Introduction

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Endothelin-1 (ET-1) is a potent vasoconstrictor and mitogen of vascular smooth muscle cells (Harrison et al., 1992; Mathew et al., 1996). Smooth muscle cells contain both the ET_A and ET_B receptor subtypes; however, the relative subtype distribution varies depending on the vessel type (Godfraind, 1993; Adner et al., 1998a). In coronary arteries the ET_A receptor subtype predominates; approximately 80% of the contractile response is attributed to the ET_A receptor, whereas ET_B is responsible for the remaining 20% (Dagassan et al., 1996; Elmoselhi and Grover, 1997).

The selective up-regulation of either subtype is enhanced with the progression of atherosclerosis, thrombosis, and cardiac hypertrophy (Wang et al., 1995; Mathew et al., 1996; Stewart, 1998). Evidence suggests either ET receptor subtype may contribute to the enhanced contractile response to ET with arterial injury (Harrison et al., 1992; Wang et al., 1995; Dagassan et al., 1996; White et al., 1998). Coronary artery disease is a leading cause of death in the U.S. population (Russell et al., 1998); therefore, it is important to understand the role of ET-1 receptors in this artery. Few investigators have studied the functional response to ET-1 in a coronary artery injury model. Dagassan et al. (1996) described an up-regulation of ET_B receptors in the left anterior descending (LAD) coronary artery from humans displaying atherosclerosis. Hasdai et al. (1997) also found enhanced ET_B-mediated vasoconstriction in the left circumflex coronary artery from hyperlipidemic pigs. In contrast, Katwa et al. (1999) and Wang et al. (1995) demonstrated increased ET_A function in the right coronary artery and the LAD coronary artery, respectively, with arterial injury. However, Godfraind (1993) found that there is a high degree of heterogeneity of ET_A and ET_B receptors along the length of the LAD. ET-1-induced contractions are mediated almost entirely by ET_A receptors at the distal end of the LAD, whereas ET_B receptors are partially responsible for the contraction at the proximal end. To eliminate this large degree of heterogeneity we used the right coronary artery because it has a predominant ET_A receptor population that is responsible for mediating vasoconstriction (Bacon and Davenport, 1996; Schiffrin and Touyz, 1998). In this study we hypothesized that arterial injury (i.e., organ culture) would enhance ET receptor-mediated vasoconstriction and myoplasmic calcium (Ca_m) responses in the porcine right coronary artery.

Organ culture is a technique that has been used to study cell proliferation in vessels (Gotlieb and Boden, 1984; Newby and Zaltsman, 1999; Voisard et al., 1999). Newby and Zaltsman (1999) recently demonstrated medial proliferation of smooth muscle cells within the organ-cultured rabbit and pig aorta, as well as human saphenous veins. They indicate that organ culture greatly parallels chemotaxis and matrixes remodeling in animal models because of the intact interactions that are present within organ culture. Recently, Voisard et al. (1999) pointed out that organ culture is a valuable model that mimics the injury response of atherosclerosis.

This study uniquely demonstrates that the Ca_m response mediated by ET_A or ET_B receptors in isolated smooth muscle cells is paralleled by the development of isometric tension in both cold-stored and organ-cultured coronary arteries. Our results indicate that the 2-fold increase in Ca_m and tension development to ET-1 with organ culture is attributed to ET_A receptors, and not ET_B receptors.

	Materials and Methods

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Organ Culture and Single-Cell Fura-2 Digital Imaging. Hearts were obtained from local abattoirs and prepared as described in our laboratory (Sturek et al., 1991). Right coronary arteries (distal end) were either cold stored for 4 days at 5°C or organ cultured for 4 days at 37°C in a 95% O₂, 5% CO₂ incubator. The arterial segments that were organ cultured or cold stored were opened longitudinally to expose the lumen and placed with the lumen facing up in a 100-mm Petri dish containing 30 ml of RPMI 1640 (Life Technologies, Grand Island, NY). The RPMI 1640 was changed every 2 days.

