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CELLULAR AND MOLECULAR
Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts (R.E.C., S.H.); and Department of Biology, Tufts University, Medford, Massachusetts (D.E.C.)
Received September 24, 2003; accepted December 16, 2003.
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Abstract |
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-tocopherol, riboflavin, and N-acetyl-cysteine) were without effect. A flavoprotein oxidase inhibitor (diphenylene iodonium) and a hydroxy radical scavenger (butylated hydroxy anisole) also displayed the effects of DHPs. In conclusion, DHPs indirectly alter NTR1 function in live cells by a mechanism that depends on the drug's ability to donate hydrogen but does not simply involve sulfhydryl reduction.
), exerts several biological effects by way of its G protein-coupled receptor NTR1 (Vincent et al., 1999) located on neurons, endocrine cells, and smooth muscle cells (Ferris, 1989; Rostene and Alexander, 1997). Because NTR1 is often coupled to Gq/11, one of the pathways activated by NT involves phosphatidylinositol-specific phospholipase C (PLC)-mediated formation of inositol phosphates (IPs) and the release of intracellular Ca2+ (Hermans and Maloteaux, 1998). NT also stimulates an influx of Ca2+ into excitable (Trudeau, 2000) and nonexcitable cells (Gailly, 1998), and as a consequence, some of its effects are inhibited by Ca2+ channel blockers (CCBs). For example, the contractile effect of NT on intestinal smooth muscle (Mule and Serio, 1997) is antagonized by nifedipine, a blocker of voltage-gated Ca2+ channels (VGCCs). However, the effects of CCBs in these systems are not well defined, and some results are controversial. In guinea pig atria, nifedipine alters the ionotropic response to NT but investigators question whether the effect depends on Ca2+ influx (Golba et al., 1995). Also inconsistent is the fact that NT inhibits, rather than stimulates, VGCC currents in frog melanotrophs (Belmeguenai et al., 2002). These contradictory findings have led us to hypothesize that CCBs can alter NT signaling by exerting effects that do not involve Ca2+ channels.
It is well established that CCBs in the 1,4-dihydropyridine (DHP) class, such as nifedipine and nimodipine, can affect multiple targets (Triggle, 2003). Used in the submicromolar range, DHPs are relatively specific VGCC blockers; however, in the micromolar range, they block store-operated Ca2+ channels (SOCCs) (Harper et al., 2003), voltage-dependent K+ channels (Hatano et al., 2003), Na+ channels (Yatani et al., 1988), and ligand-gated ion channels (Lopez et al., 1993). In addition, DHPs are powerful antioxidants that can inhibit lipid peroxidation (Diaz-Araya et al., 1998) and protect cells against oxidative injury (Mak et al., 2002).
In the first article of this series (Carraway et al., 2003), we reported that a variety of CCBs dose responsively enhanced the binding of 125I-NT to NTR1 in prostate cancer PC3 cells, whereas they inhibited NT-induced IP formation. The effects were drug-specific, receptor-specific, and indirect, suggesting an involvement of selective cellular mediators and a requirement for cellular metabolism or architecture. Implicating Ca2+ channel(s) and/or Ca2+-dependent step(s) was the fact that IP formation required Ca2+ in the medium and that NT caused an influx of Ca2+ into the cells. The most potent agents were VGCC blockers (nifedipine and nimodipine); however, their effects on NTR1 function occurred in a dose range shown by others to block SOCC. Because NT was shown to release Ca2+ from internal stores and to stimulate SOCC-mediated store refilling, this suggested that CCBs altered NT binding and NT-induced IP formation by blocking SOCC function. Although several relatively selective SOCC blockers were also able to alter NTR1 function, the rank order of potency (nifedipine > SKF-96365 > miconazole > trifluoperazine) did not agree with published results for inhibition of SOCC conductance. Furthermore, the effect of nifedipine on NT binding occurred without Ca2+ in the medium. We concluded that a number of possible explanations should be considered: 1) Ca2+ channel occupation per se, not Ca2+ influx, might have mediated the effects of DHPs on NTR1 function; 2) the effects of DHPs could have involved effects on Na+ or K+ channels, because NT binding is modulated by these ions (Carraway et al., 1993); and 3) the antioxidative property of DHPs might have been the basis for these actions, because NT binding is sulfhydryl-dependent (Mitra and Carraway, 1993).
