Potentiation by endothelin-1 of Ca2+ sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery

Pergamon

0M6-3623(94)00258-4

Gen. Pharmac.Vol. 26, No. 4, pp. 855-864, 1995 Copyright © 1995ElsevierScienceLtd Printed in Great Britain.All rights reserved 0306-3623/95$9.50+ 0.00

Potentiation by Endothelin-1 of C a 2 + Sensitivity of Contractile Elements Depends on Ca 2+ Influx through L-type Ca 2÷ Channels in the Canine Cerebral Artery YOSHIO TANAKA, HIROMI ISHIRO, TOHRU NAKAZAWA,* MICHIHIRO SAITO, KUNIO ISHII and KOICHI NAKAYAMA Department of Pharmacology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City, Shizuoka 422, Japan [Tel: (+ 81) 54-264-5694; Fax: (+ 81) 54-264-5696] (Recewed21 June 1994)

Abstract--1. Endothelin-I (ET-1) contracted canine cerebral artery in a concentration-dependentmanner with an increase in intraceUular Ca 2÷ concentration ([Ca2÷]i), and at higher concentrations it produced a greater contraction with a smaller increase in [Ca2+]~. 2. Ca 2÷ channel antagonist such as d-cis-diltiazem inhibited the tension more effectively than the [Ca2+]~ increased by ET-I. 3. In Ca2+-free solution containing 0.2 mM EGTA, ET-I elicited a transient increase in [Ca2+]~ and tension. 4. In the Staphylococcus aureus a-toxin-permeabilized artery, ET-1 shifted the p Ca-tension relationship leftwards in the presence of GTP. 5. These findings suggest that ET-I contracts the canine cerebral artery by increasing not only the Ca 2+ influx through L-type Ca 2+ channels, but also Ca 2+ release from the intracellular storage sites, and also Ca 2÷ sensitivity of contractile elements. The degree of Ca 2+ sensitivity is strongly affected by [Ca2+]iwhich is increased by the Ca 2+ influx through L-type Ca 2+ channels. Key Words: Endothelin-1, canine cerebral artery, intracellular Ca 2+ concentration ([CaZ+]i), transmembrane Ca 2+ influx, Ca 2÷ sensitivity, Ca 2÷ channels, Ca 2+ channel antagonists, fura-2

INTRODUCTION Endothelin-1 (ET-1), a member of the endothelin (ET) family which consists of 21 amino acids (Yanagisawa et al., 1988), elicits strong and longlasting vascular contractions. In addition to the direct vascular contractile activity, ET-1, especially at a low concentration range ( p M - n M ) corresponding approximately to the normal plasma level of the peptide in h u m a n beings (Ando et al., 1989), potentiates the actions of other vasoconstrictor agonists including biogenic amines such as 5-hydroxytryptamine (5-HT) (Nakayama et al., 1991). Therefore, when endothelium is functionally or structurally damaged, ET-1 per se and/or synergistically with other endogenous spasmogens may enhance the contractile activity of *To whom all correspondence should be addressed.

vascular smooth muscle, which leads to the arterial spasm in the circulatory system. Cerebral arteries isolated from various animal species including eats and dogs also respond to ET-1 with potent and sustained contractions (Saito et al., 1989, 1991; Asano et al., 1989; F u k u d a et al., 1991; Kamata et al., 1991; Encabo et al., 1992; Tanoi et al., 1992; Salom et al., 1992) which are more susceptible to Ca 2+ antagonists such as nifedipine, d-cisdiltiazem and verapamil in comparison with the contractions obtained in other peripheral arteries such as the aorta and mesenteric artery (Saito et al., 1989, 1991; Tanoi et al., 1992). These findings suggest that the intracellular Ca 2+ concentration ([Ca2+]i) increased by the Ca 2+ influx through L-type Ca 2+ channels is a main determinant factor for ET-Iinduced contraction in this clinically important vascular tissue such as cerebral artery. By contrast, in 855

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rat aorta (Sakata et al., 1989), porcine coronary artery (Nakayama et aL, 1991) and rabbit mesenteric artery (Nishimura et al., 1992), other Ca 2÷ handling mechanisms such as increase in Ca 2÷ sensitivity of contractile elements and Ca 2÷ release from intracellular storage sites as well as Ca 2÷ influx have been shown to be involved in ET-l-induced contractions. However, the interactions of each determinant factor for vascular contraction have not been well elucidated. Therefore, it is still unclear whether the suppression by Ca 2÷ antagonists of ET-l-induced cerebral artery contraction is exclusively due to the inhibition of Ca z+ influx per se, or other determinant factors, particularly Ca 2+ sensitivity of contractile elements modified by the Ca 2* influx are also involved in the response. To further elucidate the Ca 2÷ handling mechanisms underlying ET-1-induced cerebral arterial contraction and its inhibition by Ca 2+ antagonists, we measured simultaneously the [Ca2+]i and tension during ET-1induced contraction in a canine cerebral artery loaded with fura-2, and compared them with those in a high KCl-induced contraction. Furthermore, to examine whether ET-I augments the myofilament Ca 2÷ sensitivity as well as Ca 2÷ release from the intracellular storage sites, the artery was permeabilized with Staphylococcus aureus s-toxin (Nishimura et al., 1988; Kitazawa et al., 1989) and incubated in Ca 2÷free solution, respectively. We also studied on the role of endothelium in the ET-l-induced contraction. A part of the present work has appeared elsewhere in a I preliminary form (Tanaka et al., in press).

