INTRODUCTION

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THE SARCOPLASMIC RETICULUM (SR) participates in the regulation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in smooth muscle. Two types of Ca²⁺ release receptors have been identified in the SR. One class (2, 6) is sensitive to D-myo-inositol 1,4,5-trisphosphate (IP₃), produced in response to agonists such as norepinephrine (NE), 5-hydroxytryptamine (5-HT; serotonin), and arginine vasopressin (AVP). A second class (5, 11) of receptors is sensitive to caffeine and ryanodine and appears to be involved in the regenerative control of [Ca²⁺]_i via Ca²⁺-induced Ca²⁺ release.

The functional role of the SR is probably related to its ability both to release Ca²⁺ in response to receptor activation and to accumulate Ca²⁺ via its Ca²⁺-activated ATPase activity. In addition to raising intracellular Ca²⁺ levels in response to appropriate stimuli, the SR may contribute to Ca²⁺ elimination from the cytoplasm by virtue of the release of Ca²⁺ into a restricted subplasmalemmal space, thus facilitating extrusion over the cell membrane via Na⁺/Ca²⁺ exchange (13, 18). Increased [Ca²⁺]_i is expected to lead to increased Ca²⁺ accumulation in the SR and, if long-standing, possibly increased capacity of the SR to accumulate and release Ca²⁺. Such effects could contribute to the understanding of its functional role, because the response to agents acting on the SR would be amplified.

Increased Ca²⁺ release from the SR of smooth muscle has been described in animal models of hypertension and bladder hypertrophy (12, 20). These changes could be hypothesized to be due to persistent activation, leading to increased [Ca²⁺]_i and thus increased load on the SR. To specifically investigate the role of increased [Ca²⁺]_i for this response, an in vitro culture approach would be useful, because this allows the control of experimental conditions to an extent not attainable in vivo. Dispersed smooth muscle cells in culture rapidly modulate from a contractile to a synthetic phenotype (3), confounding the interpretation of results with respect to the physiological mechanisms operating in intact tissue. Thus an in vitro culture method preserving the contractile phenotype is needed. The culture of whole vascular tissue, where smooth muscle cells are kept in their normal extracellular matrix environment, preserves contractility and structural integrity for periods of several days (4, 14, 22). In cultured rat tail artery, we found that the presence of FCS causes decreased contractility, which may primarily be a consequence of an increased Ca²⁺ load and which can be separated from the growth stimulation caused by FCS (14). In the present study, the long-term effects of the Ca²⁺ load on SR function in rat tail arterial rings kept in culture for 4 days were investigated.

	MATERIALS AND METHODS

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Tissue preparation and culture. Female Sprague-Dawley rats, weighing ~200 g, were killed by cervical dislocation. The tail artery was dissected free under sterile conditions and transferred to culture medium (DMEM/Ham's F-12; 1:1) containing 1.08 mM CaCl₂, 100 U/ml penicillin, and 100 µg/ml streptomycin. Attached connective tissue was removed, and the artery was cut into rings under a dissecting microscope. The width and luminal diameter of the rings did not vary substantially and were ~0.6 and 0.5 mm, respectively. Rings were transferred to culture dishes containing the above medium with or without FCS (10%). To some dishes verapamil (1 µM), EGTA (1 mM), or KCl and CaCl₂ (26 and 1.42 mM, respectively) were added. The dishes were placed in a water-jacketed cell incubator at 37°C under 5% CO₂ in air.

Experimental procedure. After 4 days in culture, the rings were bathed in a nominally Ca²⁺-free Krebs solution of the following composition (in mM): 4.7 KCl, 15.5 NaHCO₃, 1.2 KH₂PO₄, 1.2 MgCl₂, 11.5 glucose, and 122 NaCl. To remove the endothelium, a thin needle was passed through the lumen of the ring. The rings were mounted on two stainless steel wires (diameter 0.2 mm), one connected to a force transducer (AE 801; SensoNor, Horten, Norway) and the other connected to an adjustable support. The rings were stretched to a passive tension of 1.2 ± 0.1 mN/mm, which is close to the optimal preload for these preparations. Rings were immersed in warm physiological salt solution (PSS) containing (in mM) 135.5 NaCl, 5.9 KCl, 2.5 CaCl₂, 1.2 MgCl₂, 11.6 HEPES, and 11.5 glucose. High-K⁺ (140 mM) solution was prepared by replacing NaCl with KCl. The solution was contained in 0.4-ml Plexiglas cups fitted into thermostatted (37°C) metal blocks. For Ca²⁺ measurements, a 125-µl chamber was perfused by solution at a rate of 1 ml/min. In all experiments, the rings were allowed to equilibrate for at least 35 min before being stimulated with high-K⁺ solution. After 5 min, the rings were transferred to Ca²⁺-free PSS containing 1 mM EGTA. After an additional 5 min, Ca²⁺-releasing agents were added. This protocol was repeated with another releasing agent after 15 min of equilibration in normal PSS or ended with a maximal reference contraction in a high-K⁺ solution supplemented with 10 µM NE. In some experiments Ca²⁺ release was also induced without a preceding contraction in high-K⁺ solution. The width of each ring was measured at the end of the experiments with a dissecting microscope equipped with an ocular scale. Wall tension was expressed in units of millinewtons per millimeter by dividing peak force by twice the ring width.

