Introduction

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Although store-operated Ca⁺⁺ entry has been demonstrated in a number of vascular smooth muscle preparations (Xuan et al., 1992; Skutella and Rüegg, 1997; for a review see Daniel et al., 1995), the relationship between Ca⁺⁺ entry via this pathway and contraction remains unclear. Indeed, it was recently suggested that the elevated [Ca⁺⁺]_i level that results from inhibition of SR Ca⁺⁺-ATPase, a process dependent on store-operated Ca⁺⁺ entry (Putney, 1990), is dissociated from contraction (Abe et al., 1996; Nomura et al., 1997). In support of this suggestion are the observations that low concentrations of cyclopiazonic acid (1 µM; CPA), a selective inhibitor of SR Ca⁺⁺-ATPase (Seidler et al., 1989), elevated [Ca⁺⁺]_i but did not induce contraction in femoral artery from Wistar-Kyoto and spontaneously hypertensive rats (Nomura et al., 1997). Furthermore, although higher CPA concentrations further elevated [Ca⁺⁺]_i and induced contraction in these vessels, as well as in carotid arteries from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats (Sekiguchi et al., 1996), high CPA concentrations did not elicit contraction in rat mesenteric resistance arteries despite elevating [Ca⁺⁺]_i (Naganobu and Ito, 1994). Others have also demonstrated that, depending on the vessel, SR Ca⁺⁺-ATPase inhibitors did not induce, or variably induce contraction (Shima and Blaustein, 1992; Kwan et al., 1994; Low et al., 1996). Thus, the relationship between [Ca⁺⁺]_i elevation due to store-operated Ca⁺⁺ entry and contraction may be further complicated by the vessel under study.

An additional observation in support of the suggestion that elevated [Ca⁺⁺]_i due to store-operated Ca⁺⁺ entry is dissociated from contraction is the inability of 10 µM CPA, which greatly elevated [Ca⁺⁺]_i, to enhance contractions to norepinephrine and KCl in ferret portal vein (Abe et al., 1996). In possible contrast, Naganobu and Ito (1994), demonstrated that 10 µM CPA elevated [Ca⁺⁺]_i and potentiated the contraction to phenylephrine in rat mesenteric resistance arteries. These results may again suggest that, in some vessels, and possibly related to vessel size and SR function, CPA-elevated [Ca⁺⁺]_i may be coupled to contraction. Alternatively, the potentiation may have resulted from inhibition of the Ca⁺⁺ buffering capacity of the SR by CPA (Naganobu and Ito, 1994; see van Breemen et al., 1995, for review).

The purpose of this study, therefore, was to further test whether elevated [Ca⁺⁺]_i due to store-operated Ca⁺⁺ entry is restricted to a compartment dissociated from contraction. To test this hypothesis, we investigated in rat aorta, using simultaneous measurements of [Ca⁺⁺]_i and contraction, the 1) concentration-response relationship between CPA-elevated [Ca⁺⁺]_i and contraction; 2) ability of CPA to enhance contractions to PDB, KCl and U46619, a stable thromboxane A₂ receptor agonist and 3) effects of verapamil, an L-type Ca⁺⁺ channel blocker and Ni⁺⁺, a nonselective cation channel blocker, on these [Ca⁺⁺]_i-contraction relationships. Some of these results have been submitted as an abstract (Tosun et al., 1998a).

	Materials and Methods

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Rats (Sprague-Dawley, male, 250-350 g) were asphyxiated with CO₂ and the thoracic aorta removed and cleaned of extraneous fatty tissue. Each aorta was cut into helical strips (2 × 10 mm), the endothelium removed, and the strip mounted vertically on a holder attached to an isometric force transducer. Preliminary results demonstrated that U46619-induced contraction, as well as EC₅₀ values, were similar in strips and ring segments normalized to cross-sectional area (see also Tosun et al., 1997).

