Differential modulation by the endothelium of contractile responses to 5-hydroxytryptamine, noradrenaline, and histamine in the rabbit isolated basilar artery

Gen. Pharmac. Vol. 28, No. 5, pp. 681-687, 1997 Copyright @ 1997 Elsevier Science Inc. Printed in the USA.

ISSN 0306-3623/97 $17.00+.00 Pll S0306-3623(96)00352-7 All rights reserved ELSEVIER

Differential Modulation by the Endothelium of Contractile Responses to 5-Hydroxytryptamine, Noradrenaline, and Histamine in the Rabbit Isolated Basilar Artery Akira Ohnuki and Yasuo Ogawa* DEPARTMENT o r PHARMACOLOGY,JUNTENDO UNIVERSITYSCHOOL OF MEDICINE,

2-1-1 HoNoo, BUNKVO-KU,TOKYO 113, JAPAN [TEL: 81-3-5802-1034; Fax: 81-3-5802-0419] ABSTRACT. 1. To assess modification by endothelium, we determined the contractile responses of intact rings from rabbit basilar artery to histamine, noradrenaline and 5-hydroxytryptamine (5-HT) and compared them with Triton X-lOO-treated and NO-nitro-L-arginine (L-NNA)-treated preparations. 2. Among the agonists examined histamine caused the strongest contraction of vascular smooth muscles and noradrenaline the weakest one. The present results are consistent with the basal release of endotheliol-derived relaxing factor (EDRF). 3. 5-HT caused a much greater modification than the one expected from basal release of EDRF, suggesting that the release is induced by 5-HT, in contrast to the previous results. Our findings also indicate that 5-HT could induce the release of both contracting factor (EDCF) and EDRF from the endothelium, tEN PHAe.MAC28;5:681--687, 1997. © 1997 Elsevier Science Inc. KEY WORDS. Rabbit basilar artery, endothelium, 5-hydroxytryptamine, histamine, noradrenaline, N Gnitro-L-arginine (L-NNA), endothelial-derived relaxing factor (EDRF), endothelial-derived contractile factor (EDCF) INTRODUCTION Recent investigations have revealed that the vascular endothelium exerts an important influence on blood vessel tone: a vasodilating action by the release of endothelium-derived relaxing factor (EDRF) (Furchgott and Zawadzki, 1980) and endothelium-derived hyperpolarizing factor (EDHF) (Komori and Suzuki, 1987), and a vasoconstricting action by the release of endothelium-derived contractile factor (EDCF) (Hickey et al., 1985), for which candidates are endothelins, angiotensin II, free radicals, prostaglandins, and related metabolites of arachidonates. The critical influence of EDRF on the vascular tone was first demonstrated by Furchgott and Zawadzki (1980). EDRF has been identified as NO or a closely related compound produced from the amino acid L-arginine by NO synthase (Furchgott, 1988; Ignarro, 1990; Ignarro et al., 1988; Moncada et al., 1991; Palmer et al., 1987, 1988). Release of NO from the endothelial cells is subtly modulated by the shear stress caused by blood flow on the endothelial cells, as well as by vasoactive agents. NO synthase activity has now been described not only in endothelial cells but also in neurons, glia, and other types of cells, and knowledge of NO synthesis has grown (Bredt and Snyder, 1994; Garthwaite and Boulton, 1995; Ignarro, 1990; Moncada et al., 1991). To assess the extent of the involvement of endothelial cells in vasocontraction by such agonists as histamine, noradrenaline, and 5-hydroxytryptamine (5-HT), we compared the contractions of intact rings of the basilar artery with those of Triton X-100-treated and NC-nitro-t-arginine (L-NNA)-treated preparations. In similar experiments, several investigators (Brian and Kennedy, 1993; Con*To whom all correspondence should be addressed. Received 29 May 1996, Accepted 12 July 1996.

