INTRODUCTION

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THE PRESENCE OF ALL COMPONENTS of the renin-angiotensin system in neonatal rat cardiomyocytes (NRC) is consistent with its role in maintaining cardiovascular homeostasis (3, 5). Angiotensin II (ANG II) regulates cardiac contractility and growth via stimulation of angiotensin type 1 (AT₁) receptors (3). Stimulation of AT₁ receptors leads to Ca²⁺ mobilization through the activation of phospholipase C (PLC), resulting in the generation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃] and diacylglycerol (12, 27, 31, 32). The mechanisms governing the regulation of ANG II-evoked increases in cystosolic free calcium concentration ([Ca²⁺]_i) levels are not fully understood. Besides enhancing the [Ca²⁺]_i level, ANG II elicits complex intracellular signaling events that include the production of superoxide anions and the activation of several kinases as well as the alteration of cyclic nucleotides and nitric oxide levels (31). In addition, ANG II activates phospholipase A₂ and D, resulting in elevation of arachidonic acid (AA)-derived metabolites (6, 15, 19, 22, 24, 36). In rat vascular smooth muscle cells, the hypertrophic responses to ANG II are suggested to be at least partially linked to generation of noncyclooxygenase-derived AA metabolites (6, 22). Recently, we and others have demonstrated that ANG II-evoked vasoconstrictor responses in rat aortic rings and perfused rat mesenteric vascular bed were reduced by 2-[12-hydroxydodeca-5,10-diynyl]-3,5,6-trimethyl-p-benzoquinone (AA-861), a selective blocker of 5-lipoxygenase (5-LO), or 3-[[[3-[2-(7-chloro-2-quinlinyl)ethinyl]phenyl][[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-(E) sodium salt (MK-571), a selective cysteinyl leukotriene (CysLT)₁ antagonist (29, 30). These data suggested that ANG II may enhance the production of the AA-derived 5-LO metabolite CysLT. However, whether ANG II promotes CysLT production in vascular smooth muscle cells and cardiomyocyte has not been investigated.

CysLT include leukotriene D₄ (LTD₄), C₄ (LTC₄), and E₄ (LTE₄). They exert their actions via activation of at least two pharmacologically defined G protein-coupled receptors (CysLT₁ and CysLT₂) that are linked to PLC-mediated Ca²⁺ mobilization (8, 16). In addition to their well-known bronchiolar smooth muscle spasmogenic effect, these inflammatory mediators have been shown to enhance contractile responses in smooth muscle preparations or [Ca²⁺]_i levels in their target cells (4, 10, 20, 21, 23, 29, 30). Recently, with the use of a variety of techniques including in situ hybridization, Northern blotting, and RT-PCR, the presence of CysLT₁ and/or CysLT₂ transcripts in cardiac tissue has been identified (8, 10, 16, 21). CysLT has been shown to exert potent effects on the heart, contributing to heart failure (2, 10). Leukotriene A₄ hydrolase plays a critical role in the generation of CysLT, and its expression is elevated in the heart of ANG II-induced hypertensive rats, suggesting that ANG II may promote cardiac CysLT production (9). Moreover, low concentrations of CysLT, specifically LTD₄ and LTC₄, have been shown to promote a positive inotropic effect in the rat heart (11). Despite these reports, there are no studies to demonstrate that CysLT, namely, LTD₄ and LTC₄, promotes Ca²⁺ mobilization and that ANG II-evoked Ca²⁺ mobilization is linked to CysLT generation in cardiomyocytes. In the present study, using adherent NRC in primary cultures, we attempted to address these issues.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cardiomyocyte cultures. The care and use of animals conforms to the regulations stipulated by the University Animal Care Committee. The details of isolation and primary culture of ventricular myocytes from newborn (3 days old) Sprague-Dawley rats have been previously described (14, 34, 35). After removal of mesenchymal cells, the cell suspension was layered on either six-well culture plates for CysLT assay or on glass coverslips for fluorescence measurement. Bromodeoxyuridine (0.1 mM) was included in the medium to ensure selective suppression of mesenchymal cells to facilitate purified myocyte-rich (>95%) primary cultures of NRC (14).

