INTRODUCTION

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THE ENDOTHELIUM plays an important role in the regulation of vascular tone via synthesizing various vasoactive substances, such as nitric oxide (NO), prostacyclin, and endothelin. Exercise protocols, either chronic or acute, have profound effects on the release of these vasoactive substances or on the endothelium-dependent control of vascular tone (3-8, 10, 20, 21). For example, acute exercise augments vasodilating responses to ACh and decreases vasoconstrictive responses to phenylephrine (PE), possibly by increasing NO release (8, 21). Like many receptor-mediated agonists, ACh affects cellular function via generating intracellular calcium concentration ([Ca²⁺]_i) signals (2, 18, 24). Besides, endothelial NO synthase (eNOS) is known to be a calcium-dependent enzyme (15, 19). Therefore, the ACh-evoked endothelial calcium signaling is likely to be involved in the exercise-induced vascular adaptation.

To explore the cellular mechanism of exercise effects, it is desirable to monitor the [Ca²⁺]_i level in the intact vascular endothelium because cultured endothelial cells (EC) may lose their in vivo properties and become adapted to the environment in vitro. It has been reported that ACh receptors are no longer expressed in culture (23). Perhaps due to technical difficulties, [Ca²⁺]_i measurements in the endothelium of dissected vessels using either patch-clamp labeling of a single EC (2) or spectrofluorimeter to monitor a group of ECs as a whole (24) have not been very popular. Recently, we developed an in situ EC [Ca²⁺]_i imaging method that allows simultaneous visualization of large numbers of ECs with single cell resolution (13). Thus ACh-induced aortic EC [Ca²⁺]_i elevation between control and exercised rats could be compared at the tissue/cellular level.

With the use of this newly developed method, we found that chronic exercise enhances the ACh-evoked EC [Ca²⁺]_i response (9). However, whether a single bout of strenuous exercise has a similar effect is still unclear. Besides, whether the enhancement of ACh-induced vasorelaxation by acute exercise is coupled to the change in the EC [Ca²⁺]_i elevation is unknown. We therefore examined the effect of acute exercise on ACh-induced EC [Ca²⁺]_i elevation and vasorelaxation simultaneously in the present study. Possible role of calcium influx in the exercise effects was evaluated by the removal of extracellular calcium or by the addition of Gd³⁺, an inward Ca²⁺ current inhibitor. To investigate the role of NO in exercise-enhanced vasorelaxation and EC [Ca²⁺]_i responses to ACh, some experiments were performed using specimens pretreated with N-nitro-L-arginine (L-NNA), a NOS inhibitor.

	MATERIALS AND METHODS

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Animals and the acute exercise protocol. This study was conducted in conformity with the policies and procedures detailed in the Guide for the Care and Use of Laboratory Animals. Eight- to ten-week-old male Wistar rats were randomly assigned to either the control or exercise group after a 1-wk familiarization period. The rats were housed in groups of three or four per cage in an environmentally controlled room (temperature, 25 ± 1°C; 12:12-h light-dark cycle). They were fed standard rat chow and water ad libitum. The exercise rats ran on a treadmill as previously described (8). Briefly, they ran on a motor-driven treadmill (model T408E, Diagnostic and Research Instruments; Taoyuan, Taiwan) with a speed of 0.25 m/s at the beginning. The running speed was progressively increased by 0.05 m/s increments every 3 min until the animals were exhausted (usually to the speed of 0.65 m/s). The sedentary control groups were placed in the treadmill without running for 10 min before euthanization.

Vessel preparation and fura 2 loading. Immediately after exercise, the animals were anesthetized with ether inhalation and decapitated. The thoracic aorta was excised and cut into rings (5 mm long), which were stored in an organ chamber containing Krebs-Ringer solution bubbled with 95% O₂-5% CO₂ (22°C, pH 7.4). This solution had the following composition (in mM): 118.0 NaCl, 4.8 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 24 NaHCO₃, 0.03 Na₂-EDTA, and 11.0 glucose. Aortic rings were fluorescently labeled by incubation with 10 µM fura 2-AM and 0.025% Pluronic F-127 in Krebs-Ringer solution for 1 h (24). Extracellular fura 2-AM was washed away afterward.