nor and Feniuk, 1989; Nakagomi et al., 1988; Trezise et al., 1992), have reported a basal release of EDRF and a 2 to 3 times attenuation by the endothelium. Here, we present evidence, although indirect, for 5-HT-stimulated release of EDRF and the result that the influence of the endothelium is much greater than the results obtained so far. We also found that the integrity of the intact endothelium is maintained only with enormous difficulty in in vitro experiments. MATERIALS A N D METHODS Materials

The composition of Krebs physiological salt solution (PSS) was as follows (mM): 118.9 NaC1, 4.7 KCI, 1.2 MgClz, 1.2 CaC12, 1.2 KH2PO4, 24.9 NaHCO3, and 5.6 glucose. The composition of the high K+Krebs solution was (raM): 123.34 K-methansulfonate, 0.26 KC1, 1.2 MgC12, 1.2 CaCl2, 1.2 KH2PO4, 24.9 KHCO3, and 5.6 glucose where the product of K+C1 - was kept constant (Aizu and Ogawa, 1992). The following drugs were used: 5-HT, L-NNA, and carbamylcholine chloride from Sigma Chemical (St. Louis, MO, USA); histamine dihydrochloride, ascorbic acid, and Triton X-100 from Wako Pure Chemical (Osaka, Japan); (+)noradrenaline hydrochloride from Aldrich Chemical (Milwaukee, WI, USA); methansulfonic acid from Tokyo Kasei (Tokyo, Japan), and sodium pentobarbitone from Abbott Laboratories (North Chicago, IL, USA). 5-HT and L - N N A were dissolved in 0.1 N and 0.2 N HC1, respectively, for their stock solutions; further dilutions were made in distilled water. Control experiments demonstrated that the amount of HCI that was finally carried over into the bathing solution had no effect on buffer pH or tension development. Noradrenaline was dissolved in 0.1% ascorbic acid to prevent spontaneous oxidation. Solutions were protected from light before application. All other drugs were dissolved in distilled water for stock solutions, and further dilu-

682 tion was made with Krebs solution before use. A stock solution of Triton X-100 (10%) was prepared in distilled water and kept refrigerated between uses.

A. Ohnuki and Y. Ogawa a

Tissue preparation Male Japan white rabbits (body weight 2.5-3.0 kg) were anesthetized with sodium pentobarbitone (50 mg/kg) and sacrificed by exsanguination from the common carotid arteries. The brain was rapidly removed and placed in ice-cold PSS. Hereafter, unless described otherwise, all procedures were conducted at 4°C. The basilar artery was carefully dissected from the brain stem and cleaned of extraneous connective tissue under a binocular microscope, then peffused with PSS through stainless steel tubing (0.2 mm in diameter) to remove any residual hematoma. A segment of the basilar artery was peffused intraluminally with Triton X-100 (0.03% in PSS, 0.3 ml/ min for 3 rain) to abolish endothelial function, and then with PSS for 1 rain to remove Triton X-100. Control preparations were taken from the adjacent segment, which was treated similarly with PSS alone. The artery was cut into ring segments 1 mm in width, and these segments were placed in 2 ml organ baths containing PSS at 4°C, and bubbled with 95% 02 and 5% CO2 (pH 7.4-7.5). The temperature was gradually increased to 33-+0. I°C where force measurements were carried out. The temperature of 33°C, rather than 37°C, was adopted because of longer tissue viability and much weaker rundown of the preparations.

