VILLANI, G.M., JOTHIANANDAN, D. & FURCHGOTT, R.F. (1985). Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta. J. Pharmacol. Exp. Ther., 232, 708-716. McNICOLAS, L.F., MARTIN, W.R., SLOAN, J.W. & NOZAKI, M. (1980)
Innervation of the spinal cord by sympathetic fibers. Exp. Neurol., 69, 383-394. MONCADA, S., HERMAN, A.G., HIGGS, E.A. & VANE, J.R. (1977). Differential formation of prostacyclin (PGX or PGI2) by layers of arterial wall. An explanation for the antithrombic properties of vascular endothelium. Thromb. Res., 11, 323-344.
NIELSEN, K.C. & OWMAN, C. (1967) Adrenergic innervation of pial arteries related to the circle of Willis in the cat. Brain Res., 6, 773-776. NIELSEN, K.C., OWMAN, C. & SPORRONG, B. (1971). Ultrastructure of autonomic innervation apparatus in the main pial arteries of rats and cats. Brain Res., 27, 25-32. OHGUSHI, M. (1968). Adrenergic fibers to the brain and spinal cord vessels in the dog. Arch. Jpn. Chir., 37, 294-303. POOLE, J.C.F., SANDERS, A.G. & FLOREY, H.W. (1958). The regeneration of aortic endothelium. J. Path. Bact., 75, 133-143. RAPER, A.J., KONTOS, H.A., WEI, E.P. & PATTERSON, J.L. (1972). Unresponsiveness of pial precapillary vessels to catecholamines and sympathetic nerve stimulation. Circ. Res., 31, 257-266. SAKAKIBARA, Y., FUJIWARA, M. & MURAMATSU, I. (1982) Pharmacological characterization of the alpha adrenoceptors of the dog basilar artery. Naunyn-Schmiedebergs Arch. Pharmacol., 319, 1-7. SAKAMOTO, T., SHIMAZAKI, S. & MONAFO, W.W. (1988). [14C]butanol distribution: a new method for measurement of spinal cord blood flow. Am. J. Physiol., 255, H953-H959. SANDLER, A.N. & TATOR, C.H. (1976). Regional spinal cord flow in primates. J. Neurosurg., 45, 647-659. SATO, S. & SUZUKI, J. (1975). Anatomical mapping of the cerebral nervi vasorum in the human brain. J. Neurosurg., 43, 559-568. SKARBY, T.V.C., ANDERSSON, K.-E. & EDVINSSON, L. (1983). Pharmacological characterization of postjunctional a-adrenoceptors in isolated feline cerebral and peripheral arteries. Acta Physiol. Scand., 117, 63-73. SMITH, A.L., PENDER, J.W. & ALEXANDER, S.C. (1969) Effects of PCO2 on spinal cord blood flow. Am. J. Physiol., 216, 1158-1163. TODA, N. (1983). Alpha adrenergic receptor subtypes in human, monkey and dog cerebral arteries. J. Pharmacol. Exp. Ther., 226, 861-868. TODA, N. & FUJITA, Y. (1973). Responsiveness of isolated cerebral and peripheral arteries to serotonin, norepinephrine, and transmural electrical stimulation. Circ. Res., 33, 98-104. TODA, N. & MIYAZAKI, M. (1978). Regional and species differences in the response of isolated arteries to angiotensin II. Jpn. J. Pharmacol., 28, 495-497. TODA, N. & MIYAZAKI, M. (1981). Angiotensin-induced relaxation in isolated dog renal and cerebral arteries. Am. J. Physiol., 240, H247-H254. TSUKAHARA, T., USUI, H., TANIGUCHI, T., SHIMOHAMA, S., FUJIWARA, M. & HANDA, H. (1986). Characterization of muscarinic cholinergic receptors in human and dog cerebral arteries. Stroke, 17, 300-305. WHITE, R.P. & HAGEN, A.A. (1982). Cerebrovascular actions of prostaglandins. Pharmacol. Ther., 18, 313-331.