As previously described (Tosun et al., 1997, 1998b), the holder containing the strip was then placed in a cuvette containing Krebs-Ringer bicarbonate solution (Rapoport, 1987), plus 0.2 mM neostigmine, 1 mM probenecid, 0.02% pluronic F-127 and 5 µM fura-2/AM. Tissue was placed under 20 mN resting tension and was incubated in the dark for 2.5 to 3 hr at room temperature with sonication applied external to the cuvette. The cuvette was then placed in a water-jacketed holder (37°C) and resting tension readjusted to 20 mN. The tissue was perfused (12 ml/min) with 37°C gassed Krebs-Ringer bicarbonate solution containing 3 µM indomethacin and 1 mM probenecid, and allowed to equilibrate for 30 min before agent addition.

The intimal surface of the fura-2 loaded tissue was subjected to excitation wavelengths of 340 and 380 nm. Emitted fluorescence was measured at 510 nm using a PTI Deltascan-1 spectrofluorometer configured for front-face fluorescence (Photon Technology International, South Brunswick, NJ). The ratio of 340 to 380 nm excitation (R340/380) is reported as a relative measure of free [Ca⁺⁺]_i. Absolute levels of [Ca⁺⁺]_i are not reported due to the uncertainty of the conventional calibration method in intact tissue. However, assuming an apparent K_d of the fura-2:Ca⁺⁺ complex of 224 nM (Grynkiewicz et al., 1985), the basal level of [Ca⁺⁺]_i was ~25 to 50 nM and increased to ~200 nM with maximal U46619 stimulation (Tosun et al., 1997). Sf₂/Sb₂ (Grynkiewicz et al., 1985) was 2.1 after background subtraction (Tosun et al., 1998b). Contractile force was measured simultaneously with [Ca⁺⁺]_i and reported in mN.

Statistical methods. Statistical significance between means was determined with analysis of variance followed by the Newman-Keuls test unless otherwise indicated. Significance was accepted at P = .05. Shown are means ± S.E. N represents the number of animals.

Materials. Reagent sources were as follows: Biomol Research Laboratories, Inc. (Plymouth Meeting, PA): verapamil; Molecular Probes (Eugene, OR): fura-2/AM, pluronic F-127; Sigma Chemical Co. (St. Louis, MO): chelerythrine, CPA, BHQ, indomethacin, ionomycin, neostigmine methyl sulfate, nickel chloride, norepinephrine hydrochloride, PDB, probenecid; Pharmacia & Upjohn (Kalamazoo, MI): U46619 (gift).

	Results

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CPA and BHQ. CPA and BHQ, inhibitors of sarcoplasmic reticulum Ca⁺⁺-ATPase, induced concentration-dependent increases in [Ca⁺⁺]_i (fig. 1, A-D). Elevated [Ca⁺⁺]_i due to 1 µM CPA and BHQ, the lowest concentration tested, was not accompanied by significant contraction (fig. 1, A, B and D). In contrast, 10 nM U46619 and 33.2 mM KCl elevated [Ca⁺⁺]_i to a similar magnitude as 1 µM CPA and BHQ, and induced considerable contraction (fig. 1D).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effects of Ni++ and verapamil on CPA-, BHQ-, KCl- and U46619-induced [Ca++]i elevation and contraction in rat aorta. Shown are tracings (A-C) and cumulative data (D) of simultaneously recorded CPA- and BHQ-induced [Ca++]i elevation (R340/380) and contractile force (mN) in the presence of verapamil and Ni++. Also shown (D) are the cumulative data on the effects of U46619 and KCl on simultaneously recorded [Ca++]i elevation and contraction. A, CPA was added cumulatively (1-10 µM) followed by 10 µM verapamil and then 0.1 mM Ni++; B, 1 µM CPA was added followed by 10 µM verapamil, 0.1 mM Ni++ and then 1 µM norepinephrine (NE); C, 10 µM CPA was added followed by 1 and then 10 µM verapamil, and then cumulative concentrations of Ni++ (0.1-1 mM). Tissues were then challenged with cumulative concentrations of U46619 (0.01-1 µM) and then 1 µM NE (data also described in Tosun et al., 1998b, in press); D, cumulative data on the effects of CPA, BHQ, 10 nM U46619 and 33.2 mM KCl on [Ca++]i elevation and contractile force. Shown are means ± S.E.; N = 3 in each case, except N = 16, 9 and 11 for 10 µM CPA, 10 nM U46619 and 33.2 mM KCl, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Effects of CPA on KCl-induced [Ca++]i elevation and contraction in rat aorta. Shown are tracings (A) and cumulative data (B) of simultaneously recorded [Ca++]i elevation (R340/380) and contractile force (mN) due to 33.2 mM KCl followed by 1 µM CPA. Shown are means ± S.E.; N = 3 in each case. Shown for comparison are the effects of 1 µM CPA on [Ca++]i elevation and contraction (open bars; from fig. 1D).