To clarify this issue, we have now performed studies to elucidate the mechanism(s) by which DHPs alter NTR1 function in PC3 cells. The role of Ca2+ and Ca2+ channels was examined by testing the effects of agents that altered cellular [Ca2+] and/or perturbed Ca2+ channel structure. We tested the possibility that Na+ and/or K+ channels participated by varying the concentrations of these ions. The importance of redox activity was assessed by comparing the effects of a series of DHPs with known antioxidative ability and by testing other antioxidants. Our results support the hypothesis that DHPs alter NTR1 function via an indirect, redox-sensitive mechanism that does not seem to involve reduction of sulfhydryl groups.
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Materials and Methods |
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-tocopherol, 
-carotene, ascorbic acid, riboflavin, thiamine, pyridoxine, menadione, and all other chemicals were from Sigma-Aldrich (St. Louis, MO). Compound 1, 1-ethyl-1,4-dihydro-2,6-dimethyl-4-(4-methoxyphenyl)-3,5-pyridinedicarboxylic acid dimethyl ester, was a generous gift from Dr. Juan Arturo Squella (University of Chile, Santiago 1, Chile).
Binding to PC3 Cells. PC3 cells, obtained from American Type Culture Collection (Manassas, VA), were maintained by our tissue culture facility (Seethalakshmi et al., 1997). Cells were grown to 95% confluence in 24-well culture plates. High-performance liquid chromatography-purified monoiodinated NT (125I-NT) at 2000 Ci/mmol was prepared and binding was performed as described by us (Carraway et al., 2003). In brief, cells were washed with 2 ml/well of Hepes-buffered Locke-BSA (Locke): 148 mM NaCl, 5.6 mM KCl, 6.3 mM Hepes, 2.4 mM NaHCO3, 1.0 mM CaCl2, 0.8 mM MgCl2, 5.6 mM glucose, and 0.1% BSA, pH 7.4. Stock solutions of each agent in Locke or in dimethyl sulfoxide (10 mM) were prepared just before use and were diluted to give 
1% dimethyl sulfoxide final. Equilibrium binding at 37°C was performed for 25 min using 105 cpm/ml 125I-NT in 1.0 ml of Locke with varying amounts of NT. The reaction was stopped on ice for 15 min, the medium was aspirated, and the cells were washed twice with 2 ml and once with 4 ml of ice-cold saline. Total cellular binding was assessed by measuring radioactivity (Packard 10-well gamma-counter) and protein (Bio-Rad assay; BSA standard; Bio-Rad, Hercules, CA) in cells extracted in 0.6 ml of 0.2 M NaOH. Specific binding, defined as that displaceable by 1 µM NT, was 95%. Binding displacement curves were constructed for each set of treatments and binding parameters were determined by Scatchard analysis and by using the Cheng-Prusoff equation Ki = IC50/1 + [L]/Kd, where Kd and [L] are the dissociation constant and the concentration of the ligand, respectively. The sucrose buffer used in some experiments was identical to Locke except that 296 mM sucrose was substituted for the NaCl and NaHCO3. The K+ depolarization buffer used in some experiments was identical to Locke except that 60 mM KCl was substituted for 60 mM NaCl.
Binding to PC3 Cell Membranes. PC3 cell membranes were prepared and collected by centrifugation at 30,000g as described by us (Seethalakshmi et al., 1997). Binding of 125I-NT (105 cpm) to membranes (10–50 µg) was performed at 20°C for 60 min in 10 mM Tris-HCl (pH 7.5), containing 1 mM MgCl2, 1 mM dithiothreitol, 0.1% BSA, and protease inhibitors as described previously. Membranes were collected and washed onto glass fiber (GF-B) filters using a cell harvester (Brandel Inc., Gaithersburg, MD), and the filters were counted (Carraway et al., 1993).
