Introduction

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Although the agonist-induced influx of extracellular Ca⁺⁺ via L-type (voltage-gated) Ca⁺⁺ channels plays an important role in contraction of vascular smooth muscle, some evidence suggests that Ca⁺⁺ influx via channels other than L-type may also mediate contraction. For example, blockers of L-type Ca⁺⁺ channels only partially inhibited norepinephrine-induced contraction in rabbit ear artery (Casteels and Droogmans, 1981; see also references in Bolton, 1979). In addition, Morel and Godfraind (1991) suggested that Ca⁺⁺ influx via non-L-type Ca⁺⁺ channels may contribute to agonist-induced contraction, because nisoldipine, an L-type Ca⁺⁺ channel blocker, only partially inhibited norepinephrine-induced ⁴⁵Ca⁺⁺ influx and the associated contraction in rat aorta. Ca⁺⁺ influx via nonselective cation channels also apparently plays a role in contraction, at least with respect to contraction in response to endothelin-1, because the contraction was inhibited by Ni⁺⁺, a blocker of these channels (Blackburn and Highsmith, 1990; Chen and Wagoner, 1991; Shetty and DelGrande, 1994; Zuccarello et al., 1996).

This study was undertaken based on two related issues. First, the relationship between agonist-elevated [Ca⁺⁺]_i via non-L-type Ca⁺⁺ channels and contraction has not been reported with indicators of [Ca⁺⁺]_i. Thus, the effects of blockers of cation channels on agonist-elevated [Ca⁺⁺]_i levels have not been clarified. Second, whether extracellular Ca⁺⁺ influx contributes to TxA₂ receptor-mediated contraction in rat aorta is controversial (Dorn and Becker, 1993; Kurata et al., 1993). Thus, this study tests the hypothesis that Ca⁺⁺ influx via L-type, as well as non-L-type Ca⁺⁺ channels, is partly responsible for TxA₂ receptor-mediated contraction in vascular smooth muscle. This hypothesis was investigated by simultaneously measuring changes in [Ca⁺⁺]_i and contraction in rat aorta in response to the selective TxA₂ receptor agonist, U46619. Our results indicate that TxA₂ receptor-mediated contraction of vascular smooth muscle depends on the influx of extracellular Ca⁺⁺ via both L-type and non-L-type Ca⁺⁺ channels, as well as a mechanism independent of [Ca⁺⁺]_i elevation.

	Materials and Methods

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Rats (Sprague-Dawley, male, 250-350 g) were asphyxiated with CO₂, and the thoracic aorta was 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.

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. Neostigmine was added to prevent the cleavage of the acetoxymethyl group of fura-2/AM by extracellular esterases and, thus, rendering the fura-2 membrane impermeable (Grynkiewicz et al., 1985; Gilbert et al., 1991). Probenecid was used to block fura-2 sequestration into cytosolic organelles and leakage out of the cell (Di Virgilio et al., 1989). Pluronic F127 was added to increase the solubility of fura-2/AM in the incubation solution (Poenie et al., 1986).

Tissue was placed under 20 mN resting tension, under which maximal contraction to 1 µM U46619 and 1 µM norepinephrine was achieved, and was incubated in pregassed Krebs-Ringer bicarbonate solution in the dark for 2.5 to 3 h at 25°C with sonication applied external to the cuvette (without perfusion). 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 (pH 7.4-7.5) containing 3 µM indomethacin and 1 mM probenecid, and allowed to equilibrate for 30 min before addition of the agent. Indomethacin was included to prevent the release of cyclooxygenase products by U46619 (Jeremy and Dandona, 1989).

The procedures/agents involved with fura-2/AM loading, as well as probenecid in the perfusion buffer, did not alter contraction, because the EC₅₀ values for U46619-induced contraction of fura-2-loaded tissue and of control tissue (not subjected to loading and not exposed to probenecid during perfusion) were similar (4.6 and 6.1 nM, respectively). Contractile force was measured simultaneously with [Ca⁺⁺]_i (see below) and reported in milliNewtons.

For the measurement of [Ca⁺⁺]_i, 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 by use of a PTI Deltascan-1 spectrofluorometer configured for front-face fluorescence (Photon Technology International, South Brunswick, NJ). The ratio of 340 to 380 nm excitation (R_340/380) is reported as a relative measure of free [Ca⁺⁺]_i.

