Mechanisms of the thapsigargin-induced Ca(2+) entry in in situ endothelial cells of the porcine aortic valve and the endothelium-dependent relaxation in the porcine coronary artery

Abstract

The mechanisms of the thapsigargin (TG)-induced capacitative Ca(2+) entry in in situ endothelial cells and its role in the regulation of arterial tone were investigated using front-surface fluorimetry and fura-2-loaded strips of porcine aortic valve and coronary artery. In the presence of extracellular Ca(2+), TG induced an initial rapid and a subsequent sustained elevation of cytosolic Ca(2+) concentration ([Ca(2+)](i)) in valvular strips. In the absence of extracellular Ca(2+), TG induced only a transient increase in [Ca(2+)](i). The TG-induced sustained elevation of [Ca(2+)](i) in endothelial cells was inhibited completely by 1 mM Ni(2+) and partly by 10 microM econazole and 30 microM ML-9, but not by 900 ng ml(-1) pertussis toxin or 100 microM wortmannin. Therefore, cytochrome P450 and protein phosphorylation are suggested to be involved in the TG-induced Ca(2+) influx in in situ endothelial cells. TG induced an endothelium-dependent large relaxation consisting of an initial and a late sustained relaxation in coronary arterial strip precontracted with U46619 (a thromboxane A2 analogue). Indomethacin alone had no effect, while indomethacin plus N(omega)-nitro-L-arginine (L-NOARG) markedly inhibited the sustained phase and slightly inhibited the initial phase of the TG-induced relaxation. TG induced a smaller but sustained relaxation during the 40 mM K(+)-induced precontraction than that seen during the U46619-induced precontraction. This relaxation was completely abolished by the pretreatment with indomethacin plus L-NOARG. In conclusion, both nitric oxide (NO) and endothelium-derived hyperpolarizing factor were suggested to mediate the TG-induced relaxation, while NO plays a major role in the sustained relaxation. The TG-induced sustained [Ca(2+)](i) elevation in endothelial cells was thus suggested to be mainly linked to the sustained production of NO.

Figure 1

Effect of thapsigargin on [Ca²⁺]_i in in situ endothelial cells of the porcine aortic valve. (a,b) Representative recordings of changes in [Ca²⁺]_i induced by 10 μM ATP and 1 μM thapsigargin (TG) in endothelial cells in the presence (a) and absence (b) of extracellular Ca²⁺. In (a), 1 mM NiCl₂ was applied during a sustained phase of the TG-induced elevation of [Ca²⁺]_i. In (b), the valvular strip was exposed to the 2 mM EGTA-containing Ca²⁺-free media for 10 min before and during the application of 1 μM TG. The level of [Ca²⁺]_i elevation induced by 10 μM ATP was assigned to be 100%. (c) Concentration-dependent effect of TG on [Ca²⁺]_i in endothelial cells in situ. TG was applied in a cumulative manner. Data are the mean±s.e.mean (n=5).

Figure 2

Characterization of the thapsigargin-induced Ca²⁺ entry in in situ endothelial cells of the porcine aortic valve. (a) Effect of econazole on the thapsigargin (TG)-induced sustained [Ca²⁺]_i increase, as evaluated by the [Ca²⁺]_i level obtained at 30 min after the application of 1 μM TG. Econazole (10 μM) was applied 5 min before the application of TG (pre) or 15 min after the application of TG (post). (b) Effects of pretreatment with myosin light chain kinase inhibitors, ML-9 and wortmannin on the [Ca²⁺]_i level obtained at 30 min after the application of TG. Inhibitors were applied 10 min before and during the stimulation with 1 μM TG. (c) Effect of pertussis toxin (IAP) on the TG-induced sustained increase in [Ca²⁺]_i. The valvular strips were treated with 900 ng ml⁻¹ IAP for 3 h. All data are the mean±s.e.mean (n=5). The level of the control [Ca²⁺]_i elevation induced by 10 μM ATP was considered to be 100%. ^*P<0.05, ^**P<0.01 compared with the control values. N.S., not significantly different.

Figure 3

Effect of thapsigargin on smooth muscle [Ca²⁺]_i and tension in the porcine coronary arterial strips with and without endothelium. (a) A representative recording of the changes in [Ca²⁺]_i (upper trace) and tension (lower trace) induced by 1 μM thapsigargin (TG) under resting conditions in the strips without endothelium. (b, c) Representative recordings of changes in [Ca²⁺]_i and tension induced by 1 μM TG during contraction induced by 100 nM U46619 in coronary arterial strips without (b) and with (c) endothelium. TG was applied 10 min after the initiation of the precontraction. The responsiveness to 118 mM K⁺-depolarization was recorded at the beginning of each measurement. The levels of [Ca²⁺]_i and tension seen in normal (5.9 mM K⁺) PSS and 118 mM K⁺-PSS were designated to be 0 and 100%, respectively.

Figure 4

Effect of indomethacin, N^ω-nitro-L-arginine (L-NOARG) and high K⁺-depolarization on the thapsigargin-induced endothelium-dependent relaxation. (a) A representative recording of the changes in [Ca²⁺]_i (upper trace) and tension (lower trace) of smooth muscle induced by 1 μM thapsigargin (TG) during contraction induced by 100 nM U46619 in the strips with endothelium that were pretreated with 10 μM indomethacin and 100 μM L-NOARG. TG was applied 10 min after the initiation of the precontraction. (b) A representative recording of changes in [Ca²⁺]_i and tension of smooth muscle induced by 1 μM TG during the 40 mM K⁺-induced contraction in the strip with endothelium. (c) A representative recording of changes in [Ca²⁺]_i and tension of smooth muscle induced by 1 μM TG during the 40 mM K⁺-induced contraction in the presence of 10 μM indomethacin and 100 μM L-NOARG in strips with endothelium. The responsiveness to 118 mM K⁺-depolarization was recorded at the beginning of each measurement.

Figure 5

Summary of the effect of thapsigargin on [Ca²⁺]_i and tension during the contraction induced by U46619 or 40 mM K⁺, either in the presence or absence of indomethacin and L-NOARG. The levels of [Ca²⁺]_i (upper panel) and force (lower panel) were obtained at peak relaxation and at 25 min (sustained phase of relaxation) after the initiation of precontraction by U46619 and 40 mM K⁺, either in the presence or absence of 10 μM indomethacin+100 μM L-NOARG. During the control precontraction obtained in the absence of indomethacin and L-NOARG (thapsigargin (−), indomethacin+L-NOARG (−)), the level of [Ca²⁺]_i and force sustained for more than 25 min. For the precontraction obtained in the presence of indomethacin and L-NOARG (thapsigargin (−), indomethacin+L-NOARG (+)), therefore, only the data obtained at 10 min after the initiation of precontraction are shown. The levels of [Ca²⁺]_i and tension seen in normal (5.9 mM K⁺) PSS and 118 mM K⁺-PSS were considered to be 0 and 100 %, respectively. When the effects of indomethacin and L-NOARG were evaluated, the 0 and 100% levels were determined in their presence. The data are the mean±s.e.mean (n=3–4). ^*P<0.05, ^**P<0.01 compared with the value obtained at the corresponding time point without thapsigargin.