Determination of [Ca²+]_i using fura 2 fluorescence in adherent NRC. After NRC grown on glass coverslips attained confluence (3 days), the cells were maintained in serum-free medium for 24 h. The cells were washed twice in Krebs-HEPES buffer [composed of (in mM) 145 NaCl, 5 KCl, 1.8 CaCl₂ · 2H₂O, 1.2 MgCl₂ · 6H₂O, 10 glucose, and 10 HEPES with 0.2% BSA; pH 7.4]. Cells were loaded in the dark with fura 2-AM (final concentration 5 µM) for 30 min, followed by three buffer washes. Coverslips were inserted into a microcuvette containing 500 µl buffer at 37°C. The excitation signals (340/380 nm) were determined using a fluorimeter designed to monitor fura 2 fluorescence (JASCO CAF-100 Ca²⁺ Analyzer, Japan Spectroscopic; Tokyo, Japan). Details of calibration and determination of basal and agonist-evoked increases in [Ca²⁺]_i levels have been described previously (14, 34). The concentration-peak [Ca²⁺]_i response (CR) curves to each agonist [ANG II, arginine vasopressin (AVP), endothelin (ET-1), and LTC₄ or LTD₄] were evaluated using fresh coverslips of fura 2-loaded cells for each challenge. CR determinations for LTC₄ and LTD₄ were performed in the buffer medium devoid of BSA. The CR determinations to agonists were also performed in the presence of optimal concentration(s) of the 5-LO inhibitor AA-861 [either 10 or 30 µM (29, 30, 33)], the CysLT₁ antagonist MK-571 [100 nM (16, 26, 29, 30)], the dual CysLT₁/CysLT₂ antagonist BAY-u9773 [100 nM (8)], the AT₁-selective antagonist losartan [1 µM (3, 12, 27, 28)], the AT₂-selective antagonist PD-123319 [1 µM (3, 12, 7, 27, 28, 32)], and the cell-permeant Ins(1,4,5)P₃ blocker 2-aminoethoxydiphenyl borate [2-APB; 50 µM (18)]. The concentrations of all these agents were carefully chosen to ensure their selectivity of inhibition/blockade as validated by previous reports. Each agent was maintained in the cuvette for 3 min before the agonist challenge. In select experiments, the fura 2-loaded cells on coverslips were washed and placed in Ca²⁺-free buffer with 1 mM EGTA (pH 7.4) in the cuvette for 15 min before the agonist challenge and fluorescence measurements.

Single cell fura 2 imaging. NRC grown on glass coverslips (Delta T Dish 0.15 nm, Bioptechs; Butler, PA) were maintained in DMEM for 3 days and changed to serum-free medium for 24 h. The cells were washed in Krebs-HEPES buffer and subjected to fura 2 loading and washing. The dish was mounted on the stage of an inverted Olympus I ×70 epifluorescence microscope fitted with the specification of UApo20×/340.5 objective that has the capability to monitor the apochromat-reflected light fluorescence of fura 2. The fura 2 fluorescence images were acquired using a fast monochromatic integral 125-W xenon light source (SpectraMaster Monochromator, Life Science Resources, Perkin Elmer; Gaithersburg, MD) with a shutter speed for monitoring alternate 340- and 380-nm excitation signals every 200 ms. Ratiometric signals at 340/380 nm were acquired at a rate of 3 images/s. The emission signal at 510 nm was collected using a charge-coupled device camera (Astrocam; Cambridge, UK). The digitized signals were stored and processed using UltraVIEW Imaging System software (Wallac Imaging, Perkin Elmer). ANG II (50 nM) was added to the coverslips after images were acquired for the first 30 s to determine basal fura 2 fluorescence. Interacting agents were added to fura 2-loaded cells for 3 min before imaging. The respective Ca²⁺-saturated and Ca²⁺-free 340-to-380-nm fluorescence ratio values were determined using the Ca²⁺ ionophore bromoA-23187 (50 µM) and then by quenching with 50 µl of Tris (50 mM)-EGTA (100 mM) solution (pH 8.5) at the end of each experiment. Once these values were entered, the software program employed provided the absolute [Ca²⁺]_i values for each cell using the Grynckiewicz equation.