6

DNA Imaging. Cold-stored and organ-cultured arteries were fixed in 4% paraformaldehyde and incubated for 24 h in 30% sucrose at (20°C). Arteries were freeze mounted and cut into 7-µm sections. Cross sections were stained with 2.5 × 10⁷ M 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) for 20 min at 37°C to quantitate single-cell DNA content (Kapuscinski, 1995). Imaging of DAPI fluorescence was done using a widefield epifluorescent microscope (Nikon Diaphot, Garden City, NY). Images at three focal planes 0.5 µm apart in the z-axis were acquired for deconvolution analysis. Out-of-focus fluorescence was removed by deconvolution software equipped with a digital signal processing board that used the nearest neighbor algorithm (Vaytek, Inc., Fairfield, IA). DAPI fluorescence was quantitated using Image Pro Plus 3.0 software (Media Cybernetics, Silver Springs, MD).

Isometric Tension Measurements. Similar to the single-cell imaging experiments, porcine right coronary arteries were sectioned and placed in a Petri dish containing RPMI 1640 for cold storage or organ culture. After 4 days, vessel segments were sectioned into 5-mm rings and the endothelium removed using a toothpick. Rings were also further studied within 3 h after the sacrifice of the pig. The absence of an intact endothelium was confirmed by the lack of a relaxation response to 1 × 10⁷ M bradykinin. Rings were mounted via two stainless steel wire supports in 25 ml of isolated organ baths maintained at 37°C, and the physiological salt solution (PSS) aerated with a 95% O₂, 5% CO₂ mixture. The two support wires were connected to an isometric force transducer (Grass Medical Instruments, Quincy, MA) and a linear displacement micrometer (Mitutoyo, MTI Corp., Paramus, NJ). Force generation was amplified by a custom-designed amplifier (Technical Resources Core Facility, Dalton Cardiovascular Research Center), and the data acquired by a computer equipped with an analog-to-digital converter and Labtech Acquire software (Laboratory Technologies Corp., Wilmington, MA). Rings were set near their optimum length-tension relationship by progressively lengthening each ring and subsequently contracting it with the addition of a 60 × 10³ M KCl solution to the organ bath. This procedure was repeated until the active force generated was no more than 10% greater than at the previous length. Rather than stretching all vessel rings to the same passive tension, the method of setting each vessel at optimal length was chosen to reduce variability and maximize the response of each vessel ring. Rings were initially exposed to an 80 × 10³ M KCl solution (80K) before exposing them to ET-1. In those experiments using antagonists, cells were incubated for 45 min with the appropriate concentration of the antagonist before generating a cumulative concentration-response relationship to ET-1 in half-log increments (3 × 10¹⁰-3 × 10⁷ M).

Solutions. Isolated smooth muscle cells within the superfusion chamber were continually superfused with PSS containing 138 × 10³ M NaCl, 5 × 10³ M KCl, 2 × 10³ M CaCl₂, 1 × 10³ M MgCl₂, 10 × 10³ M HEPES, 10 × 10³ M glucose, titrated to pH 7.4 (with NaOH). The depolarizing solution (80K or 25 × 10³ M KCl solution) was composed of 65 × 10³ M NaCl, 80 or 25 × 10³ M KCl, 2 × 10³ M CaCl₂, 1 × 10³ M MgCl₂, 10 × 10³ M HEPES, 10 × 10³ M glucose, titrated to pH 7.4 (with NaOH). For isometric tension measurements the PSS was similar to that for single cells except that 24 × 10³ M NaHCO₃ was added and NaCl was reduced by 24 × 10³ M. The stock solution of potassium chloride (Sigma Chemical Co.) was prepared in distilled water. Fura-2-acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR), BQ123 (Peptides International, Louisville, KY), PD145065 (Sigma Chemical Co.), and BQ788 (Peptides International) were prepared in dimethyl sulfoxide (Sigma Chemical Co.). DAPI (Molecular Probes, Inc.) and endothelin-1 (Peninsula Labs, Inc., San Carlos, CA) were prepared in N,N-dimethyl-formamide (Sigma Chemical Co.) and 0.01 N acetic acid, respectively. Bosentan was a gift from Roche Pharmaceuticals (Nutley, NJ). Drugs to be used within the superfusion system were diluted from stock solutions into PSS and superfused at a rate of approximately 2 ml/min.

