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Journal of Physiology (2001), 534.3, pp. 701-711
© Copyright 2001 The Physiological Society

Ca²⁺ influx in the endothelial cells is required for the bradykinin-induced endothelium-dependent contraction in the porcine interlobar renal artery

Eikichi Ihara, Dmitry N. Derkach, Katsuya Hirano, Junji Nishimura, Hajime Nawata * and Hideo Kanaide

	ABSTRACT

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1. To determine the mechanism of bradykinin-induced production of endothelium-derived contracting factors, we monitored the changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in in situ endothelial cells in porcine aortic valvular strips and the changes in [Ca²⁺]_i of smooth muscle cells and force in porcine interlobar renal arterial strips using front-surface fluorometry of fura-2.
2. In the presence of N-nitro-L-arginine methyl ester, bradykinin caused an endothelium-dependent transient elevation of [Ca²⁺]_i and contraction in smooth muscle in the interlobar renal artery. This contraction was completely inhibited by a prostaglandin H₂/thromboxane A₂ receptor antagonist.
3. In the absence of extracellular Ca²⁺, bradykinin failed to induce contraction. However, replenishing extracellular Ca²⁺ to 0.75 mM and higher induced an instantaneous contraction. However, replenishing Ca²⁺ per se did not induce any contraction in the absence of bradykinin. Pretreatment with either 10^-5 M 1-(-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl)-1H-imidazole hydrochloride (SKF96365) or 0.2 mM Ni²⁺ abolished the contraction induced by bradykinin in the presence of extracellular Ca²⁺.
4. Treatment with 10^-5 M indomethacin completely inhibited the contractile response induced by Ca²⁺ replenishment, regardless of the timing of its application, before or after the application of bradykinin.
5. In endothelial cells in the valvular strips, bradykinin caused a transient [Ca²⁺]_i elevation in the presence of 1.25 mM extracellular Ca²⁺, but [Ca²⁺]_i returned to the resting level within 10 min. Neither 10^-5 M SKF96365 nor 0.2 mM Ni²⁺ had any effect on the peak [Ca²⁺]_i elevation, but decreased [Ca²⁺]_i in the declining phase. In the absence of extracellular Ca²⁺, bradykinin induced a transient [Ca²⁺]_i elevation to a level similar to that seen in the presence of 1.25 mM extracellular Ca²⁺. However, [Ca²⁺]_i then rapidly returned to the prestimulation level within 5 min. Subsequent Ca²⁺ replenishment to 0.75 mM and higher in the presence of bradykinin elevated [Ca²⁺]_i to significantly higher levels than the resting level seen in the media containing 1.25 mM Ca²⁺.
6. In conclusion, Ca²⁺ influx in the endothelial cells is essential for bradykinin to induce endothelium-dependent contraction in the porcine interlobar renal artery.

	INTRODUCTION

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By producing and releasing various vasoactive substances, endothelial cells regulate vascular tone (for review see Furchgott & Vanhoutte, 1989). A number of studies have been conducted on endothelium-dependent relaxation in a wide variety of blood vessels (Shepherd & Katusic, 1991). Nitric oxide (NO) (Ignarro et al. 1987), hyperpolarizing factors (EDHF) (Chen & Suzuki, 1989) and prostacyclin (PGI₂) are major mediators of endothelium-dependent relaxation. Endothelium-dependent contraction has also been reported in limited combinations of agonists and types of blood vessel (Katusic & Shepherd, 1991). Prostaglandin H₂ (PGH₂) (Kato et al. 1990), thromboxane A₂ (TXA₂) (Ihara et al. 1999) and endothelin (Yanagisawa et al. 1988) have been shown to mediate endothelium-dependent contraction. It is well established that the intracellular Ca²⁺ signal in endothelial cells plays a primary role in the production of NO (Fleming et al. 1997), EDHF (Fukao et al. 1997) and PGI₂ (Adeagbo & Henzel, 1998). Elevation of [Ca²⁺]_i due to Ca²⁺ influx has also been reported to be necessary for the production of endothelin (Russell & Davenport, 1999). Therefore, the Ca²⁺ signal may also play an important role in the production of contracting as well as relaxing factors in endothelial cells. However, the precise mechanisms regarding the production of contracting factors remain to be elucidated.

We previously reported that bradykinin induced triphasic regulation of vascular tone, consisting of an initial transient relaxation, a subsequent transient contraction and a sustained relaxation, during the phenylephrine-induced precontraction in the porcine interlobar renal artery (Ihara et al. 2000). The contracting phase was completely abolished by a PGH₂/TXA₂ receptor antagonist or a cyclo-oxygenase inhibitor, and was partly blocked by a TXA₂ synthase inhibitor, thus suggesting the involvement of PGH₂/TXA₂ in the bradykinin-induced contraction. We reported that bradykinin increased [Ca²⁺]_i in the endothelial cells by activating Ca²⁺ release from the intracellular store and Ca²⁺ influx from the extracellular space (Aoki et al. 1994). We also showed that activation of capacitative Ca²⁺ influx by thapsigargin induced a triphasic response in vascular tone similar to that observed with bradykinin in the porcine interlobar renal artery (Ihara et al. 1999). However, little is known about the correlation between the Ca²⁺ signals and the production of contracting factors in endothelial cells. In the present study, we investigated the underlying mechanism of bradykinin-induced vasoconstriction by determining the relationships between the intracellular Ca²⁺ signal and production of PGH₂ and TXA₂ in the porcine renal artery. To this end front-surface fluorometry (Kanaide, 1999) was used to monitor the changes in [Ca²⁺]_i in in situ endothelial cells of the fura-2-loaded strips of porcine aortic valve, and the changes in [Ca²⁺]_i in smooth muscle cells and force were monitored in the fura-2-loaded strips of porcine interlobar renal artery. It was difficult to selectively load the endothelial cells with fura-2 and monitor changes in [Ca²⁺]_i without interference from the signal from the smooth muscle in the arterial strips (Kuroiwa et al. 1995; Ihara et al. 1999). Furthermore, we found that cultured endothelial cells established from porcine renal artery lost responsiveness to bradykinin (data not shown). Therefore, we used strips of porcine aortic valve to investigate Ca²⁺ signalling in in situ endothelial cells, as previously described (Aoki et al. 1994). The endothelial cells of the valvular strips are active in uptake of acetylated low density lipoprotein (Kuroiwa et al. 1995), and express endothelial constitutive nitric oxide synthase and produce NO (Mizuno et al. 1998). In front-surface fluorometry, the strips are illuminated by excitation light through quartz optic fibres placed in front of the tissue facing its surface, and the fluorescence originating from the tissue surface is collected by the glass optic fibres which are also placed in front of the tissue. The quartz and glass optic fibres are arranged in a concentric inner circle and outer circle, respectively, at the end facing the tissue surface.