Total CysLT measurement. NRC grown on six-well culture plates for 3 days (~0.3-0.5 × 10⁶ cells/well) were maintained for the last 24 h in 2 ml of medium devoid of serum. The medium was replaced with Krebs-HEPES buffer before stimulation with ANG II (100 nM) for varying time intervals (from 5 to 360 s). CR determinations for ANG II (100 pM-1 µM)-evoked increases, AVP (100 pM-1 µM)-evoked increases, and ET-1 (100 pM-1 µM)-evoked increases in CysLT levels were determined 1 min after the addition of the respective agonist(s). All these assays were performed in duplicate. The responses to ANG II were also determined in the presence of AA-861 (10 µM), losartan (1 µM), or PD-123319 (1 µM). A 450-µl aliquot of culture medium was stored in siliconized tubes at 80°C. Total CysLT levels were determined within 10 days of storage by enzyme immunoassay following the protocol provided by Cayman Chemicals (Ann Arbor, MI). Total CysLT levels were assayed spectrophotometrically (405 nm for measurement of acetylcholinesterase activity) as outlined in the kit using a Anthos HT1 96-well microplate reader (Anthos Labtec Instruments; Salzburg, Austria). The lowest detection limit was 4.0 pg/ml, and the 50% (B/B₀) ratio was 40 pg/ml. The intra- and interassay coefficients of variation were 7.3 ± 2.5% and 5.8 ± 2.9%, respectively. Cells were counted, and the data were normalized to express the values as CysLT per milliliter per million cells.

Materials and supplies. Fura 2-AM and pluronic acid F-127 were from Molecular Probes (Eugene, OR). Culture media, DMEM, serum, and trypsin were from GIBCO-BRL (Life Technologies; Grand Island, NY). AVP, ANG II, and ET-1 were from Bachem (Torrance, CA). Bromodeoxyuridine, AA-861, PD-123319, 2-APB, and analytic grade salts for the preparation of Krebs buffer were purchased from Sigma Chemical (Oakville, Ontario, Canada). BAY-u9773 was obtained from BioMol Research Laboratories (Plymouth Meeting, PA). MK-571 and losartan were provided by Merck-Frosst Canada. LTC₄, LTD₄, and enzyme immunoassay kits for the estimation of CysLTs were obtained from Cayman Chemicals.

Statistical analysis. Experimental values are reported as means ± SE of a minimum of five separate experiments performed on different days using different batches of NRC. Comparison of mean values was performed by ANOVA (Super ANOVA software). Simultaneous multiple comparisons were assessed using Scheffé's F-test, and the concentration of agonist required to produce 50% of the maximal response (EC₅₀) and the maximal increase in [Ca²⁺]_i (E_max) values attained for each agonist were derived from log CR curves.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The basal [Ca²⁺]_i levels in normal Ca²⁺ buffer were 90 ± 12 nM in multiple cells (n = 54) and 121 ± 18 nM in single cells (n = 23), and the differences in resting levels observed by both methods were not significant. None of the interacting agents (AA-861, MK-571, BAY-u9773, 2-APB, losartan, or PD-123319) affected the basal fura 2 fluorescence ratio. A representative tracing of ANG II-evoked increases in the fura 2 fluorescence ratio performed on the same day in the presence or absence of either AA-861 (10 µM) or MK-571 (100 nM) is shown in Fig. 1. The addition of ANG II led to a rapid concentration-dependent increase in the ratio of fura 2 fluorescence in multiple cells, with maximal increases observed between 30 s and 1 min; at 2 min after stimulation, the fluorescence ratio decreased to a steady state above the basal level. The CR curves to ANG II determined in either normal Ca²⁺ (1.8 mM) or Ca²⁺-free buffer is shown in Fig. 2. The addition of AA-861 (10 µM) or MK-571 (100 nM) led to a significant reduction in ANG II-evoked increases in peak [Ca²⁺]_i values (P < 0.01) in both normal and Ca²⁺-free buffer. Moreover, the addition of AA-861 or MK-571 did not affect either the time to attain the peak response or the time for reduction in peak Ca²⁺ to steady-state levels for varying concentrations of ANG II (Fig. 1). The effect of blockade on ANG II responses was similar when AA-861 was increased to 30 µM. Although the E_max values for ANG II were relatively lower in Ca²⁺-free medium, both AA-861 and MK-571 evoked a similar degree of blockade (percent reduction in E_max) of ANG II responses (Fig. 2, right). These data suggest that both AA-861 and MK-571 decrease the E_max with no change in EC₅₀ values for ANG II-evoked peak [Ca²⁺]_i responses (Table 1). In contrast, a high concentration of either AA-861 (30 µM) or MK-571 (100 nM) failed to affect the CR curves to AVP or ET-1 (Table 1).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Representative experiment of angiotensin II (ANG II)-evoked changes in the ratio of fura 2 fluoresence (340/380 nm) in adherent neonatal rat cardiomyocytes (NRC). Primary cultures of NRC grown on coverslips and loaded with fura 2 were stimulated with increasing concentrations of ANG II (10 pM-1 µM) either in the absence (control; A) or presence of AA-861 (10 µM; B) or MK-571 (100 nM; C). These agents were maintained in the cuvette in Krebs buffer (pH 7.4) containing normal Ca2+ (1.8 mM) at 37°C for 3 min before challenge with the indicated concentrations of ANG II. Note that each coverslip containing fura 2-loaded NRC was stimulated only once with a single concentration of ANG II. Excit, excitation.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of the 5-lipoxygenase (5-LO) inhibitor AA-861 and cysteinyl leukotriene (CysLT)1-selective antagonist MK-571 on peak cytosolic free Ca2+ concentration ([Ca2+]i) responses to ANG II in NRC. Primary cultures of NRC were stimulated with increasing concentrations of ANG II in either the absence (control; ) or presence of AA-861 (10 or 30 µM; ) or MK-571 (100 nM; ) in Krebs buffer (pH 7.4) at 37°C. ANG II concentration-peak [Ca2+]i response (CR) curves were determined with either Ca2+ being present (1.8 mM; left) or absent (0 mM Ca 2+ + 1 mM EGTA, pH adjusted to 7.4; right) in the buffer. Each CR curve was determined eight times using different batches of NRC. *P < 0.05 and **P < 0.01 compared with AA-861- and MK-571-treated cells.