Force recordings The ring segments were suspended between two stainless steel hooks (0.14 mm in diameter) inserted into the lumen of a vessel with minimal injury to the endothelium to record isometric tension changes. One wire was firmly attached to the tissue bath and another was connected to a force-displacement transducer (Minebea UL-2GR), whose signal was amplified by an amplifier (Nihon Denki Sanei AS1202) and recorded on a chart recorder (Sekonic SS-100). After each ring segment was maintained unstretched on the wire for 30 minutes, it was gradually adjusted to a resting tension of 0.15 raN. This resting tension, which is lower than the optimum for active tension development, enables the ring segment to show most stable and reproducible contractile responses during determinations. After an hour's equilibration in the normal PSS, the response to a highK + ( 149.7 mM) solution was determined to assess the tissue viability and to calibrate the following responses to an agonist as % K + contracture. Once the contractile response to the high-K + solution reached a plateau, tissues were washed by PSS until the force returned to baseline. After a 20-min equilibration the integrity of the endothelium was assessed by a relaxant response of the tissue precontracted by 1 ~M histamine to carbachol (30 IzM), an endothelium-dependent vasodilator. A n intact endothelium was denoted by a relaxation of more than 80%; destruction of endothelium, in contrast, was denoted by no relaxation or by contraction (Fig. 1). Preparations showing intermediate responses were rejected. After washing with PSS and a subsequent 20-min incubation in PSS, the preparations with and without functional endothelium were exposed to histamine, noradrenaline, or 5-HT in a cumulative manner to obtain their concentration-response curves. Only a single agonist was examined with one preparation. L-NNA-treated preparations were obtained by 50-min incubation in a PSS containing 10 IxM L - N N A after the initial high-K + contraction. Loss of EDRF with these preparations was confirmed when no relaxation was observed after 30 IxM carbachol, as was the case with Triton X-100 treated ones.

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F I G U R E 1. Criteria for integrity of endothelial function. The integrity of endothelial function was assessed by examining the relaxant response to 30 ~ M carbachol (~) when a stable contraction had been obtained by 1 ~tM histamine (13. (a) Whole course of contractile response to histamine in nontreated preparation. (b) Nontreated preparation with intact endothelium. (c) Triton X-100-treated, endothelium-impaired preparation. Open arrows represent washout by PSS. Some endothelium-intact preparations showed rapid relaxation followed by a slow one on application of 30 lutM carbachol as shown in (b); others showed only rapid relaxation, as in Fig. 5a.

Statistical analysis In all examinations contractile responses to agonists were expressed as % K + contracture, which will enable comparison among different preparations. Results shown in the table and figures were expressed as arithmetic mean"+SD. They were analysed according to two-way repeated-measures analysis of variance ( A N O V A ) with adjustment by Fisher's, Scheffe's, or Bonferroni-Dunn's methods under the assumption of balanced data using StatView version 4.02 for Macintosh. There were no differences in conclusion among the 3 methods of adjustment. If the group sizes were made equal, conclusions were not changed. Comparison at a specified concentration of an agonist between preparations, which, as mentioned, proved to be significantly different, was made by Student's t-test for unpaired data. Differences between data were considered significant when P
Basilar Artery and 5-HT ing that the function of the endothelium was lost. L-NNA-treated preparations that failed to synthesize N O or related substance also showed similar contraction to the high-K + solution. As shown later, the efficacy in vasoconstriction is variable among the three agonists. Contractions by agonists were calibrated with the tension induced by 149.7 mM K+(% K + contracture). The high-K + contracture should be done in a high-K + medium, where the product of K + by C1- is kept constant. Otherwise, swelling of smooth-muscle cells (and probably also endothelial cells) may occur by redistribution of ions and H20 molecules, a process is driven by Donnan equilibrium, as in skeletal muscle fibers (Aizu and Ogawa, 1992). To inhibit the endothelium function, Triton X-100 treatment (Verrecchia et al., 1986) was found to be more suitable than mechanical treatment (Trezise et al., 1992) or gas perfusion (Nakagomi et al., 1988), because the latter methods damaged smooth muscles to a greater or lesser extent, as evidenced by the decrease to about 80% in high-K + contracture. It is necessary, however, to modify the condition for Triton X-100 treatment for rabbit basilar artery. The concentration of Triton X-100 of 0.1% for dog basilar artery (Brian and Kennedy, 1993; Connor and Feniuk, 1989) was too high for rabbit basilar artery. We found that treatment with 0.03% Triton X-100 for 3 min at 4°C was most appropriate.