Ca²⁺ measurements. After equilibration, rings were incubated for 90 min at 22°C with the fluorescent probe fura 2-AM (13.3 µM; Molecular Probes, Eugene, OR). The Ca²⁺ release protocol was the same as that described above. EGTA-containing solution, caffeine, and NE were applied through a pipette located 1 mm from the ring. This was done to ensure rapid exposure to the agents necessary to resolve Ca²⁺ transients but precluded heating the applied solution. Therefore, after 30 min of equilibration at 37°C, the rest of the experiment was performed at room temperature (22°C). In situ calibration was performed at the end of each experiment by permeabilization with ionomycin (50 µM) as described by Himpens et al. (9). [Ca²⁺]_i was estimated from the ratio of fluorescence at 510 nm for excitation at 340 and 380 nm, as described by Grynkiewicz et al. (8), with a value of 224 nM for the dissociation constant of Ca²⁺ binding to fura 2.

Statistics. Summarized data are expressed as means ± SE. Student's t-test was used to evaluate statistical significance. For multiple comparisons, the Bonferroni correction was used.

	RESULTS

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The effects of various Ca²⁺-releasing agents on rat tail arterial rings were tested after 4 days of culture. Identical experiments were run on fresh preparations from the same animal. Transient force responses were elicited by the addition of agonists after incubation in Ca²⁺-free solution. Because Ca²⁺ release should be highly dependent on the level of Ca²⁺ store filling, we standardized Ca²⁺ loading by exposing the rings to high-K⁺ (140 mM K⁺) solution for 5 min before switching to Ca²⁺-free solution and inducing Ca²⁺ release. After this procedure, responses to releasing agents were clearly greater than after incubation (>30 min) in normal PSS (caffeine: 145 ± 13%; NE: 141 ± 5%; n = 8). In all experiments the releasing agents were applied after 5 min in Ca²⁺-free solutions containing 1 mM EGTA to eliminate the inflow of extracellular Ca²⁺. This treatment does not appreciably deplete the SR, because SR Ca²⁺ exchanges very slowly in these preparations. To estimate the rate of loss, we evaluated NE-evoked force transients after variable time periods in EGTA-buffered solution. The transient amplitude decreased linearly with time at a rate of 1.7 ± 0.3%/min (n = 4).

The amplitude of the agonist-evoked force transient was taken as an indicator of intracellular Ca²⁺ release. The maximal force output was decreased by culture in the presence of FCS. Therefore, tension transients were quantitated relative to the maximal force response of the individual ring (Fig. 1). Responses to all agents were enhanced after culture. This effect of culture varied between agonists, but there was no apparent difference between caffeine- and IP₃-mediated responses. In contrast to what was found for all IP₃-inducing agents, responses to caffeine were significantly greater in rings cultured in the presence of 10% FCS than in control rings cultured in the absence of serum. This increase was accompanied by an increased responsiveness to caffeine at low concentrations, as revealed by dose-response relationships (Table 1). However, at the standard concentration of 20 mM used in this study, the responses relative to the maximum concentration used (40 mM) were equal (0% FCS: 83.7 ± 7.2%; 10% FCS: 79.9 ± 8.0%). Therefore, differing sensitivities do not account for the increased peak forces in response to caffeine after culture with FCS. There were no significant effects of FCS on the sensitivity to NE, 5-HT, or AVP after culture, and accordingly the concentrations giving maximal tension were used in Ca²⁺ release experiments. Culture itself caused a sensitization to NE, as previously described (15), probably because of a degeneration of adrenergic nerves (22). In fresh preparations, caffeine responses were too small to give an adequate measure of sensitivity.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   A: tension responses to maximal activation [high-K+ solution + 10 µM norepinephrine (NE)] in freshly prepared and cultured tail arterial rings. B: force transients in response to Ca2+-releasing agents [10 µM NE, 10 µM 5-hydroxytryptamine (5-HT), 0.1 µM arginine vasopressin (AVP), and 20 mM caffeine]; n  6 for all determinations. * P < 0.05 and ** P < 0.01.