(Received February 19,1990 Revised April 17,1990 Accepted April 20, 1990)
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Macmillan Press Ltd, 1990 I
Contractile and relaxant responses of the canine isolated spinal artery to vasoactive substances K. Shirai, Y. Kawai & 'T. Ohhashi 1st Department of Physiology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390, Japan
1 Effects of vasoactive substances were investigated in the canine isolated spinal branch of the intercostal artery (SBICA).
2 Addition of angiotensin II (All), vasopressin, noradrenaline (NA), adrenaline, 5-hydroxytryptamine (5HT), and dopamine each produced concentration-dependent contraction in the SBICA, whereas prostaglandin F2., histamine, and tyramine caused only slight contraction. The decreasing order of the potency of contractile agents was All > vasopressin = NA > 5-HT > adrenaline > dopamine. 3 Although the pD2 value for phenylephrine (5.31 0.36) was smaller than that for NA (6.48 + 0.13), there was no significant difference in EmaX value between these two agonists in the SBICA. On the other hand, xylazine produced only a slight contraction, the pD2 value being 3.59 + 0.08. Phentolamine (10-810-6 M) and prazosin (10-810-6 M) competitively inhibited the NA-induced contraction, while yohimbine (10-8-10-6M) did not. 4 Acetylcholine (ACh), sodium nitroprusside (SNP), ATP, ADP, and adenosine caused concentrationdependent relaxations in SBICA following contraction with NA. On the other hand, isoprenaline up to 1i-4M did not produce any relaxation. The decreasing order of potency of the relaxant agents was ACh > SNP > ATP = ADP = adenosine. 5 The ACh-induced relaxation was competitively inhibited by atropine and was abolished by mechanical removal of the endothelium. Aspirin (5 x 10- 5M) did not affect the relaxant response to ACh, while oxyhaemoglobin (10- 5 M) and methylene blue (10- 5 M) produced significant attenuation. 6 These results suggest that NA produces contraction of the isolated canine SBICA which is mainly mediated via a1-adrenoceptors and that ACh causes a relaxation of the SBICA due to release of endothelium-derived relaxing factor (EDRF) from the endothelial cells.
Introduction
Methods
The neural and humoral regulation of regional blood flow in the central nervous system is of both physiological and clinical interest. Many studies, both in vivo and in vitro, have been carried out to evaluate the mode of regulation of cerebral circulation (see Edvinsson & MacKenzie, 1977; Heistad & Kontos, 1983 for review). On the other hand, studies on the regulation of spinal cord blood flow have been limited. Blood flow in the spinal cord has been measured by means of in vivo techniques including hydrogen clearance (Korbine et al., 1974), 133Xe clearance (Smith et al., 1969; Griffiths, 1973), the microsphere method (Marcus et al., 1977), and [14C]-iodoantipyrine autoradiography (Sandler & Tator, 1976). Furthermore, new methods such as Laser-Doppler flowmetry (Sakamoto et al., 1988) and the ['4C]-butanol 'indicatorfractionation' technique (Lindsberg et al., 1989) have been validated for quantitating spinal blood flow in vivo. These studies have suggested that neural, chemical, metabolic, and autoregulatory factors may play important roles in the regulation of spinal cord blood flow. In contrast to the in vivo studies, there are few in vitro studies that have examined directly the effects of vasoactive substances on isolated blood vessels which supply blood to the spinal cord. The spinal cord receives its blood supply from arteries which are branches of either the vertebral artery, intercostal artery, lumbar artery, or iliolumbar artery. The present study was undertaken to compare the effects of various vasoactive substances on the isolated spinal branch of the intercostal artery (SBICA) of the dog, and to investigate pharmacologically the mechanisms of smooth muscle contraction and relaxation induced by noradrenaline and acetylcholine, respectively.