Histamine.induced contraction Histamine was used to precontract vessels to assess the integrity of the endothelium (Seager et al., 1992). This suggests that there might be little endothelium involvement in the action of histamine. Therefore, concentration-dependent contractions by histamine were determined with nontreated, Triton X-100-treated and L-NNA-treated segments of rabbit basilar arteries. As shown in Fig. 2, the determinations were carried out in a cumulative manner. Sufficient time was allowed for the effect of each concentration to become fully established before adding a higher concentration. To facilitate comparison among preparations, the results were calibrated with the response to 149.7 mM K + contracture (% K + contracture). The accumulated results are summarized in Fig. 3. The maximal responses of the contraction were similar among nontreated, Triton X-100-treated, and L-NNA-treated preparations, and were as large as 130 to 140% K + contracture. With less than 1 btM of histamine, however, Triton X-100-treated and L - N N A treated preparations showed significantly (P<0.05) larger contraction than the control. The difference between Triton X-100-treated and L - N N A - t r e a t e d preparations was not significant. The involvement of endothelium is clearly shown at low concentrations of histamine, whereas the depressing effect of N O cannot be seen at high concentrations of the drug. This may result in the apparent leftward shift of the concentration-response curves with changed steepness for endothelium-impaired preparations.

Response to noradrenaline and 5 , h y d r o x y t r y p t a m i n e The concentration-response curves for noradrenaline and 5-HT were obtained as for histamine (Figs. 4a, 4b). The control arterial rings contracted concentration-dependently in response to noradrenaline (Fig. 4a). The maximum response to 0.1 mM noradrenaline was about 16% K + contracture. Treatment with Triton X-100 or L - N N A increased the contractile response to noradrenaline over the whole range of 0.1 to 100 p~M. There was no significant difference in the concentration-response curves between Triton X-100treated and L-NNA-treated preparations. Their maximum responses

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Histamine (-IogM) FIGURE 2. Concentration-dependent effect of histamine by cumulative addition on (a) nontreated, (b) Triton X-100-treated, and (c) L - N N A - t r e a t e d rabbit isolated basilar arteries. Solid arrows indicate the application of each concentration of the agent. Numbers represent the negative logarithms of concentrations of histamine (-log M). Open arrows represent washout by PSS. Vertical bar: 5 mN. Horizontal bar: 5 min. were about 50% K + contracture. At 0.1 mM noradrenaline, the enhancement on removal of the effect of EDRF was about 3-fold. The maximum contraction by 10 IxM 5-HT of the nontreated segments was as small as 13% K + contracture, which was similar to that by noradrenaline (Fig. 4b). The contractile response to 5-HT of the nontreated control preparations was much smaller than that obtained in previous results (Brian and Kennedy, 1993; Connor and Feniuk, 1989; Nakagomi et al., 1988; Trezise et al., 1992). O n the other hand, taking the difference between the 149.7 mM K + contracture and the 30- to 40-mM K + contracture into consideration, the maximum responses of the Triton X-100-treated and L - N N A treated preparations were comparable to those in the published resuits. Therefore, the maximal responses of Triton X-100-treated and L-NNA-treated segments that failed to produce N O or its related substances were as large as approximately 6 and 8 times that

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A. Ohnuki and Y. Ogawa

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FIGURE 3. Concentration-responsecurve for histamine of rabbit isolated basilar artery. O, control, nontreated (n=6); A, Triton X100-treated (n=5); D, L-NNA-treated (n=5). Determinations were carried out as shown in Fig. 2. L-NNA (30 I~M) was added to the organ bath 50 min before construction of the agonist concentration-response curve. Abscissa: agonist concentration expressed as (-log M). Ordinate: contractile response to histamine expressed as % K + contracture. Statistical analysis was made as described in Materials and Methods. Probability values calculated according to two-way repeated-measures ANOVA with adjustment by Bonferroni-Dunn's method were 0.0057, <0.0001, and 0.0455 between the control and Triton X-100-treated or L-NNA-treated ring and between the two treated rings, respectively, when the significant Pvalue was 0.0167 or less. The other methods of adjustment gave the same conclusion. Each point represents the arithmetic mean value and the vertical bar the SD. **P<0.05, **P<0.01. See also Table 1.

