Angiotensin II, a unique vasoactive agent dissociates myosin light chain phosphorylation from contraction.

Angiotensin II (100 nM) induced bi-phasic increases in cytosolic Ca2+ level ([Ca2+]i) through the activation of angiotensin II type 1 receptor. Pharmacological examinations using 10 µM verapamil, 30 µM La3+, and 1 µM thapsigargin indicated that the first phase of the [Ca2+]i-increase was mediated by Ca2+ release from sarcoplasmic reticulum (SR) and Ca2+ influx independently of voltage dependent Ca2+ channel (VDC). In contrast, the second phase of [Ca2+]i-increase was mediated by Ca2+ influx through VDC. Although both [Ca2+]i and myosin light chain (MLC)-phosphorylation at the first phase was apparently exceeded the threshold for contraction as estimated by high K+-induced responses, there was no appreciable contraction, indicating the dissociation between MLC phosphorylation and force during this phase. In contrast, the second phase of [Ca2+]i was associated with the increases in both MLC phosphorylation and force. These results suggest that angiotensin II is a unique agonist which dissociates MLC-phosphorylation from muscle force during the Ca2+ releases from SR.

Angiotensin II is one of the most potent vasoconstrictors and vascular smooth muscle cell mitogens [5, 10]. Many studies on vascular biology and pharmacology revealed that angiotensin II plays a pivotal role in the physiology and pathology of the vessel wall [2, 8, 21, 22].

Signal transduction pathway of angiotensin II-stimulation has been well examined using vascular smooth muscle cells [11, 14]. Activation of vascular smooth muscle cells by angiotensin II, which mainly binds to angiotensin II type 1 (AT1) receptors, results in the phospholipase C-mediated generation of two second messenger, inositol triphosphate (IP3) to release Ca2+ from sarcoplasmic reticulum (SR) and diacylglycerol (DG) to activate protein kinase C (PKC). Angiotensin II also induces protein tyrosine kinase activation, which in turn activates phospholipase Cγ [19] and other protein kinases, and phosphorylates contractile proteins [25, 30].

In in vitro studies, angiotensin II has been shown to elicit relatively small contraction distinct from other vascular contractile agents since it induces only a transient and smaller amplitude of contraction during the continuous incubation irrespective of a very small EC50 values. Due to such smaller responses, not so large quantity of papers have been reported so far. Furthermore, among the papers, only a few reports have analyzed the relationship between the changes in cytosolic Ca2+ level ([Ca2+]i) and myosin light chain (MLC) phosphorylation in the angiotensin II-stimulated vascular smooth muscles [9, 23]. Thus, the purposes of this study was to correlate the muscle force with [Ca2+]i and MLC-phosphorylation in the angiotensin II-stimulated rat aorta. We found that phasic contraction induced by angiotensin II was composed of two phases and the first phase of [Ca2+]i transient, which is mediated by Ca2+ release form stored site, and the subsequent MLC phosphorylation was not associated with force generation.

MATERIALS AND METHODS

Preparations, solutions and measurement of muscle force and [Ca2+]i simultaneously

All animal procedures were performed in accordance with the guidelines of the University of Tokyo. Male Wistar rats (200–250 g) were euthanized by a sharp blow on the neck and exsanguination. Segments of the thoracic aorta were isolated and cut into helical strips (2 mm wide, 10 mm long) for measurement of [Ca2+]i. The isolated muscle rings or strips were placed in physiological salt solution (PSS) containing (in mM); NaCl, 136.9; KCl, 5.4; CaCl2, 1.5; MgCl2, 1.0; NaHCO3, 23.8 and glucose, 5.5. Ethylene diamine tetraacetic acid (EDTA, 1 µM) was also added to remove contaminating heavy metal ions, which catalyze oxidation of organic chemicals in PSS. The endothelium was removed by gently rubbing the intimal surface with the flat face of a pair of forceps moistened with PSS. The high concentration of KCl (72.7 mM) solution was prepared by replacing NaCl with equimolar KCl in PSS. All solutions had a pH of 7.4 when saturated with a 95% O2–5% CO2 mixture at 37°C. Muscle tension was recorded isometrically with a force-displacement transducer. Each muscle strips were attached to a holder in a special organ bath (2 ml) with resting tension of 10 mN, and equilibrated for 30 to 60 min to obtained a stable contractility induced by 72.7 mM KCl.