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Effects of CPA on KCl-, U46619-, and PDB-induced contraction in rat aorta. Absolute increases in contractile force (mN) induced by 1 µM CPA in the presence of 33.2 mM KCl, 1 µM CPA in the presence of 10 nM U46619 plus 10 µM verapamil (Verap.) and 1 or 10 µM CPA in the presence of 10 nM PDB plus 10 µM verapamil. Data were derived from figs. 2 and 4 to 6. Shown are means ± S.E.; N = 3 in each case, except N = 4 for U46619. * Significantly less than other values;  significantly greater than KCl and U46619;  significantly greater than other values.

Verapamil and Ni⁺⁺. Verapamil (10 µM) had little effect on 1 µM CPA-elevated [Ca⁺⁺]_i (fig. 1B). In contrast, in the presence of verapamil, 0.1 mM Ni⁺⁺ abolished the elevated [Ca⁺⁺]_i due to 1 µM CPA (fig. 1B).

PDB. We then investigated whether 1 µM CPA, which did not by itself induce contraction, elicits contraction in the presence of PKC activation by 10 nM PDB. In contrast to the lack of effect of 1 µM CPA on contraction (fig. 1, B and D), 1 µM CPA in the presence of PDB and 10 µM verapamil contracted tissues to 95 ± 14% (mean ± S.E.; N = 3) of the 33.2 mM KCl-induced contraction (figs. 3 and 4). PDB alone elicited minimal contraction and [Ca⁺⁺]_i elevation in the presence of verapamil (fig. 4). A higher PDB concentration (20 nM) induced considerable Ca⁺⁺ sensitization, as 20 nM PDB (N = 2) contracted tissues to 250% of the 33.2 mM KCl-induced contraction, although the R340/380 was elevated to only 126% of the R340/380 elevation due to 33.2 mM KCl. PDB (20 nM) maximally contracted tissues because 100 nM PDB contracted tissues to a similar magnitude as 20 nM PDB (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effects of 1 µM CPA on [Ca++]i and contraction in the presence of PDB and verapamil in rat aorta. Shown are tracings (A) and cumulative data (B) of simultaneously recorded [Ca++]i elevation (R340/380) and contractile force (mN) due to 1 µM CPA in tissues pretreated with 10 nM PDB followed by 10 µM verapamil. After 1 µM CPA, tissues were challenged with cumulative concentrations of Ni++ (0.1-1 mM). Differences between PDB plus verapamil plus CPA-treated tissues and PDB plus verapamil (1) or PDB plus verapamil plus CPA plus Ni++-treated tissues (2) are shown by the hatched bars. Shown are means ± S.E.; N = 3 in each case. Shown for comparison are the effects of 1 µM CPA on [Ca++]i elevation and contraction (open bars; from fig. 1D). * Significantly greater than PDB plus verapamil (closed bar);  significantly greater than other values (closed bar);  significantly greater than 1 µM CPA (open bar).

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effects of 10 to 20 µM CPA on [Ca++]i and contraction in the presence of PDB and verapamil. Shown are tracings (A-C) and cumulative data (D) of simultaneously recorded [Ca++]i elevation (R340/380) and contractile force (mN) due to 10 to 20 µM CPA in the presence of PDB and verapamil. A, 10 nM PDB was added followed by 10 µM verapamil, 20 µM CPA, and then cumulative concentrations of Ni++ (0.1-1 mM); B, 10 µM CPA was added followed by 10 µM verapamil, 10 nM PDB and then cumulative concentrations of Ni++ (0.1-1 mM); C, 20 µM CPA was added followed by 10 nM PDB, 10 µM verapamil and then cumulative additions concentrations of Ni++ (0.1-2 mM); D, cumulative data on the effects of 10-20 µM CPA in the presence of 10 nM PDB and 10 µM verapamil. Differences between PDB plus verapamil plus CPA-treated tissues (2), and PDB plus verapamil (1) or PDB plus verapamil plus CPA plus Ni++-treated tissues are shown by the hatched bars. Shown are means ± S.E.; N = 3 in each case. Shown for comparison are the effects of 10-20 µM CPA on [Ca++]i elevation and contraction (open bars; 10 µM CPA data from fig. 1D). * Significantly greater than other values (closed bar);  significantly greater than PDB, and PDB plus verapamil (closed bar);  significantly less than 1 and 10-20 µM CPA (open bar); ¶ significantly less than 1 and 2.