Measurement of EC [Ca²+]_i in the dissected thoracic aorta. The basic setup for EC [Ca²⁺]_i imaging was similar to that described in our previous reports (9, 13). Fura 2-loaded vessel rings were opened longitudinally and pinned to the base plate of a tissue flow chamber. The chamber was mounted on an inverted microscope with epifluorescence attachments (Diaphot 300, Nikon; Tokyo, Japan). The 510-nm fluorescence images excited at 340 or 380 nm were recorded by a high-sensitivity SIT camera (model C2400-08, Hamamatsu; Hamamatsu, Japan). Axon image workbench software (Axon Instruments; Foster City, CA) was used to acquire, digitize, and store the experimental results for off-line processing. The average value of EC [Ca²⁺]_i was calculated by monitoring a large area in the mainstream region of an opened aortic segment covering ~0.15 mm² of tissue surface, or >200 cells. At the end of each experiment, the calcium concentration was calibrated by the established methods (11-13). Briefly, the calcium concentration was calibrated by applying ionomycin (5 µM) in the presence of 5 mM EGTA, followed by 10 mM CaCl₂. All signals were corrected for autofluorescence, which was determined by exposing the tissue to 5 mM manganese to quench cytosolic fura 2 at a 360-nm excitation wavelength. Endothelial [Ca²⁺]_i was estimated after subtracting the background autofluorescence using the following equation: [Ca²⁺]_i = K_d[(R  R_min)/(R_max  R)]B, where K_d is the dissociation constant (~224 nM), R is the ratio of 340 over 380 nm during measurements, R_max is the ratio of 340 to 380 nm in the presence of saturating calcium levels, R_min is the ratio in calcium-free solution, and B is the ratio of the fluorescence at 380 nm with calcium-free solution to that of saturated CaCl₂ solution. All experiments were conducted at room temperature.

Simultaneous measurements of ACh-induced EC [Ca²+]_i responses and vasorelaxation in PE-precontracted aortic segments. To allow simultaneous measurement of vascular EC [Ca²⁺]_i and vascular smooth muscle contraction, the vessel mounting procedure for EC [Ca²⁺]_i measurement was modified (12). One side of the longitudinally opened vessel segment was fixed in the direction of blood flow with insect pins. The corners on the opposite side were passively stretched and pinned onto the base plate. This arrangement allows free movement of the central portion of the specimen when vasoactive chemicals were added. After the vessel was mounted, the tissue flow chamber was placed on the microscope stage and perfused with Krebs-Ringer buffer at 30°C under a constant flow rate of 0.7 ml/min for an equilibration period of 1 h. The dose responses of ACh-induced EC [Ca²⁺]_i elevation and vascular displacement were determined in the PE (10⁷ M)-precontracted vessel segment by subsequent exposure to cumulative ACh (10⁸-10⁵ M). The relative movement of endothelial cells was used as an index of vascular tone, whereas fluorescence ratio images from fura 2-labeled endothelial cells provided quantitative information about EC [Ca²⁺]_i.

4

Role of calcium influx in exercise-enhanced EC [Ca²+]_i response to ACh. To further verify the possible role of calcium influx in acute exercise-enhanced EC [Ca²⁺]_i responses to ACh, we applied Ca²⁺-free buffer or Gd³⁺ in our tissue flow chamber system. However, these treatments affected PE-evoked smooth muscle contraction; we therefore performed this part of the experiments in noncontracted vessel segments. To assure that the exercise effect on ACh-evoked calcium responses also occurred in noncontracted vessel segments, dose responses of ACh-induced EC [Ca²⁺]_i elevation were determined in the absence of PE. Our pilot study indeed showed that acute exercise increased ACh-evoked calcium responses in noncontracted vessel segments as well (data not shown). Therefore, we executed the following experiments in noncontracted vessel specimens.

6

EC [Ca²+]_i responses and vasorelaxation to ATP or sodium nitroprusside in PE-precontracted aortic segments. To study whether the enhancement of endothelium-dependent vasorelaxation and calcium response after acute exercise is specific to ACh, we also measured vascular responses evoked by ATP (10⁵ M), another endothelium-dependent vasodilator. Furthermore, we also monitored vasorelaxing responses to sodium nitroprusside (SNP; 10⁸ M), a NO donor, to examine the possibility of exercise-altered reactivity in vascular smooth muscles.