Mongrel dogs of either sex, weighing 6-13 kg, were anaesthetized with sodium pentobarbitone (25mgkg-1, i.v.) and killed by bleeding. Spinal branches of the 10th, 11th, and 12th intercostal arteries (0.6-0.8mm outer diameter) were quickly dissected out. After the arteries were cleaned of fat and connective tissue, they were cut into cylindrical segments (2mm long) under a dissecting microscope. Two tungsten wires were run through the lumen of the arterial segment. The preparations were suspended in 10 ml organ baths which were perfused with Krebs solution at a constant rate of 4 ml min -. The composition of the solution (mM) was as follows: NaCl 120.0, KCl 5.9, NaHCO3 25.0, NaH2PO4 1.2; CaCI2 2.5, MgCl2 1.2 and glucose 5.5. The solution was maintained at 36.0 + 0.50C and aerated with a gas mixture of 95% 02 and 5% CO2 to give a pH of 7.3-7.4. The upper end of the preparation was connected to a force-displacement transducer (Shinko Tsushin UL-10), and the lower end was fixed to the bottom of the bath. The resting tension was adjusted to 1.0g, which was optimal for obtaining maximum contractile response to 80 mm KCl solution.
' Author for
correspondence.
Experimental protocol All arterial segments were allowed to equilibrate for 90min. After the equilibration period, cumulative concentrationresponse curves for vasoconstrictor agents except angiotensin II (All) and vasopressin were obtained by stepwise increase in concentration of the drug by a factor of 10, as soon as a steady response to the previous administration had been achieved. Concentration-response curves for All and vasopressin were obtained by the addition of single concentration of the peptides every 30-120min, long enough to avoid tachyphylaxis. At the beginning and end of each experiment, the
RESPONSES OF ISOLATED SPINAL ARTERY
contractile response to 80 mm KCl solution was obtained. The extent of contraction induced by an agonist was expressed as a percentage of the KCl-induced contraction in each preparation. When a-adrenoceptor antagonists were tested, the drugs had been dissolved in oxygenated Krebs solution and perfused at least 30min before the concentration-response curves for a-adrenoceptor agonists were determined. Controls were always run with no antagonist to observe timedependent changes in the sensitivity of the preparations to the agonists. When responses to vasorelaxant agents were observed, the preparations were contracted by 5 x 10-6-10-5M noradrenaline (NA), which resulted in 40-60% of the 80mM KClinduced maximum contraction in each preparation. After the contraction reached a plateau, cumulative concentrationresponse curves for the relaxants were obtained in the same way as for constrictor agents. The extent of relaxation was expressed as a percentage of maximum relaxation induced by 10- M sodium nitroprusside. In some experiments, the effects of atropine, aspirin, methylene blue, and oxyhaemoglobin on the relaxant responses to acetylcholine (ACh) were investigated. These inhibitors had been perfused at least 30min before ACh was added. Oxyhaemoglobin was prepared by the technique described by Martin et al. (1985). The responses to ACh were also examined in preparations from which the endothelial cells had been removed by mechanically rubbing the intimal surface with cotton sticks. In four rubbed canine SBICA preparations, the absence of endothelial cells was confirmed histologically by the silver staining procedure (Poole et al., 1958).
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Figure 1 The contractile responses of canine isolated spinal branches of intercostal arteries to angiotensin II (*), vasopressin (K), noradrenaline (A), 5-hydroxytryptamine (0), adrenaline (A), dopamine (V), prostaglandin F2. (-), histamine (El), and tyramine (0). On the ordinate scale, contractions induced by 80mM KCl solution were taken as 100% in each experiment. All points represent the mean of 5 observations, and the vertical lines indicate the standard error of the mean.
Drugs The following drugs were used: acetylcholine chloride (Daiichi Seiyaku), atropine sulphate (Tanabe), ATP disodium (Wako), isoprenaline hydrochloride (Nikken Kagaku), methylene blue (Merck), phentolamine mesylate (Ciba-Geigy), prazosin hydrochloride (Pfizer), prostaglandin F2. (Ono Yakuhin), sodium nitroprusside (Merck), and xylazine hydrochloride (Bayer). (-)-Adrenaline bitartrate, angiotensin II, aspirin, dopamine hydrochloride, haemoglobin, histamine dihydrochloride, 5hydroxytryptamine creatinine sulphate, (-Y)noradrenaline bitartrate, tyramine hydrochloride, vasopressin, and yohimbine hydrochloride were obtained from Sigma Chemical Co. Adenosine, ADP disodium, and phenylephrine hydrochloride were purchased from Kowa Co. The concentrations of drugs are expressed in log molar terms.
