INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE ENDOTHELIUM REGULATES vascular tone via the synthesis of various vasoactive substances, such as various endothelium-derived relaxing factors (EDRFs) and endothelin. Exercise protocols, either chronic or acute, have profound effects on the release of these vasoactive substances as well as on the endothelium-dependent control of vascular tone (7, 10, 11, 23). For example, exercise training augments the vasodilating response to acetylcholine (ACh) and decreases the vasoconstrictive response to norepinephrine, possibly by increasing the endothelium-derived nitric oxide (NO) release (5, 6, 8, 9). Moreover, the gene expression of endothelial NO synthase (eNOS) is also increased after chronic exercise (24, 29). Because many agonist-evoked responses are coupled to intracellular calcium concentration ([Ca²⁺]_i) elevation (3, 21, 26), endothelial calcium signaling is likely to be involved in the exercise-induced vascular adaptation.

Cultured endothelial cells may lose their in vivo properties and become adapted to the in vitro environment. For example, the expression of muscarinic receptor mRNA of bovine endothelial cells diminishes in the culture condition (25). To explore the cellular mechanism of exercise effects, it is desirable to monitor the [Ca²⁺]_i level in intact endothelium. Perhaps because of technical difficulties, [Ca²⁺]_i measurements in the endothelium of excised vessels, either by patch-clamp labeling of a single endothelial cell (3) or by spectrofluorimeter to monitor a group of endothelial cells (26), have not been very popular. Recently, we developed an in situ calcium-imaging method that allows simultaneous visualization of large numbers of endothelial cells with single-cell resolution (13). With the use of this technique, agonist-induced aortic endothelial [Ca²⁺]_i elevations at the tissue/cellular level were directly compared between control and exercise-trained rats.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals and exercise-training protocol. This study was conducted in conformance with the policies and procedures described in the Guide for the Care and Use of Laboratory Animals. Four-week-old male Wistar rats were randomly assigned to either control or exercise groups after a 1-wk familiarization period. The rats were housed in an environmentally controlled room (temperature 25 ± 1°C; 12:12-h light-dark cycle) in groups of three or four per cage and were fed a standard rat chow and water ad libitum. The exercise groups were trained for 10 wk as described previously (5, 6). Briefly, they ran on a motor-driven drum exerciser (Drex; Columbus Instruments) at an intensity of ~60% of maximal oxygen consumption for 60 min/day, 5 day/wk, for 10 wk. The training speeds began at 0.16 m/s and reached 0.30 m/s by the end of the experiments. The sedentary control groups were placed in the drum exerciser without running for 10 min/day.

Measurements of resting systolic blood pressure and heart rates. Resting systolic blood pressure (SBP) and heart rates (HR) were measured by using a tail-cuff method (Narco Bio-Systems, Houston, TX) to evaluate the training effects on these two parameters. For 1 wk before the measurements were taken, the animals were restrained in the measuring cages for 30 min/day to avoid novel effects. Thereafter, resting SBP and HR were determined weekly.

Vessel preparation and fura 2 loading. To avoid the acute effects of exercise, animals were killed >48 h after training. They were anesthetized by ether inhalation and decapitated. The thoracic aorta was immediately obtained, cut into rings (5 mm long), and stored in an organ chamber containing Krebs-Ringer solution gassed with 95% O₂-5% CO₂ (22°C, pH 7.4). The composition (in mM) of the solution was 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 for 1 h with 10 µM of fura 2-acetonymethyl ester (AM) and 0.025% Pluronic F-127 in Krebs-Ringer solution, and the excess fura 2-AM was washed out (13).