U46619. We then tested whether TxA₂ receptor-mediated contraction was also enhanced by 1 µM CPA. The contraction to 10 nM U46619 was slightly, but significantly, enhanced in tissues exposed to 1 µM CPA and verapamil (2.4 ± 0.4 mN; mean ± S.E.; N = 4; figs. 3 and 6). The small magnitude of potentiation was not due to an inability of the tissue to further contract, as maximal force elevation in response to U46619 was 26.3 ± 3.3 mN (mean ± S.E.; N = 3; as reported in Tosun et al., 1998b). Ni⁺⁺ (0.1 mM) abolished the enhanced contraction and [Ca⁺⁺]_i elevation due to U46619 in the presence of verapamil (fig. 6).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Effects of CPA on [Ca++]i and contraction in the presence of U46619 and verapamil. Shown are tracings (A) and cumulative data (B) of simultaneously recorded [Ca++]i elevation (R340/380) and contractile force (mN) due to 10 nM U46619 followed by 10 µM verapamil and then 1 µM CPA. Tissues were then challenged with 0.1 mM Ni++. Shown are means ± S.E. N = 4 in each case. Shown for comparison are the effects of 1 µM CPA on [Ca++]i elevation and contraction (open bars; from fig. 1D). * Significantly greater than U46619 plus verapamil and U46619 plus verapamil plus CPA plus Ni++;  significantly greater than U46619 plus verapamil using Student's paired t test.

	Discussion

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This study suggests that, while [Ca⁺⁺]_i elevated as a result of store-operated Ca⁺⁺ entry is restricted to a noncontractile compartment, [Ca⁺⁺]_i within this compartment can be coupled to contraction by PKC activation (see working model of fig. 7). In support of this conclusion are the observations that 1 µM CPA 1) induced an insignificant amount of contraction, despite elevating [Ca⁺⁺]_i to a similar level as the vasoconstrictors KCl (33.2 mM) and U46619 (10 nM) (fig. 1D) and 2) was capable of eliciting contraction in the presence of PDB (figs. 3 and 4).

View larger version (65K): [in this window] [in a new window]   Fig. 7.   Working model of the mechanisms underlying sarcoplasmic reticulum Ca++-ATPase inhibitor-induced [Ca++]i elevation and contraction. A, Control: Ca++ () leak from the peripheral sarcoplasmic reticulum (SR;  or   ) is taken back up into the SR by Ca++-ATPase (  ). Ca++ extrusion, and exchange processes at the sarcolemma (SL;  ) also assist in maintaining low [Ca++]i. B, CPA (1 µM): Partial inhibition of SR Ca++-ATPase (  ) results in the partial depletion of SR Ca++ and resultant opening of store-operated channels (SOC-1; process ). Ca++ influx via SOC-1 results in the filling of subsarcolemmal restricted compartments bound by SL and peripheral SR (I and II). Ni++ (0.1 mM) blocks Ca++ influx via SOC-1. C, CPA (10 µM): Severe inhibition of SR Ca++-ATPase (  ) results in an even greater SR depletion causing not only the opening of SOC-1 (process 1), but also the opening of SOC-2 (process ) and, thus, further [Ca++]i elevation in compartment II. Ni++ (1 mM) blocks Ca++ influx via SOC-2. In addition, the greater depletion of SR Ca++ can cause sensitization of the contractile elements (process ; see text) which, along with Ca++ within compartment I, results in contraction. The large amount of [Ca++]i elevation due to SR depletion can also cause membrane depolarization, resulting in the opening of voltage operated (L-type) Ca++ channels (VOCC; process ), Ca++ influx, and contraction. D, CPA (1 µM) plus PDB and verapamil: In addition to the processes described in "B", phorbol dibutyrate (PDB) activates PKC within compartment I. This activation sensitizes the contractile elements (process ) which, along with Ca++ within compartment I, results in contraction. E, CPA (10 µM) plus PDB and verapamil: Processes described in C and D occur. F, CPA (10 µM) plus verapamil, PDB and Ni++ (0.1 mM): 0.1 mM Ni++ abolishes contraction due to CPA plus PDB, despite only partially decreasing the elevated [Ca++]i. See text for further explanation.