Figure 2 demonstrates concentration-response curves of aadrenoceptor agonists in five preparations. Phenylephrine (a1-agonist) produced a concentration-dependent contraction. Although the pD2 value for phenylephrine was smaller (P < 0.05) than that for NA, there was no significant difference in the Emx values of these two agonists (Table 1). On the other hand, xylazine (Ox2-agonist) produced only a small contraction (Figure 2). The pD2 and Em.x values for xylazine were significantly smaller (P < 0.01) than those for NA (Table 1). Phentolamine (10-8-10-6M) and prazosin (10-8-10-6M) inhibited the responses to NA in a concentration-dependent manner (Figure 3a,b). On the other hand, yohimbine in the concentration range 10 8M-106 M did not affect the contrac50 r
Statistics Experimental values shown in the text, figures, and tables are mean + standard error of the mean (s.e.mean). The negative logarithm of ED50, the concentration of agonists causing one half of the maximum contraction, is expressed as the pD2 value. Statistical analyses were performed by use of Student's t test or one-way analysis of variance (ANOVA) followed by Dunnett's test. The difference in means were considered significant when P < 0.05.
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Results Contractile responses of isolated spinal arteries Administration of angiotensin II (AII), vasopressin, noradrenaline (NA), 5-hydroxytryptamine (5-HT), adrenaline, and dopamine produced concentration-dependent contractions in spinal branches of intercostal arteries (SBICA) of the dog (Figure 1). The order of potency of the contractile agents was AII > vasopressin = NA > 5-HT > adrenaline > dopamine. Prostaglandin F2, (PGF2G), histamine, and tyramine caused only very small contractions in these preparations (Figure 1).
0 9
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-log [agonists] (M) Figure 2 The contractile responses of canine isolated spinal branches of intercostal arteries to noradrenaline (A), phenylephrine (0), and xylazine (0). The ordinate scale is the same as in Figure 1. All points represent the mean of 5 observations, and the vertical lines indicate s.e.mean.
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Table 1 pD2 and Em.. values for various a-adrenoceptor agonists in the canine spinal branch of the intercostal artery
-log [Agonists] (M) 9
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E mnax a
8 a
.
7
6
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4
0i
Agonist
n
pD2
(%)
Noradrenaline Phenylephrine Xylazine
5 5 5
6.48 + 0.13 5.31 + 0.36* 3.59 + 0.08**
43.4 + 5.6 45.7 + 6.9Ns 3.7 + 1.6**
a Maximum contraction produced by a-agonists as a percentage of KCI response. * Significantly different from the value for noradrenaline, P < 0.05, ** P SNP > ATP = ADP = adenosine. Addition of isoprenaline ranging from 10 -8M to I0- M did not produce any relaxation in these preparations (Figure 4). Atropine (10--1i0-6M) caused a parallel shift to the right of the concentration-response curve to ACh. Schild plot
analysis showed that the slope was 1.11 + 0.12, which did not differ from unity and the pA2 value was 10.6 + 0.54 (n = 6). As shown in Figure 5b, addition of ACh (10 1- -10-3 M) did not cause relaxation in preparations without endothelium, but SNP 10- 5M did. Pretreatment with 5 x 10-5M aspirin did not affect ACh-induced relaxation (Figure Sc). On the other hand, the effect of ACh was significantly attenuated by pretreatment with 10- 5M methylene blue (Figure Sd). Pretreatment with oxyhaemoglobin 10- M increased the threshold concentration and decreased the maximum relaxation produced by ACh. These results are summarized in Figure 6.
Table 2 Slope and pA2 values of a-adrenoceptor antagonists against noradrenaline in canine spinal branch of intercostal artery n
Slope
pA2
Phentolamine Prazosin Yohimbine
5 5 5
1.07 + 0.12s 1.07 + 0.14S No effect
8.79 + 0.39 10.20 + 0.47 No effect
L
Figure 4 The relaxant responses to acetylcholine (El), sodium nitroprusside (U), ATP (A), ADP (0), adenosine (A), and isoprenaline (*) in canine isolated spinal branches of intercostal arteries contracted by 5 x 10--0-10`M noradrenaline. On the ordinate scale, relaxations induced by 10- M sodium nitroprusside were taken as 100% in each experiment. All points represent the mean of 5 observations, and the vertical lines indicate s.e.mean.