Measurement of IP Formation. Formation of [3H]IP in response to NT was measured as described previously (Carraway et al., 2003). Briefly, PC3 cells in 24-well plates were incubated 48 h with myo-[3H]inositol (2.5 µCi/ml) in medium 199, 5% fetal calf serum. After washing in Locke, cells were preincubated 10 min with varying concentrations of test agent in Locke, 15 mM LiCl. After aspiration, fresh Locke with test agent was added, and reactions were started by adding NT or control. After 30 min at 37°C, medium was aspirated, ice-cold 0.1 M formic acid in methanol (1 ml) was added and plates were placed at–20°C overnight. Samples were adsorbed to AG-1 x 8 (formate form; Bio-Rad), which was washed five times in 5 mM myo-inositol and eluted in 1.5 M ammonium formate, 0.1 M formic acid. Scintillation counting was performed in Ecoscint (National Diagnostics, Manville, NJ).
Statistics. Statistical comparisons were made using the Student's t test. Data were calculated as mean ± S.E.M., and p < 0.05 was considered significant.
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Results |
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), we reported that CCBs, particularly those in the DHP class, dose-responsively enhanced the binding of 125I-NT to PC3 cells (as much as 3-fold) and inhibited NT-induced IP formation (as much as 70%). The fact that NT caused an influx of 45Ca2+ in PC3 cells that was inhibited by nifedipine suggested that DHPs might alter NTR1 function by blocking Ca2+ movement. To test this hypothesis, we examined the effects of Ca2+ chelators and Ca2+ ionophores on NT binding to PC3 cells, and on the ability of nifedipine to enhance NT binding. Blocking Ca2+ influx with 2 mM EGTA enhanced NT binding (38 ± 6% increase; p < 0.05), but the effect was small relative to the 200% increase by nifedipine. In addition, the ability of nifedipine to enhance NT binding persisted in the absence of extracellular Ca2+, was not reversed by 20 µM ionomycin, and was not altered by chelation of intracellular Ca2+ using 50 µM BAPTA-AM (Fig. 1).
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These results indicated that the enhancement of NT binding by DHPs was not due to a change in Ca2+ influx. In contrast, the inhibition of NT-induced IP formation by DHPs might have involved an effect on Ca2+, because our earlier work showed that this response was Ca2+-dependent (Carraway et al., 2003).
Ca2+ Channel Agonists versus Antagonists. The VGCC agonist (-)-BayK-8644 and the antagonist nifedipine enhanced NT binding to a similar extent (Fig. 2A). Both compounds enhanced binding by increasing NTR1 affinity, not by altering receptor number (Fig. 2B; Table 1). Another VGCC agonist, FPL-64176, known to act at a unique non-DHP site (Zheng et al., 1991), was also active, although less potent (Fig. 2A). In addition, the agonists (-)-BayK-8644 and FPL-64176 shared with the antagonist nifedipine an ability to inhibit NT-induced IP formation (Fig. 2C). For each agent, the NT dose-response relationship was shifted downward, indicating that the efficacy of NT was decreased, not its potency (Fig. 2D).
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These results were consistent with the possible involvement of SOCC, but not VGCC, in the effects of DHPs on NTR1 function. Because IP formation was Ca2+-dependent, the fact that these agents decreased the efficacy of NT was in keeping with their known ability to diminish SOCC conductance.
Agents in Combination Gave Additive Effects. We wondered how combinations of CCBs would interact with NTR1, especially in regard to agonist/antagonist combinations and mixtures of CCBs that bind at different sites on Ca2+ channels. To test this, dose-response studies were performed using combinations of antagonist nimodipine and agonist FPL-64176, which are known to bind at discrete sites. Whereas NT binding was enhanced in an additive manner at low concentrations of each drug, the results were less than additive at high concentrations (Fig. 3A). NT-induced IP formation was inhibited in an analogous manner and at high concentrations, it reached a limit at 
70% inhibition (Fig. 3B). Similar studies were performed using various combinations of nifedipine, verapamil, and diltiazem (all antagonists). Again, when low doses of these drugs were combined, additive effects were observed for the enhancement of NT binding and for the inhibition of NT-induced IP formation, whereas at high doses the effects were less than additive (data not shown). No potentiative or antagonistic effects were observed. Together, these results indicated that the drugs tested, whether Ca2+ channel agonists or antagonists, seemed to act in a similar manner to alter NTR1 function.