Measurement of in situ endothelial [Ca²⁺]_i. The basic setup for endothelial [Ca²⁺]_i imaging was similar to our previous setup used for single-platelet [Ca²⁺]_i measurements (15). After fura 2 loading was completed, vessel rings were cut open and pinned to the base plate of a flow chamber (13, 16). There was a 0.28-mm gap between the vessel lumen and the cover glass to allow flow passage. The chamber was mounted on an inverted microscope with epifluorescence attachments (Diaphot 300; Nikon, Tokyo, Japan). The excitation light from a xenon lamp was filtered with a high-speed rotating filter wheel (Lambda 10-2; Sutter, Novato, CA) to provide wavelengths of 340 and 380 nm. The fluorescence images at 510 nm were recorded by a high-sensitivity silicon-intensified target 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. Depending on the objective magnification, Ca²⁺ images for up to 400 endothelial cells could be recorded simultaneously. The histogram of [Ca²⁺]_i was calculated from 100 randomly selected cells. The average value of [Ca²⁺]_i for each preparation was also calculated by monitoring a tissue surface area of ~0.15 mm² or >200 cells. At the end of each experiment, the Ca²⁺ 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 cytosol fura 2 at a 360-nm excitation wavelength. Endothelial [Ca²⁺]_i was estimated after the background autofluorescence was subtracted by using the following equation (12): [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 nm to 380 nm (340/380) during measurements, R_max is the ratio in the presence of saturating Ca²⁺ levels, R_min is the ratio in Ca²⁺-free solution, and B is the ratio of the fluorescence at 380 nm in Ca²⁺-free solution to that in saturated CaCl₂ solution. All experiments were conducted at room temperature. Figure 1 shows a fluorescent image of the rat aortic endothelium labeled with fura 2-AM.

View larger version (175K): [in this window] [in a new window]   Fig. 1.   Fluorescence micrograph of the luminal face of a fura 2-loaded rat aorta using a ×20 ultraviolet lens at excitation and emission wavelengths of 340 and 515 nm, respectively.

[Ca²⁺]_i elevation responses to ACh. After the vascular endothelial cells had been focused properly, 3 ml of fresh Krebs-Ringer buffer were perfused through the chamber at a flow rate of 0.05 ml/min. At the same flow rate, dose responses of ACh-induced [Ca²⁺]_i elevation were determined by subsequent applications of ACh (from 10⁸ to 10⁵ M). Between each ACh application, the chamber was washed with fresh buffer for at least 3.5 min to recover the basal [Ca²⁺]_i level. The results between control and exercise groups were compared by off-line image analysis.

[Ca²⁺]_i elevation responses to ATP. Endothelial [Ca²⁺]_i responses to ATP (10⁵ M), another endothelium-dependent vasodilator, were studied and compared by using the same methods described for ACh study.

Role of Ca²⁺ influx. To elucidate the role of Ca²⁺ influx in our study, [Ca²⁺]_i responses to ACh (10⁵ M) were determined in the presence or absence of 50 µM of SKF-96365, a membrane Ca²⁺ channel blocker, or by replacing the normal buffer with Ca²⁺-free solution. In addition, we estimated the calcium influx by measuring the rate of fluorescence quench by manganese (28). Ca²⁺-free bath solution containing 150 µM MnCl₂ was used to perfuse the chamber to quench endothelial fluorescence at rates proportional to the endothelial Ca²⁺ permeability. The slope of the fluorescence quench curve in the presence of ACh was normalized by that in the presence of ionomycin and served as an indicator of ACh-evoked Ca²⁺ influx rate.

5

Role of membrane potential. Endothelial Ca²⁺ influx can be depressed by potassium-induced membrane depolarization (2). To evaluate whether the exercise training effect is linked to the alteration of membrane potential, we examined the ACh-induced Ca²⁺ responses under membrane potential-manipulated conditions. Some experiments were carried out either in the presence of a high concentration of extracellular K⁺ (60 mM), to substitute Na⁺, or by the administration of 50 µM of the K⁺ ionophore valinomycin.

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 Pluronic F-127 and SKF-96365, which were purchased from Molecular Probes (Eugene, OR) and Biomol Research Laboratories (Plymouth, PA), respectively.

Statistical analysis. Results are expressed as means ± SE. The sample size (N) represents the number of animals used. Dose responses of ACh-induced [Ca²⁺]_i elevation were analyzed by ANOVA with a repeated measures design. Differences between control and exercise-trained groups were compared by using unpaired Student's t-tests with P < 0.05 considered as statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Resting SBP and HR. The resting SBP and HR are shown in Table 1. These parameters were significantly lower in trained animals than in control ones. These results confirmed the effectiveness of our training protocol.