Statistical Analysis. Data were expressed as the mean ± S.E. for the number (n) of single cells (fura-2 imaging) or animals (isometric tension) within each group. Analysis of data was done by either a one-way ANOVA or a Kruskal-Wallis one-way ANOVA followed by Bonferroni's test or Dunn's test, respectively, when more than two groups were present. A paired or unpaired Student's t test was used when comparing only two groups. Smooth muscle cells that were identified as "responders" to an agonist were defined as those cells whose response to an agonist was at least three standard deviations above the baseline for 5% of the time exposed to the agonist (P < .01). The percentage of responders was analyzed using the chi square distribution. Statistical analyses of the data were performed using SigmaStat (Jandel Scientific Software, San Rafael, CA). The pD₂ values (log EC₅₀) were calculated and analyzed using GraphPad Prism 2.0 (GraphPad Software Inc., San Diego, CA). Significance was defined as P < .05.

	Results

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A concentration-response relationship (3 × 10¹⁰-3 × 10⁷ M) to ET-1 was generated in fresh (n = 8) and cold-stored arterial rings (n = 18). The pD₂ values (log EC₅₀) in fresh and cold-stored rings (7.96 ± 0.23 and 7.60 ± 0.13, respectively) were not significantly different. Additionally, isometric tension development (96 ± 12 and 121 ± 8%, respectively) to a maximal concentration (T_max) of ET-1 (3 × 10⁷ M) was not significantly different between fresh and cold-stored rings. We have previously demonstrated (M. Sturek, B. Hill, and B. Wamhoff, unpublished observations) using isolated single smooth muscle cells from fresh and cold-stored arteries that there is no difference in the basal Ca_m concentration or the peak Ca_m response to 5 × 10³ M caffeine or 80K between groups, suggesting cold-stored arteries serve as an adequate paired time control for those arteries that were organ cultured.

A cumulative concentration-response relationship to ET-1 was generated from 3 × 10¹⁰ to 3 × 10⁷ M in arterial rings that had been organ cultured (n = 20). As demonstrated in Fig. 1, organ-cultured rings demonstrated a significant increase (227 ± 20%) in their T_max to 3 × 10⁷ M ET-1 compared with cold-stored rings (121 ± 8%; n = 18). However, there was no difference in potency (pD₂) to ET-1 between cold-stored and organ-cultured rings (7.60 ± 0.13 and 7.56 ± 0.03, respectively). The 2-fold increase in the T_max of organ-cultured rings indicates organ culture may enhance the physiological function of ET receptors to elicit a contraction. To identify whether either the ET_A or ET_B subtype contributes to this increased tension development, a concentration-response relationship was generated to ET-1 in the absence and presence of bosentan (ET_A and ET_B antagonist), PD145065 (ET_A and ET_B antagonist), BQ123 (ET_A antagonist), and BQ788 (ET_B antagonist).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Organ-cultured arterial rings display an enhanced contractile response to ET-1 compared with cold-stored rings. ET-1-induced tension development is expressed as a percentage (%) of the maximum contractile response generated by 80K. *, represents a significant (P < .05) difference from the cold-stored rings.

Bosentan (10⁶ M), a nonselective ET antagonist, inhibited isometric tension development to ET-1 in organ-cultured and cold-stored arterial rings as indicated by the significant rightward shift of the concentration-response relationship to ET-1 (Fig. 2; Table 1). In cold-stored rings, the pD₂ value in the absence of bosentan was 7.95 ± 0.10. The pD₂ could not be calculated in cold-stored rings in the presence of bosentan because a sigmoidal concentration-response relationship was not generated. In organ-cultured rings, the pD₂ values in the absence and presence of bosentan were 7.57 ± 0.05 and 6.78 ± 0.14, respectively. Bosentan did not significantly affect the T_max in organ-cultured and cold-stored rings. In addition, another ET_A and ET_B antagonist, PD145065 (10⁶ M), similarly shifted the ET-1 concentration-response relationship to ET-1 to the right in both cold-stored and organ-cultured rings (data not shown).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Antagonism of contractions to ET-1 by bosentan in organ-cultured (OC) and cold-stored (CS) arterial rings. A cumulative concentration-response relationship was generated to ET-1 (3 × 1010-3 × 107 M) in the absence or presence of 106 M bosentan in organ-cultured and cold-stored rings. The ET-1-induced tension development is expressed as a percentage (%) of the maximum contractile response generated by 80K. *, indicates a significant (P < .05) difference in tension development from OC + bosentan at each respective ET-1 concentration. , indicates a significant difference in tension development from CS + bosentan.