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Effects of Ca2+ Channel Pertubation. Because evidence for direct "conformational coupling" of Ca2+ channels with some receptors existed (Grazzini et al., 1996), it was conceivable that Ca2+ channels might interact directly with NTR1. To examine this hypothesis, we tested treatments expected to perturb Ca2+ channel structure for effects on NTR1 function. VGCC can respond to membrane depolarization and Ca2+ feedback (Catterall, 2000). Agents that elevated cellular [Ca2+], either via release of internal Ca2+ stores (thapsigargin and UTP) or by enhancing Ca2+ influx (ionomycin), increased NT binding by 30% (Fig. 4A). BAPTA-AM, which would decrease cellular [Ca2+], had little effect. Because thapsigargin, ionomycin, and UTP did not change NT binding to PC3 cell membranes (data not shown), their effects in intact cells were indirect. Cell membrane K+-depolarization also increased NT binding (increment, 15%), and this was associated with an inhibition of NT-induced IP formation (Fig. 4B). The dose-response relationship was shifted downward by K+ depolarization, indicating that the efficacy of NT was decreased by 35% (Fig. 4B).
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The inhibition of NT-induced IP formation by treatments expected to perturb Ca2+ channels implicated VGCC and/or SOCC in this response and was consistent with the Ca2+ dependence of PLC (Carraway et al., 2003). However, these effects were relatively small (1/3 that of DHP), as were the effects of these treatments on NT binding (1/5 that of DHP). Thus, we were drawn to the idea that the effects of DHPs were, to a large degree (as much as 80%), attributable to some other property that was not necessarily related to the ability to alter Ca2+ channel behavior.
DHP Effects Were Not Na+-Dependent. Because DHPs can inhibit Na+ channels and because NT binding to PC3 cell membranes is decreased by Na+ (Seethalakshmi et al., 1997), we considered the hypothesis that DHPs enhanced cellular binding of NT by interfering with the inhibitory effect of Na+. Substituting sucrose for NaCl in the Locke buffer increased NT binding and shifted the NT displacement curve to the left; however, nifedipine caused a further shift to the left, even in the absence of Na+ (Fig. 5A). Scatchard analyses showed that each of these effects was due to an increase in NTR1 affinity, without a change in NTR1 number (Table 2). Careful comparison showed that the effect of 50 µM nifedipine on NT binding (2-fold increase) and on the Ki for displacement of NT binding (2-fold decrease) was unaffected by removal of Na+ (Table 2). Similarly and in accordance with the binding data, NT was 2 fold more potent in stimulating IP formation in the absence of Na+ than in its presence (EC50: Locke, 1.1 ± 0.1 nM; sucrose 0.5 ± 0.1 nM; three experiments; p < 0.05). However, the ability of nifedipine to inhibit NT-induced IP formation was independent of Na+ (Fig. 5B). These results indicated that nifedipine altered NTR1 function by mechanism(s) that did not require Na+ in the buffer.
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DHP Effects Were Not K+-Dependent. Because DHPs can inhibit K+ channels and because K+ inhibits NT binding to cell membranes, we performed experiments similar to those described above to assess the K+ dependence of the effects on NTR1 function. Substitution of 60 mM KCl for NaCl in the Locke buffer did not alter the ability of nifedipine to enhance NT binding (EC50: control, 22 ± 3 µM; 60 mM K+, 24 ± 3 µM; three experiments) and to inhibit NT-induced IP formation (IC50: control, 15 ± 2 µM; 60 mM K+, 14 ± 2 µM; three experiments). Thus, these effects of nifedipine were not K+-dependent.