	METHODS

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Tissue preparation

The porcine interlobar renal arterial strips with and without endothelium, and the porcine aortic valvular strips were prepared as previously described (Aoki et al. 1994; Ihara et al. 2000). In brief, porcine kidneys and aortic valves were obtained at a local slaughterhouse. The interlobar arteries were excised and the adventitia were mechanically removed under the binocular microscope using micro-scissors. The aortic leaflets were cut into strips in an axial direction.

Fura-2 loading

Renal arterial strips with and without an endothelium and the valvular strips were loaded with fura-2 as previously described (Aoki et al. 1994; Ihara et al. 1999). After loading with fura-2, both arterial and valvular strips were equilibrated in normal physiological saline solution (PSS; for composition see Drugs and solutions) for at least 60 min before starting the experimental protocols as previously described (Ihara et al. 2000).

Simultaneous measurement of smooth muscle [Ca²⁺]_i and force in the arterial strips

Experiments on arterial strips were carried out at 37 °C. The strips were mounted vertically onto a force transducer, TB-612T (Nihon Koden, Japan), in a quartz organ bath filled with normal PSS. Changes in smooth muscle [Ca²⁺]_i in the arterial strips were monitored as previously described (Ihara et al. 1999), using a front-surface fluorometer, CAM-OF-3 (JASCO, Tokyo, Japan). The fluorescence intensities (500 nm) at 340 nm (F₃₄₀) and 380 nm (F₃₈₀) excitation and their ratio (F₃₄₀/F₃₈₀), which indicated smooth muscle [Ca²⁺]_i, were continuously monitored. The resting load was adjusted to 100 mg. At the beginning of each protocol, the responsiveness of each strip to 10^-6 M phenylephrine was recorded, and stimulated once with 118 mM K⁺ to refill the intracellullar Ca²⁺ stores. This sustained contraction induced by 10^-6 M phenylephrine was used as a reference response in the porcine renal artery as previously described (Ihara et al. 1999). The levels of [Ca²⁺]_i and the force at rest and during the 10^-6 M phenylephrine-induced sustained contraction were designated as 0 and 100 %, respectively. When we examined the effects of the removal and replenishment of extracellular Ca²⁺, the strips were incubated in the Ca²⁺-free PSS containing 2 mM EGTA for 10 min, and then in the Ca²⁺-free PSS without EGTA.

Measurement of [Ca²⁺]_i of in situ endothelial cells in the aortic valvular strips

The fura-2 fluorometry of the strips of aortic valve was carried out in a manner similar to that used for arterial strips. However, the experiments were performed at 25 °C to prevent the leakage of the fluorescent dye, as previously described (Aoki et al. 1994). The [Ca²⁺]_i levels of in situ endothelial cells at rest and the peak [Ca²⁺]_i elevation induced by 10^-5 M ATP were designated as 0 and 100 %, respectively.

Drugs and solutions

The composition of normal PSS was (mM): 123 NaCl, 4.7 KCl, 15.5 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgCl₂, 1.25 CaCl₂ and 11.5 D-glucose. PSS was aerated with 95 % O₂ and 5 % CO₂, with a resulting pH of 7.4. PSS containing high K⁺ was prepared by replacing NaCl with equimolar KCl. Ca²⁺-free solution was Ca²⁺-free PSS. Fura-2 AM and EGTA were purchased from Dojindo Laboratories (Kumamoto, Japan). Indomethacin was purchased from Wako (Osaka, Japan). DMEM was purchased from Gibco (Life Technologies, Rockville, MD, USA). ONO-3708, a TXA₂/PGH₂ receptor antagonist, and OKY-046, a TXA₂ synthase inhibitor, were kindly donated by the Ono Pharmaceutical Co. (Osaka, Japan). N-nitro-L-arginine methylester (L-NAME), probenecid and phenylephrine were purchased from Sigma (St Louis, MO, USA). Bradykinin was purchased from the Peptide Institute, Inc. (Osaka, Japan). ATP was purchased from Boehringer Mannheim (Germany). SKF96365 (1-(-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenethyl)-1H-imidazole hydrochloride) was purchased from Calbiochem (La Jolla, CA, USA).

Data analysis

All data are expressed as means ± S.E.M. One strip obtained from one animal was used for each experiment, and therefore the number of experiments (n value) indicates the number of animals. Student's t test was used to determine statistical significance between the two groups. P values of less than 0.05 were considered to be significant. All data were collected using a computerized data acquisition system (MacLab; Analog Digital Instruments, Australia).