Reagents. All chemicals for the preparation of Krebs-Ringer solution were purchased from Merck (Darstadt, Germany). Other reagents were obtained from Sigma (St. Louis, MO) except for Pluronic F-127, which was purchased from Molecular Probes (Eugene, OR).

Statistical analysis. Results are expressed as means ± SE; n = sample sizes. Dose responses of ACh-induced EC [Ca²⁺]_i elevation or vasorelaxation were analyzed by ANOVA with a repeated measures design. Differences of other variables between control and exercise groups were compared by using unpaired Student's t-test, with P < 0.05 considered statistically significant.

	RESULTS

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Comparison of ACh-induced calcium responses and vasorelaxation in PE-precontracted aortic segments between control and exercise groups. In response to PE (10⁷ M), vasoconstriction reached a steady state within a few minutes without disturbing the EC [Ca²⁺]_i level (Fig. 1). The PE-evoked vascular displacement was larger in the control than in the exercise group (54 ± 6 vs. 35 ± 4 pixels, respectively, n = 5, P < 0.05). These results are consistent with a previous study (21) that examined the effects of acute exercise on PE-induced vasoconstriction in vivo. The basal endothelial [Ca²⁺]_i levels before drug administration were assessed. There was no significant difference in the basal levels of endothelial [Ca²⁺]_i between the two groups (97 ± 24 and 94 ± 9 nM for the control and exercise groups, respectively, n = 5).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   ACh-induced endothelial cell (EC) intracellular Ca2+ concentration ([Ca2+]i) elevation and vasorelaxation in a control sample. Both EC [Ca2+]i elevation (A) and vascular displacement (B) in a rat aortic en face preparation were monitored simultaneously. Whereas phenylephrine (PE; 107 M) induced only vasoconstriction, ACh induced both EC [Ca2+]i elevation and vasorelaxation in a dose-dependent manner.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Comparison of dose-response relations of ACh-induced EC [Ca2+]i elevation (A) and vasorelaxation (B) between acute exercise and control groups. Acute exercise significantly enhanced the ACh-induced EC [Ca2+]i response and vasorelaxation (n = 5, *P < 0.05). N-nitro-L-arginine (L-NNA) pretreatment significantly inhibited the ACh-induced EC [Ca2+]i response and vasorelaxation in the exercise group (n = 5, #P < 0.05). In comparison, L-NNA pretreatment significantly inhibited the ACh-induced vasorelaxation (n = 5, +P < 0.05) without altering the ACh-induced EC [Ca2+]i response in the control group. Results were analyzed by repeated-measures ANOVA.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Correlations between ACh-evoked EC [Ca2+]i elevation and vasorelaxation. Data from Fig. 2 were plotted by logarithmic curve fitting. There was no difference in the curve shapes between control and exercise groups, although higher values of EC [Ca2+]i and vasorelaxation could be reached in the exercise group.

Role of calcium influx in exercise-enhanced calcium response to ACh. To examine the role of EC calcium influx in the exercise effects, specimens were exposed to Ca²⁺-free buffer in the absence of PE. Figure 4 demonstrates that Ca²⁺-free buffer replacement diminished ACh-evoked EC [Ca²⁺]_i elevation, which could be reversed by subsequent exposure to the original Ca²⁺-containing buffer. Moreover, the exercise effects on ACh-induced EC [Ca²⁺]_i elevation was abolished when extracellular Ca²⁺ was removed (Fig. 5A). Administration of Gd³⁺, a blocker of inward Ca²⁺ current, partially inhibited ACh-induced EC [Ca²⁺]_i elevation in the exercise group but not in the control group (Fig. 5B). Gd³⁺ treatment also abolished the exercise effects on ACh-induced EC [Ca²⁺]_i elevation. The percentage of inhibition in ACh (10⁶ M)-evoked EC [Ca²⁺]_i elevation after various treatments to inhibit Ca²⁺ influx are summarized in Table 1. The inhibitory effects on EC [Ca²⁺]_i elevation from either Ca²⁺-free buffer replacement or Gd³⁺ administration were more pronounced in the exercise group than in the control group.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   A tracing obtained from an exercise sample demonstrating that replacement of Ca2+-free buffer partially inhibits the ACh (106 M)-evoked EC [Ca2+]i response, which could be reversed by normal buffer. It was also noted that, whereas the exposure of Ca2+-free buffer caused a minor decrease of basal EC [Ca2+]i level, the replenishment of extracellular Ca2+ induced a transient EC [Ca2+]i elevation.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Effects of Ca2+-free buffer (A) or Gd3+ pretreatment (B) on ACh (106 M)-evoked EC [Ca2+]i elevation (n = 5 and 4 for A and B, respectively). Before the treatments (left), acute exercise (solid bars) drastically increased ACh-induced calcium responses compared with controls (open bars) (**P < 0.01). After the treatments (right), either Ca2+-free buffer or Gd3+ (200 µM), the difference between control and exercise groups was abolished.