Relationship to Antioxidant Activity. Because DHPs exhibit antioxidant ability (Mak et al., 2002), we wondered how this related to the effects on NTR1 function. DHPs inhibited Fe3+/ascorbate-stimulated lipid peroxidation in rat brain slices with activity order nicardipine > nimodipine > nifedipine (Diaz-Araya et al., 1998). The same activity order was found when these agents were compared for ability to enhance NT binding (Fig. 2A) and to inhibit NT-induced IP formation (Fig. 2C). In both systems, nicardipine was 2- to 4-fold more potent than nifedipine (Table 3). We also tested felodipine, which was 2- to 4-fold more active than nicardipine (Table 3). Although felodipine was reported to be inactive in the rat brain assay mentioned above, it was more active than nicardipine in a similar assay using myocardial membranes (Janero and Burghardt, 1989). In addition, the relative chemical reactivity of DHPs with superoxide anion was reported to be felodipine > nimodipine > nifedipine > compound 1 (Ortiz et al., 2003). For these substances, the potency to alter NT binding correlated to antioxidant activity, giving r2 = 0.89 (Table 3). Compound 1, a DHP analog with N-ethyl in place of the NH moiety, was reported by Ortiz et al. (2003) to have a greatly reduced reactivity with superoxide (<10% that of felodipine). Here, we found that it displayed 4% the activity of felodipine and 20 to 50% the activity of nifedipine in altering NTR function (Table 3). These results suggested that DHPs might act by some reaction(s) involving hydrogen donation.
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). Testing vitamin-like antioxidants on NT binding in PC3 cells, we found -carotene, thiamine, riboflavin, pyridoxine, ascorbic acid, -tocoferol, and tetrahydro-biopterin to be ineffective (used at 20–180 µM; n = 3), whereas vitamin K (menadione) had a small effect at 180 µM (percentage of control: 168 ± 8; n = 3; p < 0.05). Other antioxidants without effect included N-acetyl cysteine, glutathione, and sodium borohydride (used at 1–3 mM; n = 3); trolox, ellagic acid, (+)-catechin, (-)-epigallocatechin gallate, and rutin (used at 10–100 µM; n = 3). In striking contrast were the results for the polyphenolic antioxidants luteolin (a flavonoid) and resveratrol, which displayed effects that were indistinguishable from those of DHPs. Luteolin and resveratrol enhanced NT binding (Fig. 6A), and the effect involved an increase in NTR1 affinity without a significant change in NTR1 number (Fig. 6B; Table 1). These antioxidants also inhibited NT-induced IP formation (Fig. 6C), and the effect involved a dose-dependent decrease in NT efficacy (Fig. 6D). When tested together for effects on NT binding, the response to luteolin plus nimodipine and resveratrol plus nimodipine was additive at low doses of each agent, whereas they were less than additive at high doses (data not shown). Thus, polyphenolic antioxidants mimicked the effects of DHPs and seemed to act via the same pathway.
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Involvement of Sulfhydryl Groups. Because some antioxidants act by reducing sulfhydryl groups on proteins and because NT binding requires sulfhydryl groups associated with NTR1 (Mitra and Carraway, 1993), we tested the hypothesis that DHPs increase NT binding by maintaining sulfhydryl group(s) in a reduced state. Confirming the importance of sulfhydryl groups, we showed that sulfhydryl chelators, Ni3+ (IC50 of 50 µM) and Cd2+ (IC50 of 600 µM), inhibited NT binding to PC3 cells, and that their effects were inhibited by 2 mM DTT (data not shown).
However, in the basal state, the sulfhydryl group(s) required for NT binding to PC3 cells was primarily reduced, because NT binding was increased only slightly by 1 mM ascorbic acid (114 ± 7%; n = 4), 2 mM DTT (125 ± 8%; n = 4), and 5 mM N-acetyl-cysteine (107 ± 6%; n = 4). In addition, 2 mM DTT did not alter the NT displacement curve (Fig. 5A) and did not inhibit the effects of nifedipine. NT binding was enhanced similarly by 50 µM nifedipine in the presence and absence of 2 mM DTT (control, 246 ± 10%; DTT, 231 ± 11%; n = 4). NT-induced IP-formation was inhibited similarly by nifedipine in the presence and absence of DTT (Fig. 5B). Thus, DHPs acted by an antioxidant mechanism that did not involve the reduction of sulfhydryl groups in NTR1.