Simultaneous measurement of muscle force and [Ca2+]i was performed as reported by Sato et al. [24] with fluorescent Ca2+ indicator, fura-PE3, which was less dye-leakage than fura-2. Rat aortic strips were treated with PSS containing acetoxymethylester of fura-PE3 (fura-PE3/AM, 5 µM) and less cytotoxic detergent, cremophor EL (0.02%), for 4–5 hr at room temperature. After loading of fura-PE3/AM, the muscle strips were washed with PSS at 37°C for 20 min to remove uncleaved fura-PE3/AM and held horizontally in a temperature-controlled organ bath (7 ml). One end of muscle strips was connected to a force-displacement transducer to monitor muscle force. Experiments were performed with a bio-fluorimeter (CAF-110, Japan Spectroscopic, Tokyo, Japan), and the ratio of 500 nm fluorescence excited at 340 nm (F340) to that excited at 380 nm (F380) (F340/F380) was used as indicator of [Ca2+]i. The resting [Ca2+]i and the [Ca2+]i at 5 min after application of 72.7 mM KCl were taken as 0% and 100%, respectively.

MLC-phosphorylation

The amount of MLC-phosphorylation was measured according to the modified method described by Word et al. [31]. Strips of rat aorta were held isometrically under a resting tension force of 10 mN on stainless steel holders and quickly frozen in dry-ice acetone with 10% trichloroacetic acid (TCA). The strips were then incubated for several hours in the acetone/TCA solution until the temperature of the solution became around 0°C, and were then crushed in the liquid nitrogen. The crushed muscle powder was suspended in10% TCA and 10 mM dithiothreitol (DTT) (200–400 µl). The homogenate was centrifuged at 10,000 × g for 10 min and the pellet was washed with 500 µl diethyl ether several times to remove TCA. The pellet was then suspended in urea-glycerol buffer (25–75 µl) containing 20 mM Tris, 23 mM Glycine, 8 M urea, 10 mM DTT and 0.04% Bromophenol blue, and was centrifuged at 10,000 × g for 15 min. The supernatant was used as a sample.

The phosphorylated MLC was separated using Glycerol polyacrylamide gel electrophoresis (PAGE), the MLC or phosphorylated Ser19 MLC was measured by western blot using anti-MLC antibody (donated by Dr. JT Stull).

Chemicals

The following chemicals were used: angiotensin II, thapsigargin (Sigma-Aldrich, St. Louis, MO, U.S.A.), cremophor EL, DTT, TCA, EDTA (Nacalai Tesque, Kyoto, Japan), KRH594 (gently donated by KISSEI Pharmaceutical Co., Ltd., Nagano, Japan) and fura-PE3/AM (Texas fluorescence laboratories, Austin, TX, U.S.A.).

Stastics

The numerical data were expressed as mean ± standard error. Differences between mean values were evaluated by Student’s t-test and, where appropriate, analysis of variance (one-way ANOVA; Bonferroni’s test) was performed.

RESULTS

As shown in Fig. 1, angiotensin II (100 nM) induced bi-phasic increases in [Ca2+]i in rat aorta. The first phasic increase in [Ca2+]i was accompanied by a very slow-rise in muscle force. After the first phase of the increase in [Ca2+]i, the second phase of [Ca2+]i-increase with spontaneous oscillation was observed. The second phase was accompanied by a large and sustained increase in muscle force. After 6–15 min, spontaneous oscillatory increases in [Ca2+]i suddenly stopped and the sustained level of [Ca2+]i decreased toward the resting level of [Ca2+]i even in the continuous presence of angiotensin II. Angiotensin II (10 nM or 100 nM)-induced contractions were completely inhibited by selective AT1 receptor antagonists, 1 µM losartan or 10 nM KRH-594 (data not shown).