A representative tracing of ANG II-, LTD₄-, and LTC₄-evoked [Ca²⁺]_i responses is shown in Fig. 3. Both LTC₄ and LTD₄ evoked rapid increases in peak [Ca²⁺]_i levels (E_max: LTD₄ >> LTC₄) with a similar time course but much lower E_max compared with ANG II. The peak responses were attained at 40, 43, and 47 s after the addition of ANG II, LTD₄, and LTC₄, respectively. The plateau phase that sustained at levels slightly above their respective baseline values were reached between 120 and 180 s after the addition of respective agonist(s). There were no significant differences between them (Fig. 3). The inclusion of MK-571 significantly attenuated the E_max and increased the EC₅₀ value for LTD₄, but it failed to evoke a significant reduction in the weak [Ca²⁺]_i signals evoked by LTC₄. On the contrary, the inclusion of a nonselective CysLT antagonist, BAY-u9773, completely blocked the [Ca²⁺]_i responses to both LTD₄ and LTC₄ (Fig. 4). The analysis of data obtained from several CR curves is summarized in Table 2. The Ins(1,4,5)P₃ blocker 2-APB and the AT₁-selective antagonist losartan significantly attenuated the responses to ANG II, whereas PD-123319 had no effect on either basal or ANG II-evoked peak [Ca²⁺]_i responses (Fig. 5, left). 2-APB also attenuated the maximal [Ca²⁺]_i responses, with a significant reduction in E_max values for LTD₄ (Fig. 5, right).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Representative experiment of ANG II- and leukotriene D4 (LTD4)- and C4 (LTC4)-evoked changes in the ratio of fura 2 fluoresence (340/380 nm) in NRC. Primary cultures of NRC grown on coverslips and loaded with fura 2 were stimulated with either ANG II (100 nM), LTD4 (100 nM), or LTC4 (100 nM). These agents were added to the cuvette in Krebs buffer (pH 7.4) containing normal Ca2+ (1.8 mM) at 37°C. Note that each coverslip containing fura 2-loaded NRC was stimulated only once with a single agonist.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Effect of the CysLT1-selective antagonist MK-571 and the dual CysLT1/CysLT2 antagonist BAY-u9773 on peak [Ca2+]i responses to LTD4 and LTC4 in NRC. Adherent NRC were stimulated with increasing concentrations of LTD4 (left) or LTC4 (right) in either the absence (control; ) or presence of MK-571 (100 nM; ) or BAY-u9773 (100 nM; ) in Krebs buffer (pH 7.4) containing normal Ca2+ (1.8 mM) maintained at 37°C. Each CR curve was determined six times using different batches of NRC. **P < 0.01 compared with MK-571- and BAY-u9773-treated cells (left) or BAY-u9773-treated cells (right).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of losartan, PD-123319, and 2-aminoethoxydiphenyl borate (2-APB) on peak [Ca2+]i responses to ANG II and LTD4 in NRC. CR curves to ANG II (left) were determined in normal Ca2+ (1.8 mM) buffer in either the absence (control; ) or the presence of the angiotensin type 1 (AT1) receptor antagonist losartan (1 µM; ), or the AT2 receptor antagonist PD-123319 (1 µM; ), or the inositol (1,4,5)-trisphosphate antagonist 2-APB (50 µM; ). CR curve determination for LTD4 (right) was restricted to studies in the presence or absence of 2-APB (50 µM) alone because losartan and PD-123319 failed to alter the control CR curve to LTD4 in preliminary experiments. Each curve was determined six times using different batches of NRC. *P < 0.05 and **P < 0.01 compared with losartan- and 2-APB-treated cells.