Involvement of Flavoprotein Dehydrogenase(s). Hypothesizing that DHPs might act by scavenging ROS produced by cellular flavoprotein dehydrogenases, we tested the effects of diphenylene iodonium (DPI), an inhibitor of these enzymes. DPI mimicked the effects of DHPs on NT binding (Fig. 6A) and NT-induced IP formation (Fig. 6C). The hydroxy-radical scavenger butylated hydroxy anisole (BHA) was also effective (Table 3). These results suggest that flavoprotein dehydrogenases and/or ROS produced by these enzymes participate in the effects of DHPs on NTR1 function.
Comparisons of Chemical Structures. The chemical structures of the DHPs and polyphenols were similar, each possessing aromatic ring structures with redox capability (Fig. 7). The order of potency (Table 3) for ability to alter NTR1 function (felodipine > nitrendipine 
nicardipine > nimodipine > nifedipine > luteolin > resveratrol) seemed to relate to donor group acidity (NH > OH) and to the number of conjugated double bonds. For DHPs, chloro substituents in the adjacent phenyl ring gave the highest activity (felodipine), whereas nitro in the meta position was less effective (nitrendipine, nicardipine, and nimodipine) and nitro in the ortho position was least effective (nifedipine). Luteolin and resveratrol contained conjugated 
-bonded rings, which could potentially support the stability of radicals and cations (Solomons, 1994). By donating hydrogen(s), DHPs could conceivably form pyridinium or pyridine analogs with an even greater number of conjugated double bonds and potential to support radical and cation formation. The very high membrane partition coefficients displayed by DHPs (Mason et al., 1999) could determine their ability to accumulate at target site(s).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). However, this effect may have been made unimportant by the overriding effects of DHPs on NT binding, which were clearly derived from the antioxidant property. Some of the DHP effect on NT binding (20%) was reproduced by treatments aimed to perturb Ca2+ channel structure (elevation of cellular [Ca2+] and membrane depolarization). Although this might have indicated that Ca2+ channels interact with NTR1, the simplest explanation was that these manipulations also acted along the antioxidant pathway.
Based on our finding that the enhancement of NT binding by nifedipine did not require Ca2+ and was not reversed by ionomycin, we concluded that DHPs did not act by diminishing Ca2+ influx. The direction of the Ca2+ flux was also unimportant, because DHP Ca2+ channel agonists and antagonists had similar effects. A second hypothesis considered was that Ca2+ channel occupation per se was sufficient to promote these effects, because Ca2+ channels were known to interact with G proteins (De Waard et al., 1997) and receptors (Grazzini et al., 1996). Postulating that perturbation of VGCC (Catterall, 2000) might alter NTR1 function, we tested the effects of K+ depolarization and agents known to alter cellular [Ca2+]. The effects observed were relatively small and it was unlikely that the far more robust responses to DHPs could be explained on this basis. Therefore, we hypothesized that DHPs altered NTR1 function by mechanism(s) not necessarily involving Ca2+ channels.
DHPs are commonly used at concentrations as high as 10 µM to block VGCC, although they are specific for this purpose only in the nanomolar range (Triggle, 2003). Above 1 µM, DHPs disrupt SOCC (Harper et al., 2003), Na+ channels (Yatani et al., 1988), and K+ channels (Hatano et al., 2003) and inhibit lipid peroxidation (Diaz-Araya et al., 1998). All of these were possible targets for the effects observed here, given that the IC50 value for inhibition of NT-induced IP formation ranged from 1 µM (felodipine) to 15 µM (nifedipine). Because Na+ was known to inhibit NT binding to cell membranes (Carraway et al., 1993), we tested the hypothesis that DHPs enhanced NT binding by blocking Na+ channels. When sucrose was substituted for NaCl, NT binding was enhanced, but the effects of nifedipine persisted. In agreement with the binding data, NT was more potent in promoting IP formation in the absence of Na+; however, this had no effect on the ability of nifedipine to inhibit the response to NT. This work and similar studies with K+ indicated that Na+ and K+ were not involved in the effects of nifedipine on NTR1 function, although a conformational coupling involving Na+ or K+ channels was still possible.