Absolute levels of [Ca⁺⁺]_i are not reported because of the uncertainty of the conventional calibration method in intact tissue. However, to compare our results with those in the literature, we reported previously, based on a limited series of experiments, that basal and 0.1 µM U46619-elevated [Ca⁺⁺]_i were approximately 50 and 200 nM, respectively (Tosun et al., 1997). [Ca⁺⁺]_i was calculated as follows. Maximal and minimal R_340/380 were determined by addition of 10 µM ionomycin, followed by Ca⁺⁺-free solution containing 2 mM EGTA, respectively. MnCl₂ (5 mM) was added at the end of each experiment to determine autofluorescence which was subtracted from the experimental values. [Ca⁺⁺]_i was then calculated assuming an apparent dissociation constant (K_d) of the fura-2/Ca⁺⁺ complex of 224 nM and using the formula [Ca⁺⁺]_i = K_d × (R  R_min)/(R_max  R) × Sf2/Sb2 (Grynkiewicz et al., 1985). Sf2/Sb2 is a correction factor equal to the ratio of the fluorescence intensities at 380 nm excitation wavelength of the Ca⁺⁺-free fura-2 in the presence of the Ca⁺⁺ chelator, EGTA and Ca⁺⁺-saturated fura-2 in the presence of the Ca⁺⁺ ionophore, ionomycin (Grynkiewicz et al., 1985). This value was determined separately for each experiment and was 2.10 ± 0.08 (mean ± S.E.M.; n = 10).

Statistical methods. Statistical significance between multiple and two means was determined with analysis of variance followed by the Newman-Keuls test, and unpaired Student's t test, respectively. Significance was accepted at P = .05. Shown are means ± S.E.M. n represents the number of animals. Values of maximal effect (E_max) and 50% effective concentration (EC₅₀) were derived for each cumulative concentration-response curve with an iterative nonlinear least squares program (KaleidaGraph by Synergy Software, Reading, PA). Geometric means of the EC₅₀ values (pD₂) were compared.

Materials. Reagent sources were as follows: Biomol Research Laboratories Inc. (Plymouth Meeting, PA), verapamil; Calbiochem-Novabiochem (San Diego, CA), SKF96365; Molecular Probes (Eugene, OR), fura-2/AM, pluronic F-127; Sigma Chemical Co. (St. Louis, MO), cyclopiazonic acid, indomethacin, neostigmine methyl sulfate, nickel chloride, norepinephrine hydrochloride, probenecid; Pharmacia & Upjohn (Kalamazoo, MI), U46619 (gift).

	Results

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Typical tracings of simultaneous changes in [Ca⁺⁺]_i and contraction in response to cumulative concentrations of U46619 are shown in figure 1. EC₅₀ values for U46619-induced [Ca⁺⁺]_i elevation and contraction were similar [EC₅₀ values: 5.5 (pD₂ = 8.25 ± 0.02) and 6.1 nM (pD₂ = 8.21 ± 0.02), respectively; means ± S.E.M.; n = 3; figure 2A]. U46619 (0.1 µM) maximally elevated [Ca⁺⁺]_i, whereas U46619 concentrations greater than 0.1 µM appear necessary to elicit maximal contraction (figs. 1 and 2A). Maximal contraction was 26.3 ± 3.3 mN (calculated by nonlinear curve fitting; mean ± S.E.M.; n = 3).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Tracings of the effects of cumulative U46619 concentrations on [Ca++]i and contraction in rat aorta. Shown are tracings of simultaneous changes in [Ca++]i and contraction in response to cumulative concentrations of U46619. [Ca++]i and contraction are expressed as R340/380 and mN, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Concentration-response relationship for U46619-induced [Ca++]i elevation and contraction in rat aorta. (A) Aorta was challenged with cumulative U46619 concentrations and simultaneous changes in [Ca++]i and contraction were recorded. (B) The data in panel A is replotted as the relationship between [Ca++]i and contraction. The arrow represents a linear fit of the [Ca++]i elevation and contraction caused by 1 to 7 nM U46619. [Ca++]i and contraction are expressed as percents of the maximal response (Emax). Shown are means ± S.E.M. n = 3 in each case.

The relationship between U46619-elevated [Ca⁺⁺]_i and contractile force is shown in figure 2B. U46619 concentrations at the lower portion of the concentration-response relationship (< 10 nM; fig. 2A) yielded an approximately linear relationship between [Ca⁺⁺]_i elevation and contractile force (fig. 2B). At higher U46619 concentrations (10 nM), greater contractile force was achieved than predicted by the linear relationship between [Ca⁺⁺]_i elevation and the contractile force observed at the lower U46619 concentrations (fig. 2B).