Selective inhibition of isometric tension development was determined using the selective ET_A antagonist BQ123 (Table 1). In cold-stored rings, only 10⁵ M BQ123 (Fig. 3A) significantly inhibited the concentration-response relationship to ET-1; no inhibition was demonstrated using 10⁶ M BQ123 (Fig. 3B). In contrast, both 10⁶ M (Fig. 3B) and 10⁵ M (Fig. 3A) BQ123 shifted the concentration-response relationship (i.e., pD₂ values) to the right 5- and 7-fold, respectively, in organ-cultured rings. As shown by Fig. 4, selective inhibition of the ET_B receptor with BQ788 (10⁵ or 10⁶ M BQ788) did not alter the pD₂ value or the T_max in the cold-stored and organ-cultured arterial rings (Table 1). Using cardiac fibroblasts we have confirmed the antagonistic activity of BQ788, which contain both ET_A and ET_B receptors (data not shown).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Antagonism of contractions to ET-1 by BQ123 in OC and CS arterial rings. A, inhibition of ET-1-induced tension development by 105 M BQ123 in organ-cultured and cold-stored rings. B, inhibition of ET-1-induced tension development by 106 M BQ123 in organ-cultured rings. A cumulative concentration-response relationship was generated to ET-1 (3 × 1010-3 × 107 M) in the absence or presence of either 105 or 106 M BQ123 in organ-cultured and cold-stored rings. The ET-1-induced tension development is expressed as a percentage (%) of the maximum contractile response generated by 80K. *, indicates a significant (P < .05) difference in tension development from OC + BQ123 at each respective ET-1 concentration. , indicates a significant difference in tension development from CS + BQ123.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   There is no antagonism of ET-1-induced contractions by BQ788 in OC and CS arterial rings. A cumulative concentration-response relationship was generated to ET-1 (3 × 1010-3 × 107 M) in the absence or presence of 10 5 M BQ788 in organ-cultured and cold-stored rings. The ET-1-induced tension development is expressed as a percentage (%) of the maximum contractile response generated by the 80K.