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5-HT of the control preparations, respectively. The magnitude of the reduction by the presence of functional endothelium was much greater than in previous results, which were reported to be only 2to 3-fold (Brian and Kennedy, 1993; Connor and Feniuk, 1989; Nakagomi et al., 1988; Trezise et al., 1992). The treatment with LNNA or Triton X-100 of the arterial segments increased the response to 5-HT over all concentrations (1 nM-10 FtM). With 0.1 ~M 5-HT or higher doses, L-NNA-treated preparations showed significantly (P<0.01) larger contractions than those treated with Triton X-100. The results of contractile responses to histamine, noradrenaline, and 5-HT are summarized in Table 1. To determine why the contractile response of the nontreated control preparations was so small in comparison with the earlier reported results, (Brian and Kennedy, 1993; Connor and Feniuk, 1989; Nakagomi et al., 1988; Trezise et al., 1992) we determined the

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FIGURE 4. Concentration-response curves for (a) noradrenaline and (b) 5-HT of rabbit isolated basilar artery. Contractile responses to agonists in nontreated (O) ( n = 4 for a and b), Triton X-100-treated (A) ( n = 4 and 7 for a and b, respectively), and L-NNA-treated preparations ([]) ( n = 6 and 8 for a and b, respectively) were determined as for Figs. 2 and 3. Statistical analysis was made as described in Materials and Methods. The probability values according to Bonferroni-Dunn's adjustment with noradrenaline (a) were 0.0002, 0.0001, and 0.9159 between the control and Triton X-100-treated or L-NNA-treated rings and between the two treated rings, respectively. The probabilities with 5-HT (b) were <0.0001, <0.0001 and 0.0020, respectively, in the same sequence, when the significant P value was 0.0167 or less. The other methods of adjustment gave the same conclusions in both a and b. Significant differences between Triton X-100- and L-NNA-treated arteries are marked with daggers (*P<0.01), and those between endothelium-intact and impaired ones with asterisks (**P<0.01). See also Table 1.

TABLE 1. ECs0 values and maximal responses produced by histamine, noradrenaline, and 5-hydroxytryptamine in nontreated, Triton X-100-treated and L-NNA-treated rabbit isolated basilar arteries -log (EC50)

Histamine Noradrenaline 5-HT

Maximal response (%)

Nontreated

TX- 100

L-NNA

Nontreated

TX- 100

L-NNA

6.35 _+ 0.36 (6) 5.30 _+ 0.08 (4) 7.68 -+ 0.52 (4)

6.85 _+ 0.14 (5) 5.96 _+ 0.30 (4) 7.66 _+ 0.23 (7)

6.98 + 0.12 (5) 5.94 -+ 0.51 (6) 7.62 -+ 0.30 (8)

138.4 -+ 3.40 (6) 16.5 -+ 5.50 (4) 13.2 _+ 6.40 (4)

132.7 -+ 10.1 (5) 49.9 -+ 9.50 (4) 73.5 -+ 10.2 (7)

143.6 + 17.8 (5) 52.4 + 5.70 (6) 102.8 + 14.2 (8)

Results expressed as means -+ sd (number of determinations).

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FIGURE 5. Contractile responses to repetitive applications of 1 ~M 5-HT. (a) Nontreated preparation. (b) Triton X-100-treated preparation. Upward solid arrows indicate additions of 1 ~M 5-HT. Open arrows indicate washout with PSS. The solid downward arrow shows addition of 100 ~M carbachol.