L-type Ca⁺⁺ channels. We then investigated the role of extracellular Ca⁺⁺ influx via L-type Ca⁺⁺ channels in TxA₂ receptor-mediated contraction. Verapamil (1 µM), an L-type Ca⁺⁺ channel blocker, inhibited U46619-induced contraction and [Ca⁺⁺]_i elevation (figs. 3 and 4). At a lower U46619 concentration (0.01 µM), verapamil inhibited the resulting [Ca⁺⁺]_i elevation and contraction to similar magnitudes (~50%; fig. 5). At higher U46619 concentrations (0.1 and 1 µM), although verapamil induced a smaller inhibitory effect on [Ca⁺⁺]_i elevation (~30%), an even smaller relaxant effect was observed (~5-10%; fig. 5). Increasing the verapamil concentration to 10 µM did not induce further inhibition of the 0.01, 0.1 and 1 µM U46619-induced [Ca⁺⁺]_i elevation and contraction (data not shown).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Tracings of the effects of verapamil treatment before or after 0.01 µM U46619 challenge on [Ca++]i elevation and contraction in rat aorta. Shown are tracings of simultaneous changes in [Ca++]i and contraction in aorta (A) challenged with 0.01 µM U46619, followed by 1 µM verapamil, 1 mM Ni++ and then 1 µM norepinephrine, and (B) treated with 1 µM verapamil for 30 min before challenge with 0.01 µM U46619, followed by 1 mM Ni++, and then 1 µM norepinephrine. Tracings shown in A and B were obtained from strips from the same aorta. [Ca++]i and contraction are expressed as R340/380 and mN, respectively.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Effects of verapamil treatment before or after U46619 challenge on [Ca++]i elevation and contraction in rat aorta. As in figure 3B, aorta was treated with 1 µM verapamil (pre-Ver.) for 30 min, followed by 0.01 µM U46619. After attainment of plateau increases in [Ca++]i and contraction, tissues were challenged with 1 mM Ni++. As in figure 3A, other vessels were exposed to 0.01 µM U46619, and after attainment of plateau increases in [Ca++]i and contraction, challenged with 1 µM verapamil (post-Ver.), followed by 1 mM Ni++. [Ca++]i and contraction are expressed as R340/380 and mN, respectively. Shown are means ± S.E.M. n = 4-6. *Significantly greater than verapamil-, and verapamil plus Ni++-treated tissue.  Significantly less than corresponding verapamil-treated tissue.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Effects of verapamil and Ni++ on U46619-induced [Ca++]i elevation and contraction in rat aorta. Aorta was exposed to 0.01, 0.1 and 1 µM U46619 (see fig. 3A). After attainment of plateau increases in [Ca++]i and contraction, tissue was challenged with 1 µM verapamil, followed by 1 mM Ni++. [Ca++]i and contraction are expressed as percents of the plateau U46619-induced increases in [Ca++]i and contraction before verapamil challenge. Shown are means ± S.E.M. n = 3 in each case. *Significantly greater than verapamil-treated tissue. Significantly greater than verapamil plus Ni++-treated tissue.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Tracings of the effects of verapamil on KCl-induced [Ca++]i elevation and contraction in rat aorta. Shown are tracings of simultaneous changes in [Ca++]i and contraction in response to 33.2 mM KCl (final concentration), and the effects of 10 µM verapamil on these changes. [Ca++]i and contraction are expressed as R340/380 and mN, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   Effects of EGTA on U46619-induced [Ca++]i elevation in rat aorta. Shown is a tracing of changes in [Ca++]i elevation in aorta in response to 0.1 µM U46619 2 min after addition of 5 mM EGTA. After EGTA exposure, tissue was washed with normal Krebs-Ringer bicarbonate solution () and then exposed to 1 µM SQ29548, a TxA2 receptor antagonist (Dorn et al., 1992), as indicated.