To further investigate the altered contractile function of ET receptors, smooth muscle cells were enzymatically isolated from both cold-stored and organ-cultured arteries, and the Ca_m response measured to 3 × 10⁸ M ET-1 in the absence and presence of 10⁵ M bosentan, BQ123, and BQ788 (Fig. 5, A and B). We used 3 × 10⁸ M ET-1 because this was the approximate EC₅₀ value (pD₂ = 7.5) for the concentration-response relationship generated to ET-1 in cold-stored and organ-cultured arterial rings. Previous observations indicate that not all cells respond to ET-1 in the absence of an antagonist; however, in evaluating the effect of antagonists on the Ca_m response all cells (both responders and nonresponders) were pooled. All cells were pooled because it would not be known whether the nonresponders to ET-1 inherently did not respond to ET-1, or whether the antagonist effectively inhibited the Ca_m response to ET-1. Because our reported results include both responders and nonresponders to ET-1, the reported Ca_m response to ET-1 will be lower (due to the "dilution" of the Ca_m response to ET-1 by the nonresponders) than if calculated for just those cells responding to ET-1. Similar to the development of isometric tension in arterial rings, cells from organ-cultured arteries had a significantly increased Ca_m response to ET-1 (0.50 ± 0.05 ratio units; n = 20) compared with those cells from cold-stored arteries (0.19 ± 0.03 ratio units; n = 24). Bosentan significantly decreased the ET-1-induced Ca_m response in cells from both cold-stored (0.09 ± 0.02 ratio units; n = 29) and organ-cultured (0.10 ± 0.03 ratio units; n = 14) arteries compared with the paired control cells without bosentan (0.19 ± 0.03 and 0.50 ± 0.05 ratio units, respectively). In cells from organ culture, BQ123 also significantly decreased the Ca_m response (0.13 ± 0.04 ratio units; n = 14) to similar levels as bosentan. BQ788 had little effect (P > .05) on the ET-1 response (0.37 ± 0.05 ratio units; n = 18) in cells from organ culture. In cells from cold-stored arteries, both BQ123 (0.19 ± 0.04 ratio units; n = 19) and BQ788 (0.27 ± 0.03 ratio units; n = 20) did not significantly decrease the Ca_m response to ET-1.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Inhibition of ET-1-induced increases in myoplasmic calcium by several selective ET-1 antagonists. A, representative tracing from a single smooth muscle cell isolated from an organ-cultured artery and exposed to 3 × 108 M ET-1. An example of smooth muscle cells to be digitally imaged is demonstrated in the inset. The black line surrounding a single cell represents the enclosed "area of interest" (i.e., cell area) to be measured and analyzed. B, antagonism of ET-1-induced increases in myoplasmic calcium in isolated smooth muscle cells from cold-stored and organ-cultured arteries. In those experiments using antagonists, cells were incubated for 1 h with 105 M of the antagonist before exposing them to 3 × 108 M ET-1. *, indicates a significant (P < .05) difference from the no antagonist condition within the organ-cultured or cold-stored group. , indicates a significant difference from the cold-stored, no-antagonist group.

In agreement with data generated by both isometric tension recordings and single-cell Ca_m responses to ET-1 in the presence of BQ788, the selective ET_B receptor agonist sarafotoxin 6C (10⁸ M), did not significantly increase Ca_m above basal levels in isolated cells from cold-stored and organ-cultured arteries (Fig. 6). Sarafotoxin 6C has been extensively used as an ET_B agonist in vascular smooth muscle (Adner et al., 1998a; White et al., 1998). We previously found that 10⁸ M sarafotoxin 6C elicited a similar Ca_m response as 10⁷ M sarafotoxin 6C. Previously, we determined that not all cells respond to ET-1 and sarafotoxin 6C; therefore, the Ca_m response was determined only in those cells that responded to the agonists (Fig. 6). In cells isolated from organ-cultured arteries, 75% (15 of 20) responded to ET-1, whereas only 14% (5 of 35) responded to sarafotoxin 6C. However, in cells from cold-stored arteries, 33% (4 of 12) and 35% (9 of 26) of the cells responded to ET-1 and sarafotoxin 6C, respectively. As shown in Fig. 6, ET-1 elicited a significantly increased Ca_m response of 0.47 ± 0.10 ratio units above baseline in isolated cells from organ culture compared with the Ca_m response in cells from cold-stored arteries (0.07 ± 0.03 ratio units). In contrast to ET-1, the Ca_m response to sarafotoxin 6C was significantly decreased with organ culture (0.04 ± 0.03 ratio units) compared with cold storage (0.11 ± 0.04 ratio units).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Organ culture enhances and attenuates the myoplasmic calcium responses to ET-1 and sarafotoxin 6C, respectively, compared with cold storage. Isolated smooth muscle cells from organ-cultured or cold-stored arteries were either exposed to 3 × 108 M ET-1 or 108 M sarafotoxin 6C. *, indicates a significant (P < .05) difference from the organ culture group exposed to ET-1. , indicates a significant difference from the organ culture groups exposed to sarafotoxin 6C.