[Ca²⁺]_i elevation responses to ACh. The basal endothelial [Ca²⁺]_i levels were assessed before ACh administration. There was no significant difference between the basal endothelial [Ca²⁺]_i of the control and that of the trained groups (65.7 ± 6.4 and 64.9 ± 11.2 nM, respectively). Typical tracings of [Ca²⁺]_i elevation responses of ~200 cells to different doses of ACh in one vessel segment of each group are shown in Fig. 2. The general pattern is composed of an initial peak and a subsequent plateau that lasted for at least 4-5 min until ACh removal. As a whole, the trained group had greater [Ca²⁺]_i elevations in response to ACh stimulation than the control group (Fig. 3; P < 0.05).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Typical tracings of acetylcholine (ACh)-evoked intracellular Ca2+ concentration ([Ca2+]i) elevation responses in a control and a trained (exercise) rat thoracic aorta. ACh concentrations are expressed as log M.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Dose-response relations of ACh-evoked [Ca2+]i elevation ([Ca2+]i) in control (, N = 12) and exercise-trained (, N = 9) groups. *P < 0.05 (ANOVA with repeated measures design).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Typical histograms of ACh-induced [Ca2+]i responses in 100 cells randomly selected from a control or an exercise-trained sample. Ca2+ responsiveness of these cells was proportional to the ACh concentration, and the dose-dependent shift is more profound in the exercise group than in the control group.

[Ca²⁺]_i elevation responses to ATP. The ATP-induced endothelial [Ca²⁺]_i elevation curves were similar to the ACh-induced ones. Moreover, chronic exercise enhanced ATP-induced endothelial [Ca²⁺]_i responses. The ATP-evoked endothelial [Ca²⁺]_i elevations were 155 ± 26 and 370 ± 81 nM for the control and exercised groups, respectively (P < 0.05).

Role of Ca²⁺ influx. The percentage of changes of ACh (10⁵ M)-evoked [Ca²⁺]_i elevation after various treatments to inhibit Ca²⁺ influx are shown in Table 2. SKF-96365, Gd³⁺ administration, or Ca²⁺-free buffer replacement has greater inhibition in trained compared with control animals. The differences between control and trained groups disappeared (e.g., before SKF-96365 treatment: 363 ± 17 vs. 630 ± 125 nM; after treatment: 292 ± 34 vs. 228 ± 48 nM for the control and trained groups, respectively).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   An example of Mn2+ quench to determine Ca2+ influx. Vessel segments were incubated in the presence of MnCl2 (150 µM) to quench endothelial fura 2 at its isofluorescence excitation wavelength of 360 nm. ACh (10 µM) and ionomycin (5 µM) were added subsequently. ACh-induced Ca2+ influx rate was estimated by taking the ratio of slope a over slope b.

Role of membrane potential. When the membrane potential was depolarized with high K⁺, the endothelial [Ca²⁺]_i responses to ACh were partially inhibited in both control and trained groups. However, the degree of inhibition was greater in the trained group than in the control group (Table 2). When the membrane potential was hyperpolarized with valinomycin, the endothelial [Ca²⁺]_i levels gradually increased in both control and trained groups. A subsequent addition of ACh could induce hardly any significant [Ca²⁺]_i elevation (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our study is the first to report the exercise training effects on endothelial [Ca²⁺]_i signaling in freshly dissected rat vessel segments. We found that 1) the average basal endothelial [Ca²⁺]_i levels were the same in control and exercise groups (65 nM); 2) chronic exercise enhanced the ACh- or ATP-induced endothelial [Ca²⁺]_i elevation; 3) ACh-evoked endothelial [Ca²⁺]_i elevation was heterogeneous among individual endothelial cells in either group; and 4) the exercise effect on ACh-evoked endothelial [Ca²⁺]_i elevation was mostly due to elevated Ca²⁺ influx.

Our results of ACh-induced [Ca²⁺]_i changes in vascular endothelium are similar to those observed by Usachev and co-workers (26). On the other hand, Carter and Ogden (3) have applied patch-clamp techniques to load another fluorescent dye, furaptra (500 µM), into single endothelial cells in isolated rat aortas. In their study, ACh evoked a large initial [Ca²⁺]_i peak, in the range of 6-35 µM, which was followed by repetitive spikes of amplitude ranging from 2 to 18 µM. However, we have not observed any [Ca²⁺]_i peak of this magnitude or any single cell with repeated spikes from ACh-treated vessel specimens. Repeated [Ca²⁺]_i spikes with maximal initial peak values of ~1-2 µM were occasionally observed in certain histamine-treated endothelial cells (data not shown). This is consistent with a previous report (14) in which a fluorimeter was used to measure [Ca²⁺]_i levels in cultured human endothelial cells.