Non-L-type Ca⁺⁺ channels. Ni⁺⁺ (1-2 mM), a nonselective blocker of cation channels, abolished the U46619-elevated [Ca⁺⁺]_i that remained after administration of verapamil (figs. 3-5). Ni⁺⁺ also further inhibited, but did not abolish, the U46619-induced contraction that remained after administration of verapamil (figs. 3-5). Approximately 20, 60 and 60% of the 0.01, 0.1 and 1 µM U46619-induced contraction remained, respectively, after verapamil plus Ni⁺⁺ treatment (fig. 5). Norepinephrine (1 µM) still elicited transient [Ca⁺⁺]_i elevation and contraction in the presence of verapamil plus Ni⁺⁺ and after 0.01 µM U46619 challenge (fig. 3), which suggests that the inhibitory effects of Ni⁺⁺ were not caused by decreased Ca⁺⁺ release. SKF96365 (2 µM), a purported blocker of receptor-operated Ca⁺⁺ entry (Merritt et al., 1990), induced inhibitory effects similar to 1 mM Ni⁺⁺ on the [Ca⁺⁺]_i elevation and contraction caused by U46619 (fig. 8).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Tracings of the effects of verapamil and SKF96365 on U46619-induced [Ca++]i elevation and contraction in rat aorta. Shown are tracings of simultaneous changes in [Ca++]i elevation and contraction in aorta challenged with 0.01 µM U46619, followed by 1 µM verapamil, and 1 and then 2 µM SKF96365 (SKF). [Ca++]i and contraction are expressed as R340/380 and mN, respectively.

Ca⁺⁺ sensitization. Because contractions induced by higher U46619 concentrations were associated with smaller increases in [Ca⁺⁺]_i (fig. 2B), and in the presence of verapamil plus Ni⁺⁺, and verapamil plus SKF96365, U46619-induced contraction was still present whereas [Ca⁺⁺]_i elevation was abolished (figs. 3-5 and 8), we tested whether Ca⁺⁺ sensitization might play a role in the U46619-induced contraction. To prevent U46619-induced [Ca⁺⁺]_i elevation, vessels were pretreated with 10 µM cyclopiazonic acid, which depletes agonist-releasable Ca⁺⁺ through inhibition of sarcoplasmic reticulum Ca⁺⁺-ATPase(Seidler et al., 1989; Uyama et al., 1993), followed by (final concentrations) 10 µM verapamil and 1 mM Ni⁺⁺. It was necessary to add cyclopiazonic acid because preliminary results demonstrated that 10 nM U46619 induced a small increase in [Ca⁺⁺]_i in tissues pretreated with verapamil and Ni⁺⁺ (data not shown). Treatment with cyclopiazonic acid elevated [Ca⁺⁺]_i and variably induced contraction, and subsequent exposure to verapamil and Ni⁺⁺ abolished these changes (Tosun et al., 1998, in press). Despite pretreatment with cyclopiazonic acid, verapamil and Ni⁺⁺, U46619 still induced contraction, although the magnitude of contraction was less than that of untreated (control) tissues (approximately 6, 19 and 23% of the contraction in untreated tissues challenged with 0.01, 0.1 and 1 µM U46619, respectively; fig. 9).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Effects of pretreatment with cyclopiazonic acid, verapamil and Ni++ on U46619-induced [Ca++]i elevation and contraction in rat aorta. Aorta was pretreated with 10 µM cyclopiazonic acid (CPA) for 20 min, followed by cumulative addition of 1 and then 10 µM verapamil. After 50 min, tissues were also exposed to cumulative addition of 0.1, 0.3 and then 1 mM Ni++. Tissues then were challenged cumulatively with the indicated U46619 concentrations. Tissues unexposed to cyclopiazonic acid, verapamil and Ni++ served as controls. Control data are the same as those used to derive the results of figure 2. Shown are means ± S.E.M. Control tissues, n = 3; treated tissues, n = 4. * Significantly less than corresponding control tissue.

	Discussion

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The present study, through the use of simultaneous measurements of contraction and [Ca⁺⁺]_i, and blockers of Ca⁺⁺ entry, clearly assists in the resolution of the controversy regarding the role of extracellular Ca⁺⁺ in TxA₂ receptor-mediated contraction in rat aorta. Specifically, the present results suggest that TxA₂ receptor-mediated contraction depends on Ca⁺⁺ influx. This conclusion is based on the demonstration that blockers of L-type Ca⁺⁺ channels and nonselective cation channels, verapamil and Ni⁺⁺, respectively, abolished TxA₂ receptor-mediated [Ca⁺⁺]_i elevation at both submaximal and maximal U46619 concentrations, and decreased a significant percentage of the associated contractile response (figs. 3-5 and 8). It is unlikely that the inhibitory effects of verapamil were the result of a nonselective action, because similar partial inhibitory effects on TxA₂ receptor-mediated [Ca⁺⁺]_i elevation and contraction were observed at 1 and 10 µM verapamil (present results). Furthermore, the inhibitory effects of Ni⁺⁺ on the [Ca⁺⁺]_i elevation and contraction were not caused by a direct intracellular action, because Ni⁺⁺ concentrations as high as 5 mM did not gain access to the cytosol (Merritt et al., 1989).