	RESULTS

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Bradykinin-induced endothelium-dependent contraction at rest in the interlobar renal artery

In order to investigate the mechanism of the bradykinin-induced, PGH₂/TXA₂-mediated contraction, the contractile response was observed by applying bradykinin at a resting state in the presence of L-NAME without phenylephrine-induced precontraction. Figure 1A shows the representative recordings of the changes in smooth muscle [Ca²⁺]_i and force following application of 10^-8 M bradykinin at rest. After a short time lag (~1 min) with a slight decrease in [Ca²⁺]_i, bradykinin caused a transient elevation of [Ca²⁺]_i and force. Their levels reached a peak at ~3 min and returned to the prestimulation level within 10 min despite the presence of bradykinin. The [Ca²⁺]_i level during bradykinin-induced contraction was significantly smaller than that obtained with 118 mM K⁺ whereas the force level was larger than that observed with 118 mM K⁺. Figure 1B summarized the concentration-dependent changes in [Ca²⁺]_i and force induced by bradykinin. No change in either [Ca²⁺]_i or force was observed at 3 10^-9 M and lower concentrations of bradykinin whereas significant increases in both [Ca²⁺]_i and force were observed at 10^-8 M and higher concentrations of bradykinin. The levels of [Ca²⁺]_i obtained with 10^-8 and 10^-7 M bradykinin were 35.1 ± 6.1 and 35.8 ± 8.9 %, respectively (n = 6), and the levels of force were 163.7 ± 23.5 and 181.8 ± 26.2 %, respectively (n = 6). The levels of [Ca²⁺]_i and force obtained with 10^-8 M bradykinin did not significantly differ from those obtained with 10^-7 M bradykinin. Therefore, 10^-8 M bradykinin was used in the following experiments to investigate the role of the Ca²⁺ mobilization in the bradykinin-induced production of contracting factors in renal endothelial cells.

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Figure 1. Endothelium-dependent contraction induced by applying bradykinin at a resting state in the porcine interlobar renal artery A, the representative recordings of changes in [Ca2+]i (fluorescence ratio) and force induced by 10-8 M bradykinin applied at rest in the strips with endothelium (control protocol). After recording reference responses to 10-6 M phenylephrine (Phe) and depolarization with 118 mM K+, the experimental protocol was performed. Both 10-6 M captopril and 10-5 M L-NAME were applied 10 min before the application of bradykinin. B, concentration-response curves for the bradykinin-induced elevation of [Ca2+]i of smooth muscle () and force (). Data are means ± S.E.M. of four independent experiments. The levels of [Ca2+]i and force at rest and during the phenylephrine-induced sustained contraction were designated as 0 and 100 %, respectively.

Effect of bradykinin on [Ca²⁺]_i of in situ endothelial cells in the presence of extracellular Ca²⁺

Figure 2A shows the representative recordings of changes in [Ca²⁺]_i following the application of 10^-8 M bradykinin in the presence of 10^-6 M captopril and 1.25 mM extracellular Ca²⁺ in the strips of the aortic valve. The strips were treated with captopril 10 min before and during the stimulation of bradykinin. The [Ca²⁺]_i level reached its peak around 1 min, and thereafter gradually declined to the prestimulation level within 10 min. The endothelial cells are known to lack the voltage-operated Ca²⁺ channels, and therefore the Ca²⁺ influx in the endothelial cells is resistant to such organic Ca²⁺ channel blockers as dihydropyridines, benzodiazepines and phenylalkylamines (Uchida et al. 1999). SKF96365 is widely used to inhibit the Ca²⁺ influx in the endothelial cells (Schilling et al. 1992), and we previously observed that SKF96365 inhibited the Ca²⁺ influx induced by thapsigargin and vascular endothelial cell growth factor in porcine aortic valvular strips (Kawasaki et al. 1999, 2000). On the other hand, NiCl₂ is a non-selective inorganic inhibitor of Ca²⁺ entry (Jones & Sharpe, 1994). We, therefore, examined the effects of SKF96365 and NiCl₂ on bradykinin-induced [Ca²⁺]_i elevation in valvular endothelial cells. Pretreatment with either 10^-5 M SKF96365 or 0.2 mM NiCl₂ significantly inhibited the [Ca²⁺]_i level obtained 3, 5 and 10 min after the application of 10^-8 M bradykinin, while they had no effects on the peak level of [Ca²⁺]_i (Fig. 2B). In the absence of extracellular Ca²⁺ (Fig. 3A), bradykinin induced a sharp transient elevation of [Ca²⁺]_i. The peak level of [Ca²⁺]_i induced by 10^-8 M bradykinin in the presence of extracellular Ca²⁺ was 114.8 ± 11.4 % (control) (n = 6). The peak levels of [Ca²⁺]_i observed in the presence of SKF96365 and NiCl₂ were 109.3 ± 13.1 and 94.1 ± 10.1 %, respectively (n = 6), and that observed in the Ca²⁺-free PSS was 118.9 ± 15.1 % (n = 6). There were no significant differences between these four values. Collectively, the initial increase in [Ca²⁺]_i induced by bradykinin was mainly caused by the Ca²⁺ release from the intracellular stores, and the late phase of the [Ca²⁺]_i increase seen in the presence of extracellular Ca²⁺ was due to the Ca²⁺ influx from the extracellular spaces.

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Figure 2. Bradykinin-induced [Ca2+]i elevation in in situ endothelial cells in the presence of extracellular Ca2+ A, the representative recordings of changes in [Ca2+]i (fluorescence ratio) induced by 10-8 M bradykinin in the presence of 1.25 mM extracellular Ca2+ in in situ endothelial cells. Captopril (10-6 M) was applied 10 min before the application of bradykinin. B, a summary of the time courses of changes in [Ca2+]i induced by 10-8 M bradykinin in the absence of any inhibitors () and presence of 10-5 M SKF96365 () and 0.2 mM Ni2+ (). Data are the means ± S.E.M. of six independent experiments. The strips were treated with either SKF96365 or 0.2 mM Ni2+ 10 min before and during the stimulation of bradykinin. * Significantly different (Student's t test, P < 0.05); N.S., not significantly different (Student's t test, P > 0.05).