(Nakayama and Tanaka, 1988; Nakayama and Tanaka, 1989; Nakayama et al., 1991). The artery segments in ring form were loaded with 5/tM fura-2/ AM for 4-5 hr at room temperature in normal Tyrode solution (in mM: NaCI 158.3, KCI 4.0, NaHCO3 10, NaH2PO4 0.42, CaC! 2 2.0, MgCI 2 1.05, and glucose 5.6) containing 5 mg/ml bovine serum albumin (BSA) and a nontoxic detergent, cremophor EL (0.05%). After fura-2 loading, the artery was rinsed with normal Tyrode solution for 15rain, mounted in a quartz glass chamber containing 7 ml of Tyrode solution bubbled with 97% Oz and 3% CO2 and maintained at a pH of 7.35 at 35°C with two L-shaped tungsten wires (150#m diameter) inserted through the lumen of the ring. One wire was attached to a supporting hook, and the other was connected to the level of a mechanoelectric transducer (model T7-8-240, Orientec, Tokyo, Japan). Cytosolic Ca 2+ signals and isometric tension development were recorded, the former with a fluorimeter (CAF-100, Japan Spectrophotometric, Tokyo, Japan) and the latter with a force-displacement transducer. Ultraviolet light obtained from a xenon high-pressure lamp for excitation (340 nm (F340) and 380 nm (F380), each with + 5 nm) was focused on the artery and the corresponding emission signals (500 -4- 10 nm) as well as the ratio signal (F34o/F38o), referred to R340/380, a measure of [Ca2÷]i, were monitored. Absolute [Ca2+]i was calculated by the ratio method (Grynkiewicz et al., 1985; Himpens et al., 1989) from the following equation:

MATERIALS AND M E T H O D S

Healthy mongrel dogs of either sex weighing 7-15kg were used. Each animal was maintained on a diet of standard dog chow (Oriental Food, Funabashi, Japan) and looked after for at least 10 days before being used experimentally. The animals were anesthetized with sodium pentobarbital (30 mg/kg, i.v.) and exsanguinated by bleeding from the carotid arteries. A cylindrical segment of the basilar artery 2 cm long with an outer diameter of about 1 mm was removed from the proximal portion near the communicating artery (circle of Willis). The arteries were cleared of connective tissue and adventitia under a dissection microscope and cut into ring segments about 2 mm wide. The average wet weight of the ring segment was about 0.5 mg.

where Kd is the dissociation constant of fura-2 for Ca :÷, assumed in vivo to be 224 nM (Grynkiewicz et aL, 1985); B is the ratio of F380in Ca2+-free solution to that in Ca 2+ containing solution; R is the fluorescence ratio at F34o/F38o, Rmin is obtained by addition of 10 -5 M ionomycin after superfusion with a Ca 2+free depolarizing solution (140 mM KC1) containing EGTA (2 mm) for 20 min, and Rm~x is obtained by addition of excess Ca 2+ (10 mM) after determination of Rmin. Changes in [Ca2+]i and those in tension were expressed as percentages of the differences between basal values and those obtained with a 10 min stimulation with 80 mM KCI. In the present study, the initial and maintenance phases of the [Ca2+]~ and tension of the canine cerebral artery were referred to as those that appeared within 30sec and at 10-20rain after the stimulations of the artery, respectively.

Recording o f [Ca2+]~ and isometric tension

Permeabilization with Staphylococcus aureus or-toxin

[Ca2+]i and tension were simultaneously measured according to procedures reported previously

Ring segments of canine basilar artery about 1.0 mm wide were mounted in a chamber containing

Isolation o f cerebral artery

[Ca2+]i = Kd × B x [(R - Rmin)/(Rma x - R)]