DHPs are antioxidants that inhibit lipid peroxidation and impart cytoprotective effects (Mak et al., 2002). Testing a series of DHPs with known antioxidant ability, we found that the activity order for ability to alter NTR1 function (felodipine > nicardipine > nimodipine > nifedipine) was similar to that reported for inhibition of lipid peroxidation (nicardipine nimodipine > nifedipine; Diaz-Araya et al., 1998) and for chemical reactivity with superoxide (felodipine > nimodipine > nifedipine; Ortiz et al., 2003). In addition, the effects of DHPs were mimicked by antioxidant polyphenols (luteolin and resveratrol), a hydroxy radical scavenger (BHA), and an inhibitor of flavoprotein oxidases (DPI). The IC50 values determined for these agents (Table 3) were in good agreement with values for antioxidant effects in other systems, e.g., luteolin (Hendricks et al., 2003), resveratrol (Leonard et al., 2003) and DPI (Brar et al., 2002). These findings support the hypothesis that DHPs act on NTR1 by a redox-sensitive mechanism, although the target(s) remain to be identified.
For each DHP and antioxidant that enhanced NT binding, there was an associated ability to inhibit NT-induced IP formation. The potency order for these drugs was the same in the two assays (Table 3) and the potency values were correlated (r2 = 0.58), indicating that NT binding and IP formation were similarly sensitive to the chemical properties of these drugs. DHPs might exert two separate effects (one to increase NT binding and another to inhibit IP formation), each having the same drug dependence. Alternatively, they could exert one effect (e.g., altering the state of NTR1) that determines the ability to bind NT and to activate signaling. It is also possible that by inhibiting IP formation, DHPs produce feedback effects on NT binding. Given that DHPs altered NTR1 function at concentrations near to the blood levels (0.2 µM) in patients receiving these drugs therapeutically (Palma-Aguirre et al., 1995) and below those used for in vitro work (Lopez et al., 1993; Triggle, 2003), these effects could be of clinical and pharmacological importance.
There are multiple mechanisms by which antioxidants might alter NTR1 function. Because NT binding to cell membranes is sulfhydryl-dependent, antioxidants might enhance NT binding by maintaining essential sulfhydryl groups in a reduced state. However, this hypothesis is not supported by our finding that cell-permeable sulfhydryl reducing agents (DTT and N-acetyl-cysteine) have little effect on NTR1 function and do not interfere with responses to nifedipine. A second possibility is that antioxidants disrupt signaling cascades that modulate NTR1 activity, for example, by protein phosphorylation. Phosphorylation of NTR1 can desensitize the receptor (Hermans and Maloteaux, 1998), and inhibition of this process might give enhanced binding. In some systems, phosphorylation of Gq is required for activation of PLC (Umemori et al., 1997). Inhibition of this process might diminish NT-induced IP formation. A third idea is that antioxidants might disrupt mitochondrial ATP production, causing secondary effects on GTP/GDP exchange or on kinases involved in NTR1 action.
Although the targets for the actions of DHPs on NTR1 function are not known, the scavenging of ROS is one possibility. Major sources of ROS include mitochondrial enzymes involved in oxidative metabolism (Kamata and Hirata, 1998) and plasma membrane NAD(P)H oxidases involved in signaling by tyrosine kinase receptors (Bae et al., 1997) and G protein-coupled receptors (Seshiah et al., 2002). Thus, ROS scavengers not only protect against oxidative injury but also they inhibit signal transduction involved in inflammation and cell growth (Lassegue and Clempus, 2003). Because NT can induce inflammation and regulate cell growth (Seethalakshmi et al., 1997), it is possible that NT signaling involves ROS. Preliminary data shows that DHPs and polyphenols inhibit NT-induced DNA synthesis (S. Hassan and R. E. Carraway, unpublished data). It also may be important that PKC, which mediates some NT effects (Vincent et al., 1999), is implicated in the activation of NAD(P)H oxidases (Brodie and Blumberg, 2003). Because polyphenolic antioxidants can inhibit protein kinase C (Ferriola et al., 1989), one can imagine multiple mechanisms by which these drugs might disrupt NTR1 function.