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Figure 3. Effect of the removal and replenishment of extracellular Ca2+ on the bradykinin-induced [Ca2+]i transient in endothelial cells and the endothelium-dependent contraction in the interlobar renal artery A and B, representative recordings showing changes in [Ca2+]i in endothelial cells in valvular strips (A) and changes in [Ca2+]i of smooth muscle cells and in force of smooth muscle in the arterial strips during endothelium-dependent contraction (B) induced by 10-8 M bradykinin. The effects of removing and replenishing extracellular Ca2+ on the bradykinin-induced [Ca2+]i transient in valvular strips and the endothelium-dependent contraction in arterial strips were examined in the same experimental protocol as follows: after treating strips with Ca2+-free PSS containing 2 mM EGTA for 10 min, 10-8 M bradykinin was applied. Extracellular Ca2+ (1.25 mM) was replenished 5 min after the application of bradykinin. C, the representative recordings of changes in [Ca2+]i of smooth muscle cells and force induced by 10-7 M U46619, a TXA2 analogue, in Ca2+-free PSS containing 2 mM EGTA in arterial strips. In endothelial cells in the valvular strips (A), the levels of [Ca2+]i (fluorescence ratio) obtained at rest and with 10-5 M ATP were designated as 0 and 100 %, respectively. In the arterial strips (B and C), the levels of [Ca2+]i (fluorescence ratio) and force at rest and during the phenylephrine-induced sustained contraction were designated as 0 and 100 %, respectively.

Effect of the removal and replenishment of extracellular Ca²⁺ on bradykinin-induced [Ca²⁺]_i elevation in endothelial cells and endothelium-dependent contraction in the interlobar renal artery

To determine whether Ca²⁺ influx was required for the bradykinin-induced endothelium-dependent contraction in renal endothelial cells, we examined the effects of removal and replenishment of extracellular Ca²⁺ on the bradykinin-induced [Ca²⁺]_i transient in endothelial cells and the endothelium-dependent contraction in arterial strips (Fig. 3). When the valvular strips were exposed to Ca²⁺-free medium containing 2 mM EGTA, [Ca²⁺]_i gradually decreased to the new steady-state level within 10 min. The following application of 10^-8 M bradykinin in Ca²⁺-free medium without EGTA induced a transient elevation of [Ca²⁺]_i which reached its peak (118.9 ± 15.1 %, n = 6) at ~1 min, and then rapidly returned to the prestimulated level within 3 min. After [Ca²⁺]_i returned to the prestimulation level, the replenishment of extracellular Ca²⁺ to 1.25 mM 5 min after the addition of bradykinin caused an instantaneous elevation of [Ca²⁺]_i (107.8 ± 8.2 %, n = 6) to above the resting [Ca²⁺]_i level seen in normal PSS (containing 1.25 mM Ca²⁺). Thereafter the [Ca²⁺]_i level gradually declined to a steady-state level (58.6 ± 8.6 %, n = 6) which was significantly higher than the resting level. In the absence of bradykinin, however, Ca²⁺ replenishment caused only a slow recovery of [Ca²⁺]_i to the resting level, and no [Ca²⁺]_i elevation above the resting level was observed (data not shown).

In the arterial strips, stimulation with bradykinin in Ca²⁺-free medium did not induce any changes in the smooth muscle [Ca²⁺]_i or force (Fig. 3B), although [Ca²⁺]_i in endothelial cells increased to a level similar to that seen in the presence of extracellular Ca²⁺ as previously shown (Fig. 3A). Direct activation of PGH₂/TXA₂ receptors by U46619, a TXA₂ analogue, caused an apparent contraction even in the absence of extracellular Ca²⁺ (Fig. 3C). A significant contraction was observed at a U46619 concentration as low as 10^-8 M. Therefore, bradykinin did not appear to induce a PGH₂/TXA₂-mediated contraction in the Ca²⁺-free medium to a level which could be detected as a contractile response in the arterial strips. However, the replenishment of extracellular Ca²⁺ to 1.25 mM at 5 min after the application of bradykinin did induce instantaneous increases in [Ca²⁺]_i and force without any time lag after the stimulation (Fig. 3B). The level of this force development was 180.6 ± 53.5 % (n = 6), which was comparable to that obtained in the presence of extracellular Ca²⁺ (Fig. 1A). Replenishment of Ca²⁺ per se (without bradykinin stimulation) did not induce any contraction (data not shown).

We examined the concentration-dependent effects of Ca²⁺ replenishment on bradykinin-induced elevation of [Ca²⁺]_i in endothelial cells and [Ca²⁺]_i and force in the arterial strips. When extracellular Ca²⁺ was replenished to 0.25 and 0.5 mM, the [Ca²⁺]_i of the endothelial cells gradually increased, but did not reach the resting level seen in normal PSS containing 1.25 mM Ca²⁺ by 20 min (Fig. 4A). When the extracellular Ca²⁺ was replenished to 0.75 mM and higher, [Ca²⁺]_i increased to a higher level than the resting level (Fig. 4A). In the arterial strips, a significant elevation of [Ca²⁺]_i and force above the resting levels was observed when the extracellular Ca²⁺ was replenished to 0.75 mM and higher. The extracellular Ca²⁺ concentration required to induce endothelium-dependent contraction under bradykinin stimulation in arterial strips was similar to that required to induce elevation of [Ca²⁺]_i to above the resting level in endothelial cells. There was a close correlation among the levels of [Ca²⁺]_i elevation in endothelial cells, [Ca²⁺]_i elevation in smooth muscle and the extent of force development in the arterial strips (Fig. 4B).

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Figure 4. Correlation between the peak [Ca2+]i elevation in the valvular strips and force development in the arterial strips induced by the Ca2+ replenishment under stimulation with bradykinin A, concentration-dependent effects of Ca2+ replenishment on the bradykinin-induced [Ca2+]i elevation in endothelial cells in the valvular strips () and on the bradykinin-induced [Ca2+]i elevation of smooth muscle () and the contraction in the arterial strips (). The maximal levels of [Ca2+]i elevation in endothelial cells and that of [Ca2+]i elevation of smooth muscle and force development in the arterial strips obtained with Ca2+ replenishment under stimulation with bradykinin at the indicated concentrations of extracellular Ca2+ are shown. * Significantly higher than the resting level (Student's t test, P < 0.05); N.S., not significant (Student's t test, P > 0.05). B, the correlation between the peak levels of [Ca2+]i in the valvular strips and force in the arterial strips obtained with Ca2+ replenishment under the stimulation with bradykinin was reconstructed from the data shown in A.