Ca²⁺ measurements verified that the differences in force in response to caffeine and NE observed after culture reflect differences in the release of intracellular Ca²⁺ (Fig. 2 and Table 2). This series of experiments was run at room temperature (see MATERIALS AND METHODS), which probably accounts for the low force development compared with that in the mechanical experiments shown in Fig. 1. The apparent dissociation between [Ca²⁺]_i and force on stimulation by NE, in contrast to what was found for high-K⁺ solution or caffeine, is consistent with its Ca²⁺-sensitizing effect, first described by Morgan and Morgan (17). The force elicited by either high-K⁺ solution alone or a mixture of high-K⁺ solution and NE was lower after FCS culture, whereas the [Ca²⁺]_i response was not (Table 2). This confirms that the normalization of transient responses to maximal force (Fig. 1) is valid in terms of indicating the magnitudes of agonist-induced Ca²⁺ transients. The kinetics of the Ca²⁺ transients in response to the two agents were compared. As shown in Table 3, rings cultured with FCS showed a significantly faster rising phase of responses to caffeine but there was no difference in responses to NE. The rate of decline of the transient was about five times lower than the rate of rise, and independent of agonist or culture condition.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Simultaneous registrations of tension (mN/mm) and intracellular Ca2+ concentration ([Ca2+]i; nM) in rings cultured in presence of 10% FCS (A) or absence of FCS (B). Responses to caffeine (caff) and NE are indicated. K+, high-K+ solution.

Exposure to FCS increases [Ca²⁺]_i in the tail artery (14). Because fluorometric measurements of [Ca²⁺]_i cannot be done during the actual period of incubation, the [Ca²⁺]_i-raising effect of FCS was verified in rings loaded with fura 2 after culture with FCS and equilibrated in PSS. The addition of FCS raised [Ca²⁺]_i, and this effect was antagonized by the addition of EGTA or verapamil (Fig. 3). Thus no apparent desensitization to the [Ca²⁺]_i-raising action of FCS occurred during culture, and [Ca²⁺]_i is likely to have been increased throughout the culture period. Similar results were obtained with depolarizing high-Ca²⁺ medium (30 mM KCl, 2.5 mM CaCl₂; data not shown). In contrast to the direct effect of added FCS, it was found that basal [Ca²⁺]_i values in rings loaded with fura 2 were not different after culture either with or without FCS when the rings were subsequently incubated in normal FCS-free PSS (Table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Simultaneous measurements of tension and [Ca2+]i in one ring after culture for 4 days in 10% FCS. FCS (10%), EGTA (1 mM), and verapamil (1 µM) were added as indicated; n = 3.

To test the hypothesis that altered SR Ca²⁺ handling is due to increased [Ca²⁺]_i during culture, culture media were supplemented with verapamil (1 µM) or EGTA (1 mM) to lower [Ca²⁺]_i (Fig. 3). Both of these treatments significantly inhibited the effects of FCS on force transients evoked by caffeine but did not affect NE-evoked transients (Fig. 4A). Furthermore, the effects of FCS on the Ca²⁺ release pattern could be mimicked by culture in depolarizing high-Ca²⁺ medium. The effects of culture with depolarizing medium and with FCS on maximal tension development were also similar, and both effects were reversible by EGTA treatment (Fig. 4B).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   A: relative tension of caffeine- and NE-induced transients expressed as percentage of contraction induced by high-K+ medium + 10 µM NE. Culture conditions are indicated, as are additions of verapamil (V; 1 µM) and EGTA (E; 1 mM) to medium; n > 12 for all determinations; * P < 0.05. B: effects of culture with EGTA (1 mM) on maximal tension development in rings treated with 0 or 10% FCS or depolarizing, high-Ca2+ medium. * P < 0.05 and ** P < 0.01.

The effect of culture with FCS on agonist-induced force transients was persistent. It remained after the rings were washed for 30 min with cold (8°C) Ca²⁺-free PSS, and thereafter for at least 2.5 h in PSS without FCS (data not shown). Pretreatment with ryanodine or the SR Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) abolished the responses to both caffeine and NE. Ryanodine induced a sustained plateau in force (29 ± 2% of a contraction induced by high-K⁺ solution; n = 4), whereas the contractile response to CPA was more variable. In contrast to the response to ryanodine, which is irreversible, responses to caffeine reappeared at their original levels after CPA was washed out (Fig. 5). This indicates that the increased caffeine responses after culture reflect an actual increase in the Ca²⁺ storage and/or release capacity of the SR and not merely a persistent shift in ionic distributions after culture.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Force registration from one ring (of five) cultured in presence of 10% FCS, showing effect of cyclopiazonic acid (CPA; 10 µM) on caffeine responses.