contractile responses to repeated applications of 1 IzM 5-HT (Fig. 5). The first contraction of the control arterial ring was very small (Fig. 5a); subsequent contractions gradually increased after each application and constant responses were obtained after the fifth or sixth application of 1 txM 5-HT. The time course of contraction also was modified. Phasic contractions were prominent in early challenges, but tonic contractions following phasic ones were more marked and augmented in later challenges. The fifth contraction was nearly 6 times that of the first and similar to the contraction in the endothelium-impaired preparation (Fig. 5). In contrast, the Triton X-100-treated preparation showed a consistently large contraction with each application of 1 txM 5-HT (Fig. 5b). The time course of each contraction also was similar, unlike the case with the endothelium-intact preparation. With the L-NNA-treated preparation, similar unchanged repetitive contractions were observed (data not shown). It might be argued that the 5-HT receptor on the endothelial cells that causes EDRF release is desensitized by repeated applications of 5-HT. However, this is unlikely because the contraction remained small in cumulative experiments up to 10 tzM as shown in Fig. 4b. The possibility that 5-HT might contract the basilar artery by a mechanism that is more susceptible to EDRF can be excluded on the basis of the similar sequences of the logic. A reasonable explanation for the response of the preparation with intact endothelium may be that the endothelium is partly impaired during mechanical movements of the repeated cycles of contraction and relaxation. It should be noted, however, that 100 IzM carbachol caused rapid relaxation during the 6th contraction by 5-HT, indicating that the endothelium still remained functional. DISCUSSION The contractile responses of rabbit basilar artery to vasoconstrictive agents (histamine, noradrenaline, and 5-HT) were determined in endothelium-intact and endothelium-impairedpreparations (Triton X-100-treated and L-NNA-treated) to assess modification by endothelium. The maximum responses of the control (nontreated ring)

to the three agonists were in descending order: histamine ("q40% K+ contracture), noradrenaline (~16% K+ contracture), and 5-HT (~13% K+ contracture). Vascular contraction by agonists is composed of smooth-muscle contraction by agonists and its modification by factors that are released from endothelial cells. Among the three agonists used here, the efficacy in smooth-muscle contraction was in descending order: histamine (~130% K+ contracture), 5-HT (~75% K+ contracture), and noradrenaline (,'.,50% K + contracture) on the basis of maximal contractions of Triton X-100-treated preparations that have lost endothelium function. Among the three, histamine has the weakest action on the endothelium and 5-HT the strongest. Histamine at low concentrations gave rise to larger contractions with endothelium-impaired preparations, whereas maximum contractions were similar with the three preparations. This indicates that the depressing effect of EDRF depends on the concentrations of the agonist (i.e., the strength of its contracting effect) and that the influence was overcome by strong contraction of arterial smooth muscle in the presence of very high concentrations of histamine. This also is likely to be the case with high-K+ contracture, consistent with the idea that histamine induces only EDRF basal release. These findings are not incompatible with the results of endothelium-independentcontraction of pig basilar arteries (Miyamoto and Nishio, 1993), although Usui et aI. (1993) reported endothelialdependent contraction of the canine basilar arteries. In contrast to histamine, noradrenaline and 5-HT caused more marked endothelial-dependent contraction. The magnitudes of contractions of Triton X-100-treated and L-NNA-treated preparations by noradrenaline were about 50% K + contracture, whereas 5-HT gave rise to the maximum responses of approximately 75% and 100% K+ contracture with Triton X-100-treated and L-NNA-treated preparations, respectively. The degrees of stimulation by these treatments would correspond to a 3- and 6- to 8-time increase for noradrenaline and 5-HT respectively, over those of the nontreated preparations. Although the factor of augmentation for noradrenaline was similar to that seen in previous results (Nakagomi et al., 1988; Trezise et al.,