Effects of SKF96365 and NiCl₂ on bradykinin-induced contraction in the interlobar renal artery

The contribution of Ca²⁺ influx in endothelial cells to the bradykinin-induced contraction was further investigated by examining the effects of SKF96365 and NiCl₂ on a contraction similar to that shown in Fig. 1A (Fig. 5). SKF96365 and NiCl₂ were shown to inhibit bradykinin-induced Ca²⁺ influx without affecting Ca²⁺ release in the endothelial cells (Fig. 2B). The strips were treated with either 10^-5 M SKF96365 or 0.2 mM NiCl₂ in normal PSS for 10 min, and were then stimulated with 10^-8 M bradykinin. Treatment with SKF96365 decreased the resting [Ca²⁺]_i, while having no effect on the resting force (Fig. 5A). NiCl₂ had no effect on either the resting [Ca²⁺]_i or force (Fig. 5B). In the presence of SKF96365 or NiCl₂, bradykinin failed to induce any contraction (Fig. 5A and B). PGH₂/TXA₂ receptor stimulation with U46619 caused an apparent contraction even in the presence of SKF96365 or NiCl₂ (Fig. 5C and D). In the absence of SKF96365 and Ni²⁺, U46619 induced an abrupt elevation of [Ca²⁺]_i which peaked at ~1 min (Fig. 5E). The [Ca²⁺]_i then decreased abruptly to a level above the resting level where it remained for more than 20 min. The force immediately increased at ~1 min and then reached a sustained level. The levels of [Ca²⁺]_i and force at 1 min after application of U46619 were 178.4 ± 8.9 % and 245.2 ± 25.4 % (n = 6), respectively, and those at 20 min were 103.2 ± 9.8 % and 332.7 ± 37.8 % (n = 6), respectively. In the presence of SKF96365, [Ca²⁺]_i and force at 1 min were 102.6 ± 7.6 % and 164.1 ± 12.7 % (n = 6), respectively, while those at 20 min were -25.9 ± 10.0 % and 78.7 ± 7.3 % (n = 6), respectively. In the presence of NiCl₂, [Ca²⁺]_i and force at 1 min were 151.1 ± 15.5 % and 194.8 ± 16.8 % (n = 6), respectively, and those at 20 min were 99.7 ± 8.3 % and 272.5 ± 23.4 % (n = 6), respectively. The response of [Ca²⁺]_i and force to U46619 was significantly inhibited by SKF96365 (P < 0.05), but it was not affected by NiCl₂ (P > 0.05). These observations indicated that any production of contracting factors by bradykinin in the presence of SKF96365 or Ni²⁺ can be detected as [Ca²⁺]_i elevation or force development. It is thus unlikely that the abolition of the bradykinin-induced endothelium-dependent contraction by SKF96365 and Ni²⁺ was due to the inhibition of Ca²⁺ influx in smooth muscle.

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Figure 5. Effect of SKF96365 and NiCl2 on the bradykinin-induced contraction in the interlobar renal artery A and B, the representative recordings of changes in [Ca2+]i (fluorescence ratio) and force induced by 10-8 M bradykinin in the presence of 10-5 M SKF96365 (A) and 0.2 mM Ni2+ (B) in renal arterial strips with endothelium. Captopril, L-NAME, SKF96365 and Ni2+ were applied 10 min before the application of bradykinin. C-E, the representative recordings of the changes in [Ca2+]i of smooth muscle cells and force induced by 10-7 M U46619, a TXA2 analogue, in the presence of 10-5 M SKF96365 (C), 0.2 mM Ni2+ (D) and in their absence (E) in renal arterial strips with endothelium. The strips were treated with either SKF96365 or 0.2 mM Ni2+ 10 min before and during the stimulation of U46619.

Effect of a cyclo-oxygenase inhibitor on bradykinin-induced contraction in the interlobar renal artery

In order to determine whether the Ca²⁺ influx in endothelial cells was required for either the production or release of contracting substances, we examined the effects of indomethacin, a cyclo-oxygenase inhibitor, on the contraction induced by the Ca²⁺ replenishment under stimulation with bradykinin. We previously demonstrated that pretreatment with 10^-5 M indomethacin for more than 10 min completely abolished the endothelium-dependent contraction induced by 10^-8 M bradykinin during phenylephrine-induced contraction in normal PSS (Ihara et al. 2000). When 10^-5 M indomethacin was applied 10 min before the addition of bradykinin in Ca²⁺-free PSS, replenishment of extracellular Ca²⁺ did not induce any contraction (Fig. 6A). When 10^-5 M indomethacin was applied 5 min after the application of bradykinin the contraction induced by the replenishment of extracellular Ca²⁺ (Fig. 6B) was also inhibited. However, in the control experiment without indomethacin (Fig. 6C), Ca²⁺ replenishment 15 min after stimulation with bradykinin caused a contraction (181.8 ± 33.6 %, n = 6) similar to that seen with Ca²⁺ replenishment 5 min after bradykinin stimulation (Fig. 3B).

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Figure 6. Effects of indomethacin, a cyclo-oygenase inhibitor, on the contraction induced by the Ca2+ replenishment under stimulation with bradykinin Representative recordings showing changes in the force induced by the Ca2+ replenishment under stimulation with bradykinin in the presence (A and B) and absence (C) of 10-5 M indomethacin. The interlobar renal arterial strips were exposed to the Ca2+-free PSS containing 2 mM EGTA for 10 min, and then stimulated with 10-8 M bradykinin without EGTA. Indomethacin was applied 10 min before (A) and 5 min after (B) the stimulation of bradykinin. The extracellular Ca2+ was replenished 5 min after (A) and 15 min after (B and C) the application of bradykinin. The levels of [Ca2+]i (fluorescence ratio) and force at rest and during the phenylephrine-induced sustained contraction were designated as 0 and 100 %, respectively. Similar results were observed in at least three independent experiments.