The present conclusion that TxA₂ receptor-mediated contraction depends on Ca⁺⁺ influx is in contrast to the conclusion of Dorn and Becker (1993), who suggested that Ca⁺⁺ influx was not necessary to elicit maximal U46619 (0.1 µM)-induced contraction in this vessel and, moreover, depended on Ca⁺⁺ release from internal stores. These discrepancies likely result from the procedures used in the latter study. First, to investigate the dependence of U46619-induced contraction on extracellular Ca⁺⁺, Dorn and Becker (1993) added ethylenediaminetetraacetate to the bathing solution. This procedure did not inhibit U46619-induced maximal contraction (Dorn and Becker, 1993). However, it is not clear whether this procedure in practice lowered the extracellular [Ca⁺⁺] sufficiently to block contraction depending on extracellular Ca⁺⁺. Second, to investigate the dependence of U46619-induced contraction on Ca⁺⁺ release, Dorn and Becker (1993) transiently exposed aorta to ethylenediaminetetraacetate plus ionomycin. Although this procedure inhibited U46619-induced maximal contraction (Dorn and Becker, 1993), it is not clear again whether this procedure depleted Ca⁺⁺ stores in the intact tissue, or whether this procedure may have nonspecifically inhibited contraction.

Consistent with the present conclusion that U46619-induced contraction in rat aorta depends on extracellular Ca⁺⁺, as based on the use of Ca⁺⁺ entry blockers, is the previous demonstration that removal of extracellular Ca⁺⁺ in the presence of EGTA inhibited U46619-induced contraction (Kurata et al., 1993). However, exposure to Ca⁺⁺-free solution containing EGTA can inhibit U46619-induced Ca⁺⁺ release, as demonstrated in intact rat aorta (fig. 7 and Kurata et al., 1993), and cultured rat aorta smooth muscle cells (Furci et al., 1991; Dorn and Becker, 1993). Furthermore, it has been suggested that Ca⁺⁺-free solution containing even low EGTA concentrations may reduce Ca⁺⁺_i stores by removing superficially bound Ca⁺⁺ and, thus, subsequently extracting Ca⁺⁺_i located at the cell membrane surfaces (Guan et al., 1988). Thus, the possibility should be considered that decreased release of Ca⁺⁺ may also contribute to the inhibition of agonist-induced contraction observed after removal of extracellular Ca⁺⁺.

Although the present results demonstrate that verapamil plus Ni⁺⁺ abolished U46619-elevated [Ca⁺⁺]_i, and decreased the associated contraction, the percent of the contractile response depending on Ca⁺⁺ influx is greater at lower magnitudes of contraction. Specifically, contraction at EC_60-70 (0.01 µM U46619) and EC₁₀₀ (1 µM U46619) levels were inhibited by verapamil plus Ni⁺⁺ by 80 and 40%, respectively (fig. 5).

The dependence of the U46619-induced contraction on Ca⁺⁺ influx via L-type Ca⁺⁺ channels is also greater at lower magnitudes of contraction, because verapamil inhibited 0.01 and 1 µM U46619-induced contraction by 45 and 10%, respectively (fig. 5). Furthermore, the verapamil inhibition of the 0.01 and 1 µM U46619-induced contraction was associated with decreased elevations of [Ca⁺⁺]_i of 50 and 30%, respectively, although these values were not significantly different (fig. 5). Although the decreases in [Ca⁺⁺]_i elevation are approximations because of the nonlinear relationship between R_340/380 and [Ca⁺⁺]_i (Williams et al., 1985), the deviation from linearity over the [Ca⁺⁺]_i range of the present study (basal and 0.1 µM U46619-elevated [Ca⁺⁺]_i of ~50 and 200 nM, respectively; Tosun et al., 1997) is minimal. Indeed, we have observed in a cell-free system titrating 1 µM fura-2 acid with Ca⁺⁺ buffer (with appropriate combinations of Ca⁺⁺EGTA and K₂EGTA) that the R_340/380/[Ca⁺⁺]_i relationship is essentially linear over this Ca⁺⁺ concentration range (Tosun M, Paul RJ and Rapoport RM, unpublished observation).