Endothelin-l-produced cerebral artery contraction about a 0.5 ml volume of HEPES-buffered solution by means of two tungsten wires passed through the lumen of the ring; one wire was attached to a supporting hook, and the other was attached to a force transducer (Miniature Load Cell, Kulite Semiconductor Products Inc., Leonia, NJ, U.S.A.) mounted on a micromanipulator (Narishige, Tokyo, Japan). The composition of HEPES-buffered solution for the experiments was (mM) NaC1, 158.3; KCI, 4; CaCI 2, 2; MgCI 2, 1.05; glucose, 5.6; and HEPES, 10.4 neutralized with NaOH to pH 7.4 at about 20°C. The tissue was stretched to an optimal length (about 140% of the initial muscle length = 100%) to give the maximum active tension development in response to 162.3 mM KCI-HEPESbuffered solution (replacing NaC1 totally with an equimolar KC1). After measuring steady contractions induced by 162.3mM KC1-HEPES-buffered solution, the ring segments were exposed for 45 min to Ca2÷-free HEPES-buffered solution containing 2 mM EGTA, and then incubated for 10o20 min in a relaxing solution [in mM: KCH3SO3, 128.7; Mg(CH3SO3) 2 5.1; Na2ATP, 4.2; EGTA, 2; PIPES, 20, and neutralized to pH 7.0 with KOH at 20°C]. The artery was then treated for 10-15min with Staphylococcus aureus or-toxin (s-toxin) (10#g/ml) in the normal relaxing solution. Thereafter, the functions of sarcoplasmic reticulum to accumulate and release Ca 2+ were eliminated by a Ca 2+ ionophore, A23187 (10 -5 M) for 20 min in the relaxing solution. In the activating solution, 10 mM EGTA was used, and a specified amount of Ca(CH3SO3) 2 was added to give a desired concentration of free Ca 2+. Ionic strength was kept constant at 0.2 M by adjusting the concentration of KCH3SO 3. In the present study, all the values of stability constants for complexes in solutions were calculated as previously reported (Horiuti, 1992). For the tension development after permeabilization with c~-toxin, exogenous ATP was required absolutely, thus the majority of the cells seemed to be permeabilized by the procedure described above. The potentiation phenomena described here means sensitization, an apparent increase in Ca -'+ potency which is manifest as an increase in pCa.

Drugs The drugs used in the present study were as follows: human endothelin-1 (ET-1), bradykinin (Peptide Research Institute, Osaka, Japan), NG-nitroe-arginine (L-NNA) (Aldrich Chem. Company Inc., Milwaukee, WI, U.S.A.), ionomycin calcium (Hoechst Japan, Tokyo, Japan), Fura-2/AM, ethyleneglycol-bis-(fl-aminoethyl ether)-N,N,N',N'GP 26/4--N

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tetraacetic acid (EGTA) (Dojindo Laboratories, Kumamoto, Japan), Staphylococcus aureus s-toxin (GIBCO BRL, Gaithersburg, MD, U.S.A.), d-cisdiltiazem hydrochloride, papaverine hydrochloride, prostaglandin F2~ (PGF2~) (Wako, Osaka, Japan), indomethacin, 2-nitro-4-carboxyphenyl-N,Ndiphenylcarbamate (NCDC) and calcium ionophore A23187 (Sigma, St Louis, MO, U.S.A.). Endothelin-I and bradykinin were prepared in a phosphate buffered-saline (pH 7.4) containing 0.05% bovine serum albumin (BSA). PGF2~, NCDC, A23187 and ionomycin calcium were dissolved in ethanol to make a 10 mM stock solutions and were diluted to the desired concentrations with distilled water. Indomethacin was dissolved in 0.1 N NaHCO 3 at 10 mM and diluted to the desired concentrations with distilled water. All other drugs were prepared with distilled water. Drugs were added directly to the organ bath and expressed as molar concentration (M) in the medium.

Statistical analysis The data are shown as the mean ___S.E.M. Statistical analysis was made by a paired or unpaired Student's t-test. Differences with a P value less than 0.05 were considered significant.

RESULTS

Contraction of canine basilar artery in response to ET-I and effects of L-NNA and indomethacin Cumulative application of ET-1 (10-~°-3 × 10-SM) contracted the canine basilar artery in a concentration-dependent manner, and the concentration required to induce 50% contraction (ECs0) and maximum contraction were 7 × 10-~°M and 122% of the tonic contraction produced by 80 mM KC1 [4.64 + 0.64 g, n = 6 ( = 100%)] (Table 1). When L-NNA (10 -s M) or indomethacin (3 x 10 -6 M) was administered into the organ bath in which a basilar artery was incubated for 20 min before the application of ET-I, the concentration-response relationship for ET-1 was not significantly affected. Furthermore, ET-1, up to the concentration of l0 --7 M, did not dilate the endothelium-intact artery which was precontracted with PGF2~ (10-SM) (=100%), whereas it was relaxed by 10-TM bradykinin, an endothelium-dependent vasorelaxant (86.1 + 5.2%, n =4), indicating that ET-I did not produce any apparent endothelium-dependent relaxation in the canine cerebral artery. Therefore, in the following study, we used endothelium-intact artery segments.