Single cell Ca²⁺ imaging studies revealed qualitatively similar results. A typical experiment performed with ANG II stimulation (50 nM) on three different coverslips loaded with fura 2 on the same day using the same batch of cardiomyocytes is shown in Fig. 6. Basal fluorescence levels before stimulation are shown in Fig. 6, A-C [control (A), AA-861 (B), and MK-571 (C)]. Figure 6, E and F, shows the effect of ANG II at 30 s in the presence of either AA-861 (E) or MK 571 (F). The responses are compared with images determined for ANG II in the absence of these interacting agents [control (Fig. 6D)]. Single cell fluorescence determination from several experiments (n = 5) gave the following absolute [Ca²⁺]_i values: control 585 ± 34 nM, AA-861 400 ± 27 nM (P < 0.01), and MK-571 460 ± 21 nM (P < 0.01). Although single cell E_max values for ANG II were higher compared with data obtained with multiple cells, a similar pattern of blockade in the presence of AA-861 or MK-571 was evident. Between 2- and 3-min intervals, the cells had reached steady-state fluorescence close to the basal value, suggesting the fluorescence changes are consistent with [Ca²⁺]_i changes and that the results gathered were not due to photobleaching (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Fura 2 Ca2+ imaging in a single NRC stimulated with ANG II (50 nM). Ca2+ images were acquired at a rate of 3 images/s using three different coverslips loaded with fura 2 under identical conditions on the same day. A-C: basal fura 2 fluorescence images (340-to-380-nm excitation ratio) acquired before the addition of ANG II in the absence (A) or presence of AA-861 (B) or MK-571 (C). D-F: changes in fura 2 fluorescence at 30 s after the addition of ANG II in the same cells. D: ANG II alone; E: ANG II in the presence of AA-861 (10 µM); F: ANG II in the presence of MK-571 (100 nM). The aggregate [Ca2+]i values in single cells obtained were as follows (in nM): 110 (A), 124 (B), 117 (C), 710 (D), 470 (E), and 530 (F). At the end of 2 min, the fluorescence values were <200 nM in all cells (data not shown). Similar response patterns were recorded in five separate experiments.