	DISCUSSION

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We recently reported that bradykinin induced not only endothelium-dependent relaxation but also contraction in the porcine interlobar renal artery (Ihara et al. 2000). The endothelium-dependent contraction was mediated by PGH₂ and TXA₂ as shown in the present study and as previously reported (Ihara et al. 2000). In the present study, we demonstrated for the first time that the [Ca²⁺]_i elevation in endothelial cells due to Ca²⁺ influx was necessary for bradykinin to induce a PGH₂/TXA₂-mediated endothelium-dependent contraction in the porcine interlobar renal artery. However, elevation of [Ca²⁺]_i due to Ca²⁺ release was not sufficient for bradykinin to induce a PGH₂/TXA₂-mediated contraction. These conclusions were based on the following observations. (1) In the absence of extracellular Ca²⁺, bradykinin induced a sharp transient elevation of [Ca²⁺]_i to a level similar to that seen in the presence of 1.25 mM extracellular Ca²⁺. However, in the arterial strips, bradykinin failed to induce an endothelium-dependent contraction in the absence of extracellular Ca²⁺. (2) The subsequent replenishment of extracellular Ca²⁺ under stimulation with bradykinin induced an instantaneous increase in [Ca²⁺]_i in endothelial cells, and contraction in the arterial strips. (3) The concentration of extracellular Ca²⁺ upon replenishment required to induce contraction in the arterial strips closely correlated with that required to induce [Ca²⁺]_i elevation above the resting level in endothelial cells. (4) In the presence of either SKF96365 or NiCl₂, bradykinin failed to induce any contraction in the arterial strips, while it did cause a sharp transient elevation of [Ca²⁺]_i in endothelial cells.

The bradykinin-induced Ca²⁺ release in endothelial cells was not sufficient to induce endothelium-dependent contraction in the arterial strips. However, there was a possibility that [Ca²⁺]_i elevation due to Ca²⁺ release induced production of PGH₂ and TXA₂, but that [Ca²⁺]_i elevation due to Ca²⁺ influx was necessary to trigger their release. However, as indomethacin inhibited the contraction induced by Ca²⁺ replenishment under bradykinin stimulation regardless of the timing of application, i.e. before or after the transient [Ca²⁺]_i elevation due to the bradykinin-induced Ca²⁺ release in endothelial cells, this possibility was ruled out. As a consequence, we concluded that the Ca²⁺ influx is necessary for the production and release of PGH₂ and TXA₂ in endothelial cells. The level of [Ca²⁺]_i elevation induced by bradykinin seen in the Ca²⁺-free medium was as high as that seen in the presence of extracellular Ca²⁺, thus indicating that the production and release of the contracting factors does not depend merely on the level of [Ca²⁺]_i elevation. Subcellular compartmentalization or duration of the Ca²⁺ signal is suggested to be a critical determinant of the production of the contracting factors.

The key enzyme for production of PGH₂ and TXA₂ is phospholipase A₂ (PLA₂)_. Mammalian cells express structurally diverse forms of PLA₂ including secretory PLA₂, Ca²⁺-independent PLA₂ and cytosolic PLA₂ (cPLA₂) (Balsinde & Dennis, 1997; Kramer & Sharp, 1997; Tischfield, 1997). Among these PLA₂s, cPLA₂ is the only known PLA₂ that is likely to be involved in receptor-mediated eicosanoid production and is also regulated by intracellular signalling molecules such as phospholipase C, mitogen-activated protein kinase and phosphatidylinositol 3-kinase. cPLA₂ binds to membranes in a Ca²⁺-dependent fashion (Kramer & Sharp, 1997). The translocation of cPLA₂ places the enzyme in close proximity to its substrate phospholipid, and thus could be a prerequisite for the production of arachidonic acid. Ca²⁺ release was shown to be sufficient to induce translocation of cPLA₂ to the membrane (Hirabayashi et al. 1999). It is thus possible that bradykinin-induced Ca²⁺ release may cause a translocation of cPLA₂. However, our results suggest that additional mechanisms other than translocation are required for the activation of the enzyme. The activity of cPLA₂ is also Ca²⁺ dependent (Kramer & Sharp, 1997). Compartmentalization of the Ca²⁺ signal could be one of the additional mechanisms required for the activation of cPLA₂. Ca²⁺ influx but not Ca²⁺ release was shown to cause an elevation of [Ca²⁺]_i in the submembranous region (Kargacin et al. 1991), which can efficiently activate cPLA₂ localized at the membrane. Hirabayashi et al. (1999) reported that [Ca²⁺]_i elevation lasting longer than 5 min was required to induce a prolonged translocation of cPLA₂ and the release of arachidonic acid (Hirabayashi et al. 1999). The long lasting elevation of [Ca²⁺]_i could be another mechanism required for the activation of cPLA₂. However, our results showed that the production of PGH₂ and TXA₂ occurred within 1 min after the application of bradykinin in normal PSS (Fig. 1A). In addition, in the absence of extracellular Ca²⁺, the contraction was observed immediately after Ca²⁺ replenishment. These findings suggested that subcellular compartmentalization of the Ca²⁺ signal plays a critical role in the production of contracting factors. Such subcellular compartmentalization of the Ca²⁺ signal could be achieved by Ca²⁺ influx.

We previously reported that thapsigargin caused a large sustained [Ca²⁺]_i elevation due to a capacitative Ca²⁺ influx in endothelial cells, and thereby induced endothelium-dependent contraction by releasing mainly TXA₂ in the same interlobar renal arterial strips as those used in the present study (Ihara et al. 1999). Taken together with the findings of the present study, Ca²⁺ influx is thus suggested to be necessary and sufficient for the production and release of contracting factors in endothelial cells. Although [Ca²⁺]_i elevation in endothelial cells induced by thapsigargin was much larger than that induced by bradykinin, the force development of the arterial strips induced by thapsigargin was smaller than that obtained with bradykinin. This finding suggests that the activity of cPLA₂ is also regulated by factors other than Ca²⁺. The catalytic activity of cPLA₂ was reported to be increased by phosphorylation (Kramer & Sharp, 1996; Borsch-Haubold et al. 1998), especially phosphorylation of Ser-505 by mitogen-activated protein kinase (Lin et al. 1993). Bradykinin has been shown to activate the divergent intracellular signal transduction pathways in addition to Ca²⁺ signalling. It includes the activation of inositol 1,4,5-trisphosphate, tyrosine phosphorylation, phospholipase

C and mitogen-activated protein kinase (Naraba et al. 1998). It is thus possible that bradykinin caused a phosphorylation of cPLA₂, and thereby caused a greater activation of cPLA₂ and the production of the contracting factor for a given [Ca²⁺]_i elevation than thapsigargin.