Yoshio Tanaka et al.

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Effects of ET- 1 assessed by simultaneous recording of [Ca ;+] and tension Figure 1 shows typical tracings which represent the tension measured simultaneously with [Ca2+]i in a canine basilar artery loaded with fura-2. When the KCI concentration in the medium was increased from 4 to 80 nM, the basilar artery showed an increase in basal tension, the so-called KC1 contracture. The time course of [Ca2+]i was dissociated from that of tension: [Ca2+]i increased immediately within 30 sec, and after reaching the peak value, it decreased gradually and reached a plateau level. On the other hand, tension development induced by 80 mM KCI was always preceded 1-2 sec by fluorescence changes and continued to increase further after the stimulation and reached the peak level in about 10min. The mean [Ca2+]i at the basal tone of a selected number of canine basilar arteries was 84.8_ 5.4nM (n = 12), whereas that during the tonic phase of 80 mM KCIinduced contraction at 10min was 299.8 + 31.6nM (n = 12). When the KC1 concentration in the medium was decreased from 80 mM to 4 mM, elevated [Ca2+]i and tension were restored to each resting level. The tension development produced by ET-I (10 -s M) was also preceded by a change in [Ca2÷]i. In this case, the time course of [Ca2+]~ was also dissociated from that of tension: after reaching the peak level within 30 sec, [Ca2÷]i was decreased gradually. On the other hand, tension reached its peak within 5-10 rain and it remained relatively stable for at least 40min. Papaverine (10 -4 M) abolished the tension increased by ET-1 and decreased [Ca2+]i below the resting level. Figure 2A shows the time courses of [Ca2+]i and tension when the canine basilar artery was contracted by high KCI (20-120 mM). [Ca2÷]i and tension were dependent on the concentration of KCI in the medium. Figure 2B shows the corresponding timedependent changes in [Ca2+]i-tension relationship. The [Ca2÷]i-tension relationship changed counter-

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Fig. I. Typical tracings showing [Ca2+]i-tension relationship in a canine basilar artery ring segment during high KCI (80mM)- and ET-1 (10 -8 M)-induced contractions.

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[Ca~],(*/.) Fig. 2. Simultaneous measurements of [Ca2+]i and tension during high KCl-induced contraction. (A) Time-courses of [Ca2÷]i (a) and tension (b) of the basilar artery stimulated with high KC1 (20-120raM). The responses to a single application of KC1 were plotted on the ordinate as % changes of 80 mM KCl-produced responses at 10 min obtained before start of the experiments. Each point with bars represents mean ___SEM of 4-5 experiments. (©) 20 mM; (A) 30raM; (I-q) 40raM; (0) 80mM; (&) 120mM. (B) Relationship between [Ca2+]i (abscissa) and tension (ordinate) of the basilar artery during high KCl-induced contraction. (O) 20mM; (A) 30mM; (I-q) 40mM; ( 0 ) 80 mM; (&) 120 mM. Points of each counterclockwise curve correspond with the time (min) elapsed which are indicated in the figure after stimulation with various concentrations of KC1 (20-120 mM). clockwise in all cases. The dissociation of time courses between [Ca:+]i and tension was prominent especially at the initial phase of contraction when the artery was stimulated with higher KC1 (80-120 mM). In the maintenance phase of contraction at 10 min, increases in [Ca2+]i and tension were in a parallel manner, d-cis-Diltiazem (10 -5 M), a Ca 2+ antagonist, abolished both [Ca2+]~ and tension increased by 80 mM KCI. Figure 3 shows the time courses of [Ca2+]i and tension (Fig. 3A) and the corresponding timedependent [Ca2+]i-tension relationship (Fig. 3B)

859

Endothelin-l-produced cerebral artery contraction

//" when the canine basilar artery was stimulated with single applications of ET-I (10-t°-10-TM). The changes in [Ca:+]i and tension were dependent on the concentrations of ET-1. As in the case of high KCl-induced responses, the [Ca2+]i-tension relationship changed counter-clockwise (Fig. 3B). At 10--20 min after the application of ET-1 (10 -9 M), [Ca2+]i and tension were increased in a parallel manner, whereas ET-I at higher concentrations (10-8-10 -7 M) produced a greater contraction with a small increase in [Ca2÷]i, so that the [Ca2÷]i-tension relationship shifted upwards.

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Fig. 4. Relationship between [Ca:+]i and tonic tension elicited by ET-I at 20min and the inhibitory effects of d-cis-diltiazem (10 5M). The responses to a single application of various concentrations of ET-1 were plotted in the absence (O) and in the presence (O) of d-cis-diltiazem (10-5 M), and those to 80 mM KCI were taken as 100% on the abscissa and ordinate. Each points with bars indicates mean + SEM of 3-4 preparations. Numbers indicated in the figure represent the negative logarithm of the molar concentrations of ET-1 applied.