Although the mechanism underlying the PKC-mediated coupling between [Ca⁺⁺]_i restricted to this noncontractile compartment and contraction is not entirely clear, the coupling is dependent on [Ca⁺⁺]_i within this compartment. This conclusion is based on the observations that, in the presence of verapamil, 0.1 mM Ni⁺⁺ 1) abolished the 1 µM CPA plus PDB-induced contraction and [Ca⁺⁺]_i elevation (fig. 4) and 2) abolished the 10 and 20 µM CPA plus PDB-induced contraction, and decreased the magnitude of [Ca⁺⁺]_i elevation by the same amount as the decrease due to 0.1 mM Ni⁺⁺ in 1 µM CPA-challenged tissue exposed and unexposed to PDB (figs. 4 and 5). It is unlikely that 0.1 mM Ni⁺⁺ inhibited the contractions through a direct intracellular effect because, although Ni⁺⁺ quenched fura-2 fluorescence in human neutrophils treated with ionomycin, Ni⁺⁺ 1) at concentrations as high as 5 mM did not quench fura-2 fluorescence in untreated cells and 2) prevented Mn⁺⁺ quenching of fura-2 fluorescence (Merritt et al., 1989).

We further propose that the PKC-mediated coupling between contraction and the 0.1 mM Ni⁺⁺-sensitive CPA-induced [Ca⁺⁺]_i elevation observed in a noncontractile compartment may result from colocalization of PKC within this compartment (fig. 7). This suggestion is based on the consideration that if PKC were not restricted to this compartment, then the CPA plus PDB-induced contraction would have been maintained by the [Ca⁺⁺]_i remaining after 0.1 mM Ni⁺⁺ exposure in 10 and 20 µM CPA-challenged tissues (fig. 5).

Furthermore, the PKC-mediated coupling between this compartmentalized [Ca⁺⁺]_i and contraction does not result from enhanced [Ca⁺⁺]_i because 1) despite the enhanced [Ca⁺⁺]_i elevation due to 1 µM CPA in the presence of PDB (fig. 4), 3 µM CPA alone elevated [Ca⁺⁺]_i to a similar level as 1 µM CPA plus PDB, and yet induced minimal contraction (figs. 1, A and D); 2) the 0.1 mM Ni⁺⁺-sensitive components of the 1 µM CPA-elevated [Ca⁺⁺]_i observed in the presence and absence of PDB were of similar magnitudes (figs. 1B and 4B) and 3) PDB, in the presence of verapamil, potentiated the 10 µM CPA-induced contraction without further elevating [Ca⁺⁺]_i (figs. 5, B and D).