The Ca²⁺ sensitivity of the contractile apparatus increased during bradykinin-induced contraction in comparison with that seen during the 118 mM K⁺-induced contraction. U46619, a stable agonist of the TXA₂/PGH₂ receptor (Liel et al. 1987), enhanced the Ca²⁺ sensitivity of the contractile apparatus in the human umbilical artery (Toyofuku et al. 1995), rabbit pulmonary artery (Himpens et al. 1990), porcine coronary artery (Kawasaki et al. 1998) and porcine renal artery (Ihara et al. 1999). The present study demonstrated for the first time that endogenous TXA₂/PGH₂ enhanced the Ca²⁺ sensitivity of the contractile apparatus. However, the time course of contractions differed between endogenously produced TXA₂/PGH₂ and exogenously applied U46619. The former caused a transient contraction while the latter caused a sustained contraction (Fig. 5E). Endogenously produced TXA₂/PGH₂ is thought to degrade rapidly in renal arterial strips with an intact endothelium.

In the present study, the endothelium-dependent contraction was initiated by adding bradykinin at the resting state in the presence of L-NAME. The concentration of L-NAME used in the present study was shown to completely inhibit the sustained phase of the relaxation induced by bradykinin (Ihara et al. 2000). It is conceivable that L-NAME eliminated the NO-mediated component of relaxation. However, the relaxation, especially the initial relaxation, was mediated by endothelium-derived hyperpolarizing factor (EDHF) (Ihara et al. 2000). The contribution of EDHF to the bradykinin-induced relaxation was shown to be only transient (Ihara et al. 2000). The initial slight decrease in [Ca²⁺]_i with no obvious decrease in force observed just after the addition of bradykinin (Fig. 1A) was thus considered to be caused by EDHF.

In conclusion, bradykinin, at 10^-8 M and higher, induced an endothelium-dependent contraction mediated by both PGH₂ and TXA₂ in the porcine interlobar renal artery. The [Ca²⁺]_i elevation due to Ca²⁺ influx from the extracellular space in endothelial cells was thus found to be both necessary and sufficient for bradykinin to induce a PGH₂/TXA₂-mediated endothelium-dependent contraction. However, the [Ca²⁺]_i elevation due to Ca²⁺ release from the intracellular stores was not a prerequisite for a PGH₂/TXA₂-mediated endothelium-dependent contraction.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