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[Ca"l, (%) Fig. 3. Simultaneous measurements of [Ca2+]i and tension during ET-l-induced contraction. (A) Time-courses of [Ca2+]i (a) and tension (b) of the basilar artery stimulated with ET-1 (10-1°M-10-TM). The responses to a single application of ET-1 were plotted on the ordinate as % changes of 80 mM KCl-produced responses at 10 min obtained before start of the experiments. Each point with bars represents mean + SEM of 4-5 experiments. (O) 10-1° M; (A) 10-gM; (Q) 10-s M; (&) 10-7 M. (B) Relationship between [Ca2+]i (abscissa) and tension (ordinate) of the basilar artery during ET-l-induced contraction. (O) 10-1°M; (A) 10-gM; (O) 10-SM; (A) 10-TM. Points of each counterclockwise curve correspond with the time (min) elapsed which are indicated in the figure after stimulation with various concentrations of ET-1 (10-1°-10-7 M).

Figure 4 shows the effect of d-cis-diltiazem (10-SM) applied to the bath 40min before the addition of ET-I on [Ca2÷]i-tension relationship at the maintenance phase of ET-I-induced contraction (20 min). The inhibitory effect of d-cis-diitiazem (10 -5 M) on the tension development by ET-I was greather than that on [Ca2÷]i. [Ca2+]i and tension increased by 10-TM ET-I at 20min, which was 57.0_3.3% ( n = 4 ) and 108.9 4- 7.2% ( n = 4 ) of 80mM KCl-induced responses, respectively. On the other hand, in the presence of d-cis-diltiazem (10 -5 M), [Ca2+]i and tension increased by 10-7M ET-1, only 13.5_+2.5% ( n = 4 ) a n d 13.1_+3.0% (n = 4), respectively. Thus, d-cis-diltiazem (10 -5 M) inhibited the contraction (95.8%) more effectively than [Ca2+]i (43.5%) increased by ET-I (10 -7 M). Thereby, as shown in Fig. 4, the [Ca2+]~-tension relationships in the ET-i-induced responses after the treatment with d-cis-diltiazem were shifted leftwards and downwards. The external solution was replaced with a Ca 2+free solution containing 0.2 mM EGTA and the artery was incubated in the solution for 10 min. The Ca 2+ removal rapidly decreased [Ca2+]i which was accompanied by a decrease in tension. ET-1 (10 -7 M) transiently increased both [Ca2÷]~ and tension (Fig. 5). The [Ca2+]i and tension increased by ET-I (10 -7 M) in the Ca2+-free solution containing 0.2 mM EGTA were 53.9+9.1% (n = 4 ) and 43.7-t-3.2% (n = 4) of the 80mM KCI-induced responses, respectively. When NCDC (10-4M), a putative

860

Yoshio Tanaka et aL

inhibitor of phospholipase C (PLC), was present in the Ca2+-free solution, ET-I (10 -7 M)-elicited increases in [Ca2÷]i (23.9 + 4 . 2 % , n = 4 , P <0.01) and contraction (35.3 -t- 3.2%, n = 4, P < 0.01) were significantly inhibited. However, NCDC (10 -4 M) did not affect [Ca2+]i and tension increased transiently by caffeine (20 mM) (data not shown), which enhances the Ca2+-induced Ca 2÷ release mechanism.

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The effects of ET-I on the relationship between pCa and tension in the x-toxin-permeabilized artery were examined. After x-toxin treatment (10 mg/ml for 10--15min), the arteries developed tension when exposed to various concentrations of Ca 2+ (pCa=7.5-5.0), whereas the intact preparation which was not subjected to the treatment with x-toxin did not show any appreciable tension development. The maximum contraction at pCa 5.0 (0.63 + 0.22 g, n = 4 ) was mostly the same as that elicited by 162.3mM KC1 (0.55_+0.63g, n = 4 ) in the same preparations before permeabilization. The pECs0 value, a measure of Ca 2+ sensitivity, was 6.44 _+ 0.09 (n = 4) in the control response, and it was increased, but not significantly by further addition of GTP (10 -4 M) (6.53 +_0.02, n = 4). ET-I (10 -7 M) augmented the sensitivity to Ca 2÷ (pCa 6.75) in the presence of GTP (10-4M) (Fig. 6A), which was partly inhibited by GDPflS (10-3M). ET-1 (10-7M) shifted profoundly leftwards the pCa-tension relationship in the presence of GTP, thus p ECso was significantly increased (6.85 + 0.09, n = 4, P < 0.01 vs. the control in the presence of GTP alone).