686 1992), the factor (6-8) for 5-HT was much larger than the earlier findings of 2- to 3-fold (Brian and Kennedy, 1993; Connor and Feniuk, 1989; Nakagomi et al., 1988; Trezise et al., 1992). If the basal release of EDRF were the only cause of the modification, contractions by noradrenaline also would be affected similarly, as with histamine. In other words, we may expect that the basal EDRF will depress agonist-induced forces developed by smooth muscles following the relationship shown in Fig. 3. Maximum responses to noradrenaline were about 16% and 50% K + contracture with endothelium-intact and with EDRF-lacking preparations, respectively. Histamine (0.1 >M), which gave about a 50% K + contracture to EDRF-lacking preparations, also caused an about 10% K + contracture with endothelium-intact preparations (Fig. 3). Therefore, the degree of attenuation in the case of noradrenaline (16%/50%), which could be ascribable to EDRF, is not so much different from that seen with histamine (10%/50%). However, the maximal response to 5-HT of L-NNA-treated preparations, which were just unable to produce N O (100% K + contracture as shown in Fig. 4b), corresponded to the response of the endothelium-impaired preparations to about 0.5 IxM histamine (Fig. 3). Then, the contraction of the endothelium-intact preparations is expected to be about 60% K + contracture if the relationship shown in Fig. 3 holds. With 5HT, in contrast, it was reduced to 13% K + contracture. Therefore, attenuation by EDRF was more marked with 5-HT than with histamine. Release of EDRF, then, may be induced by 5-HT. This is at variance with the claim that contractile response to 5-HT was modified by basal release of EDRF (NO or a related substance) (Connor and Feniuk, 1989; Trezise et al., 1992). This inference also is supported by the findings of successive increase in the contractions seen in an endothelium-intact preparation after repeated applications of 5-HT, as shown in Fig. 5a. The possibility of desensitization to 5-HT was excluded. A reasonable explanation for this finding is a partial impairment of the endothelium during mechanical movements of the repeated cycles of contraction and relaxation. Under this condition, the endothelium still remains functional, although not perfectly. Furthermore, it should be noted that a very weak augmentation of successive contractions of intact preparations (not more than a 40% increase) was observed with histamine and noradrenaline (data not shown). L-NNA-treated preparations showed significantly (P<0.01) larger contractions to 5-HT (0.1 IxM or higher concentrations) than Triton X-100-treated preparations. This suggests that 5-HT may release not only EDRF (NO or its related substances), but also EDCF. This potential EDCF release might account for our failure to show relaxation of precontracted intact preparations on the application of 5-HT. Because indomethacin had no effect on contractions in our determinations (data not shown), a metabolite of arachidonate cannot be a candidate for EDCF, although Seager et al. (1992) suggested this possibility. Previous results showed the contracting effect of prostaglandin F2a (Nakagomi et al., 1988, Trezise et al., 1992), but we failed to show it, which is consistent with our conclusion. In this context, we would like to note that U T P caused contraction, but that ATP, ITP, GTP, or CTP did not. Diadenosine penta- or hexa-phosphate (ApsA or Ap6A) rather relaxed the preparation (data not shown). One main reason for the discrepancy in the assessment of the endothelium function is the contraction of the "endothelium-intact" preparation. The maximum contraction of Triton X-100-treated preparations was similar to that reported in previous results. As shown in Fig. 5a, the contraction of a nontreated preparation on the first application of 1 b~M 5-HT was very small and increased with each successive application. With the fifth application, the contrac-

A. Ohnuki and Y. Ogawa tion reached a magnitude near that of the Triton X-100 treated preparation. Furthermore, the preparation showed rapid relaxation in response to the addition of 100 txM carbachol, which suggests the presence of a functional endothelium. The magnitude of the fourth contraction would correspond to that of the "endothelium-intact" preparations reported previously (Brian and Kennedy, 1993; Connor and Feniuk, 1989; Nakagomi et al., 1988; Trezise et al., 1992). We must be careful in conducting experiments with endotheliumintact preparation, because the endothelium is vulnerable to even minute perturbations. Our present results indicate that 5-HT may induce EDRF release, whereas the effects of histamine and noradrenaline could be explained by the basal release of EDRF. The depressing effect of EDRF is intense. The effect of intact endothelium, which is subtle and vulnerable, is critical in any consideration of cerebral circulation. Under certain pathological conditions, such as subarachnoid hemorrhage, 5-HT, which is markedly endothelium-dependent as shown here, may be produced. This could explain the underlying mechanism for vasoconstriction or vasospasm observed after the episode has subsided. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan to Y. 0 . and by a special grant on Project-Research of Juntendo University to A. O. We thank Professor Kiyoshi Sato, Chairman of the Department of Neurosurgery, Juntendo University School of Medicine, for his encouragement and support, and we are indebted to Miss Naomi Ariji for her expert secretarial assistance.