This study, as well as previous studies by our lab (Wagner-Mann and Sturek, 1991, 1992), indicate that the peak Ca_m response elicited to ET-1 in isolated smooth muscle cells was predominantly due to the release of calcium from the sarcoplasmic reticulum (SR). However, our lab and others (Goto et al., 1989; Wagner-Mann and Sturek, 1991) have shown that ET-1 does appear to induce a very small amount of calcium influx (which has a negligible contribution to the peak Ca_m response) via voltage-gated calcium channels after the initial release of the SR calcium store. In our study, the minimal contribution of calcium influx to the peak Ca_m response is demonstrated by the absence of a sustained "plateau" phase after the initial transient calcium spike (due to SR calcium release; Fig. 5A) after ET-1 application in cells from both cold-stored and organ-cultured arteries. In our preparation, this lack of a measurable sustained calcium influx in response to ET-1 is due to the rapid extrusion of calcium from the cell (Rasmussen et al., 1989; Bowles et al., 1995). We demonstrated that ET-1-induced tension development is, in a large part, due to sustained calcium influx by exposing arterial rings to ryanodine (10⁵ M) for 50 min, which our lab previously documented mobilizes and fully depletes the SR calcium store (Wagner-Mann and Sturek, 1991, 1992). Subsequent to ryanodine, 5 × 10⁸ M ET-1 was applied for 25 min, which induced a significant contractile response.

Cold-stored (Fig. 7A; n = 5) and organ-cultured (Fig. 7B; n = 5) artery sections were stained with DAPI, an indicator of DNA content. DAPI fluorescence has extensively been used to measure nuclear DNA content in cells (Seiler et al., 1993; Kapuscinski, 1995). Previous investigators have confirmed that the relative DAPI fluorescence intensity is directly related to both DNA content (Seiler et al., 1993) and cell proliferation (McCaffrey et al., 1988). There was a significant increase in the amount of DAPI fluorescence in single smooth muscle cells (Fig. 7C) from the cross-sectioned, organ-cultured (164.60 ± 3.55) compared with cold-stored (96.09 ± 2.64) arteries. In addition, DAPI-stained nuclei in organ-cultured cross sections (Fig. 7B) demonstrated a lack of circumferential orientation compared with the circumferentially oriented cold-stored cells (Fig. 7A).

View larger version (64K): [in this window] [in a new window]   Fig. 7.   Arterial cross sections from organ-cultured arteries exhibit increased DNA content. Cold-stored and organ-cultured arteries were cut into 7-µm sections and incubated with 2.5 × 107 M DAPI (DNA stain). A, typical cold-stored artery section stained with DAPI. The white arrows (A and B) indicate DAPI fluorescence of single smooth muscle cells. Cells from cold-stored arteries run circumferentially as indicated by the elongated cell morphology. B, typical organ-cultured artery section stained with DAPI. Compared with A, smooth muscle cells in B are disoriented, which is characteristic of smooth muscle cell migration. C, mean DAPI fluorescence of single smooth muscle cells within each cross section.

	Discussion

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Previous investigators have demonstrated an enhanced expression and function of either the ET_A or ET_B receptor subtype in different models of arterial injury (Wang et al., 1995; Dagassan et al., 1996; Mathew et al., 1996; Hasdai et al., 1997; Stewart, 1998; Katwa et al., 1999). We hypothesized that there is an enhanced physiological function of ET receptors in an organ culture model of the porcine right coronary artery. Although no studies have directly addressed the possibility that smooth muscle cells may respond differently to ET-1 if they are isolated from the artery itself, this study uniquely demonstrates that isolated single smooth muscle cells respond similarly as those smooth muscle cells possessing cell-cell interactions within the vessel wall. Our results demonstrate that both intact arterial rings and isolated smooth muscle cells display an enhanced response (i.e., tension development and Ca_m response, respectively) to ET-1 with organ culture. Specifically, this study demonstrates that organ culture enhances the physiological function of ET_A receptors in the right coronary artery.