I 10"M ET-1 8O mM KCI Ca~- free + 0.2 mM EGTA Fig. 5. Effect of ET-1 (10 -7 M) on [Ca2+]i and tension in canine cerebral artery in the absence of external Ca2+. External solution was changed to Ca2+-free solution containing 0.2 mM EGTA, and the artery was incubated in the solution. After a 10-min incubation, 10-TM ET-1 was added.

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pCa Fig. 6. Effects of ET-1 on pCa-tension relationships obtained in the artery permeabilized with Staphylococcus aureus ~-toxin. (A) Typical tracings showing the effects of ET-I (10 -7 M) on tension development in response to pCa 6.74 solution in the presence of GTP (10-4 M). (B) Effects of ET-! on the pCa-tension relationships. (C)) control: (O) in the presence of GTP (10-4 M); (&) GTP (10 -4 M) and ET-I (10-TM). Each point with bars represents mean + SEM from 4 experiments. *P < 0.05, **P < 0.01 vs the response in the presence of GTP (10 -4 M) alone.

DISCUSSION The present study showed that ET-1 contracted a canine cerebral artery by increasing myofilament Ca :+ sensitivity as well as transmembrane Ca 2÷ influx and Ca :÷ release from the intracellular storage sites. The Ca 2÷ influx involved in the ET-l-induced contraction seems to be mainly through L-type Ca 2÷ channels, which may further potentiate the myofilament Ca 2+ sensitivity. With regard to the role of endothelium in the ET-l-induced contraction of canine cerebral artery, the peptide-induced contraction was not significantly affected by L-NNA which could inhibit EDRF/NO formation from L-arginine (Ishii et aL, 1990), or indomethacin. Furthermore, the canine basilar artery with an intact endothelium, which was precontracted with PGF2~ (10 -5 M), did not relax in response to ET-I at the concentration of up to 10 -7 M, whereas bradykinin (10 -7 M) relaxed the artery. These findings suggest that as for the canine cerebral artery, ET-I does not produce relaxation by production of endogenous EDRF/NO or prostacyclin (PGI2). The

Endothelin-l-produced cerebral artery contraction present study supported the previous findings by others obtained in cerebral arteries isolated from the cat (Saito et al., 1989) and the dog (Saito et al., 1991). By contrast, in rat aorta (Sakata et al., 1989) and mesenteric artery (De Nucci et al., 1988), low concentrations of ET-1 produced an endothelium-dependent vasorelaxation via release of EDRF/NO or PGI:. Endothelin receptors which are responsible for the functional vascular responses such as contraction and relaxation are currently classified into two subtypes, i.e. ETA and ETB receptors (Davenport and Maguire, 1994). The vasoconstriction in response to exogenous ETs may be mainly mediated by ETA and/or partly by ETB receptors, and the release of endogenous vasodilators from endothelium is mediated through ET B receptors on the endothelium (Davenport and Maguire, 1994). Since endothelium-dependent vasodilatation produced by bradykinin in the canine cerebral artery was almost completely inhibited by the treatment with L-NNA (data not shown), ETB receptors may not be expressed on the endothelium in the canine cerebral artery. The present study showed that the [Ca2+]t-tension relationship changed with time during the contraction of canine cerebral artery in both ET-1- and high KCl-induced responses. Time-dependent changes of the [Ca2÷]~-tension relationship were also reported in other smooth muscles: caffeine- or ionomycininduced contraction in frog stomach (Yagi et al., 1988) and rat aorta (Sato et al., 1988); ET-l-induced contraction in rat aorta (Sakata et al., 1988); high KCl-induced contraction in guinea pig ileum (Himpens and Casteels, 1990) or in canine stomach (Ozaki et al., 1991). Our findings showed that in the initial phase of the contractions, the increase in [Ca2+]i was greater than that in tension in both ET-1and high KCl-induced contractions. Thus, it may imply that the apparent Ca 2÷ sensitivity of the contractile elements is decreased at the initial phase of the arterial contractions. However, the greater increase in [Ca2÷]~ compared to the tension development of canine cerebral artery in response to both ET-I and high KCI at the initial phase may be merely attributable to the time lag between the change in [Ca2+]i and the subsequent intracellular events resulting in an initiation of tension development. At present, it is widely accepted that for the generation of smooth muscle active force development in response to agonistic stimuli, Ca2÷-dependent phosphorylation of the myosin light chain by myosin light chain kinase is a pivotal step (Kamm and Stull, 1985). Thus, even if [Ca2÷]i is increased by the stimulation with ET-1 or by membrane depolarization with high KC1, it may take some time to transform intracellular biochemical processes to mechanical activity.