	DISCUSSION

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Although the plasticity of intracellular Ca²⁺ handling in smooth muscle has been extensively investigated by using dispersed cells in culture, the present study is to our knowledge the first investigation of SR release in intact smooth muscle tissue cultured in vitro. Major differences in the effect of FCS on SR function in cultured tissue vs. dispersed cells are found. Most likely, these relate to the phenotypic change occurring early in cell culture as a result of the disruption of cell-cell and cell-matrix interactions (3). On the other hand, in vivo models of hypertension and smooth muscle hypertrophy show effects on SR function compatible with the findings obtained here (see below). Systemic compensatory mechanisms active in animal models do not influence long-term responses in organ culture, and a culture model provides the ability to apply powerful pharmacological tools not accessible in vivo. Morphologically, rat tail arteries are also well maintained in culture for at least 2 wk in serum-free medium (22), and thus organ culture appears to be a valuable model with which to study the long-term adaptation of SR function as well as other aspects of excitation-contraction mechanisms (7, 21).

An increased release of Ca²⁺ from intracellular stores was found by Rohrmann et al. (20) in the early phases of urinary bladder hypertrophy caused by outlet obstruction. Similar changes in SR function were also observed in vascular smooth muscle cells from spontaneously hypertensive rats (12, 23). The changes in SR function could be a secondary response to increased sarcolemmal Ca²⁺ influx or altered mitochondrial Ca²⁺ handling but could possibly also reflect the effects of growth stimulation unrelated to altered Ca²⁺ homeostasis. Notwithstanding the difficulty of relating findings for cultured muscle to in vivo hypertrophy, results with FCS can give some insight into the respective effects of growth stimulation and Ca²⁺ load on SR function. Culture with FCS results in a decrease in maximum force, which appears to be a result of structural effects related to increased [Ca²⁺]_i rather than growth stimulation as such (14), and accordingly the present investigation has focused on the effects of the [Ca²⁺]_i increase accompanying culture with FCS. In fact, the growth-stimulating effect of FCS seems severely inhibited by conditions in intact tissue, in contrast to those in dispersed cells. After culture in the presence of 10% FCS, responses to caffeine, but not to NE, 5-HT, or AVP, were increased compared with those in serum-free culture. This was clearly a Ca²⁺-dependent effect, because it could be blocked by the inclusion of verapamil or EGTA in the culture medium. Furthermore, culture in depolarizing medium (30 mM KCl, 2.5 mM CaCl₂) mimicked the effects of FCS on caffeine vs. NE responses. The similar effects of depolarizing medium and FCS on force, and their prevention by EGTA, further show that the two treatments acted through the same mechanism, namely, increased [Ca²⁺]_i.

The unexpected finding that NE-sensitive Ca²⁺ release was not affected by FCS or depolarizing medium suggests that caffeine-sensitive release mechanisms are preferentially involved in the long-term response to increased [Ca²⁺]_i. The enhanced rate of Ca²⁺ release in response to caffeine and the leftward shift of the caffeine dose-response curve after culture with FCS (Table 2 and Fig. 2) suggest that the properties or the density of the ryanodine receptors has been altered. This might reflect a specific role of the ryanodine receptor pathway in regulating the basal Ca²⁺ level, e.g., by release into a restricted subsarcolemmal space for further extrusion via the plasma membrane (13, 18). However, the relative levels of importance of the different Ca²⁺ release pathways for this mechanism are presently unknown.

Besides the effect of culture with FCS on SR Ca²⁺ handling, culture itself, with or without FCS, caused an increase in Ca²⁺ release, affecting both caffeine- and IP₃-mediated release. This does not seem to be caused by increased Ca²⁺ load, because verapamil and EGTA did not reduce the amplitudes of responses after culture in 0% FCS. Thus the effect seems to be a trophic response influencing the Ca²⁺ storage capacity of the SR, but the role of FCS for this seems to be minor.

The results presented here are in apparent contrast to some findings for isolated smooth muscle cells. A decreased ability to release Ca²⁺ in response to caffeine or ANG II was observed in vascular smooth muscle cells after they had been isolated and placed in culture and had entered a phase of rapid proliferation under stimulation by FCS (16). Ca²⁺ release in response to ANG II, but not caffeine, is restored when the cultures reach confluence. However, arrest of the cell cycle by serum-free medium restores the response to caffeine. Furthermore, basal [Ca²⁺]_i and release from the SR have been shown to vary with the stages of the cell cycle (10). However, in the cultured tail artery there does not seem to be any correlation between the effect of FCS on basal [Ca²⁺]_i and entry into the S phase of the cell cycle, because verapamil does not affect [³H]thymidine incorporation in FCS-stimulated rings (14). Thus comparison with studies of proliferating cells in culture must be made with caution, although these studies also suggest that caffeine- and IP₃-sensitive Ca²⁺ stores may be separately regulated by trophic stimuli and Ca²⁺.