The organ culture model has been extensively used to study cell proliferation and migration (Gotlieb and Boden, 1984; Newby and Zaltsman, 1999; Voisard et al., 1999). We uniquely confirmed that cells from organ-cultured arteries are undergoing cell cycling because in intact arterial cross sections there is a greater DNA content than cells from cold storage. This agrees with investigators who confirmed that the relative DAPI fluorescence intensity positively correlates with cell proliferation (McCaffrey et al., 1988). Furthermore, the DAPI staining in single cells from organ-cultured arterial cross sections demonstrated a lack of orientation compared with the circumferentially oriented cold-stored cells. The cells from organ-cultured arteries appear to be in a migrating state. Recently, organ culture has also been used to study receptor function and density within these proliferating cells, such as happens in atherosclerosis (Adner et al., 1996, 1998a,b). Therefore, we used the organ culture model to determine whether the contractile activity of coronary smooth muscle cells is altered with arterial injury.

Our results indicate that there was no difference in the potency of ET-1 if the artery was organ cultured. Our pD₂ values for cold-stored and organ-cultured rings were 7.60 ± 0.13 and 7.56 ± 0.03, respectively (P > .05). This agrees with Harrison et al. (1992) who reported a pD₂ value of 8.17 by measuring tension development in endothelium-denuded rings from pig coronary arteries. Because the pD₂ values were similar between cold-stored and organ-cultured rings, it might be assumed that a maximal concentration of ET-1 (3 × 10⁷ M) would elicit a similar contractile response between groups as was shown by Adner et al. (1995) using fresh and organ-cultured human omental arteries. However, we demonstrated an approximate 2-fold increase in isometric tension development to a maximal concentration of ET-1 in organ-cultured arterial rings compared with cold-stored rings. In addition, using the ET-1 EC₅₀ (as determined in the arterial rings) we showed that ET-1 increased the Ca_m response 2.6-fold in single cells that were isolated from organ-cultured arteries compared with those isolated from cold-stored arteries. The apparent higher sensitivity to ET-1 in isolated single smooth muscle cells than in intact arterial rings appears to be due to the source of calcium used for tension development. Our data suggest that contractile force is highly dependent on calcium influx, and not SR calcium release. This suggests that the greater contractile force generated in organ-cultured rings is partially attributed to an increase in calcium influx. Other studies have also shown that ET-1-induced calcium influx mediates tension development (Kasuya et al., 1989; Inui et al., 1999). It has been demonstrated that a sustained influx of calcium is matched by calcium extrusion (so-called "calcium cycling"), which returns bulk Ca_m to basal levels without limiting sustained tension development (Rasmussen et al., 1989; Bowles et al., 1995). Therefore, because the ET-1-induced influx of calcium is rapidly extruded from the cell (Bowles et al., 1995), ET-1-induced calcium influx is not apparent using single-cell digital imaging of bulk Ca_m. However, we used single-cell fura-2 digital imaging to measure SR calcium release (Wagner-Mann and Sturek, 1991, 1992). Our data suggest that there is a greater enhancement of the ET-1-mediated release of calcium from the SR with organ culture. Investigators have found that the transient ET-1 induced intracellular release of calcium (via inositol triphosphate) appears to be primarily linked to mitogenesis and sensitization of the myofilaments via the activation of protein kinase C (Assender et al., 1996; Schiffrin and Touyz, 1998; Suzuki et al., 1999). Both Douglas et al. (1994) and McKenna et al. (1998) have demonstrated that ET-1 promotes neointimal thickening in arterial injury models. Therefore, the enhanced release of calcium from the SR with organ culture may induce cell proliferation as is demonstrated during atherosclerotic development.

We originally hypothesized that the enhanced tension development and Ca_m increase in response to ET-1 with organ culture was due, in part, to an enhanced action of ET receptors. This was confirmed using the nonselective ET antagonist bosentan, which inhibited both tension development in arterial rings and the Ca_m response in isolated single cells. Bosentan shifted the concentration-response relationship to the right 6-fold and inhibited the Ca_m response by 80% in cells isolated from organ-cultured arteries. Therefore, our results corroborate those reports by investigators who have shown that ET-1 receptors are up- or down-regulated in response to a variety of vascular pathologies and organ culture (Wang et al., 1995; Adner et al., 1996, 1998a,b; Mathew et al., 1996; Stewart, 1998).