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The present findings suggested that Ca 2+ sensitivity of the contractile elements was increased during the tonic contraction of cerebral artery induced by high concentrations of ET-1, whereas 80 mM KCl-induced contraction was attributable to [Ca2÷]i which was increased by augmented Ca 2+ influx through L-type Ca 2+ channels. In accordance with the findings obtained in the intact artery, the leftward shift of the pCa-tension relationship by ET-1 in the ~-toxinpermeabilized artery also showed that the peptide could increase the Ca 2+ sensitivity of contractile elements. An ET-l-elicited increase in the Ca :+ sensitivity of contractile elements was also shown in various vascular tissues, including porcine coronary artery (Nakayama et al., 1991), rat aorta (Sakata et al., 1989) and rat mesenteric artery (Nishimura et al., 1992). However, the mechanism underlying the ET-l-elicited increase in Ca 2÷ sensitivity, particularly with regard to the dependence on [Ca2÷]~, seems to be variable depending on the vascular tissues. In the cerebral artery, pretreatment of the artery with d-cisdiltiazem (10 -5 M) effectively inhibited the [Ca2÷]i increased by ET-1, which suggests that the Ca 2+ infux through L-type Ca 2÷ channels is enhanced by ET-1. Moreover, the inhibition by d-cis-diltiazem of ET-Iinduced tension was much greater than that of ET-l-induced [Ca2+]i. In other words, a greater contraction can be elicited by the Ca 2+ influx during ET-l-induced contraction of the canine cerebral artery compared with the high KCl-induced contraction. Therefore, in the cerebral artery, the influx of Ca 2+ through L-type Ca 2+ channels may act not only as the major source of activator Ca 2÷ for eliciting contraction in response to ET-1 but also as a determinant factor of Ca 2÷ sensitivity of contractile elements. By contrast, in the aorta (Sakata et al., 1989) and carotid artery (Ozaki et al., 1989) of the rat, most of the contractions elicited by ET-1 still remained in spite of the disappearance of [Ca2+]i after application of Ca 2+ channel antagonist such as verapamil or the removal of extracellular Ca 2+. Therefore, in these arteries, the Ca 2+ sensitivity of the contractile elements may be increased without any increase in [Ca2+]i. Our findings together with those reported by others (Saito et al., 1991; Asano et al., 1989; Tanoi et al., 1992) indicate why ET-l-induced contraction is susceptible to Ca 2+ antagonists in a clinically important vascular region such as cerebral artery. With regard to the mechanism of the increase in Ca 2+ sensitivity of contractile elements of vascular smooth muscle stimulated by agonistic stimuli, a variety of candidates have been postulated so far: protein kinase C (PKC) (Nishimura et al., 1988; Ozaki et al., 1990), low molecule GTP-binding proteins (G-proteins) (Kitazawa et al., 1989; Himpens

862 et aL, 1989) such as rho p21 (Hirata et H-ras p21 (Satoh et al., 1993), tyrosine et al., 1993), arachidonic acid (Gong et polyamine (Nilsson et al., 1993). We

Yoshio Tanaka et al. al., 1992) and

kinase (Satoh al., 1992) and have already suggested that the activation of PKC is involved in the process leading to the increase in Ca 2+ sensitivity of contractile element during ET-l-elicited contraction in porcine coronary artery (Nakayama et al., 1991). We also reported that 12-deoxyphorbol 13isobutyrate (DPB), a phorbol ester, produced a greater contraction with only a small change in [Ca2+]i (Hanatsuka et al., 1993). Furthermore, staurosporine (10 -7 M), a putative PKC inhibitor, effectively inhibited the ET-1 (10 -7 M)-induced contraction of canine cerebral artery (data not shown). Thus, the increase in myofilament Ca 2+ sensitivity during ET-l-induced cerebral artery contraction may also partly be mediated by the activation of PKC. As shown in the present study, the inhibition of pretreatment of canine cerebral artery with d-cisdiltiazem (10 -5 M) of the [Ca2+]i increased by ET-1, was not complete. ET-1 may enhance the Ca 2+ influx through a pathway other than L-type Ca 2+ channels (Blackburn and Highsmith, 1990; Saito et al., 1991). However, we do not necessarily hypothesize the existence of such a channel stated above because of the following reasons. In the present study, we pretreated the artery with d-cis-diltiazem for 40 min in normal Tyrode solution (4 mM KC1). The concentration of d-cis-diltiazem (10-SM) used was adequate to abolish high KCI (80 mM)-induced increases in both [Ca2+]i and tension in canine cerebral artery (Nakayama et al., 1992). However, Ca 2+ channel antagonists including d-cis-diltiazem are known to act more effectively on Ca 2+ channels when the plasma membrane is depolarized (Nakayama et al., 1989). A 40 min pretreatment with d-cis-diltiazem may inadequately inhibit the L-type Ca 2+ channels in a polarized state. By contrast, our unpublished observations indicated that the same concentration of d-cis.diltiazem, when applied during the contractile responses to ET-1, completely inhibited the [Ca2+]i increased by ET-1. ET-l is shown to cause a small but significant depolarization of the plasma membrane of vascular smooth muscles such as rat mesenteric artery (Wailnofer et al., 1989) and cat cerebral artery (Kauser et al., 1990). Thus, in the canine cerebral artery, the Ca 2+ influx augmented by ET-I may also be mediated mainly through L-type Ca 2+ channels. In the solution from which extracellular Ca 2+ was removed, ET-1 (10-TM) induced only transient changes in Ca 2+ and tension, and both changes were significantly inhibited by a putative phospholipase C inhibitor, NCDC (Tanaka et aL, 1994b). These findings suggest that ET-I increases [Ca2+]i by releasing