Fig. 1.

Effect of angiotensin II (Ang II, 100 nM) on [Ca2+]i (upper trace) and muscle force (lower trace) in rat aorta. The resting [Ca2+]i and the [Ca2+]i at 5 min after application of 72.7 mM KCl were taken as 0% and 100%, respectively. Typical traces are shown from 5−7 experiments.

We then examined the relationship between [Ca2+]i, MLC-phosphorylation and muscle force in the rat aorta stimulated with 72.7 mM KCl or 100 nM angiotensin II. As shown in Fig. 2A, in the muscles stimulated by 72.7 mM KCl for 5 sec, MLC-phosphorylation and muscle force was increased in response to the increase in [Ca2+]i to 130.59 ± 9.82%. At 15 sec, MLC-phosphorylation further increased irrespective of decrease in [Ca2+]i to 109.47 ± 2.71% with a further increase in muscle force.

Fig. 2.

Relationship between [Ca2+]i (closed column), MLC-phosphorylation (hatched column) and muscle force (open column) in aorta stimulated with 72.7 mM KCl (A) and 100 nM angiotensin II (Ang II, B). Values were obtained at 5 and 15 sec after stimulation with 72.7 mM KCl or at 30 sec and 2 min after the stimulation with 100 nM angiotensin II. The resting [Ca2+]i and the [Ca2+]i at 5 min after application of 72.7 mM KCl were taken as 0% and 100%, respectively. Muscle force was normalized against the contraction at 5 min after application of 72.7 mM KCl. MLC-phosphorylation (MLC-P) was shown by % of phosphorylated MLC against total MLC. Each column represents means ± S.E.M of 4−6 experiments. **: Significantly different from resting state (P<0.01).

In the rat aorta stimulated with angiotensin II (100 nM), [Ca2+]i at the first and the second phases was almost identical (82.3 ± 6.8% at 30 sec and 80.1 ± 3.9% at 2 min) which was significantly less than high K+-induced increase in [Ca2+]i at 5 and 15 sec. In response to these [Ca2+]i increases, MLC-phosphorylation levels at 30 sec and 2 min increased to 71.2 ± 2.5% and 71.7 ± 4.1%, respectively, whose values were significantly greater than those in high K+ stimulated muscle. Unexpectedly, the level of contraction was very small 30 sec after the addition of angiotensin II (Fig. 2B).

Next, we attempted to characterize the Ca2+ source due to angiotensin II (100 nM)-stimulation. Figure 3 shows the effects of verapamil, a voltage dependent Ca2+ channel blocker, La3+, an inorganic nonselective Ca2+ channel blocker, thapsigargin, an inhibitor of capacitative Ca2+ entry and the combination of these agents on the [Ca2+]i and contraction. In the presence of 10 µM verapamil, the second phase of [Ca2+]i was disappeared. 30 µM La3+ in the presence of verapamil also eliminated the second phase of [Ca2+]i (Fig. 3C). Furthermore, the combination treatment with La3+, verapamil and 1 µM thapsigargin abolished either the first and the second phase of [Ca2+]i. As for the contractile responses, even in the absence of the transient increase in [Ca2+]i under the condition of the presence of verapamil, La3+ and thapsigargin, angiotensin II induced first slowly developed contraction followed by a small sustained contraction, suggesting that at least a sustained contraction is mediated at least by Ca2+-independent mechanism.

Fig. 3.

[Ca2+]i (upper trace) and muscle force (lower trace) increases in rat aorta induced by 100 nM angiotensin II (Ang II) in the absence (A) or presence of 10 µM verapamil (B), 10 µM verapamil + 30 µM La3+ (C), or 10 µM verapamil + 30 µM La3+ + 1 µM thapsigargin (D). The resting [Ca2+]i and the [Ca2+]i at 5 min after application of 72.7 mM KCl were taken as 0% and 100%, respectively. Typical traces are shown from 3−4 experiments.