Vascular responses to ATP or SNP. There were significant differences between control and exercise groups in either ATP (10⁵ M)-evoked vasorelaxation or EC [Ca²⁺]_i responses (vasorelaxation: 90 ± 4% for the control and 113 ± 7% for the exercise group; EC [Ca²⁺]_i elevation: 150 ± 13 nM for the control and 393 ± 71 nM for the exercise group, respectively, n = 5, P < 0.05). In contrast, no significant differences were found between the two studied groups in SNP-induced vasorelaxation (72 ± 4% and 72 ± 4%, respectively). These results support the notion that the enhancement of vascular responses by acute exercise is possibly caused by a change in the vascular sensitivity to endothelium-dependent receptor-mediated vasodilators and not by an alteration in the reactivity of vascular smooth muscle cells.

	DISCUSSION

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We show here for the first time that acute exercise affects EC [Ca²⁺]_i signaling and vasorelaxation simultaneously in freshly dissected rat aortas. With the use of a modified vascular EC [Ca²⁺]_i imaging method (12), we found that ACh-induced vasorelaxation was well associated with endothelial [Ca²⁺]_i elevation in either the control or exercised groups. In contrast, PE did not cause EC [Ca²⁺]_i elevation. Whereas acute exercise increased both ACh-induced EC [Ca²⁺]_i and vasorelaxation responses in PE-precontracted vessel segments, it did not change the relationship between these two parameters. Moreover, these exercise effects could be abolished by L-NNA pretreatment. Finally, acute exercise enhanced ACh-induced EC [Ca²⁺]_i elevation mainly due to the augmented calcium influx.

It is interesting to note that L-NNA pretreatment significantly inhibited only the ACh-evoked high levels of EC [Ca²⁺]_i signaling in vessel segments of the exercise group but not the ACh-evoked EC [Ca²⁺]_i responses under other conditions (Fig. 2A). It did not affect the basal EC [Ca²⁺]_i either. In addition, we also tested the effects of SNP, an exogenous NO donor, on the EC [Ca²⁺]_i level. SNP was not effective from 10⁷-10⁴ M. However, a very high dose of SNP (10² M) did evoke EC [Ca²⁺]_i elevation (data not shown). On the basis of these observations, it is possible that only large amounts of NO could elevate the EC [Ca²⁺]_i level. Therefore, this positive feedback, or feedforward, mechanism may only operate under high NO conditions, such as the agonist-evoked NO-mediated vasodilatation and EC [Ca²⁺]_i elevation after exercise.

It is most likely that the change of ACh-induced vasorelaxation by acute exercise (8) is coupled to an alteration of EC [Ca²⁺]_i signaling. In this study, we found that acute exercise increased both the ACh-evoked EC [Ca²⁺]_i response and ACh-induced vasorelaxation in PE-precontracted vessel segments (Fig. 2). Moreover, when the ACh-evoked increase of EC [Ca²⁺]_i was plotted against the extent of corresponding vascular relaxation, the logarithmically fitted curves from two experimental groups were almost identical (Fig. 3). It was noted that, whereas the half-maximal relaxation induced by agonist application was accompanied with a <100 nM increase of EC [Ca²⁺]_i, there was almost 100% relaxation when EC [Ca²⁺]_i elevation reached 300 nM. Thus, although acute exercise increased the vascular responses to endothelium-dependent vasodilators (such as ACh), data points from the exercise group only extended to high levels of EC [Ca²⁺]_i elevations that accompanied a little additional vasorelaxation. In separate studies, we found that chronic exercise enhances ACh-induced vasorelaxation (4, 6) and the EC [Ca²⁺]_i response (9). Perhaps the chronic exercise also affects vasorelaxation by altering EC [Ca²⁺]_i signaling.