The ability of higher U46619 concentrations to induce greater contraction that was not associated with a further increase in [Ca⁺⁺]_i (fig. 5) may result from Ca⁺⁺ sensitization mechanisms. This suggestion is supported by the present demonstration that U46619 concentrations 10 nM induced contractions that were associated with smaller increases in [Ca⁺⁺]_i (fig. 2B), and under conditions in which [Ca⁺⁺]_i elevation was not observed, i.e., pretreatment with cyclopiazonic acid to deplete agonist-sensitive Ca⁺⁺ stores (Golovina and Blaustein, 1997), and verapamil and Ni⁺⁺ to block the influx of extracellular Ca⁺⁺ (fig. 9). The inability to observe elevated [Ca⁺⁺]_i in response to 0.1 and 1 µM U46619 in the presence of cyclopiazonic acid, verapamil and Ni⁺⁺ was not caused by the lack of sensitivity of the [Ca⁺⁺]_i measurements, because contractions of similar magnitudes induced by U46619 in control tissues elicited significant increases in [Ca⁺⁺]_i (figs. 2 and 9). Others have also suggested that Ca⁺⁺ sensitization is involved in TxA₂ receptor-mediated contraction based in part on measurements of [Ca⁺⁺]_i and contraction in response to U46619 in vessels exposed to Ca⁺⁺-free solution (Himpens et al., 1990; Kurata et al., 1993).

Another major finding of the present study is the apparent contrast between the effectiveness of the Ca⁺⁺ channel blockers to decrease U46619-elevated [Ca⁺⁺]_i in intact rat aorta (present study) versus cultured rat aorta smooth muscle cells (Dorn and Becker, 1993). Although the present results demonstrate that addition of 1 µM verapamil before or after 0.01 µM U46619 challenge decreased the [Ca⁺⁺]_i elevation by 50% (figs. 3 and 4), and after 1 µM U46619 challenge decreased the [Ca⁺⁺]_i elevation by 30% (fig. 5). The elevated [Ca⁺⁺]_i caused by 2 µM U46619 in cultured rat aorta smooth muscle cells was unaltered by pretreatment with 100 µM diltiazem, and tended to be decreased by 100 µM verapamil, although this decrease was not statistically significant (Dorn and Becker, 1993). Furthermore, U46619-induced cation entry was not observed in the cultured cells, with Mn⁺⁺ as a quencher of the fura-2 signal (Dorn and Becker, 1993), whereas the present study demonstrates that Ni⁺⁺ significantly reduced U46619-elevated [Ca⁺⁺]_i in intact tissue. These contrasting results suggest that Ca⁺⁺ handling is greatly altered in cultured vascular smooth muscle cells because certain agonists are no longer able to elicit an influx of extracellular Ca⁺⁺. This alteration in Ca⁺⁺ handling may reflect a change in the smooth muscle cell from contractile to proliferative phenotype. In any case, the present results generally suggest that conclusions regarding possible roles of Ca⁺⁺ in contraction based on [Ca⁺⁺]_i measurements in cultured cells must be viewed with caution.

Finally, this study suggests that agonist-induced contraction of vascular smooth muscle is associated with Ca⁺⁺ influx via non-L-type Ca⁺⁺ channels. This suggestion is supported by the novel demonstration that nonselective blockers of cation channels, in the presence of an L-type Ca⁺⁺ channel blocker, inhibited both TxA₂ receptor-mediated contraction and [Ca⁺⁺]_i elevation (figs. 3-5 and 8). A similar conclusion previously was reached based on the inability of an L-type Ca⁺⁺ channel blocker to completely prevent both norepinephrine-induced ⁴⁵Ca⁺⁺ influx and the associated contraction in rat aorta (Morel and Godfraind, 1991). Furthermore, agonist-induced contraction was only partially inhibited by L-type Ca⁺⁺ channels blockers and/or nonselective blockers of cation channels (references in Bolton, 1979; Casteels and Droogmans, 1981; Blackburn and Highsmith, 1990; Chen and Wagoner, 1991; Shetty and DelGrande, 1994; Zuccarello et al., 1996).

In summary, the present study demonstrates that a significant component of the TxA₂ receptor-mediated contraction in rat aorta depends on the influx of extracellular Ca⁺⁺. Ca⁺⁺ influx occurs via both L-type and non-L-type Ca⁺⁺ channels, possibly including store-operated channels. Although the identity of these non-L-type Ca⁺⁺ channels is not known, whether similar downstream contractile mechanisms are triggered by Ca⁺⁺ influx via these Ca⁺⁺ channels must still be investigated.