The basal CysLT level in the culture medium was 9.7 ± 1.9 pg · ml¹ · 10⁶ cells¹. The addition of ANG II led to a rapid, time-dependent increase in the CysLT level, reaching a maximum (19.8 ± 2.6 pg · ml¹ · 10⁶ cells¹) at 1 min, remaining at the same level until 5 min, and gradually decreasing thereafter. However, the increases in CysLT levels were significantly higher (15.7 ± 1.8 pg · ml¹ · 10⁶ cells¹) even at 30 s after the addition of ANG II (Fig. 7, left). The CR determinations for CysLT generation revealed that the ANG II effect was also concentration dependent. The E_max for ANG II (100 nM)-evoked CysLT release was twofold higher than the basal level. In contrast to ANG II, the addition of either ET-1 (100 nM) or AVP (100 nM) failed to evoke a significant increase in CysLT production. The inclusion of losartan or AA-861 led to a significant reduction in ANG II-evoked total CysLT release into the culture medium, whereas the addition of PD-123319 failed to affect either the basal or ANG II-evoked increases in CysLT production (Fig. 7, right). CysLT generation was significantly higher at 30 s, and the peak [Ca²⁺]_i increase was reached at 40 s after the addition of ANG II (100 nM), suggesting that the increased CysLT generation evoked by ANG II may contribute to an elevation in [Ca²⁺]_i (Figs. 3 and 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Time dependence of ANG II-evoked increases in CysLT levels in the culture medium of NRC (left) and the effects of losartan, PD-123319, and AA-861 on ANG II-evoked increases in CysLT levels and the lack of effect of endothelin-1 (ET-1) and arginine vasopressin (AVP) on CysLT production in NRC (right). Left: total CysLT levels in the medium at different time points after stimulation with a fixed concentration of ANG II (100 nM). Right: line graphs providing a comparison of elevation in total CysLT levels attained in the medium 1 min after stimulation with increasing concentrations of ANG II in either the absence [control (ANG II alone); ] or presence of losartan (1 µM; ), PD-123319 (1 µM; ), or AA-861 (10 µM; ). Moreover, the lack of significant changes in CysLT levels in the medium 1 min after stimulation with increasing concentrations of either ET-1 () or AVP () are also shown. Each curve was determined five times using different batches of NRC. **P < 0.01 compared with losartan-, AA-861-, ET-1-, and AVP-treated cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study provides several new observations: First, the 5-LO-derived metabolites of AA, CysLT, augment [Ca²⁺]_i responses to ANG II in NRC. Second, the inclusion of the 5-LO inhibitor AA-861 (10 µM) led to significant attenuation of ANG II-evoked increases, but not ET-1- and AVP-evoked increases, in peak [Ca²⁺]_i levels and CysLT release into the culture medium. Third, ANG II-evoked increases in [Ca²⁺]_i levels and CysLT release in NRC are mediated by AT₁ receptor activation because losartan, an AT₁ antagonist, but not the AT₂-selective antagonist PD-123319, blocked the responses to ANG II. Fourth, both LTD₄ and LTC₄ evoke [Ca²⁺]_i increase in these cells; however, the responses to these mediators were much lower than those evoked by ANG II. The CysLT₁-selective antagonist MK-571 significantly abolished the [Ca²⁺]_i responses to LTD₄ but failed to inhibit the responses to LTC₄, whereas the CysLT₁/CysLT₂ antagonist BAY-u9773 completely blocked the [Ca²⁺]_i responses to both LTD₄ and LTC₄. Finally, both ANG II- and CysLT-evoked increases in [Ca²⁺]_i levels were markedly attenuated by 2-APB, an agent that blocks Ins(1,4,5)P₃-mediated Ca²⁺ release. These data confirm that both ANG II and CysLT recruit predominantly Ins(1,4,5)P₃-sensitive intracellular Ca²⁺ pools in NRC. Thus the CysLT pathway may serve as an additional amplifier pathway in sustaining direct AT₁-mediated intracellular Ca²⁺ mobilization evoked by ANG II in NRC.

AT₁ and AT₂ subtypes on NRC. The presence of two high-affinity binding sites for [¹²⁵I]-labeled ANG II has been demonstrated in membrane preparations of NRC (25). Rogers and colleagues (13, 15) proposed that the AT₁ subtype would promote [³H]Ins(1,4,5)P₃ accumulation via activation of PLC, whereas the AT₂ subtype would be linked to [³H]AA release via activation of phospholipase A₂, and that ANG II-induced alkalinization was sensitive to blockade by AT₂-selective but not AT₁-selective antagonists. Others have suggested that AT₁-mediated hypertrophy by ANG II could be opposed by AT₂-mediated antigrowth events in NRC (3). In contrast to these reports, detailed characterization of AT₁- and AT₂-specific binding sites and receptor expression studies have revealed the presence of only a single class of AT₁-specific binding sites on NRC (32). Several studies by others have also established that both the increases in [Ca²⁺]_i and all phospholipid second messenger systems evoked by ANG II in NRC are indeed mediated solely by the activation of the AT₁ subtype and not the AT₂ subtype (12, 27, 32). Others have established that ANG II can also activate phospholipase A₂ and D, leading to an elevation in AA-derived metabolites (6, 15, 19, 22, 24, 31, 36). The present study demonstrates that ANG II-evoked increases in CysLT and [Ca²⁺]_i levels were abolished by losartan but not by PD-123319. Therefore, AT₁ receptor activation and the resultant PLC/Ins(1,4,5)P₃-mediated [Ca²⁺]_i responses may also be partly mediated by AA-derived CysLT production in NRC. The focus of the present study was to examine the involvement of CysLT in the ANG II-mediated elevation in peak [Ca²⁺]_i responses.