The chemical structures of DHPs and polyphenols contain two aromatic ring systems with a number of conjugated double bonds and redox reactive NH or OH group(s) (Fig. 7). These features seem to be essential for this activity, given that equally powerful antioxidants lacking these structures are totally ineffective. Notable is the inactivity of -tocoferol, which is a standard for many assays (Mitchell et al., 1998). In general, DHPs are more potent than polyphenols in altering NTR1 function, and this may be related to the acidity of the hydrogen donor (NH > OH). The activity of compounds within each group also varies (Table 3), and this might be due to the influence of phenyl ring substituents on donor acidity and on resonance stabilization of reaction intermediates (Solomons, 1994). Another important determinant of reactivity could be the membrane partition coefficient (Mason et al., 1999).
The chemical structures of DHPs are reminiscent of NADH. The reaction scheme whereby NAD-linked dehydrogenases donate hydrogen atoms to substrates is shown in Fig. 8A. One hydrogen is transferred from NADH as a hydride ion (H-) and another is taken as H+ from the medium (Lehninger, 1982). It is tempting to speculate that DHPs can react analogously, transferring hydrogen atoms to superoxide by way of cationic (Fig. 8B) or radical intermediates (Fig. 8C) to generate pyridine derivatives and water. DHPs are known to form pyridine adducts when reacted with alkyl radicals (Nunez-Vergara et al., 2003). Because the stability of the intermediates in Fig. 8 is negatively affected by electron withdrawal, this predicts that nitro groups in the phenyl ring (especially ortho) would diminish reactivity. The order derived from such considerations (felodipine > nitrendipine nicardipine nimodipine > nifedipine) is in fair agreement with that measured by Ortiz et al. (2003) and that found here for altering NTR1 function. Because nitrendipine, nicardipine, and nimodipine each have nitro in the meta position, a near equal reactivity with superoxide is expected. The differences in their activity in our system might be attributed to the effects of other ring substituents on lipophilicity (Fig. 7), which could affect their ability to enter cells and partition into membranes.
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DHPs inhibit cardiac contractility and relax vascular smooth muscle, and their relative abilities to do so vary >10-fold. Felodipine is 10-times more vascular-selective than nifedipine (Triggle, 2003). Although this might be due to differential expression of various Ca2+ channels, it is tempting to speculate that the antioxidative effects of DHPs also contribute. For example, if the relaxant effects of DHPs on vascular smooth muscle involve ROS signaling, this might explain the enhanced activity of felodipine relative to nifedipine.
In conclusion, DHPs enhance NT binding and inhibit NT-induced IP formation by an indirect mechanism that seems to require an aromatic structure and functional groups to facilitate hydrogen atom donation. For a series of DHPs, the ability to alter NTR1 function correlates to the ability to scavenge superoxide anion. Polyphenolic antioxidants and an inhibitor of flavoprotein oxidases mimic the effects of DHPs. We propose that DHPs disrupt NTR1 function by inhibiting cellular oxidative reaction(s) or by scavenging ROS involved in receptor regulation and signal transduction.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: NT, neurotensin; NTR1, neurotensin receptor subtype 1; PLC, phosphatidylinositol-specific phospholipase C; IP, inositol phosphate; CCB, Ca2+ channel blocker; VGCC, voltage-gated Ca2+ channel; DHP, 1,4-dihydropyridine; SOCC, store-operated Ca2+ channel; DTT, dithiothreitol; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; BSA, bovine serum albumin; ROS, reactive oxygen species; DPI, diphenylene iodonium; BHA, butylated hydroxy anisole; SKF-96365, 1-{-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole; FPL-64176, 2,5-dimethyl-4-[2-(phenylmethyl)benzoyl]-1H-pyrrole-3-carboxylic acid methyl ester; (-)-BayK-8644, S(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4[2-(trifluoromethyl)phenyl]-3-pyridine-carboxylic acid methyl ester.
Address correspondence to: Dr. Robert E. Carraway, Department of Physiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA, 01655. E-mail: robert.carraway{at}umassmed.edu
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0.3 nM, IP formation for each treatment was significantly different from the control. In A, C, and D, results were from at least three experiments for each plot. B, typical result for an experiment that was repeated twice.
) forming a pyridinium cation intermediate that further donates H+ to the peroxide anion. In C, a single electron transfer is shown forming a neutral radical intermediate. In both cases, a pyridine derivative and water are the final products. For simplicity, we show the dihydropyridine ring with the attached phenyl group (Ph), but the various substituents are omitted.