We determined the contribution of the ET_A receptor subtype in mediating the enhanced contractile and Ca_m response in organ-cultured arteries using the selective ET_A antagonist BQ123. In cold-stored rings 10⁶ M BQ123 was ineffective in inhibiting the ET-1 response; only 10⁵ M BQ123 inhibited the ET-1 response. In contrast, both 10⁶ and 10⁵ M BQ123 were effective in shifting the concentration-response relationship 5- and 7-fold to the right, respectively, in organ-cultured rings. This is similar to the 6-fold shift to the right with bosentan in organ-cultured rings. Because 10⁶ M BQ123 inhibited and had no effect on organ-cultured and cold-stored rings, respectively, this suggests that the increased contractile response to ET-1 is principally due to ET_A receptors in organ-cultured arteries.

Isolated single cells demonstrated a similar sensitivity to BQ123 as displayed by organ-cultured arterial rings. Similar to bosentan, BQ123 decreased the Ca_m response approximately 80% in cells from organ culture. In cells from cold-stored arteries, BQ123 did not decrease the Ca_m response, whereas bosentan did decrease Ca_m by about 50%. In evaluating the effect of the antagonists on the Ca_m response we pooled both cells that did or did not respond to ET-1 in the absence and presence of the antagonist. Otherwise, in the presence of the antagonist, it would not be known whether the cells inherently did not respond to ET-1, or whether the antagonist effectively inhibited the ET-1 response. This dilution of the response to ET-1 may contribute to the apparent lack of ET-1 inhibition by BQ123 in cells from cold-stored arteries. Because only 33% of the cells from cold-stored arteries responded to ET-1 (no antagonist) this decreased the overall reported Ca_m response, which makes it difficult to formulate a definitive conclusion regarding the antagonism of the ET-1 Ca_m response in these cells. In contrast, because 75% of the cells from organ-cultured arteries responded to ET-1 (nonantagonist) there was little dilution of the Ca_m response to ET-1, therefore the antagonism of ET-1 is more apparent and definitive.

Unlike BQ123, the selective ET_B antagonist BQ788 did not inhibit isometric tension development or the Ca_m response to ET-1 in both intact rings and isolated cells from organ-cultured or cold-stored arteries. In response to the selective ET_B agonist sarafotoxin 6C, cells from organ-cultured arteries actually decreased their Ca_m response 60% compared with cells from cold storage. These Ca_m data suggest there may be a functional down-regulation of ET_B receptors. However, it is difficult to accurately conclude there is a decreased function of ET_B receptors with organ culture because our data demonstrate such a small Ca_m response to sarafotoxin 6C and very little contribution of ET_B-mediated tension development in both normal and organ-cultured arteries.

Our results are important because ET receptors have been implicated in atherosclerosis (Mathew et al., 1996) and neointimal formation after balloon angioplasty (Douglas et al., 1994). An oral ET_A antagonist reduced neointimal hyperplasia in a porcine model of coronary artery injury (McKenna et al., 1998). In addition, Katwa et al. (1999) found that the expression of ET_A receptors is up-regulated in porcine coronary arteries during restenosis. In contrast, Dagassan et al. (1996) described an up-regulation of ETB receptors in atherosclerotic human coronary arteries. However, Dagassan et al. (1996) used the left anterior descending coronary artery, which appears to have a greater percentage of ET_B receptors than the right coronary artery (Harrison et al., 1992; Godfraind, 1993; Elmoselhi and Grover, 1997; Hasdai et al., 1997). The right coronary artery, as was used in this study, demonstrates an ET_B receptor population that mediates little vasoconstriction (Bacon and Davenport, 1996). Therefore, the right coronary artery is a good model to use to exclusively study the function (i.e., vasoconstriction and Ca_m response) of ET_A receptors.

This study uniquely demonstrates that the Ca_m response mediated by either the ET_A or ET_B receptor in isolated smooth muscle cells parallels the development of isometric tension in both cold-stored coronary arteries and organ-cultured coronary arteries. Organ culture greatly enhanced both the Ca_m response and isometric tension development to ET-1. Our results indicate that the increased response to ET-1 is attributed to an enhanced action of ET_A receptors, and not ET_B receptors.