ANG II-CysLT interaction. Previous studies have shown that ANG II-evoked vascular hypertrophy may be mediated by noncycloxygenase-derived AA metabolites (6, 22, 36). The 5-LO inhibitor AA-861 or the CysLT₁ antagonist MK-571 reduced the vasoconstrictor responses to ANG II, suggesting that CysLT generation and the subsequent CysLT₁ receptor activation may mediate vasoconstriction to ANG II (29, 30). Therefore, it was important to provide direct evidence at the level of signal transduction for a link among ANG II, AT₁ receptor activation, CysLT generation, and CysLT receptor-mediated alterations in [Ca²⁺]_i levels. It is well known that cell surface receptors for ET-1 are several orders of magnitude higher than the receptors for ANG II in NRC (3). Previously, we have shown that ANG II evoked much greater increases in [Ca²⁺]_i than both AVP and ET-1 in NRC (34, 35). These data suggest that additional mechanisms and signal transduction events likely account for the much greater increases in the [Ca²⁺]_i response to ANG II. The present data confirm that ANG II increased CysLT generation via AT₁ receptor activation, which in turn augments the [Ca²⁺]_i response to ANG II. In contrast, AVP- and ET-1-evoked increases in [Ca²⁺]_i levels were not reduced by the inclusion of either AA-861 or MK-571. Moreover, both ET-1 and AVP failed to promote CysLT production, suggesting that only ANG II promotes CysLT production via AT₁ receptor activation. This is consistent with the view that ANG II is a pleiotropic agonist that recruits multiple signaling pathways to account for its potential role in the regulation of cardiovascular function (31). Previously, others have confirmed that ANG II-evoked increases in [Ca²⁺]_i might play a more important role than protein kinase C activation in hypertrophic responses to ANG II in NRC (28). The present study demonstrates the consistency in the time course response for CysLT generation and the subsequent Ca²⁺ increase, suggesting that CysLT generation evoked by ANG II may serve as an additional amplifier pathway in sustaining direct AT₁-mediated Ca²⁺ mobilization in NRC. Several studies in the past have established that ANG II- and AVP-evoked elevations in [Ca²⁺]_i in NRC stemmed predominantly from the release of Ca²⁺ from the intracellular stores and that removal of extracellular Ca²⁺ led to only a partial reduction in the peak increases in [Ca²⁺]_i evoked by ANG II and AVP (7, 12, 14, 32). Moreover, thapsigargin, a sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor, abolished the [Ca²⁺]_i response to ANG II or AVP (12, 14). Therefore, the ANG II-evoked responses noted in Ca²⁺-free medium or the significant attenuation of [Ca²⁺]_i responses to ANG II seen in the presence of 2-APB are consistent with observations reported earlier. Thus, besides providing a possible explanation for the higher E_max to ANG II, the present study suggests for the first time that CysLT may play a role in ANG II-evoked contractility and/or hypertrophy in cardiomyocytes.

CysLT and Ca²+ signaling. It is important to demonstrate that CysLT contributes to Ca²⁺ mobilization in NRC. There are no studies suggesting the presence of CysLT receptors in NRC. In human detrusor smooth muscle cells, it was reported that elevation in [Ca²⁺]_i levels evoked by LTD₄ were almost exclusively due to mobilization from intracellular Ca²⁺ stores (4). In other target cells, the LTD₄-evoked Ca²⁺ response was dependent on the release of Ca²⁺ from intracellular stores and enhanced Ca²⁺ influx (20, 23). The present study demonstrates that both ANG II- and CysLT-evoked increases in [Ca²⁺]_i levels were significantly attenuated by 2-APB. In addition, both AA-861 and MK-571 caused a similar degree of blockade of the ANG II response in normal as well as Ca²⁺-free buffer. Thus this is the first report to characterize CysLT-evoked increases in [Ca²⁺]_i that may be mainly due to the release of Ca²⁺ from Ins(1,4,5)P₃-sensitive intracellular SR Ca²⁺ pools in NRC.