Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

Karl Dreja and Per Hellstrand

Department of Physiological Sciences, Lund University, S-223 62 Lund, Sweden


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To investigate the Ca2+-dependent plasticity of sarcoplasmic reticulum (SR) function in vascular smooth muscle, transient responses to agents releasing intracellular Ca2+ by either ryanodine (caffeine) or D-myo-inositol 1,4,5-trisphosphate [IP3; produced in response to norepinephrine (NE), 5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptors in rat tail arterial rings were evaluated after 4 days of organ culture. Force transients induced by all agents were increased compared with those induced in fresh rings. Stimulation by 10% FCS during culture further potentiated the force and Ca2+ responses to caffeine (20 mM) but not to NE (10 µM), 5-HT (10 µM), or AVP (0.1 µM). The effect was persistent, and SR capacity was not altered after reversible depletion of stores with cyclopiazonic acid. The effects of serum could be mimicked by culture in depolarizing medium (30 mM K+) and blocked by the addition of verapamil (1 µM) or EGTA (1 mM) to the medium, lowering intracellular Ca2+ concentration ([Ca2+]i) during culture. These results show that modulation of SR function can occur in vitro by a mechanism dependent on long-term levels of basal [Ca2+]i and involving ryanodine- but not IP3 receptor-mediated Ca2+ release.

sarcoplasmic reticulum; organ culture; tail artery; D-myo-inositol 1,4,5-trisphosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

The fluorescence and force measurements were carried out simultaneously at a sampling frequency of 0.5 Hz, which was increased to 2 Hz for the resolution of transients, by using an imaging system (IonOptix) mounted on a Nikon TMD inverted microscope with a Nikon Fluor ×20 objective. To minimize the contribution of adventitial fibroblasts to the recorded signal, the video image was focused to the medial layer, 45 to 55 µm beneath the adventitial border. This layer of the arterial wall has a bundled appearance in normal light and does not contain any fibroblasts (1, 19). Acquisitions were from a zone that averaged 0.03 mm2. In most experiments a control zone of the same size was also defined. When compared, zones from the same experiment showed very small variation in ratiometric values. The [Ca2+]i values obtained just before the induced contractions were subtracted from the ensuing peak value to give the increases in Ca2+ concentration presented.

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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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.


                              
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Table 1.   EC50 values in response to Ca2+-releasing agents

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.


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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.


                              
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Table 2.   Basal [Ca2+]i and increase in [Ca2+]i and force in response to high-K+ solution and application of caffeine or NE


                              
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Table 3.   Kinetics of Ca2+ release events

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).


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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).


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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.


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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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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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+.


    ACKNOWLEDGEMENTS

We thank Bengt-Olof Nilsson and Anders Lindqvist for useful discussions.


    FOOTNOTES

This study was supported by the Swedish Medical Research Council (Project 14X-28), the Medical Faculty, Univ. of Lund, and AB Astra-Hässle, Mölndal, Sweden.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Hellstrand, Dept. of Physiological Sciences, Lund Univ., Sölvegatan 19, S-223 62 Lund, Sweden (E-mail: Per.Hellstrand{at}mphy.lu.se).

Received 29 June 1998; accepted in final form 2 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 276(5):C1115-C1120
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