Relaxant responses of isolated spinal arteries
Antagonist
50 .
a)
Discussion Contractile responses of isolated spinal arteries
NS The value does not significantly differ from unity. The values are mean + s.e.mean, and n is the number of
The present results revealed that vasoactive substances such as AII, vasopressin, NA, 5-HT, adrenaline and dopamine
observations.
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100
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Figure 3 The effects of various a-adrenoceptor antagonists on contractile respones to noradrenaline in canine isolated spinal branches of intercostal arteries; (a) control (A), 10- I M (A), 10- 7 M (V), 10-6 M (V), phentolamine (n = 7); (b) control (A), 10-8M (A), I0- I M (V), 10- I m (V), prazosin (n = 6); (c) control (A), 10- 8 M (A), 10- 7 M (V), 10-6 M (V), yohimbine (n = 6). The ordinate scale is the same as in Figure 1. The vertical lines indicate the standard error of the mean.
RESPONSES OF ISOLATED SPINAL ARTERY a
-Iog[SNPI(M) 5
b 5mm I
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7AAA
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6 514 3 - log[AChl(M)
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Figure 5 Typical responses of isolated canine spinal branches of intercostal arteries to acetylcholine; (a) control; (b) responses to acetylcholine (ACh) and sodium nitroprusside (SNP) in the preparation from which endothelial cells were removed; (c) pretreatment with 5 x I0- M aspirin; (d) pretreatment with 10- 5 M methylene blue. All concentrations in this figure are given as negative logs.
caused a concentration-dependent contraction in the canine isolated spinal branch of the intercostal artery (SBICA). Among these substances, All was the most potent vasoconstrictor (Figure 1). In general, All is regarded as a potent vasoconstrictor agent in isolated small arterial preperations. However, there is a regional difference in the response of isolated arteries to All, and it produces only a slight contraction in canine cerebral arteries (Toda & Miyazaki, 1978). In canine isolated cerebral arteries, All also causes a relaxation in preparations contracted by PGF2. (Toda & Miyazaki, 1981). This relaxation seems to be mediated mainly by the release of prostacyclin from the endothelial cells. In contrast to the cerebral arteries, pretreatment with 5 x 10-5M aspirin produced no effect on the All-induced contraction in the SBICA. Thus, there may be a difference in AII-induced release of endogenous vasodilator prostaglandins between cerebral and spinal arteries. PGF2. produces a marked contraction in isolated cerebral arteries of various species (White & Hagen, 1982). However, PGF2. up to 10-0M elicited only a small contraction of the -log[ACh](M) 11a
10 -
9
8 EI 7-II 6 a
5
4
3
0
,o1
X 50
Y y y y
100
Figure 6 The effects of aspirin 5 x lo- M (A, n = 7), methylene blue 10-5M (V, n = 12), oxyhaemoglobin 10-5M (0, n = 7) and removal of endothelium (O, n = 6) on relaxant responses to acetylcholine in isolated canine spinal branches of intercostal arteries. The control concentration-response curve to acetylcholine is represented by (-, n = 5). The vertical lines indicate s.e.mean.