It is plausible to speculate that the large amount of exercise-enhanced ACh-stimulated NO release in the intact endothelium may promote the EC [Ca²⁺]_i response, which could further activate eNOS and enhance endothelium-dependent vasorelaxation in a positive feedback way. Existing evidence not only supports that eNOS is a Ca²⁺-dependent enzyme (15, 19) and that ACh-induced vasorelaxation is coupled to EC [Ca²⁺]_i elevation (2, 12, 18), but also indicates that NO is capable of inducing intracellular calcium rise in cultured EC (1, 25). In this study, we observed that L-NNA, a NOS inhibitor, abolished the exercise effects on ACh-induced vasorelaxation and the EC [Ca²⁺]_i response. Therefore, NO-Ca²⁺ interaction should play an important role in these exercise effects.

To further investigate the possible mechanisms for the exercise effect on EC [Ca²⁺]_i signaling, we used Ca²⁺-free buffer replacement to inhibit calcium influx in noncontracted vessel specimens. Our data showed that this treatment significantly reduced the ACh-evoked EC [Ca²⁺]_i elevation in both the control and exercise groups, with the latter being more severely inhibited (Fig. 5). Moreover, in the absence of extracellular calcium, calcium responses to ACh became indistinguishable between two groups. This result clearly shows that the enhancement of ACh-induced EC [Ca²⁺]_i elevation by acute exercise is mainly due to an increase in calcium influx. This is in accordance with the hypothesis that the activation of eNOS depends on the prolonged [Ca²⁺]_i elevation, not on the transient elevation caused by intracellular calcium release from the stores (16, 26).

Furthermore, the recently discovered mechanosensitive cationic channels (17) may play an important role in causing the elevated endothelial calcium influx after acute exercise, because blood flow or shear stress is increased during exercise. When the vessel segment was pretreated with flow to simulate acute exercise, we observed an enhancement of the ACh-induced EC [Ca²⁺]_i response as well (14). In the present study, Gd³⁺, a mechanosensitive cationic channel blocker, reduced the EC [Ca²⁺]_i response to ACh by one-third in the exercise group but had little effect in the controls. This implies that acute exercise may induce the upregulation of mechanosensitive cationic channels to enhance the ACh-evoked EC [Ca²⁺]_i responses. As we have previously shown that acute exercise modulates the number/affinity of M₃ muscarinic receptors and ₂-adrenergic receptors in rat aortas (8), endothelial membrane protein upregulation appears to be one of the many ways that the body adapts to exercise.

EC [Ca²⁺]_i signaling has been proposed to serve as an integrating signal for endothelium-dependent vasorelaxation (12). In the present study, we found that acute exercise enhanced the agonist-evoked EC [Ca²⁺]_i elevation and vasorelaxation. Because eNOS is a calcium-dependent enzyme, this increased EC [Ca²⁺]_i signaling may be one of the factors responsible for the enhanced NO-dependent vasodilation after acute exercise. Although chronic exercise upregulates eNOS gene expression (22, 27), it is unlikely that acute exercise has such an effect within a short period of time. Because the release of endothelium-derived hyperpolarization factor (EDHF) is also calcium dependent, the increased EC [Ca²⁺]_i response to ACh after acute exercise may indicate a possible increase in EDHF release as well. Nevertheless, it may not be a major factor responsible for our observed exercise effects, which can be completely abolished by the NOS inhibitor L-NNA (Fig. 2).

In conclusion, acute exercise enhances agonist-stimulated vasorelaxation, which is associated with augmented EC [Ca²⁺]_i elevation. NO and NO-Ca²⁺ interaction should be involved in these exercise effects. Furthermore, the augmented ACh-induced EC [Ca²⁺]_i elevation response after acute exercise is largely due to an increase in calcium influx.