In possible contrast to the suggestion that 1 µM CPA-elevated [Ca⁺⁺]_i is restricted to a noncontractile compartment (see also Abe et al., 1996; Nomura et al., 1997), it may be considered that the 10 to 20 µM CPA-elevated [Ca⁺⁺]_i, which was of a greater magnitude than that due to 1 µM CPA (figs. 1 and 5), may not be entirely restricted to a noncontractile compartment. This possibility is based on the observations that 10 to 20 µM CPA, unlike 1 µM CPA, frequently elicited significant contraction (13 of 20 tissues; figs. 1 and 5). However, the 10 µM CPA-elevated [Ca⁺⁺]_i per se may not support the contraction, because 1) 0.1 mM Ni⁺⁺ abolished the contraction and only partially decreased the elevated [Ca⁺⁺]_i (fig. 1A) and 2) the 0.1 mM Ni⁺⁺-sensitive 1 µM CPA-elevated [Ca⁺⁺]_i is also not coupled to contraction (see above discussion; fig. 1B). Alternatively, we suggest that, similar to the contraction due to 1 µM CPA in the presence of phorbol dibutyrate, the 10 µM CPA-induced contraction may result from both the elevated [Ca⁺⁺]_i sensitive to blockade by 0.1 mM Ni⁺⁺ and PKC activation. This suggestion is based on the observation that chelerythrine, a PKC inhibitor (Herbert et al., 1990), relaxed, albeit only partially, the 10 µM CPA contraction. It should also be noted that Ca⁺⁺ influx via L-type Ca⁺⁺ channels plays a minor role, if any, in the 10 µM CPA-induced [Ca⁺⁺]_i elevation and contraction (figs. 1, A and C and 5B; see also Mikkelsen et al., 1988; Low et al., 1991; Shimamoto et al., 1992; Xuan et al., 1992; Gibson et al., 1994; Sekiguchi et al., 1996; Nomura et al., 1997).

Our results further suggest that the CPA-induced steady-state [Ca⁺⁺]_i elevation is entirely dependent on the influx of extracellular Ca⁺⁺ and, furthermore, the influx occurs largely through store-operated channels. This conclusion is supported by the relative inability of verapamil to decrease CPA-elevated [Ca⁺⁺]_i (figs. 1 and 5B), although 1) 0.1 mM Ni⁺⁺ abolished 1 µM CPA-elevated [Ca⁺⁺]_i and 2) 0.1 mM Ni⁺⁺ partially decreased and 1 mM Ni⁺⁺ abolished 10 µM CPA-elevated [Ca⁺⁺]_i (fig. 1). Others have also demonstrated that Ni⁺⁺ inhibits store-operated Ca⁺⁺ entry in vascular smooth muscle and endothelial cells (Pacaud et al., 1993; Yamamoto et al., 1995). In addition, 0.4 mM Ni⁺⁺ abolished the 10 µM CPA-induced contraction of mouse anococcygeus (Wayman et al., 1996), and Ca⁺⁺-free solution prevented SR Ca⁺⁺-ATPase inhibitor-induced [Ca⁺⁺]_i elevation in carotid and femoral arteries from Wistar-Kyoto, spontaneously hypertensive, and stroke-prone spontaneously hypertensive rats (Sekiguchi et al., 1996; Nomura et al., 1997). The lack of extracellular Ca⁺⁺ dependence of thapsigargin-induced [Ca⁺⁺]_i elevation in rabbit inferior vena cava (Chen and van Breemen, 1993), may reflect differences in SR function in different vessels/species. In any case, the present effects of Ni⁺⁺ on CPA-induced [Ca⁺⁺]_i elevation and contraction further suggest that the influx of Ca⁺⁺ via store-operated channels may be specifically coupled to noncontractile compartments sensitive to blockade by low (0.1 mM) and high (1 mM) Ni⁺⁺ concentrations (fig. 7).

Although the contribution of [Ca⁺⁺]_i elevated as a result of store-operated Ca⁺⁺ entry to agonist-induced contraction is not clear, this possibility may be supported by the present observation that 1 µM CPA potentiated 10 nM U46619-induced contraction (fig. 6). It is unlikely that this potentiation resulted from inhibition of the buffering capacity of the superficial SR (see van Breemen et al., 1995, for review), as KCl-induced contraction was not potentiated by 1 µM CPA (fig. 2).

In summary, this study demonstrates that elevated [Ca⁺⁺]_i due to store depletion is dissociated from contraction. Furthermore, the portion of the elevated [Ca⁺⁺]_i sensitive to blockade by 0.1 mM Ni⁺⁺ can be coupled to contraction via PKC activation. Thus, the contractile response to agonists that deplete Ca⁺⁺ stores, possibly via either inositol phosphate formation and/or Ca⁺⁺-ATPase inhibition (Pernollet et al., 1995; Miwa et al., 1997), as well as activate PKC, may be mediated in part through store-operated Ca⁺⁺ entry. The portion of the elevated [Ca⁺⁺]_i remaining after 0.1 mM Ni⁺⁺ and not associated with contraction may be associated with a different function, such as cell growth.