References Aim M. and Ogawa Y. (1992) Involvement of chloride-bicarbonate exchange in cell volume regulation during potassium contracture of single isolated smooth muscle cells. Jpn. J. Pharmacol. 58, 292P. Bredt D. S. and Snyder S. H. (1994) Nitric oxide: a physiologic messenger molecule. Ann. Rev. Biochem. 63, 175-195. Brian J. E. Jr. and Kennedy R. H. (1993) Modulation of cerebral arterial tone by endothelium-derived relaxing factor. Am. J. Physiol. 264, H12451250. Connor H. E. and Feniuk W. (1989) Influence of the endothelium on contractile effects of 5-hydroxytryptamine and selective 5-HT agonists in canine basilar artery. Br. J. Pharmacol. 96, 170-178. Furchgott R. F. (1988) Studies on relaxation of rabbit aorta by sodium nitrite: the basis for the proposal that the acid-activatable inhibitory factor from bovine retractor penis is inorganic nitrite and the endotheliumderived relaxing factor is nitric oxide. In Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves and Endothelium. (Edited by Vanhoutte, P. M.) pp. 401-414. Raven, New York. Furchgott R. F. and Zawadzki J. V. (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288, 373-376. Garthwaite J. and Bouhon C. L. (1995) Nitric oxide signaling in the central nervous system. Ann. Rev. Physiol. 57, 683-706. Hickey K. A., Rubanyi G., Paul R. J., and Highsmith R. F. (1985) Characterization of a coronary vasoconstrictor produced by cultured endothelial cells. Am. J. Physiol. 248, C550-556. Ignarro L. J. (1990) Biosynthesis and metabolism of endothelium-derived nitric oxide. Ann. Rev. Pharmacol. Toxicol. 30, 535-560. Ignarro L. J., Byms R. E. and Wood K. S. (1988) Biochemical and pharmacological properties of endothelium-derived relaxing factor and its similarity to nitric oxide radical. In Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves and Endothelium. (Edited by Vanhoutte, P. M.) pp. 427-435. Raven, New York. Komori K. and Suzuki H. (1987) Electrical responses of smooth muscle cells during cholinergic vasodilation in the rabbit saphenous artery. Circ. Res. 61, 586-593. Miyamoto A. and Nishio A. (1993) Characterization of histamine receptors in isolated pig basilar artery by functional and radioligand binding studies. Life Sci. 53, 1259 1266. Moncada S., Palmer R. M. J. and Higgs E. A. (1991 ) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol. Rev. 43, 109-142. Nakagomi T., Kassell N. F., Sasaki T., Lehman R. M., Tomer J. C., Hongo K. and Lee J. H. (1988) Effect of removal of the endothelium on vaso-

Basilar Artery and 5-HT contraction in canine and rabbit basilar arteries. J. Neurosurg. 68, 757766. Palmer R. M. J., Ferrige A. G. and Moncada S. (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327, 524-526. Palmer R. M. J., Ashton D. S. and Moncada S. (1988) Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature 333, 664-666. Seager J. M., Clark A. H. and Garland C. J. (1992) Endothelium-dependent contractile responses to 5-hydroxytryptamine in the rabbit basilar artery. Br. J. Pharmacol. 105, 424428.

687 Trezise D. J., Drew G. M. and Weston A. H. (1992) Analysis of the depressant effect of the endothelium on contraction of rabbit isolated basilar artery to 5-hydroxytryptamine. Br. J. Pharmacol. 106, 587-592. Usui H., Kurahashi K., Shirahase H., Jino H. and Fujiwara M. (1993) Endothelium-dependent contraction produced by acetylcholine and relaxation produced by histamine in monkey basilar arteries. Life Sci. 52,377387. Verrecchia C., Sercombe R., Philipson V. and Seylaz J. (1986) Functional destruction of cerebral vascular endothelium by Triton X-100. Blood Vessels 23, 106.