Ca 2+ from the intracellular storage sites possibly via production of inositol 1,4,5-trisphosphate (IP3). ET-l has been reported to increase the production of IP3 in various vascular smooth muscles including porcine coronary artery (Kasuya et al., 1989). Therefore, in the canine cerebral artery, the increased formation of IP 3 by the stimulation with ET-1 may cause an increase in [Ca2+]i. Finally, we emphasize the pathophysiological role of ET-1 involved in the genesis of cerebral vasospasm after subarachnoid haemorrhage and the potential usefulness of Ca 2+ antagonistic vasodilators: (1) ET- 1 injected intracisternaily into dogs narrowed the diameter of the basilar artery (Asano et al., 1989); (2) the level ofimmunoreactive ET-1 was increased in the basilar artery of the dog when subarachnoid hemorrhage (SAH) was experimentally produced (Yamaura et al., 1992); (3) topically applied ET-I reduced the local cerebral blood flow in the rat (Robinson et al., 1991); and (4) high concentrations of ET-1 (9.1-12.0 pg/ml) in both plasma and cerebral fluid was demonstrated in patients with subarachnoid haemorrhage coincident with vasospasm (Masaoka et aL, 1989). In the present study, we have shown that d-cis-diltiazem was effective for inhibiting the cerebral artery contraction induced by ET-I. Furthermore, we have shown that brovincamine, a cerebral vessel dilator, effectively suppressed the cerebral artery contractions by inhibiting Ca 2+ influx through L-type Ca 2+ channels (Tanaka el al., 1994a). Thus, the present findings indicate the potential usefulness of the drugs to inhibit the Ca 2+ influx for the relief of enhanced cerebral vascular contraction produced by ET-1. SUMMARY Ca 2+ handling mechanisms underlying endothelin1 (ET-1)-induced cerebral artery contraction were examined with particular reference to the role of the transmembrane influx of Ca 2+. Intracellular Ca 2+ concentrations ([Ca2+]i) and tension were simultaneously measured in a canine cerebral artery loaded with fura-2. The artery obtained from the same animal species was permeabilized with Staphylococcus aureus or-toxin to determine whether the peptide potentiated the Ca 2+ sensitivity of contractile elements. ET-1 (10-10--10-7 M) contracted the canine cerebral artery in a concentration-dependent manner with the increase in [Ca2+]i. ET-i (10-J°-10-TM) increased [Ca2+]i and tension in a parallel manner, whereas at higher concentrations (over 10-8 M), it produced a greater contraction with a small increase in [Ca2+]i. A Ca 2+ channel antagonist such as dcis-diltiazem (10 -5 M) inhibited more effectively the

Endothelin-l-produced cerebral artery contraction ET-l-induced tension development than the [Ca2+]i increased by ET-I. In a Ca2+-free solution containing 0.2 m M E G T A , ET-1 (10 -7 M) only elicited a transient increase in [Ca:+]i and tension. In the ~t-toxinpermeabilized artery, ET-1 (10 -7 M) shifted leftwards the p Ca-tension relationship in the presence of G T P (10 -4 M). These findings suggest that ET°I increases [Ca2+]i by increasing the Ca 2+ influx through L-type Ca 2+ channels and releasing Ca 2+ from the intracellular storage sites in canine cerebral artery. ET-1 increases Ca 2+ sensitivity at higher concentrations during the sustained phase of contraction, but the mechanism operates only when [Ca2+]~ is increased by the augmented Ca 2+ influx through L-type Ca 2÷ channels. These findings also may explain the greater effectiveness by Ca 2÷ antagonists to cerebral artery contraction in response to ET-1. Acknowledgements--The present study was supported in part by Grants-in-Aid for Scientific Research (Nos 02304033, 02671005 and 04671360), and for Scientific Research on Priority Areas: "Vascular EndotheliumSmooth Muscle Coupling" (Nos 04263233 and 05256227) from the Ministry of Education, Science and Culture of Japan (K.N.). Y.T. is a recipient of Grants-in-Aid for Encouragement of Young Scientists (Nos 02771766, 03771794, 04772066 and 05772040) from the Ministry of Education, Science and Culture of Japan.

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