ADEAGBO A. S. & HENZEL, M. K. (1998). Calcium-dependent phospholipase A2 mediates the production of endothelium-derived hyperpolarizing factor in perfused rat mesenteric prearteriolar bed. Journal of Vascular Research 35, 27-35	[Medline]
AOKI H., KOBAYASHI, S., NISHIMURA, J. & KANAIDE, H. (1994). Sensitivity of G-protein involved in endothelin-1-induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ. British Journal of Pharmacology 111, 989-996	[Medline]
BALSINDE J. & DENNIS, E. A. (1997). Function and inhibition of intracellular calcium-independent phospholipase A2. Journal of Biological Chemistry 272, 16069-16072	[Full Text]
BORSCH-HAUBOLD A. G., BARTOLI, F., ASSELIN, J., DUDLER, T., KRAMER, R. M., APITZ-CASTRO, R., WATSON, S. P. & GELB, M. H. (1998). Identification of the phosphorylation sites of cytosolic phospholipase A2 in agonist-stimulated human platelets and HeLa cells. Journal of Biological Chemistry 273, 4449-4458	[Abstract/Full Text]
CHEN G. & SUZUKI, H. (1989). Some electrical properties of the endothelium-dependent hyperpolarization recorded from rat arterial smooth muscle cells. Journal of Physiology 410, 91-106	[Abstract]
FLEMING I., BAUERSACHS, J. & BUSSE, R. (1997). Calcium-dependent and calcium-independent activation of the endothelial NO synthase. Journal of Vascular Research 34, 165-174	[Medline]
FUKAO M., HATTORI, Y., KANNO, M., SAKUMA, I. & KITABATAKE, A. (1997). Sources of Ca2+ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery. British Journal of Pharmacology 120, 1328-1334	[Medline]
FURCHGOTT R. F. & VANHOUTTE, P. M. (1989). Endothelium-derived relaxing and contracting factors. FASEB Journal 3, 2007-2018	[Abstract]
HIMPENS B., KITAZAWA, T. & SOMLYO, A. P. (1990). Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflugers Archiv 417, 21-28	[Medline]
HIRABAYASHI T., KUME, K., HIROSE, K., YOKOMIZO, T., IINO, M., ITOH, H. & SHIMIZU, T. (1999). Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2. Journal of Biological Chemistry 274, 5163-5169	[Abstract/Full Text]
IGNARRO L. J., BUGA, G. M., WOOD, K. S., BYRNS, R. E. & CHAUDHURI, G. (1987). Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proceedings of the National Academy of Sciences of the USA 84, 9265-9269	[Medline]
IHARA E., HIRANO, K., DERKACH, D. N., NISHIMURA, J., NAWATA, H. & KANAIDE, H. (2000). The mechanism of bradykinin-induced endothelium-dependent contraction and relaxation in the porcine interlobar renal artery. British Journal of Pharmacology 129, 943-952	[Abstract/Full Text]
IHARA E., HIRANO, K., NISHIMURA, J., NAWATA, H. & KANAIDE, H. (1999). Thapsigargin-induced endothelium-dependent triphasic regulation of vascular tone in the porcine renal artery. British Journal of Pharmacology 128, 689-699	[Abstract/Full Text]
JONES K. T. & SHARPE, G. R. (1994). Ni2+ blocks the Ca2+ influx in human keratinocytes following a rise in extracellular Ca2+. Experimental Cell Research 212, 409-413	[Medline]
KANAIDE H. (1999). Measurement of [Ca2+]i in smooth muscle strips using front-surface fluorimetry. Methods in Molecular Biology, vol. 114, Calcium Signaling Protocols, pp. 269-277. Humana Press Inc., Totowa, NJ, USA	[Medline]
KARGACIN G. & FAY, F. S. (1991). Ca2+ movement in smooth muscle cells studied with one- and two-dimensional diffusion models. Biophysical Journal 60, 1088-1100	[Abstract]
KATO T., IWAMA, Y., OKUMURA, K., HASHIMOTO, H., ITO, T. & SATAKE, T. (1990). Prostaglandin H2 may be the endothelium-derived contracting factor released by acetylcholine in the aorta of the rat. Hypertension 15, 475-481	[Abstract]
KATUSIC Z. S. & SHEPHERD, J. T. (1991). Endothelium-derived vasoactive factors: II. Endothelium-dependent contraction. Hypertension 18, 86-92
KAWASAKI J., HIRANO, K., HIRANO, M., NISHIMURA, J., FUJISHIMA, M. & KANAIDE, H. (1999). Troglitazone inhibits the capacitative Ca2+ entry in endothelial cells. European Journal of Pharmacology 373, 111-120	[Medline]
KAWASAKI J., HIRANO, K., HIRANO, M., NISHIMURA, J., NAKATSUKA, A., FUJISHIMA, M. & KANAIDE, H. (2000). Dissociation between the Ca2+ signal and tube formation induced by vascular endothelial growth factor in bovine aortic endothelial cells. European Journal of Pharmacology 398, 19-29	[Medline]
KAWASAKI J., HIRANO, K., NISHIMURA, J., FUJISHIMA, M. & KANAIDE, H. (1998). Mechanisms of vasorelaxation induced by troglitazone, a novel antidiabetic drug, in the porcine coronary artery. Circulation 98, 2446-2452	[Abstract/Full Text]
KRAMER R. M., ROBERTS, E. F., UM, S. L., BORSCH-HAUBOLD, A. G., WATSON, S. P., FISHER, M. J. & JAKUBOWSKI, J. A. (1996). p38 mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets. Evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2. Journal of Biological Chemistry 271, 27723-22729	[Abstract/Full Text]
KRAMER R. M. & SHARP, J. D. (1997). Structure, function and regulation of Ca2+-sensitive cytosolic phospholipase A2 (cPLA2). FEBS Letters 410, 49-53	[Medline]
KUROIWA M., AOKI, H., KOBAYASHI, S., NISHIMURA, J. & KANAIDE, H. (1995). Mechanism of endothelium-dependent relaxation induced by substance P in the coronary artery of the pig. British Journal of Pharmacology 116, 2040-2047	[Medline]
LIEL N., MAIS, D. E. & HALUSHKA, P. V. (1987). Binding of a thromboxane A2/prostaglandin H2 agonist [3H]U46619 to washed human platelets. Prostaglandins 33, 789-797	[Medline]
LIN L. L., WARTMANN, M., LIN, A. Y., KNOPF, J. L., SETH, A. & DAVIS, R. J. (1993). cPLA2 is phosphorylated and activated by MAP kinase. Cell 72, 269-278	[Medline]
MIZUNO O., HIRANO, K., NISHIMURA, J., KUBO, C. & KANAIDE, H. (1998). Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery. European Journal of Pharmacology 351, 67-77	[Medline]
NARABA H., UENO, A., KOSUGI, Y., YOSHIMURA, M., MURAKAMI, M., KUDO, I. & OH-ISHI, S. (1998). Agonist stimulation of B1 and B2 kinin receptors causes activation of the MAP kinase signaling pathway, resulting in the translocation of AP-1 in HEK 293 cells. FEBS Letters 435, 96-100	[Medline]
RUSSELL F. D. & DAVENPORT, A. P. (1999). Secretory pathways in endothelin synthesis. British Journal of Pharmacology 126, 391-398	[Full Text]
SCHILLING W. P., CABELLO, O. A. & RAJAN, L. (1992). Depletion of the inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ store in vascular endothelial cells activates the agonist-sensitive Ca2+-influx pathway. Biochemical Journal 284, 521-530	[Medline]
SHEPHERD J. T. & KATUSIC, Z. S. (1991). Endothelium-derived vasoactive factors: I. Endothelium-dependent relaxation. Hypertension 18, 76-85
TISCHFIELD J. A. (1997). A reassessment of the low molecular weight phospholipase A2 gene family in mammals. Journal of Biological Chemistry 272, 17247-17250	[Full Text]
TOYOFUKU K., NISHIMURA, J., KOBAYASHI, S., NAKANO, H. & KANAIDE, H. (1995). Effects of U46619 on intracellular Ca2+ concentration and tension in human umbilical artery. American Journal of Obstetrics and Gynecology 172, 1414-1421	[Medline]
UCHIDA H., TANAKA, Y., ISHII, K. & NAKAYAMA, K. (1999). L-type Ca2+ channels are not involved in coronary endothelial Ca2+ influx mechanism responsible for endothelium-dependent relaxation. Research Communications in Molecular Pathology and Pharmacology 104, 127-144	[Medline]
YANAGISAWA M., KURIHARA, H., KIMURA, S., TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, Y., GOTO, K. & MASAKI, T. (1988). A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332, 411-415	[Medline]

Acknowledgements

We thank Mr Brian Quinn for comments and help with the manuscript. This study was supported in part by Grants-in-Aid for Scientific Research (no. 10557072, 11838013 and 11670687) and for Scientific Research on Priority Area (no. 12213103) from the Ministry of Education, Science, Sports and Culture, Japan, by the Research Grant for Cardiovascular Diseases (11C-1 and 12C-2) from the Ministry of Health and Welfare, Japan, and by the Kimura Memorial Heart Foundation Research Grant for 2000 and the grant from Kanzawa Medical Research Foundation.

Corresponding author

H. Kanaide: Division of Molecular Cardiology, Research Institute of Angiocardiology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.

Email: kanaide{at}molcar.med.kyushu-u.ac.jp

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