We further measured the MLC-phosphorylation level of the rat aorta stimulated with 100 nM angiotensin II in the presence or absence of Ca2+ release from SR and correlated of [Ca2+]i with force. As shown in Fig. 4, in the presence of verapamil plus La3+, angiotensin II induced phasic increase in [Ca2+]i reaching its peak (66.0 ± 5.1%) after about 30 sec. This [Ca2+]i-increase was associated with an increase in MLC phosphorylation (41.3 ± 2.8%). However, only a very small contraction was observed in response to the increase in MLC phosphorylation. Even in the absence of Ca2+ release under the condition in which tissues were treated with verapamil, La3+ and thapsigargin, angiotensin II increased MLC phosphorylation at 30 sec and 2 min. As was shown in the presence of verapamil and La3+, angiotensin II again induced only a very small contraction with a significant increase in MLC phosphorylation.

Fig. 4.

Relationship between [Ca2+]i (closed column), MLC-phosphorylation (hatched column) and muscle force (open column) in aorta stimulated with 100 nM angiotensin II (Ang II). Values were obtained at 30 sec and 2 min after the stimulation with angiotensin II in the absence 10 µM verapamil + 30 µM La3+ or 10 µM verapamil + 30 µM La3+ + 1 µM thapsigargin. The resting [Ca2+]i and the [Ca2+]i at 5 min after application of 72.7 mM KCl were taken as 0% and 100%, respectively. Muscle force was normalized against the contraction at 5 min after application of 72.7 mM KCl. MLC-phosphorylation (MLC-P) was shown by % of phosphorylated MLC against total MLC. Each column represents means ± S.E.M. of 4−6 experiments. **: Significantly different from resting state (P<0.01).

DISCUSSION

In the rat aorta, angiotensin II (100 nM) induced bi-phasic increases in [Ca2+]i. The first component consisted of a transient increase in [Ca2+]i. The second component consisted of a relatively sustained increase in [Ca2+]i on which spike-like, oscillatory increases in [Ca2+]i were superimposed. Several min after the start of the second phase, [Ca2+]i spontaneously returned to the resting level even in the presence of angiotensin II. Since the AT-receptors are well known to be rapidly down-regulated [1, 20], cessation of [Ca2+]i increase may be due to the down-regulation of AT-receptors. We also found that the angiotensin II-induced contraction was completely inhibited by the AT1 receptor antagonists, 1 µM losartan [7] or 10 nM KRH-594 [28], supporting the results by Schutzer et al. [27] in rat aortic tissue and by Castoldi et al. [6] in cultured rat aortic smooth muscle cells.

We next characterize the mechanisms of angiotensin II-induced increases in [Ca2+]i, Addition of an inhibitor of voltage dependent Ca2+ channel, verapamil, completely abolished the second phase of [Ca2+]i-increase without affecting the maximum amplitude of the first phase of [Ca2+]i increase, indicating that the second phase of [Ca2+]i increase by angiotensin II is mediated through voltage dependent Ca2+ channels. Since the combination treatment with verapamil and La3+, an inorganic Ca2+ channel blocker, decreased the duration of the first phase [Ca2+]i-increase without affecting the maximum amplitude, Ca2+ influx pathway other than voltage-dependent Ca2+ channels, such as capacitative Ca2+ entry [32], may partly be involved in the first phase of the [Ca2+]i-increase (Fig. 3B and 3C and our unpublished observation). Data also indicated that treatment with verapamil, La3+ and thapsigargin, a Ca2+ pump inhibitor, abolished the first phase of the [Ca2+]i-increase due to 100 nM angiotensin II in the absence of external Ca2+ (Fig. 3D). This result suggests that the first phase of [Ca2+]i increase is mediated by Ca2+ release from SR. Consisted with these observations, it has been reported that angiotensin II increases IP3 formation following activation of phospholipase Cβ in cultured vascular smooth muscle cells [12, 26].