To further investigate the possible mechanisms for the exercise effect on endothelial calcium signaling, we used SKF-96365 or Ca²⁺-free buffer to block Ca²⁺ influx. Our data showed that these treatments significantly reduced ACh-evoked endothelial [Ca²⁺]_i elevation in both control and trained groups, with the latter being more severely inhibited. In conjunction with Mn²⁺ quenching rate studies, the current study clearly shows that the enhancement of ACh-induced endothelial [Ca²⁺]_i elevation by chronic exercise is mainly due to an increase in Ca²⁺ 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 Ca²⁺ release from the stores (18, 27).

Furthermore, the recently discovered mechanosensitive cationic channels (20) may play an important role in causing the elevated endothelial Ca²⁺ influx after exercise training. Whereas Gd³⁺, the mechanosensitive cationic channel blocker, reduced the Ca²⁺ response to ACh by one-third in the exercised group, it had little effect in the control group. This implies that mechanosensitive cationic channels may be upregulated and may play some roles in ACh-evoked Ca²⁺ responses after chronic exercise. Endothelial membrane protein upregulation appears to be one of the many ways that the body adapts to exercise. We have previously shown that various exercise protocols modulate the number and/or affinity of M₃ muscarinic receptors and ₂-adrenergic receptors in rat aortas (4, 10).

It has been reported that ACh evokes hyperpolarization of intact aortic endothelium, probably via the Ca²⁺-activated K⁺ channels (19). In addition, the vasodilator-evoked endothelial hyperpolarization temporally coincides with a rise in [Ca²⁺]_i (26). However, agonist-evoked Ca²⁺ rise was diminished when the endothelial membrane potential was clamped in a depolarized state by elevated extracellular K⁺ (2). These results imply that Ca²⁺ influx is partially membrane potential dependent. To investigate the role of endothelial membrane potential, we determined the changes in ACh-induced Ca²⁺ responses in both groups while clamping the membrane potential in a depolarized state with high K⁺. Indeed, we found that the inhibition of Ca²⁺ response by high K⁺ was more pronounced in the trained group than in the control group, indicating that the exercise effect on ACh-induced Ca²⁺ response may be partially due to an increase in ACh-evoked endothelial membrane hyperpolarization. This hypothesis needs to be further verified by an electrophysiological method.

However, we cannot rule out the possibility that chronic exercise may alter the upstream components of Ca²⁺ signaling pathways as well. Our results showed that both ACh- and ATP-induced endothelial [Ca²⁺]_i responses were elevated in the exercised group. Although chronic exercise may upregulate endothelial ACh and ATP receptors in a similar way, it is also possible that the postreceptor common pathway for [Ca²⁺]_i signaling is altered. It has been reported (1) that with exercise-induced muscle hypertrophy, the sarcoplasmic reticulum increase proportionally with contractile protein, whereas the mitochondrial fraction does not.

It is plausible to assume that endothelial Ca²⁺ signaling is related to vasorelaxation. In the present study, we found that chronic exercise enhanced the agonist-evoked endothelial [Ca²⁺]_i elevation. Chronic exercise-enhanced ACh-evoked vasodilating responses have been attributed to an increase in the release of EDRFs, mainly by NO (6). Because eNOS is a Ca²⁺-dependent enzyme (17, 22), this increased endothelial Ca²⁺ signaling may be one of the factors responsible for the enhanced vasodilating response to ACh after chronic exercise. Recent studies (24, 29) also suggest that exercise training increases eNOS gene expression. Therefore, there appear to be multiple regulation sites responsible for the exercise-enhanced NO release. Because the release of endothelium-derived hyperpolarization factor (EDHF) is also Ca²⁺ dependent, the increased endothelial Ca²⁺ response to ACh after chronic exercise may indicate a possible increase in EDHF release as well. Nevertheless, it may not be a major factor responsible for our exercise effects on ACh-induced vasorelaxation, because the exercise-enhanced vasodilating response to ACh can be completely abolished by the NOS inhibitor N-nitro-L-arginine (6).

In conclusion, chronic exercise enhances agonist-stimulated endothelial [Ca²⁺]_i elevation, largely caused by increasing Ca²⁺ influx. However, there are multiple reasons for these exercise effects. The current findings, in conjunction with previously reported mechanisms such as elevated eNOS expression (24) and upregulation of membrane receptors (4, 10), should provide a relatively comprehensive picture showing how chronic exercise increases endothelium-dependent vasodilating responses.