SBICA in the present experiments. Thus, the response of the SBICA to PGF2. seems to be markedly different from that of cerebral arteries. Electron microscopic (Iwayama et al., 1970; Nielsen et al., 1971; Sato & Suzuki, 1975) and histochemical studies (Nielsen & Owman, 1967; Hernandez-Perez & Stone, 1974; Kawai & Ohhashi, 1986) demonstrated a dense adrenergic innervation in cerebral arteries of various animals. Howevef, electrical stimulation of the sympathetic nerves produces only slight or no contraction of cerebral arteries (Raper et al., 1972; Toda & Fujita, 1973). Furthermore, contraction of cerebral arteries, including the internal carotid artery, induced by exogenous NA is considerably smaller than that produced by other agonists such as 5-HT and PGF2. (Bohr et al., 1961; Toda & Fujita, 1973; Kawai et al., 1984). Thus, the physiological significance of sympathetic nerves in the regulation of cerebral circulation is not clear. In the present study, the addition of tyramine produced no contraction in SBICA (Figure 1). This does not imply, however, that sympathetic nerves do not play an important role in the regulation of the spinal circulation. Morphological studies demonstrated the existence of sympathetic innervation of the smaller arteries supplying the spinal cord (Ohgushi, 1968; Marcus et al., 1977; McNicolas et al., 1980). The pharmacological characteristics of the a-adrenoceptors in SBICA were investigated with selective agonists and antagonists. The results showed that NA (al- and a2-agonist) and phenylephrine (a1-agonist) caused almost the same maximum contraction, although the pD2 value for phenylephrine was smaller than that for NA (Table 1). On the other hand, xylazine (a2-agonist) produced only a small contraction (Figure 2, Table 1). These results suggest that NA and phenylephrine contract the SBICA mainly through activation of a1-adrenoceptors on the smooth muscle cells. This view is also supported by the present results that phentolamine (at- and a2-antagonist) and prazosin (a1-antagonist) competitively inhibited the NA-induced contraction but yohimbine (a2-antagonist) did not (Figure 3). Previous work has suggested that the a-adrenoceptor subtype in smooth muscle cells of canine cerebral arteries was a2 in the middle cerebral and basilar arteries (Sakakibara et al., 1982; Skarby et al., 1983; Toda, 1983), and both al and a2 in the internal carotid arteries (Kawai et al., 1988). On the other hand, NA-induced contractions are mediated by a1-adrenoceptors in canine extracranial arteries of various tissues (Sakakibara et al., 1982; Toda, 1983). Our findings suggest that the a-adrenoceptor subtype in the SBICA is similar to that in the extracranial arteries rather than in the cerebral arteries of dogs.
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Relaxant responses of isolated spinal arteries The addition of ACh, SNP, ATP, ADP, and adenosine produced concentration-dependent relaxations in SBICA which had been contracted by NA. On the other hand, isoprenaline up to IO- M did not cause any relaxation in the preparations. Stimulation of fi-adrenoceptors produces cerebral vasodilatation in vivo (Edvinsson & MacKenzie, 1977). In addition, fiadrenoceptor agonists induce a relaxation of isolated cerebral vessels when the preparations have intrinsic tone. These findings suggest that fi-adrenoceptors exist in cerebral arteries, contrasting with the present results in the SBICA. Among the vasodilators used, ACh was the most potent on the SBICA. This relaxation seems to be mediated via muscarinic receptors, since it was competitively inhibited by atropine. Relaxation induced by ACh, but not that induced by SNP, was abolished by mechanical removal of the endothelium. It is well known that endothelium-derived relaxing factor (EDRF) (Furchgott & Zawadzki, 1980) and prostacyclin (Moncada et al., 1977) are released from vascular endothelial cells and cause vascular smooth muscle cells to relax. In the present experiments, treatment with aspirin, an inhibi-
tor of cyclo-oxygenase, did not affect the ACh-induced relaxation, suggesting that prostacyclin did not play an important role in the ACh-induced relaxation. On the other hand, oxyhaemoglobin, an inhibitor of EDRF, and methylene blue, an inhibitor of guanylate cyclase, markedly suppressed the AChinduced relaxation in the spinal arteries. All these findings strongly suggest that the ACh-induced relaxation in the SBICA is mainly mediated through release of EDRF. A previous report suggests that ACh-induced endotheliumdependent relaxation is relatively small in canine cerebral arteries (Tsukahara et al., 1986). There may be a regional difference in endothelial function or sensitivity of vascular smooth muscles to EDRF between spinal and cerebral arteries. The present experimental results suggest that the pharmacological characteristics of SBICA are quite different from those of the isolated canine cerebral arteries reported in previous studies. In the canine SBICA, the addition of NA produces considerable contraction which is mainly mediated via a1-adrenoceptors. On the other hand, ACh causes a marked relaxation of the SBICA due to the release of EDRF from the endothelial cells.
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(Received February 19,1990 Revised April 17,1990 Accepted April 20, 1990)