The relationship between MLC-phosphorylation and muscle force revealed that the level of the increases in [Ca2+]i and MLC phosphorylation at 30 sec after the stimulation with angiotensin II (100 nM) was not reflected in the muscle force as shown in Fig. 2B. The same results were obtained in the conditions in which Ca2+ influx had been inhibited by the presence of verapamil and La3+ or the presence of verapamil, La3+ and thapsigargin (Fig. 4B). In contrast, in the muscle stimulated with 72.7 mM KCl, increased MLC-phosphorylation at 5 and 15 sec, whose values are smaller than those in the angiotensin II-induced responses at 30 sec, was associated with contraction. These contradictory results obtained in angiotensin II- and high K+-induced responses can not be explained by the possibility that the dissociation between MLC-phosphorylation and muscle force is due to the delay of force-transmission by elastic component, since the angiotensin II-induced responses were much slower than the high K+-induced responses.

Brizzolara-Gourdie and Webb [4] reported that angiotensin II stimulates cAMP formation to induce vasodilation in rat aorta. However, this is not the case with rat aorta, since enzyme-immunoassay showed that angiotensin II (100 nM) did not increase cAMP content (data not shown). Furthermore, angiotensin II did not inhibit 72.7 mM KCl- or 1 µM noradrenaline-induced contraction. These results suggest that MLC-phosphorylation 30 sec after stimulation with angiotensin II dissociates with muscle force by a cAMP-independent mechanisms. Tansey et al. [29] reported that phosphatase inhibitor, okadaic acid, inhibited carbachol-induced contractions without decreasing MLC phosphorylation in bovine trachea. Moreover, phorbol esters increased MLC phosphorylation and actin remodeling in bovine lung endothelium without increased contraction [3]. These results suggest the possibility that, like okadaic acid, angiotensin II may phosphorylates indirectly or directly some unknown regulatory proteins to dissociate MLC-phosphorylation and contraction in vascular smooth muscle. Further experiments are required to clarify this point.

The levels of phosphorylated MLC at 30 sec or 2 min after stimulation with angiotensin II (100 nM) were significantly higher than those of the response to 72.7 mM KCl at 5 and 15 sec. However, [Ca2+]i at 30 sec or 2 min after addition of angiotensin II was significantly lower than those of 72.7 mM KCl. In addition, [Ca2+]i-independent and MLC-phosphorylation dependent contraction was induced by angiotensin II in the presence of verapamil, La3+ and thapsigargin (Fig. 4). These results suggest that angiotensin II sensitizes the MLC-phosphorylation step at a given [Ca2+]i. This Ca2+ sensitization might be mediated by inhibition of protein phosphatase through rhoA/ROCK pathway [16] and/or through PKC/PCI-17 pathway [18].

We have previously shown the dissociation between the increase in [Ca2+]i due to Ca2+ release from SR and muscle force in the rat aorta stimulated with P2 purinergic receptors [17]. In this report, we have demonstrated that ATP-induced increase in [Ca2+]i was not associated with the increment of MLC phosphorylation, indicating the presence of localization of [Ca2+]i that is not able to couple with contractile proteins [15]. In contrast, verapamil inhibited noradrenaline-induced contraction with decreases of MLC-P in rat aorta [13], indicating that there is no dissociation between contraction and MLC-P. At present, we have no explanation for the marked difference between purinoceptor and α-adrenaline agonists and angiotensin II regarding the correlation between MLC phosphorylation and contraction, and it is also necessary to clarify this point in a future study.

In conclusion, angiotensin II induces contraction by an influx of Ca2+ through VDC but not Ca2+ release from SR. Angiotensin II is a unique receptor-agonist to dissociate MLC-phosphorylation from muscle force during the [Ca2+]i-increase due to Ca2+ releases from SR.